THE MECHANICS OF NUCLEAR SHAPING IN CELL
By
YUAN LI
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2017
© 2017 Yuan Li
To my beloved parents
4
ACKNOWLEDGMENTS
I would like to acknowledge and thank the people who supported me during this
crucial time of my life. The first and most important one is my advisor, Dr. Tanmay Lele,
who provided me guidance and support with great patience and attention during my
whole time in the lab. He helped me to improve my logical and critical thinking as an
independent researcher. His interesting ideas inspired me to build my original idea and
maintained my motivation on research projects. He did his best to offer a healthy and
collaborative environment to perform research. I will always be grateful to him.
I would like to thank my co-advisor Dr. Richard Dickinson who gave a lot of
helpful advices and comments on the research projects. He was always willing to meet
and discuss with me regarding the research questions, and his expertise in
mathematics modeling helped a lot on my project. I would also like to thank my other
committee members Dr. Alexander Ishov and Dr. Rinaldi Carlos for their support on
experiment and encouraging words. I would like to thank Dr. Gregg Gundersen, Dr.
Jeffery Nickerson and Dr. Kyle Roux with whom I collaborated on different projects.
They always responded to my questions timely with their expertise and provided
important plasmids and engineered cell lines that promoted my research.
I would like to thank my former lab mates Dr. Jun Wu, Dr. TJ chancellor and Dr.
David Lovett who trained me on all basic techniques in the lab and helped me design
experiments in the beginning of my research. I would like to thank my former and
current lab mates Dr. Srujana Neelem, Dr. Samer Alam, Dr. Ian Kent and Dr. Varun
Agrawal, Qiao Zhang, VJ Tocco, Keith Christopher and Andrew Tamashunas, with
whom I spent almost my lab time. The valuable discussions and interesting chats with
them gave me a helpful and fun lab environment to work in. I would also like to thank
5
Dr. Jun Yin and Dr. Shen-Hsiu Hung from Dr. Yiider Tseng’s Lab for providing their
experienced ideas and experiment facilities. I would also like to thank the former master
students Nandini Shekhar, Agnes Mendonca and Anirudh Ram and undergraduate
students Catherine Perez, Alaina Giacobbe, Aniruddha Shirhatti and Uday Rallabhandi
who worked with me on various projects. I would like to thank all my UF friends who
encouraged and helped me to go through this adventure of research.
Finally, I would like to thank my family members who are my strongest support to
achieve my goals. My mother Yinchi Liu always encouraged me to bravely fight with
difficulties in life and to never give up my dreams. Her support and sacrifice throughout
these years made my dream come true. My father Heng Li taught me to keep a
peaceful mind no matter what happens around you. It helped me survive from the two
hard times throughout this pursuing journey. I would also like to thank my other family
members, especially my beloved grandparents who made me feel loved and were
always proud of me.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 8
LIST OF FIGURES .......................................................................................................... 9
LIST OF ABBREVIATIONS ........................................................................................... 11
ABSTRACT ................................................................................................................... 13
CHAPTER
1 INTRODUCTION .................................................................................................... 15
2 MOVING CELL BOUNDARY DRIVES NUCLEAR FLATTENING DURING CELL SPREADING ........................................................................................................... 21
Materials and Methods............................................................................................ 22 Cell Culture, Plasmids and Drug Treatment ..................................................... 22
Cell Spreading and Trysinization Assay ........................................................... 22 Fixation and Immunocytochemistry .................................................................. 23
Protein Silencing .............................................................................................. 24
Western Blotting ............................................................................................... 24
Microscopy and Image Analysis ....................................................................... 25 Results .................................................................................................................... 25
Collapse of Apical Nuclear Surface Contributes to Nuclear Flattening during Early Cell Spreading ..................................................................................... 25
Nuclear Flattening does Not Require Actomyosin Contraction in Spreading Cells .............................................................................................................. 27
Intermediate Filaments and Microtubules are Dispensable for Nuclear Flattening in Spreading Cells ........................................................................ 29
Apical and Basal Actomyosin Bundles are Not Required for Nuclear Flattening during Initial Cell Spreading .......................................................... 30
Nuclear Flattening can be Reversed by Detachment of the Cell from the Substratum .................................................................................................... 31
The LINC Complex is Not Required for Nuclear Flattening .............................. 32 A Mathematical Model for Nuclear Flattening and Cell Spreading ................... 33
Discussion .............................................................................................................. 39
3 DYNAMIC DEFORMATION OF THE CELL PLASTICALLY SHAPES THE NUCLEUS AND AMPLIFIES CANCER NUCLEAR IRREGULARITIES.................. 71
Materials and Methods............................................................................................ 72
Cell Culture and Transfection ........................................................................... 72
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Cell Staining and Drug Treatment .................................................................... 73 Nuclear Excision ............................................................................................... 74
Cell Spreading Assay ....................................................................................... 74 Imaging and Image Analysis ............................................................................ 74 High-Content Imaging ...................................................................................... 75
Results .................................................................................................................... 75 The Deforming Cell Shape Amplifies Cancer Nuclear Abnormalities ............... 75
Disrupting Either the LINC Complex or the Cytoskeleton Dampens Cancer Nuclear Abnormality ...................................................................................... 76
The Reduction of Nuclear Abnormality by LINC Complex Disruption Impairs Cellular Motility .............................................................................................. 77
Abnormal Morphology of Cancer Nucleus do NOT Necessarily Reflect Chromatin Content ........................................................................................ 77
Nuclear Abnormality can be Inherited by Offspring .......................................... 78 Discussion .............................................................................................................. 79
Nuclear Abnormalities in Cancer. ..................................................................... 79
Mechanical Stress and Cancer Cell Migration. ................................................. 81
4 CONCLUSIONS ..................................................................................................... 92
Summary of Findings .............................................................................................. 92
Future Work ............................................................................................................ 94
APPENDIX
A COMPUTATIONAL MODEL FOR NUCLEAR DEFORMATION DURING CELL SPREADING ........................................................................................................... 99
Constitutive Model for Cytoskeletal Network Stress ......................................... 99
Model for Cell Mechanics ............................................................................... 101 Model Parameters .......................................................................................... 103
Methods for Simulating Cell Spreading .......................................................... 105
B THE INFLUENCE OF CELL GEOMETRY ON NUCLEAR VOLUME ................... 108
LIST OF REFERENCES ............................................................................................. 111
BIOGRAPHICAL SKETCH .......................................................................................... 121
8
LIST OF TABLES
Table page A-1 Parameters of Cell Spreading Model ................................................................ 107
9
LIST OF FIGURES
Figure page 1-1 Representative IF images of nuclear morphology .............................................. 19
1-2 Illustrating cartoon of LINC complex. .................................................................. 20
2-1 The dynamics of nuclear flattening during early cell spreading. ......................... 43
2-2 The nuclear deformation causes nuclear flattening but not already-flat nucleus toppling onto their side .......................................................................... 44
2-3 The dynamics of nuclear flattening against substratum is not influenced by gravity ................................................................................................................. 45
2-4 Nuclei does not flatten when cell spreading is prevented by inhibitors of actin assembly ............................................................................................................ 46
2-5 The nucleus flattens completely in a partially spread cell ................................... 47
2-6 The apical surface of cell was separated from the apical surface of nucleus in the early stage of cell spreading ......................................................................... 48
2-7 Nuclear flattening is independent of actomyosin contraction .............................. 49
2-8 The effect of myosin inhibition on nuclear shape in well spread cell ................... 51
2-9 Inhibition of myosin activity with Y-27632 does not alter the qualitative features of dynamic nuclear flattening during cell spreading ............................ 53
2-10 Blebbistatin prevents cell spreading at later times resulting in final nuclear rounding ............................................................................................................. 54
2-11 The absence of intermediate filaments does not prevent nuclear flattening during cell spreading .......................................................................................... 55
2-12 Disruption of microtubules by nocodazole rounds up the nucleus but also prevents cell spreading ....................................................................................... 56
2-13 The absence of microtubule does not prevent nuclear flattening during cell spreading ............................................................................................................ 57
2-14 Apical and basal actomyosin bundles are not required for nuclear flattening during initial cell spreading ................................................................................. 58
2-15 Nuclear flattening can be reversed by detachment of the cell from the substratum. ......................................................................................................... 60
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2-16 The LINC complex is not required for nuclear flattening during cell spreading ... 61
2-17 GFP-KASH4 overexpression slows down but does not prevent cell spreading and nuclear flattening ......................................................................................... 63
2-18 Nuclei in lamin A/C -/- MEFs flatten faster than WT cells ................................... 65
2-19 Mathematical model for nuclear deformation during cell spreading .................... 67
2-20 Snapshots of simulation results for different parameters .................................... 69
3-1 The abnormality of nucleus amplifies during cell spreading ............................... 83
3-2 Disrupting the LINC complex reduces nuclear abnormality ................................ 85
3-3 Disrupting cytoskeletal elements reduces nuclear abnormality. ......................... 86
3-4 Trajectory maps of MDA-MB-231 cells with or without LINC disruption .............. 87
3-5 LINC complex disruption impairs cellular motility ................................................ 88
3-6 The abnormal shape of nucleus does not correlate with DNA content. .............. 89
3-7 The abnormality of nuclear shape is heritable .................................................... 91
4-1 Data pool of x-z nuclear aspect ratio versus cell spreading area........................ 97
4-2 Local nuclear deformation in response to local protrusion and retraction of cell memrbane .................................................................................................... 98
B-1 The difference of nuclear volume induced by cell geometry vanishes after the removal of geometry constrain ......................................................................... 109
11
LIST OF ABBREVIATIONS
3T3 3-day Transfer, Inoculum 3x105 Cells, a mouse embryonic fibroblast cell Line
Blebb Blebbistatin
Colc Colcemid
Crtl Control
Cyto D Cytochalasin D
DBS Donor Bovine Serum
DIC Differential Interference Contrast
DMEM Dulbecco’s Modified Eagle’s Medium
EGF Epidermal Growth Factor
EGFP Enhanced Green Fluorescent Protein
FN Fibronectin
FRAP Fluorescence Recovery After Photo-bleaching
GFP Green Fluorescent Protein
IF Intermediate Filament
INM Inner Nuclear Membrane
KASH Klarsicht/Anc-1/Syne Homology
KD knockdown
KDEL Lysine/Aspartic acid/Glutamic acid/Leucine
LAP Lamin Associated Protein
Lat A/B Latrunculin A/B
LINC Linker of nucleoskeleton to cytoskeleton
MEF Mouse embryonic fibroblast
MEM Modified Eagle’s Medium
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mRNA Messenger RNA
MSD Mean Square Displacement
MT Microtubule
n Sample number
N2G Nesprin 2 giant
NA Numerical aperture
NE Nuclear envelope
NIH National Institutes of Health
nM Nanomolar
Noco Nocodazole
ONM Outer Nuclear Membrane
p p-value of statistic test
RFP Red Fluorescent Protein
SEM Standard Error of Mean
shRNA Small hairpin RNA
STD Standard deviation
SUN Sad1p, UNC-84
TAN Transmembrane Actin-associated Nuclear
Vim Vimentin
w/v Weight per volume
WT Wild type
Y27 Y27632
μM Micro molar
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
THE MECHANICS OF NUCLEAR SHAPING IN CELL
By
Yuan Li
August 2017
Chair: Tanmay Lele Major: Chemical Engineering
The nucleus has a smooth, regular appearance in normal cells, and its shape is
greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not
well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts.
Nuclei translated toward the substratum and began flattening during the early stages of
cell spreading. Initially, nuclear height and width correlated with the degree of cell
spreading, but over time, reached steady-state values even as the cell continued to
spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate
filaments, as well as the LINC complex, were all dispensable for nuclear flattening as
long as the cell could spread. Together, these results show that cell spreading is
necessary and sufficient to drive nuclear flattening under a wide range of conditions,
including in the presence or absence of myosin activity. To explain this observation, we
propose a computational model for nuclear and cell mechanics that shows how frictional
transmission of stress from the moving cell boundaries to the nuclear surface shapes
the nucleus during early cell spreading. Our results point to a surprisingly simple
mechanical system in cells for establishing nuclear shapes.
14
Consistent with the above results, researchers in the Lele lab found that
deformed shapes of nuclei are unchanged even after removal of the cell with micro-
dissection, both for smooth, elongated nuclei in fibroblasts and abnormally shaped
nuclei in breast cancer cells. The lack of shape relaxation implies that the nuclear shape
in spread cells does not store any elastic energy, and the cellular stresses that deform
the nucleus are dissipative, not static. Building on these results, we show that during
cell spreading, the deviation of the nucleus from a convex shape increases in MDA-MB-
231 cancer cells, but decreases in MCF-10A cells. Cancer nuclear abnormalities are
uncorrelated with the amount of DNA in cells. We propose that motion of cell
boundaries exert a stress on the cancer nucleus and this amplifies nuclear
abnormalities. Finally, we report the novel finding that disrupting the LINC complex,
which physically links the nucleus to the cytoskeleton, normalizes cancer nuclear shape
and decreases cancer cell migration.
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CHAPTER 1 INTRODUCTION
The nucleus was described and characterized back in the 1800s [1]. Typically,
the nucleus has a smooth elliptical appearance in healthy cells. Nuclear shape becomes
altered in a number of diseases like cancers [2-4] and laminopathies [5, 6] (Figure 1-1).
In pathology, the correlation between changed morphology of nuclei and certain
diseases has been observed for a long time [7]. Yet, the mechanism by which cell
establishes the shape of nucleus, and how the nuclear shape becomes abnormal in
pathologies has remained poorly understood.
The nuclear lamina, a complex structure of A- and B-type lamins [8] and lamin-
associated proteins (LAPs) [9], is a key mechanical shell that surrounds chromatin and
is necessary for stable nuclear shape [10, 11]. Chromatin is anchored to the nuclear
lamina either directly [12] or indirectly through binding with LAPs [13]. Changes to the
shape of the nucleus as observed in pathologies typically are accompanied by down-
regulation of nuclear lamins [14-16]. Such changes can alter the spatial configuration of
chromatin and consequently modulate gene expression [17-19]. Access of chromatin to
transcription factors may be influenced by nuclear shape which can also change gene
expression [20]. Thus, alterations of nuclear shape might themselves be contributors to
the progression of pathologies like cancer.
Cytoskeletal forces are exerted on the nucleus and can cause changes in
nuclear shape and position. The cytoskeletal network consists of intertwining filaments
and tubules throughout the cell [21]. Microfilaments or F-actin filaments are the smallest
components of the cytoskeleton with a diameter of 7 nm. Interacting with myosin II
motor proteins and cross linker proteins, actin filaments form a structure named the
16
actomyosin cytoskeleton, which generates contractile force in cells. Compression driven
by contraction of actomyosin bundles overlaying the surface of the nucleus has been
proposed to establish the flattened nuclear shapes [22] commonly observed in cell
culture. As we will show in this thesis however, such bundles are absent during nuclear
flattening, and myosin activity is not required for nuclear flattening. Other studies have
proposed that actomyosin exerts tensile force on the nucleus to modulate the nuclear
shape [23, 24]. Actin retrograde flow can also exert shear forces that move the nucleus
[25, 26].
Microtubules are tubular structures with an outer diameter of 25 nm, which
enable motor proteins to walk on and transport molecules throughout the cytoplasm.
Two motor proteins mainly interact with microtubules. Dynein walks toward the minus (-)
end of microtubules; and kinesin walks towards plus (+) end. Both these proteins bind to
the nesprin family proteins [27, 28] which are embedded in the outer nuclear
membrane. In this way, microtubule motors can exert force on the nuclear surface
which has been shown to move it as well as rotate it in [29, 30]. We show in this thesis
that microtubules are dispensable for nuclear flattening, contradicting previous reports
[31].
Cytoplasmic intermediate filaments (IFs) are stable rod-like fibers made of
vimentin in fibroblasts with a diameter of 10 nm. IFs are unable to generate intercellular
force; however, IFs can transmit force indirectly form actomyosin to the nuclear
membrane by interacting with actin filaments [32]. Also, IF networks can wrap around
the nucleus and resist any changes in nuclear shape passively [33]. We show that
intermediate filaments are not required for nuclear flattening.
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The so-called LINC (Linker of Nucleoskeleton and Cytoskeleton) complex
transmits mechanical stresses to the nuclear surface. The LINC complex mainly
includes outer nuclear membrane nesprin proteins and inner nuclear membrane SUN
proteins (Figure 1-2). KASH domains of the nesprin proteins bind to SUN trimers in the
perinuclear space, while the extra-nuclear domains of nesprins bind to cytoskeletal
elements. On the nucleoplasmic side, SUN proteins bind to the nuclear lamina and thus
complete the linkage between nucleus and the cytoskeleton [34-36]. Disrupting the
LINC complex disrupts force transmission between the cytoskeleton and nucleus [23,
37]. Therefore, it is possible that the LINC complex transmits mechanical stresses that
shape the nucleus, and others have argued that this is the case [38]. However, here we
show that disrupting the LINC complex slows down the process of cell spreading and
nuclear flattening; presumably because stresses are not efficiently transmitted to the
nuclear surface but does not prevent nuclear flattening.
Overall, in this thesis, we addressed two broad questions: 1) how is the nucleus
shaped in the cell and 2) how does the nucleus become abnormally shaped in cancer?
In chapter 2, we report the discovery of a novel mechanism for nuclear shaping that
contradicts previously explanations proposed in the field. We show that in fibroblasts,
cell spreading is necessary and sufficient to drive nuclear flattening under a wide range
of conditions. We propose a model in which frictional transmission of stress from the
moving cell boundaries to the nuclear surface shapes the nucleus during early cell
spreading. In chapter 3, we report that abnormalities in cancer nuclear shape are
amplified by stresses generated by the motion of cell boundaries during spreading, and
that disruption of the LINC complex and cytoskeleton-based forces reduce nuclear
18
shape abnormalities. We also show that LINC disruption reduces cancer cell motility.
Collectively, we have proposed a new mechanism for how the nucleus shapes the cell,
and how it becomes abnormal in pathologies.
19
Figure 1-1. Representative IF images of nuclear morphology in breast cancer cell line
(MDA-MB-231) compared with normal breast cell line (MCF-10A) and Hutchinson-Gilford progeria syndrome (HGPS) cell compared with normal cell [39]; fluorescence label is laminA and laminA/C, respectively.
20
Figure 1-2. Illustrating cartoon of LINC complex. Main components of LINC complex
are denoted in the picture with corresponding color of the structure.
21
CHAPTER 2 MOVING CELL BOUNDARY DRIVES NUCLEAR FLATTENING DURING CELL
SPREADING
The nucleus, the largest organelle in mammalian cells, has a smooth, regular
appearance in normal cells. However, nuclear shape becomes altered in a few
pathologies such as cancer [40-44] and in laminopathies [15, 39, 45, 46]. The control of
nuclear shapes for cells is particularly important because nuclear shape may directly
control gene expression [17-19]. How the cell shapes the nucleus is not understood.
Given the high rigidity of the nucleus, significant and dynamic changes in nuclear
shape are expected to require forces that far exceed thermal forces in the cell. Such
forces likely originate in the cytoskeleton, which is known to link to the nuclear surface
through the LINC complex (for linker of nucleoskeleton to cytoskeleton) [34, 47, 48].
Candidates for shaping the nucleus include microtubule motors which can shear the
nuclear surface [29, 30] and intermediate filaments that can passively resist nuclear
shape changes by packing around the nuclear envelope or transmitting forces from
actomyosin contraction to the nuclear surface [33, 49]. The actomyosin cytoskeleton
which can push [22], pull [23, 24] or shear and drag the nuclear surface [25, 26] is also
assumed to be a significant component of the nuclear shaping machinery in the cell.
Here, using a combination of experiments to disrupt the cytoskeleton and the
LINC complex, and mathematical modeling and computation, we show that the motion
of cell boundaries drives changes in nuclear shapes during cell spreading. Our results
Reprinted from Biophysical Journal, volume 109, Li, Y., Lovett, D., Zhang, Q., Neelam, S., Kuchibhotla, R.A., Zhu, R.J., Gundersen, G.G., Lele, T.P., and Dickinson, R.B., Moving Cell Boundaries Drive Nuclear Shaping during Cell Spreading, pages 670-686, Copyright (2015), with permission from Elsevier.
22
point to a surprisingly simple mechanical system in cells for establishing nuclear
shapes.
Materials and Methods
Cell Culture, Plasmids and Drug Treatment
NIH 3T3 fibroblasts were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) with 4.5g/L glucose (Mediatech, Manassas, VA) supplemented with 10% donor
bovine serum (DBS, Gibco, Grand Island, NY) and 1% Penicillin Streptomycin
(Mediatech, Manassas, VA). MEFs were cultured in DMEM, supplemented with 10%
DBS. All cells were maintained at 37°C in a humidified 5% CO2 environment with
passage at 80% confluence. For microscopy, cells were transferred onto 35 mm glass-
bottom dishes (World precision Instrument, Sarasota, FL) treated with 5 µg/ml
fibronectin (BD Biosciences) at 10% confluence. Transient transfection of plasmids into
cells was performed with Lipofectamine 2000 reagent (Life Technologies/Invitrogen,
Carlsbad, CA) in OPTI-MEM media (Life Technologies/Invitrogen, Carlsbad, CA). For
drug treatment studies, Y-27632 (EMD Millipore, Billerica, MA), Blebbistatin (EMD
Millipore, Billerica, MA) or ML-7 (Sigma-Aldrich) was added to the cells for inhibiting
myosin activity at concentrations of 25 µM, 50 µM and 25 µM, respectively. Nocodazole
or Colcemid (Sigma-Aldrich) was used to disrupt microtubules at a concentration of 0.83
µM and 0.27 µM, respectively. To disrupt F-actin polymerization, cells were treated with
2 µM Cytochalasin D (Biomol, Plymouth Meeting, Pennsylvania) and 5 µM Latrunculin A
(Cayman Chemical, Ann Arbor, Michigan).
Cell Spreading and Trysinization Assay
In the cell spreading assay, cells were trypsinized and then seeded onto
fibronectin coated glass-bottomed dishes. They were next incubated at 37°C in 5% CO2
23
for varying times and then fixed with 4% paraformaldehyde for 20 minutes. For myosin
inhibition and disruption of F-actin and microtubule, the cells were pre-treated with the
appropriate dose of drug for 1 hour, trypsinized and re-suspended in cell culture
medium containing the same dose of drug. They were next seeded in the presence of
the drug for varying time before fixation. In other drug treatment experiments, cells were
allowed to grow on FN coated glass-bottom dishes for 24 hours. They were then
incubated with drug-containing medium for 1 or 2 hours, after which the cells were fixed
and stained. For cell spreading with inverted coverslips, 18mm coverslips were coated
with FN overnight in first day and next day cells were trypsinized and seeded on the
coverslips for 5min to attach with the substratum. Then the coverslips were put into 12-
wells upside down with tweezer very carefully and stayed in the dish for certain time (for
example, 10min, 30min etc.) before fixing and staining. In the trysinization assay, cells
were transfected with GFP-histone H1.1 and RFP-LifeAct (Ibidi, Verona, WI), and
cultured on fibronectin-coated glass-bottom dishes for 24 hours. After placing the dish
onto the microscope stage, the culture medium was removed and the dish was washed
once very gently with phosphate buffered saline (PBS). Then 0.25% (w/v) trypsin (high
concentration) or 0.08% (w/v) trypsin in serum-free medium (low concentration) were
added to detach the cells from the substratum.
Fixation and Immunocytochemistry
Cells were first fixed with 4% (m/v) paraformaldehyde (Electron Microscopy
Sciences, Hatfield, PA) for 20 minutes, and then mounted with ProLong Gold Antifade
Mountant (Life Technologies). To visualize F-actin and nuclei, the fixed cells were
incubated with 1:40 Alexa Fluor 488 phalloidin (Invitrogen) and 1:100 Hoechst 33342
(Life Technologies) for 1 hour at room temperature, respectively. To immune-stain
24
microtubules, cells were first treated with microtubule extraction buffer containing 0.5%
(m/v) glutaraldehyde, 0.8% formaldehyde and 0.5% Triton X-100 in phosphate buffered
saline (PBS) for 3 minutes before fixing with 1% (m/v) paraformaldehyde for another 10
minutes. Then a freshly prepared 1% (w/v) sodium borohydride in PBS solution was
added to the cells for 10 minutes followed by blocking in 1% (m/v) BSA in PBS. The
cells were then incubated in 4°C overnight with rabbit polyclonal antibody to α-tubulin
(1:1000, Abcam, Cambridge, MA) in 1% BSA containing solution, washed with PBS and
then incubated with Goat Anti-Rabbit IgG (H+L) antibody (1:500, Life Technologies) at
room temperature for 1 hour.
Protein Silencing
Short-hairpin RNAs targeted to SUN2 (5’-tcggatcttcctcaggctatt-3’), Nesprin-2G
3’UTR (5’-gcacgtaaatgacctatat-3’) or Luciferase (5’-gtgcgttgttagtactaatcctattt-3’) were
PCR and cloned into retroviral plasmid vector. 293T cells were transfected with the
plasmid and pseudo-typed envelope proteins to make virus by calcium phosphate
transfection. Viruses were harvested 24 hrs after transfection. Then NIH 3T3 cells
(0.5x105 cells/12 well) were infected by virus (0.6 ml virus) with 4 µg/ml polybrene and
re-plated the second day. Cell culture media were changed to DBS DMEM overnight
before cell spreading assay
Western Blotting
Transfected samples were harvested with 1X Laemmli sample buffer and boiled
at 95 ˚C for 10 min. Amersham Protran Premium 0.2 NC (10600004 GE Healthcare,
Little Chalfont, United Kingdom) membrane was used for protein transference. Primary
antibodies to probe proteins of interest in Western Blot were rabbit anti-SUN2, rabbit
anti-Nesprin-2G [26], mouse anti-GAPDH (clone 6C5, Life Technologies, Carlsbad, CA),
25
LI-COR IRDye 680RD donkey anti-rabbit IgG (926-68073) and IRDye 800CW donkey
anti-mouse IgG (926-32212) were used. Signals were detected by Odyssey LI-COR
system (LI-COR Biosciences, Lincoln, NE). Images were processed by ImageJ (NIH).
Microscopy and Image Analysis
Fixed cells were imaged on a Nikon A1 laser scanning confocal microscope
system (Nikon, Melville, NY) with 60X/1.40NA oil immersion objective. The live cell
imaging was conducted on the same system within the environment of 37°C and 5%
CO2. For measuring the nuclear height, z-stacks were taken at an interval of 0.3 µm
and the x-z view projections were reconstructed using the NIS Elements application
(Nikon, Melville, NY). The maximum projection intensity analysis was applied to the x-z
images of the stained nucleus, and the top and bottom edges of the nucleus were
determined with the full width at half maximum (FWHM) technique [50] in MATLAB (The
MathWorks, Natick, MA). The height was calculated as the distance between the top
and bottom nuclear edge. Nuclear x-y dimensions (major and minor axis) were
measured using ImageJ (NIH). The aspect ratio was calculated as the height divided by
the length of the major axis in the x-y plan. The nuclear volume measurements were
performed using Volocity Demo (Perkin Elmer, Akron, Ohio).
Results
Collapse of Apical Nuclear Surface Contributes to Nuclear Flattening during Early Cell Spreading
We used x-z laser scanning confocal fluorescence microscopy of NIH 3T3
fibroblasts expressing GFP-histone to prepare time-lapse images of the nucleus as they
settled from suspension onto a fibronectin-coated glass dish. Three distinct nuclear
behaviors could in general be discerned during the spreading. First, the nucleus
26
translated toward the base of the cell and the lower surface of the nucleus began to
flatten against the substratum in the first few minutes of attachment (Figure 1-1A). The
speed of initial translation of the nucleus toward the substratum was surprisingly fast --
about twenty-fold faster than would be expected from gravitational settling (based on
the assumed cytoskeleton viscosity in Table A1). Next, the top surface of the nucleus
collapsed while the length of the flattened bottom surface stayed roughly constant. This
change in the nuclear shape happened over a duration of 5-6 minutes (Figure 1-1B). In
the third phase, the collapsed nucleus increased in width at nearly constant height
(Figure 1-1C; see Figure 1-1D for quantification of the shape in Figure 1-1A, B and C;
these findings were consistent among five other live cell imaging experiments; see
Figure 2-2 A and B for examples). By constructing x-z nuclear shapes from different
view angles, we confirmed that the flat nuclear shapes were due to deformation of the
nucleus instead of already-flat nuclei toppling onto their sides (Figure 2-2). In addition,
by inverting the substrate on which the cells spread, we confirmed that gravity did not
affect the initial nuclear translation to the substratum nor the rate of the subsequent
flattening (Figure 2-3 B-D). In addition, nuclear flattening only occurred during cell
spreading; inhibition of actin assembly and cell spreading by cytochalasin D or
latrunculin A prevented nuclear flattening, as quantified by the aspect ratio (height/major
axis length) (Figure 2-4 A-C).
Although cells spread continuously in the first 60 minutes increasing their spread
area from less than 200 μm2 to nearly 1400 μm2 (Figure 2-5A and B), the nuclei
flattened and reached a steady-state height early during cell spreading in the first 20-30
minutes, when the cell itself had only spread to less than 50% of its final area (Figure 2-
27
5A and B). Nuclear width stayed roughly constant over the first 20 minutes (Figure 2-
5C) and then increased steadily as the cell spread. As seen in Figure 2-5D, the aspect
ratio decreased to around 0.25 by 30 minutes, indicative of a ‘flat’ nucleus with a width
that is four times its height. We also observed that there was a separation between the
nuclear surface and the cell membrane (Figure 2-6A and B), which increased slightly
and then decreased over time as the cell spread and the nucleus flattened (reaching a
peak separation of around 2μm). The increase in the separation coincided with the
collapse of the top nuclear surface (compare Figure 2-5B and 2-6B).
Nuclear Flattening does Not Require Actomyosin Contraction in Spreading Cells
We pre-treated well-spread cells with three different inhibitors: Y-27632, a ROCK
inhibitor, ML-7, a myosin light chain kinase (MLCK) inhibitor or blebbistatin, a direct
inhibitor of myosin II activity. Our approach was to pretreat cells for 1 hour at the
appropriate dose, trypsinize cells, and to allow them to attach for 1 hour in medium
containing the inhibitor (Figure 2-7A). Neither blebbistatin (50 µM) nor Y-27632 (25 µM)
interfered with the nuclear flattening process observed in normal cells (Figure 2-7A-B,
E-H), suggesting that myosin II activity is not required for nuclear flattening. However,
ML-7 (25 µM) treatment interfered with both nuclear flattening and cell spreading
(Figure 2-7A and B). To understand the differential effects of the drugs on nuclear
flattening, we measured the area of cell spreading (Figure 2-7C) and correlated nuclear
aspect ratio with the cell spreading area (Figure 2-7D; correlations between nuclear
height and width with cell spreading area are in Figure 2-7G and H). The degree of cell
spreading decreased dramatically in ML-7 cells (Figure 2-7C and D). This revealed a
potential reason for the differential effects: ML-7 treatment prevented cell spreading
while Y-27632 or blebbistatin treatment altered cell shapes but did not prevent cell
28
spreading. Nuclear aspect ratios and cell spreading areas were comparable in
untreated control cells at 15 minutes (Figure 2-5D) and ML-7 treated cells (Figure 2-7B
and C). Taken together, these data suggest that the degree of cell spreading appears to
be a predictor of nuclear flattening in myosin inhibited cells. We and others have shown
in the past that inhibiting myosin activity in well-spread cells rounds the nucleus [23, 24].
In consideration of the above results, we examined the effect of the three myosin
inhibitors on nuclear height in well-spread cells. Myosin inhibition again resulted in a
rounded nucleus only when the cell was rounded by the action of the drug (Figure 2-
8A). In these experiments, blebbistatin (but not Y-27632 and ML-7) treatment resulted in
rounded cell morphologies; only blebbistatin treated cells showed rounded nuclear x-z
cross-sections (Figure 2-8 B-H, correlations between nuclear height and width with cell
spreading area are in Figure 2-8 G and H).
We next examined whether myosin inhibition altered aspects of nuclear
flattening, such as the initial collapse of the top surface. Inhibiting myosin with Y-27632
did not change the qualitative nature of the drop in nuclear height (except for an initial
lag time where the Y-27632 treated cell is unable to spread and the nucleus doesn’t
flatten in that time, Figure 2-9 A) The distance between the apical cell surface and
apical surface of the nucleus during collapse of the top surface increased significantly
more than control cells to a maximum of around 4μm (Figure 2-9 B). Hence, myosin
inhibition did not produce qualitative changes in the nuclear spreading dynamics. We
did find that inhibiting myosin with Y-27632 decreased the width of the flattened nucleus
and its volume (Figure 2-9 C and D) by a measureable amount. Thus, while the myosin
inhibition does not alter nuclear flattening, it appears that actomyosin forces may
29
contribute to some increase in nuclear volume by widening the nucleus after the initial
flattening (Figure 2-9 D).
We note that the differential effects of ML-7 on initial cell spreading (which it
prevents) and on already spread cells can be explained by its differential effects on
retrograde flow in slow versus fast moving cells as shown by Jurado et al. [51]. In
spreading cells where adhesions are relatively smaller, ML-7 treatment causes
complete disassembly of adhesions and is predicted to increase retrograde flow and
prevent spreading, while in well-spread cells, the retrograde flow would be reduced due
to a decrease in the “raking” of adhesions. Blebbistatin treatment prevented cell
spreading and nuclear flattening if cells were allowed to spread for longer time (6 hours,
Figure 2-10 A-C); thus, blebbistatin uniformly appears to decrease retrograde flow but
presumably it inhibits adhesions at a slower rate, which would explain why it does not
completely inhibit initial cell spreading at one hour.
Intermediate Filaments and Microtubules are Dispensable for Nuclear Flattening in Spreading Cells
Given that actomyosin contraction was not required for flattening, but instead
nuclear flattening correlated strongly with the degree of cell spreading, we examined the
role of the other two cytoskeletal structures in the cell: intermediate filaments and
microtubules. Nuclear aspect ratio was measured and compared between vimentin +/+
mouse embryonic fibroblasts (vim +/+ MEFs) and vim -/- MEFs. Compared to control
cells, the nucleus was more rounded (although it was still significantly flattened to an
aspect ratio of 0.3) in vim -/- MEF cells after one hour of cell spreading (Figure 2-11A
and B), but consistent with our observations above, the cell was comparatively less
spread (Figure 2-11C). Importantly, over longer time (12hours), the nucleus was
30
flattened in vim-/- cells (Figure 2-11B). Thus, while the absence of vimentin intermediate
filaments reduced the rate of nuclear flattening, it did not influence the extent of nuclear
flattening. To test if myosin activity was causing nuclear flattening in vim-/- MEFs, we
inhibited myosin activity in these cells with Y-27632. The nucleus was flattened in
myosin inhibited vim-/- MEFs (Figure 2-11B). Together, these results suggest that the
nucleus can flatten in the absence of myosin activity and intermediate filaments. The
experiments with these cells again point to a strong correlation between the spreading
area of the cell and nuclear flattening.
To determine the role of microtubules in nuclear flattening, we disrupted
microtubules with nocodazole and colcemid. At a dose of 1.65 μM nocodazole,
microtubules were eliminated from the cell, but the cell was unable to spread one hour
after seeding, and concomitantly, the nucleus did not flatten (Figure 2-12 A-C). Upon
decreasing the dose to 0.83μM, microtubules were greatly fragmented, but the cell was
not spread as well as the control cells at 1 hour. At 6 hours into the spreading process,
cells had no discernible microtubules (Figure 2-13A) but were able to spread (Figure 2-
13C) and the nuclei were flat (Figure 2-13B). We next allowed cells to spread overnight
and treated cells with 0.27μM colcemid. The treatment did not cause cell rounding
(Figure 2-13B) even though microtubules were completely disrupted (Figure 2-13A) and
the nucleus remained flat (Figure 2-13B). Together, both these results suggest that
microtubules are not required for nuclear flattening when cells are able to spread.
Apical and Basal Actomyosin Bundles are Not Required for Nuclear Flattening during Initial Cell Spreading
Actomyosin stress fibers have been implicated in shaping the nucleus [22].
Considering our results above which suggest that myosin activity is not required for
31
nuclear flattening, we examined the presence of actomyosin bundles in spreading cells.
In the first 20 minutes when the nucleus flattened significantly, the average number of
actomyosin bundles above the nucleus was found to be approximately 0.2, i.e. 1 out 5
cells have one apical bundle (Figure 2-14A). The number of basal fibers under the
nucleus coinciding with the time of nuclear flattening was about 1 ~ 2 per cell (Figure 2-
14B). We next examined the correlation between the nuclear height and the number of
apical and basal bundles at different times in the cell spreading process. Figure 2-14C
shows three examples where neither basal nor apical fibers could be discerned during
initial cell spreading, although the nucleus had been flattened to a considerable extent.
As seen in the plots, there were several cells where no fibers are discernible (apical or
basal), but the nucleus is clearly flat (Figure 2-14 D and E). These results, combined
with the myosin inhibition experiments above argue against a mechanical explanation in
which apical or basal actomyosin bundles play a significant role in flattening the nucleus
during initial cell spreading.
Nuclear Flattening can be Reversed by Detachment of the Cell from the Substratum
The different experiments described above seem to consistently indicate that
nuclear flattening is strongly correlated with the extent of cell spreading. A rounded cell
is predicted to have a rounded nuclear x-z cross-section, while a well-spread cell is
expected to have flat nucleus. To further test the relationship between the degree of cell
spreading and nuclear height, we treated well-spread cells with trypsin and measured
the nuclear x-z cross-section. At concentrations of trypsin (0.25% w/v) normally used for
cell passage, the nucleus rounded up remarkably fast (in a few seconds) coupled with
fast cell rounding (Figure 2-15A). There was a strong relationship between the degree
32
of cell rounding as measured by the contact length between the cell and the surface of
the substratum, and the nuclear height, at different times during the trypsinization
process (Figure 2-15B). We next treated cells with trypsin at reduced concentrations
(0.08% w/v). This slowed the cell rounding process significantly (several minutes).
Consistent with the expectation that nuclear height is determined primarily by the
degree to which the cell is spread, the nucleus did not round until the cell had
significantly changed its shape through release of cell-substratum adhesions (Figure 2-
15C). This occurred over several minutes (Figure 2-15D). These results strongly
support the concept that nuclear height correlates with the degree of cell spreading.
The LINC Complex is Not Required for Nuclear Flattening
The LINC (for Linker of Nucleoskeleton to the Cytoskeleton) complex has been
shown to transmit mechanical forces from the cytoskeleton to the nucleus [34]. We
therefore asked if an intact LINC complex is required for nuclear flattening. The
disruption of the LINC complex by over-expression of GFP-KASH4 (KASH4 is the
KASH domain from nesprin 4 which competitively binds to the SUN proteins, but lacks
the cytoskeletal linker domain) [27] slowed the flattening of the nucleus (Figure 2-16A
and B) but it also slowed normal spreading of the cells (see also Figure 2-16G and H).
Importantly, at 6 hours and 24 hours (Figure 2-17 A-C), GFP-KASH4 expressing cells
were well-spread and displayed flat nuclei. Similarly, the knockdown (Figure 2-17 D) of
nesprin 2G (Figure 2-16C and D) and SUN2 (Figure 2-16E and F) with shRNA
interference did not have any effect on nuclear flattening during initial cell spreading
(see Figure 2-16G and H for a statistical comparison of all the data). The data indicates
that an intact LINC complex is not required for nuclear flattening and cell spreading.
33
We next examined the effect of lamin A/C on the degree of nuclear flattening
(Figure 2-18). At 60 minutes, lamin A/C -/- MEFs had more flattened nuclei compared
to WT MEFs; however, WT MEFs did not spread significantly at 60 minutes (Figure 2-
18A). When allowed 6 hours to spread however, WT MEFs were able to spread and
flatten their nuclei. These results suggest that the absence of lamin A/C correlates with
an increased rate of nuclear flattening, leading to a flattened nucleus in lamin A/C -/-
MEFs compared to WT MEFs during cell spreading.
A Mathematical Model for Nuclear Flattening and Cell Spreading
The presence of individual cytoskeletal elements (microtubules, intermediate
filaments), myosin activity, or an intact LINC complex, which transmits forces from the
cytoskeleton to the nucleus, is not required for flattening the nucleus as long as the cell
is able to spread. We found that inhibiting F-actin polymerization which prevents cell
spreading prevented nuclear flattening (Figure 2-4A-C). Thus, nuclear flattening
correlates with the degree of cell spreading. Based on these results, we propose a
simple mechanical model which shows that stresses arising from cellular shape
changes and cytoskeletal network assembly from the apical cell cortex are sufficient to
explain nuclear translation to the surface and flattening against the substratum. We
modeled the cell’s cytomatrix, i.e. the cytoskeletal network phase connecting the
nucleus to the cell membrane, as a contractile compressible material that resists
compression/expansion and shear strains (like the approach by Dembo and coworkers
[52-54]). On the slow time scale of spreading (several minutes), only the viscous
resistance to deformation is considered relevant (i.e. elastic forces are considered
negligible given the remodeling that occurs in the cell over long time scales), such that
the stress tensor is proportional to the rate-of-strain tensor, i.e.
34
𝛔 = 2𝜇�̇� + σ𝐜𝐈 (2-1)
Here σc is the contractile stress due to myosin motor activity, 𝐈 is the unit dyadic,
ε̇ = 1
2(∇v + ∇vT) is the rate-of-strain tensor, and μ is viscosity which measures the
modulation of stress due to both expansion/compression and shear deformations of the
compressible network phase. Note that both shear and expansion/compression modes
in ε̇ are relevant since the network is assumed compressible. Equation (2-1) can be
considered a slow-flow limit of the more general two-phase reactive interpenetrating
flow models for cells developed by Dembo and coworkers ([52-54]) where network
contractile/viscous properties can be assumed to be uniform and hydrostatic pressure
gradients are assumed negligible.
Solving the momentum balance ∇ ∙ σ = 0 with the appropriate boundary
conditions yields the stress and velocity fields of the network. Before we discuss the full
general model for cell and nuclear mechanics during nuclear flattening, we show a
simple model which illustrates the key predictions of the general model. The simple
model (Figure 2-18A) is an approximate representation of the gap between the cell apex
and the nuclear apical surface when the gap is small compared to the inverse curvature
of the nucleus. The main purpose of this model is to show how movements of the top
cell membrane and flow from the membrane of network can exert a stress on the
nuclear surface. To illustrate the relevant properties of a cell containing
contractile/viscous medium obeying Equation (2-1), consider a simplified one-
dimensional case illustrated in Figure 2-18A, which represents the planar approximation
of the local gap of length L between the cell membrane and the nuclear envelope (the
exact derivation for a spherical cell is presented in in the Materials and Methods). Let
35
the gap expand at speed V by moving the cell membrane and keeping the nuclear
surface fixed, and assume new network assembles at the cell membrane (where f-actin
is primarily generated) at speed va. In the special case where V = va, there is no
network flow because the network assembles at exactly the rate required to fill the
volume behind the moving membrane. Otherwise, there will be network expansion V >
va or compression V < va, either of which will modulate the stress on the nuclear surface
at the base. As derived in the Materials and Methods, the resulting velocity and stress
fields for the 1-D approximation are:
vx = (V − va)x
L, (2-2)
σxx = σc + 2μdv
dx= σc + 2(V − va)
μ
L. (2-3)
Since stress is uniform in this case, the tensile stress on the nuclear surface is
equal to σxx. From equation (2-2) and (2-3), the following important properties regarding
transmission of stress to the nuclear surface are evident: (1) expansion of the gap V > 0
between the cell membrane and the nuclear envelope will increase the net tensile stress
σxx on the nuclear surface; and (2) compression of the gap (V<0) or assembly of
network at the cell membrane (va > 0) will decrease the net tension on the nuclear
surface. An important corollary to these predictions is that when the nuclear surface
stress is fixed (instead of fixing the nuclear surface position), the nuclear surface will
move at a speed such that the gap expansion speed V satisfies the stress balance in
Eq.(2-3). For example, if the nuclear surface stress is in balance with the network
contractile tension, such that σxx = σc, then V = va. Consequently, the nuclear surface
will move together with the membrane keeping V = 0 when va = 0, or it will move away
from the cell membrane at speed va when va > 0. In this way, the nuclear surface
36
movements will tend to follow the movements of the nearby cell membrane boundary,
but will also tend to move away from cell membrane surfaces where network is being
assembled. These are the important properties of the network that govern the more
general model that now follows for nuclear shape changes during cell spreading.
To model the case of a spreading cell (Figure 2-19B), new network is assumed to
assemble where F-actin is generated at the cortex and at the cell edge on the
substratum, but not at any other substratum-cell membrane interface (See Materials
and Methods for model and simulation details). Throughout the network phase, F-actin
and the other constituents of the cytoskeletal network (intermediate filaments,
microtubules) are assumed to assemble/dissemble to re-equilibrate the density and
mechanical properties of the network relatively quickly on the slow time scale of cell
spreading. As shown in Figure 2-19C, these assumptions and the constitutive stress
equation (Eq.2-1) are sufficient to predict cell spreading very similar to the experimental
observations, including the observed initial distension and net translation of the nucleus
toward the substratum and initial flattening against the substratum. Cell spreading is the
result of assembly of network at the contact boundary, which generates a centripetal
flow of network. Substratum adhesion hinders this centripetal flow, resulting in a net
outward expansion of the cell boundary near the substratum. Expansion near the
substratum corresponds to retraction of the upper cell surface away from the
substratum in order to preserve cell volume (assumed constant due to the cell’s osmotic
resistance to volume changes). Because of viscous resistance to network expansion
(Eq. 2-1), the movement of the cell boundaries generates stress on the nucleus as the
intervening network expands or compresses, and movements of the nucleus boundary
37
tend to follow those of the cell boundaries. Assembly of new network at the cortex has
the effect of increasing the downward compressive flow of network. Because the
network is assumed not to assemble at the cell-substratum interface (except at the
contact boundary), this flow causes an initial vertical distension of the nucleus followed
by a net translation of the nucleus toward the substratum (Figure 2-1C). While network
assembly at the cortex is required to predict the initial rapid downward translation of the
nucleus given the assumed network viscosity, cell spreading and the resulting nuclear
flattening only requires an assumption of network assembly at the contact boundary, as
discussed below. As shown in Figure 2-19D, the time-dependent nucleus height and
width predicted by this model agree well with the experimentally observed trends.
As detailed in the Materials and Methods, our mechanical model of the nucleus
accounts for resistance to compression, and a resistance to nuclear envelope
expansion which accounts for an excess of surface area of the nuclear lamin network
above that of a smooth sphere with the same volume. This excess surface area is
evident from the observed undulations in the lamin network [55]. As shown in Figure 2-
19E, the nuclear volume remained nearly constant during cell spreading, but the
apparent surface area of the nuclear envelope increased; once the surface area
expansion approached the “true” surface area, further shape changes were minimal due
to the large mechanical resistance to further surface area changes. Hence, a steady-
state nuclear shape is reached before spreading stops, consistent with our
observations, and the steady-state shape of the flattened nucleus depends primarily on
the stiffness of the nuclear lamina and the excess surface area of the initially rounded
nucleus. Without the assumption of network flow from the cortex, the nucleus is still
38
predicted to flatten as the cell spreads due to the vertical compression and horizontal
expansion arising from the moving cell boundaries (Figure 2-20A). However,
reproducing the relatively rapid approach and flattening of the nucleus against the
substratum early in cell spreading processes requires an assumption of network
assembly and flow from the apical cell cortex. Although the initial nuclear dynamics are
similar, the fully spread model cell with or without apical cortical network assembly
appear very similar at longer times. Hence, our results suggest that apical cortical
network assembly and flow is necessary to translate the nucleus to the substratum early
in spreading, but it is not required to explain the ultimate flat nuclear shapes like those
observed in experiments. Reproducing the observed flattening dynamics therefore
does not require the assumption of continued network assembly at the apical cortex at
longer time (< ~25 min).
When the substratum adhesion frictional parameter is reduced, the speed of
retrograde flow near the substratum increases (consistent with the molecular clutch
model [56]). At a sufficiently low adhesion, cell spreading slows and stops at a steady
state before the cell can fully spread, and the nucleus also stops flattening when the cell
stops spreading (Figure 2-20B). This result reinforces the prediction that nucleus shape
changes tend to follow cell shape changes.
Interestingly, the predicted dynamics of nucleus spreading do not depend
significantly on the background tension of network, as shown in the simulation results in
Figure 2-20C, where the network tension parameter σc was set to zero. The differential
stresses that cause nuclear shape changes arise primarily from the resistance to
expansion or compression of the network, not from the background contractile tension
39
of the network, which is treated here as a uniform tension that acts equally on all
surfaces. The predicted lack of dependence on contractile tension is consistent with our
experimental observation that inhibition of myosin does not prevent nucleus flattening in
spreading cells. (It should be noted, however, that if σc were to vary spatially, the
contractility gradient (∇σc) would drive local network flow in the gradient direction.)
As mentioned above, the shape of the flattened nucleus depends on the area
stiffness of the nuclear surface. When the area modulus was set to zero, the nucleus
continued to flatten as long as the cell continued spreading (Figure 2-20D). This
predicted behavior is consistent with the increased nuclear flattening in lamin A/C -/-
MEFs (Figure 2-18). Since the nucleus can flatten without changing volume, the
dynamics of nucleus flattening did not significantly depend on the value of volume (bulk)
modulus of the nucleus (see appendix for a fuller discussion of sensitivity of the
predictions to model parameters).
In summary, the key predictions of the model and simulations are (1)
distension/translation of the nucleus toward the surface is driven by assembly of actin at
the apical cortex, (2) nuclear flattening is driven by stresses caused by cytoskeletal
network expansion/compression upon movement of the cell boundaries, and (3) these
nuclear shape changes can arise without network contractile tension or stress fibers.
The model predictions therefore provide an explanation for the experimental
observations of nuclear flattening against the substratum without significant actomyosin
contractile tension.
Discussion
The flattened nucleus is a common feature of cultured cells, but the mechanisms
by which it is flattened have remained obscure. There is mounting evidence that the
40
cytoskeleton exerts forces on the nucleus to position it [29, 57-59]. In this paper
however, we show that as long as the cell was able to spread, inhibiting actomyosin
forces, microtubule-based forces and intermediate filaments, as well as the LINC
complex, did not prevent nuclear flattening. Remarkably, nuclear height correlated
tightly with the degree of cell spreading. Independent of the type of cytoskeletal force
perturbed, the nucleus is flat unless the perturbation prevents initial cell spreading, or
rounds a spread cell.
This robust feature of nuclear shaping suggests that it is the dynamic
deformation of the cell shape itself that causes nuclear flattening consistent with our
previous results reporting reversible nuclear deformation caused by proximal cell
protrusions in migrating cells [37]. The fact that the nuclear apex collapses during the
nuclear flattening, opening up a significant distance between the cell apex and the
nuclear apex (on the order of a few microns), argues against the cell cortex directly
compressing the nucleus downward. The near complete absence of apical actomyosin
bundles argues against any explanation for flattening that requires a downward
compressive force on the nuclear apex by large actomyosin bundles (for example ref.
[60]). That apical fibers do not participate in the flattening process does not argue
against later distortion of the nucleus by fully developed actomyosin bundles as
reported by others [61].
Neither intermediate filaments nor microtubules are required for flattening.
Finally, disruption of the LINC complex via KASH4 over-expression failed to prevent
nuclear flattening. It slowed the rate of cell spreading, suggesting perhaps that a
coupled nuclear-cytoskeleton is required for rapid F-actin polymerization, but it did not
41
prevent flattening over longer times. Given that myosin activity, MTs and vimentin IFs
are not required, that the LINC complex is dispensable is perhaps not surprising. We
have shown before that KASH4 over-expression results in rounded nuclear shapes in
cells on polyacrylamide gels [62]. This difference may be due to possibly different cell
spreading dynamics on gels versus glass. We note that the cell spreading area in
KASH4 cells was lower on gels, suggesting that the relationship between nuclear
flattening and cell spreading is conserved on other types of surfaces.
Our computational model demonstrates that expansive/compressive stresses
arising from movement of the cell boundaries and centripetal flow of cytoskeletal
network from the cell membrane is sufficient to explain translation of the nucleus toward
the substratum and subsequent flattening against the substratum. The fact that the
experimentally observed flattening dynamics could be closely reproduced using one
constitutive equation (Eq.2-1), a simple model for cell mechanics, and one “fitted”
parameter 𝑣𝑎 provides strong support for the validity of the model assumptions.
Moreover, the model successfully predicts several experimental findings: the approach
to a steady-state flattened nuclear shape despite continued cell spreading, nuclear
flattening in the absence of actomyosin tension, increased nuclear flattening in the
absence of lamin A/C, and the cessation of nuclear flattening upon the cessation of cell
spreading.
Consistent with the assumption that the nucleus is under tension, we found that
the volume of the nucleus decreased in myosin-inhibited cells. However, our
experiments show that flattening is not a consequence of tension. As explained by the
computational model, flattening can instead arise from the motion of the cell boundary
42
transmitting stresses to the nuclear surface because the intervening cytoskeletal
network resists expansion or compression. As a result, the nuclear shape changes tend
to mimic changes in cell shape during cell spreading.
Interestingly, the presence of actomyosin contraction in normal cells does not
alter the dynamics of nuclear shape changes during cell spreading. In the presence of
contraction, the net stress on the nuclear surface in the absence of any F-actin
assembly from the membrane (such as in serum-starved cells) is likely tensile.
However, even if this stress in the network is net compressive (such as when myosin is
inhibited), the differential stresses between apex and sides of the nucleus that drive
nuclear shape dynamics during cell spreading are predicted to be similar (Figure 10C).
In summary, our results support a surprisingly simple mechanical system in cells
for establishing nuclear shapes. Our computational model suggests that nuclear shape
changes result from transmission of stress from the moving cell boundary to the nuclear
surface due to frictional resistance to expansion/compression of the intervening
cytoskeletal network. Nuclear shaping are thus driven by cell shape changes.
43
Figure 2-1. The dynamics of nuclear flattening during early cell spreading. A-C). Shown are vertical cross-sections of a nucleus in a cell that settles and spreads on the substratum. The images were captured using x-z laser scanning confocal fluorescence microscopy; the nucleus is expressing GFP-histone H1. Three phases were discernible in the nuclear flattening process: A). A settling phase where the basal surface of the nucleus contacted the basal cell surface and started to spread, the height was roughly constant during this time. B). A collapse of the top surface where the basal surface of the nucleus did not spread much, and C). A widening phase where the basal surface of the nucleus continued to spread and contributed to nuclear widening; the height was roughly constant in this phase. Scale bar is 10 µm. D. Plot shows nuclear height and contact length in A-C with time.
44
Figure 2-2. The nuclear deformation causes nuclear flattening but not already-flat
nucleus toppling onto their side. Images show x-z view of the nuclear shape from two different view angles in two cells at different time. Scale bar is 5 µm.
45
Figure 2-3. The dynamics of nuclear flattening against substratum is not influenced by
gravity. A) Nuclear shape changes during cell spreading are shown for normal (top) and inverted samples (bottom) at different time points in x-z view. For the inverted case, cells were allowed to first settle and attach for 5min before inverting the sample. Neither nuclear aspect ratio B) nor cell spreading area C) has a significant difference between control and inverted
(n ≥ 32). Scale bar is 10 µm. All data are shown as Mean ± SEM
46
Figure 2-4. Nuclei does not flatten when cell spreading is prevented by inhibitors of
actin assembly. A) Images show the x-y and x-z view of control cell and cell treated with cytochalasin-D or latrunculin-A. B) Nuclear aspect ratio (height divided by the length of the major-axis in the x-y plane) increases significantly after 1hr spreading in the presence of the drugs, which corresponds to the decreases of cell spreading area C). (n ≥ 31, * indicates p<0.05; all comparisons are with untreated control). Scale bar in (A) is 20 µm in the x-y view and 5 µm in x-z view. All data are shown as Mean ± SEM.
47
Figure 2-5. The nucleus flattens completely in a partially spread cell. A) Images show
cells at different stages in the spreading process. Nuclei (blue) were completely flattened at roughly 20-30 minutes when the cells (green F-actin) had not spread completely. Scale bar is 20 µm for the x-y views, and 10 µm for the x-z views. Also shown are average nuclear heights B), average nuclear widths C) and nuclear aspect ratio (height/width) D) at different times during the cell spreading process, and the corresponding areas of cell spreading. The nuclear heights reach an approximate steady state at around 30 minutes, when the cells spread to about 50% of the final
area (n ≥ 32 cells).
48
Figure 2-6. The apical surface of cell was separated from the apical surface of nucleus in the early stage of cell spreading. A) Images of nucleus (blue) and cell (green) show the gap between them in the vertical cross-sections at different time of cell spreading. Scale bar is 10 µm. B) The gap between the top cell
surface and the top nuclear surface plotted with time (n ≥ 25 cells). The gap
increased at the beginning reflecting the collapse of the nuclear surface and
then decreased over time to near zero levels. All data are shown as Mean ±
SEM.
49
Figure 2-7. Nuclear flattening is independent of actomyosin contraction. A) Images show cells pre-treated with drug for 1 hour, trypsinized, and then seeded onto substrates for 1 hour in the presence of the drug. Cells in the presence of Y-27632 and blebbistatin showed clear effects on the cell morphology compared to the control, but the nucleus was still flattened as evident from the x-z cross-section. ML-7 treatment on the other hand prevented the spreading of the cell as well as the flattening of the nucleus. Scale bar for x-y views is 20 µm, for x-z views is 5 µm. B) Nuclear aspect ratio was larger in ML-7 treated cells reflecting unflattened nuclei, consistent with the fact that the cells were unable to spread in presence of ML-7 C). Minor differences in aspect ratio on Y-27632 or blebbistatin treatment reflect minor effects on degree of cell spreading. D) Aspect ratio correlates with the degree of cell spreading. In ML-7 treated cells, the cells were unable to spread (blue diamonds) corresponding to the large aspect ratio; none of the other treatments prevented nuclear flattening but concomitantly, they did not prevent cell spreading. Bar plots shown are nuclear height E) and width(F) during initial cell spreading under myosin inhibition and the corresponding
scatter plot G) and (F), (n ≥ 31 for all conditions, * indicates p<0.05; all
comparisons are with untreated controls).
50
51
Figure 2-8. The effect of myosin inhibition on nuclear shape in well spread cell. The experiments in A-D) show the results on treating well-spread cells (cultured overnight on fibronectin coated glass-bottom dishes) with myosin inhibitors. Blebbistatin treatment rounded up spread cells, and caused rounded nuclear shapes. Images are shown in A), while the scatter plots of aspect ratio versus cell spreading area are shown in D). B) and C) show the average aspect ratio and areas for the different myosin inhibitors. Shown are nuclear height E) and width F) of well-spread cell with myosin inhibition, and their corresponding scatter plot with cell spreading area G) and H). Scale bar in A is 20 µm for the
x-y view and 5 µm for the x-z view. (n ≥ 24, * indicates p<0.05; all
comparisons are with untreated control. All data are shown as Mean ± SEM)
52
53
Figure 2-9. Inhibition of myosin activity with Y-27632 does not alter the qualitative
features of dynamic nuclear flattening during cell spreading. As seen in A),
the time-dependent changes in nuclear height (n ≥ 33) still occur on Y-
27632 pre-treatment while the cells spread (an initial lag time in the flattening of the nucleus is attributable to an initial lag time in the spreading). Y-27632 treatment increased the separation between the apical
cell and nuclear surfaces B) (n ≥ 29). The decrease in nuclear width C)
and volume D) in Y-27632 treated cells suggests that myosin contraction
plays a role in the nuclear widening process (n ≥ 29). ( * indicates p<0.05;
all comparisons are with untreated control. Scale bar is 10 µm in both x-y view and 5 µm for the x-z view. All data are shown as Mean ± SEM)
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Figure 2-10. Blebbistatin prevents cell spreading at later times resulting in final
nuclear rounding. A) At 1 hr images show the spreading of blebbistatin-
treated cells (n ≥30) in x-y view and the associated nuclear height in x-z
view. Given more time (6 hours), the area of cell spreading decreased significantly C) and the nucleus rounded up to the similar level of well spread cell treat with blebbistatin. Consistent with this, nuclear aspect ratio at 6 hours was higher than control B). ( * indicates p<0.05; all comparisons are with untreated control. Scale bar is 10 µm in both x-y view and 5 µm for the x-z view. All data are shown as Mean ± SEM.)
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Figure 2-11. The absence of intermediate filaments does not prevent nuclear flattening during cell spreading. A) Intermediate filaments are not required for nuclear flattening during cell spreading. The nucleus is flattened in vim-/- cells similar to vim+/+ cells at 12 hours after cell seeding although vim-/- nuclei are slightly rounded at 1 hour into the spreading process. Y-27632 treatment in vim-/- cells did not prevent nuclear flattening. Scale bar in x-y view is 20 µm, in the x-z view is 5 µm. B) and C) show the average aspect ratio and spreading areas; all the differences in aspect ratio can be attributed to the corresponding (inversely related) differences in the degree
of cell spreading.( * indicates p<0.05, n ≥ 30)
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Figure 2-12. Disruption of microtubules by nocodazole rounds up the nucleus but also prevents cell spreading. (A) Immunofluorescence images are shown of 1.65 µM nocodazole treated cells in two kinds of experiments, one in which the drug was treated during initial cell spreading, and the other in which cells that were well-spread were treated with nocodazole. For both experiments, no distinct microtubules were visible, but nuclei were not flat as the cells had rounded morphologies. The quantifications of nuclear aspect ratio (B) and cell spreading area (C) again show that the nuclear
rounding is correlated with the extent of cell spreading.(n ≥ 30, * indicates
p<0.05; all comparisons are with untreated control. Scale bar is 10 µm in both x-y and x-z view. All data are shown as Mean ± SEM)
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Figure 2-13. The absence of microtubule does not prevent nuclear flattening during cell
spreading. A) Shown is the effect of nocodazole (0.83 μM) on nuclear flattening and cell spreading, and the effect of colcemid (0.27μM) on well spread cells. At 6 hours, the nocodazole-treated cells were spread and had flattened nuclei, while no microtubules were visible. Likewise, colcemid treatment for 1 hour disrupted microtubules in originally well-spread cells but did not alter nuclear height. Collectively the data suggests that microtubules are not required to establish or maintain a flattened nucleus. Measurements of aspect ratio B) and spreading area of the cells C) under various conditions.
n ≥ 30, * indicates p<0.05; all comparisons are with untreated control. Scale
bar in (E) is 10 µm for x-y view and 5 µm for x-z view. All data are shown as Mean ± SEM.
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Figure 2-14. Apical and basal actomyosin bundles are not required for nuclear flattening during initial cell spreading. A) Apical actomyosin bundles were counted above the nucleus in spreading cells (actin cables were visualized by phalloidin staining). Apical bundles appear only after 30-40 minutes by which
time the nucleus has flattened completely (n≥30). B) In spreading cells, one
basal actomyosin bundle on average appeared under the nucleus by around 15 minutes. Basal cables were only counted if they ran beneath the nucleus.
Times represent time after initial seeding, (n≥30). C) Images show examples
at different times after seeding of apical and basal F-actin stained cells (green) that lack actomyosin bundles, but have significantly flattened nuclei (blue). Scale bar is 10 µm for both panels. D) and E) show plots of the nuclear height with the number of apical and basal actomyosin bundles at 15, 20 and 30 minutes. A number of examples can be seen where there are zero apical
or basal actomyosin bundles but the nucleus is still significantly flattened (n≥
30 cells). All data are shown as Mean ± SEM.
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Courtesy David Lovett
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Figure 2-15. Nuclear flattening can be reversed by detachment of the cell from the
substratum. Trypsinization of cells rounded the nucleus A) in a remarkably short time of a few seconds. Importantly, the nuclear rounding closely followed the cell rounding- the dynamics of height changes (gray circles) and changes in contact length of the basal cell surface (black circles) are similar B). Scale bar is 10 µm for both panels in A. This concept was tested further in C) by trypsinizing cells at 1/3 the dose of the trypsin concentration used in A. The nucleus rounded much more slowly (several minutes) and closely reflected the rounding up of the cell body (the nuclear height and cell contact length are shown in D)). Thus, the degree of cell spreading determines the degree of nuclear rounding during cell detachment. Scale bar is 20 µm for both panels. All data are shown as Mean ± SEM.
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Figure 2-16. The LINC complex is not required for nuclear flattening during cell spreading. A) GFP-KASH4 expression in cells prevented flattening of nuclei at 1 hour, but also prevented cell spreading. 6 hours into the spreading, the nucleus did flatten in GFP-KASH4 expressing cells. Scale bar in x-y and x-z views are both 20 µm. B) shows the scatter plots of nuclear aspect ratio versus cell spreading area in GFP expressing (control) versus GFP-KASH4 expressing cells; GFP-KASH4 expressing cells did not spread well at 1 hour after seeding, which correlated with the expected response of a lack of nuclear flattening. C), D) Nesprin 2 knockdown did not produce any effects on aspect ratio nor the degree of cell spreading. E), F) SUN2 knockdown produced no effects on aspect ratio and degree of cell spreading. Scale bar in x-y view is 20 µm and x-z view is 5 µm in C and E. G and H show comparisons of average aspect ratio, and cell spreading area at the different conditions. Only when the cell is not able to spread does the nucleus remain
unflattened (* indicates p<0.05, n ≥ 35). All data are shown as Mean ±
SEM.
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Figure 2-17. GFP-KASH4 overexpression slows down but does not prevent cell spreading and nuclear flattening. A) Images show GFP-KASH4 cells after one hour of spreading have unflattened nuclei and not so-well spread cells; but nuclei become flat at long times (6 and 24 hours) with fully spread cells. The changes of nuclear aspect ratio and cell spreading area in B) and C) respectively, indicate that the rounded nucleus in cells expressing GFP-KASH4 at 1 hour is correlated with a slower cell spreading rate. D). Western blots of indicated proteins immunoprecipitated by SUN2 or Nesprin 2G
antibodies from NIH 3T3 cell lysates. (n ≥ 37, * indicates p<0.05). Scale bar
is 20 µm in both x-y and x-z view. All data are shown as Mean ± SEM.
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Figure 2-18. Nuclei in lamin A/C -/- MEFs flatten faster than WT cells. Nuclei in WT MEFs are less flattened at 1 hour after cell seeding A) compared to lamin A/C -/- cells which have extremely flat nuclei. The degree of cell seeding is small in WT at 1 h and increases by 6 h; lamin A/C -/- cells however are well-spread at 1 h. Scale bar in the x-y view is 20 µm, in the x-z view it is 10 µm. The lack of cell spreading, higher nuclear heights and lower nuclear widths at 1 h in WT cells compared to lamin A/C -/- cells is evident in the scatter plots of nuclear aspect ratio versus cell spreading area B), as well in the average values of nuclear aspect ratio and spreading area (C and D). (* indicates
p<0.05; n ≥ 43; all comparisons are with WT cells at 1 hour spreading). All
data are shown as Mean ± SEM
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Figure 2-19. Mathematical model for nuclear deformation during cell spreading. A) Predictions of a simplified one-dimensional model for the cytoskeletal network spanning the gap between the nuclear surface and the cell membrane. Movement of the membrane relative to the nuclear surface (speed V) or assembly of network at the surface and resulting retrograde flow (speed va) results in expansion or compression of the intervening network, thereby generating a stress on the nucleus surface. B) Key components of the model for a spreading cell. The model cell accounts for (i) resistance of the nucleus to volume expansion /compression; ii) resistance of the nuclear surface (lamina) to area expansion; iii) cell membrane tension; and iv) the cytoskeletal network phase of the cytoplasm, which is assembled at the cell cortex and at the contact boundary with the substratum. Centripetal flow of network and the frictional resistance to shear and to volume expansion/compression causes movement of the cell membrane and nuclear surfaces (surface velocities of cell and nuclear surfaces are shown by blue vectors). C) Snapshots from a simulation of cell spreading and nuclear shape changes showing the three phases of nuclear deformation observed experimentally: i) vertical distension and translation of the nucleus toward the substratum, driven by flow of network from the apical membrane where it is generated, ii) initial flattening against the substratum with a decrease in nuclear height and little change in nuclear width as the cell begins to spread and the nuclear is compressed vertically by the lowering upper cell surface; and iii) widening of the nucleus with lesser change in height, as the widening cell boundary pulls the nucleus laterally. Widths and height are plotted in D). As in the experimental observation, the nucleus quickly flattens vertically early in the process of cell spreading, then widens more slowly as the cell continues to spread. E) Plot of nuclear area and volume versus time. While the nuclear volume remains nearly constant, the area expands until the assumed excess area is smoothed and the stiffer “true” surface area is reached, at which point the surface area starts to level off toward a constant value.
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Figure 2-20. Snapshots of simulation results for different parameters. A). Simulation for case of no cytomatrix assembly at the cortex (assembly occurs only at the contact boundary). Network assembly at the cortex is required for translation toward the substratum. B). Simulation showing the effect of reduced adhesion. Reducing adhesion allows retrograde flow at the substratum; ultimately the flow speed matches the speed of network assembly, yielding steady-state cell and nucleus shapes. The nucleus shape changes cease when spreading stops. C). Simulation with no contractile stress within the network (σc = 0)). Nuclear flattening is predicted to arise from flow alone without requiring actomyosin contractile stresses in the network, consistent with the observation of flattening under myosin inhibition. D). Simulation of a cell with no resistance to nuclear lamina area expansion. Without area stiffness, the nucleus continues to flatten at a nearly constant volume. E). Plot of nuclear height (blue line), width (black line), and cell spreading area (green line) versus time for the cases show in A-D. F). Plot of apparent nuclear surface area (blue line) and volume (green line) for cases A-E.
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CHAPTER 3 DYNAMIC DEFORMATION OF THE CELL PLASTICALLY SHAPES THE NUCLEUS
AND AMPLIFIES CANCER NUCLEAR IRREGULARITIES
The aberrant morphology of nuclei is a distinct feature observed in diverse types
of cancer [63-65] used for diagnosis and prognosis [66, 67]. These features include
lobes, invaginations and folds in the nuclear lamina. Given the physical linkage between
the nuclear lamina and chromatin [68, 69], such alterations in nuclear shape can affect
chromatin compaction and consequently gene expression [17, 70-72], causing
alterations of cellular functions such as migration [73] and mitosis [74, 75].
The mechanisms for abnormal shapes of cancer nuclei have remained unclear.
Some papers have suggested that chromosomal instabilities correlate with nuclear
shape abnormalities [74, 76] in cancer cells. Other studies suggest that spatial
inhomogeneity in the nuclear lamins contribute to abnormal cancer nuclear shapes [77].
Based on the new mechanism identified in chapter 2, here we hypothesized that cancer
nuclear abnormalities are caused by stresses generated by the dynamic process of cell
spreading. To test this hypothesis, we performed 3D confocal imaging of cancer nuclear
shapes during the process of cell spreading. Using a convex hull method to quantify
three dimensional nuclear shape abnormalities, we found that nuclear abnormality is
amplified during the dynamic process of cell spreading in breast cancer cells. The
nuclear deformation driven by cell shape changes are also irreversible as no elastic
Reprinted from Journal of Cellular Physiology, in press, Tocco, V., Li, Y., Christopher, K., Matthews, J., Aggarwal, V., Paschall, L., Luesch, H., Licht, J., Dickinson, R., and Lele, T., The nucleus is irreversibly shaped by motion of cell boundaries in cancer and non-cancer cells. Copyright (2017), with permission from Wiley.
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relaxation of nucleus is observed after isolating nucleus from cytoplasm in fibroblasts
and cancer cells.
As the LINC complex transmits mechanical stresses generated by cell spreading
to the nuclear surface, we next asked if abnormal nuclear shapes can be ‘reverted’ to
normal shapes by disrupting the LINC complex. Disrupting the LINC complex by
overexpression of GFP-Sun1L-KDEL or GFP-KASH4 caused a decrease in the nuclear
shape abnormalities in MDA-MB-231 cells. Importantly, the motility of breast cancer
cells decreased significantly upon LINC disruption. We further quantified the amount of
DNA in cancer cells using a high-content imaging method. Upon comparing MCF10A
and MDA-MB-231 cells, we found no significant differences between the dependence of
nuclear shape abnormalities on DNA content. However, we found that abnormalities in
nuclear shape are heritable from mother to daughter breast cancer cells.
Taken together, our results are consistent with a model of cancer nuclear
shaping in which motion of the cell boundary transmits a viscous (dissipative) force on
the nuclear surface to amplify irregularities of nuclear shape.
Materials and Methods
Cell Culture and Transfection
All cells were maintained in a humidified incubator at 37°C and 7% CO2. Human
breast cancer cells (MDA-MB-231) were cultured in 4.5g/L glucose Dulbecco’s modified
Eagle’s medium (DMEM) without HEPES and L-glutamine (Invitrogen, Carlsbad, CA),
supplemented with 10% (v/v) donor bovine serum (DBS, Gibco, Grand Island, NY), 1%
(v/v) 100x MEM non-essential amino acid (Mediatech, Manassas, VA), 1% (v/v) 200mM
L-glutamine (Fisher Scientific, Hampton, NH) and 1% Penicillin Streptomycin
(Mediatech, Manassas, VA). Human breast epithelial cells (MCF-10A) were cultured in
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DMEM/F12 (Invitrogen, Carlsbad, CA), supplemented with 5% (v/v) horse serum
(Invitrogen, Carlsbad, CA), 1% Penicillin Streptomycin (Mediatech, Manassas, VA), 20
ng/mL epidermal growth factor (EGF, Peprotech, Rocky Hill, NJ), 0.5 µg/mL
hydrocortisone (Sigma-Aldrich, St. Louis, MO), 100 ng/mL cholera toxin (Sigma-Aldrich,
St. Louis, MO) and 10 µg/mL insulin (Sigma-Aldrich, St. Louis, MO). Cells were seeded
onto 35 mm glass-bottom dishes (WPI, Sarasota, FL) treated with 5 µg/ml fibronectin
(BD Biosciences, San Jose, CA) for imaging. Transfections of were performed with
Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA) in OptiMEM serum-free
media (ThermoFisher) following the manufacturer’s protocols
Cell Staining and Drug Treatment
For fixed-cell experiments, cells were fixed in 4% paraformaldehyde at room
temperature (25°C) for 10 min, washed with PBS, and stained with Hoechst 33342 and
fluorescent phalloidin to label DNA and F-actin, respectively. For microtubule
immunostaining, cells were first treated with microtubule extraction buffer containing
0.5% (w/v) glutaraldehyde, 0.8% formaldehyde and 0.5% Triton X-100 in phosphate
buffered saline (PBS) for 3 minutes before fixing with 1% (w/v) paraformaldehyde for
another 10 minutes. Then a freshly prepared 1% (w/v) sodium borohydride in PBS
solution was added to the cells for 10 minutes followed by blocking in 1% (w/v) BSA in
PBS. The cells were then incubated in 4°C overnight with rabbit polyclonal antibody to
α-tubulin (1:1000, Abcam, Cambridge, MA) in 1% BSA-containing solution, washed with
PBS and then incubated with Goat Anti-Rabbit IgG (H+L) antibody (1:500, Life
Technologies, Carlsbad, CA) at room temperature for 1 hour. For treatment with specific
drug, cells were pretreated for 1hr and imaged in the media containing the drug with the
same concentration
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Nuclear Excision
The work was performed by Keith Christopher. In short, the nucleus was
removed from MDA-MB-231 cells by using a 0.5 m micropipette tip (Femtotip;
Eppendorf North America, Hauppauge, NY) as a chisel. The micropipette tip was
controlled with an Eppendorf InjectMan micromanipulator system (Eppendorf North
America, Hauppauge, NY). The change of nuclear shape was recorded before and after
nuclear isolation.
Cell Spreading Assay
The seeding experiments were performed as described in the paper [78]. In brief,
cells were trypsinized and seeded onto FN coated glass-bottomed dishes in the
environment of 37°C and 5% CO2 before fixation with paraformaldehyde at different
time. In the dynamic study, cell was maintained in an environmental chamber with 37 C,
5% CO2 and imaged up to 1hr.
Imaging and Image Analysis
Imaging was done using a Nikon A1 laser scanning confocal microscope (Nikon,
Melville, NY) with a 60x/1.4NA oil immersion objective. For live cell imaging, cells were
maintained at 37 °C and 5% CO2 in a humidified chamber. Z-stacks were acquired with
a 0.3 µm axial step size for fixed cell imaging, and 1–2 µm axial step size for live cell
imaging. Fiji software [79] was used for image processing and all measurements.
For nuclear measurements, the nuclear outline was either traced by hand (for
experiments without fluorescence) or the nuclear parameters determined automatically
from applying an intensity threshold to maximum intensity XY nuclear projections of
fluorescent nuclei. Nuclear contour ratio of cancer cells was calculated as 4πA/P2,
where A is the area and P is the perimeter of the maximum intensity projection.
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To measure the three-dimensional abnormality, z-stacks were taken at an
interval of 0.3 and 0.75 µm, for fixed and live cells respectively, and X-Y position of each
pixel along the nuclear edge in each z-stack was detected with threshold determined by
Ostu method and recorded with Image J (NIH). Meanwhile, the nuclear volume was
measured with same threshold by Image J (NIH). The coordinate data of pixels in all z-
stacks were imported into Python software to calculate the volume of the convex hull
covering the whole nucleus with its integrated convex hull function. The three-
dimensional abnormality is calculated by dividing the difference between measured
nuclear volume and convex hull volume with measured nuclear volume. The intensity of
GFP-NLS signal in nucleus and cytoplasm was measured by Image J (NIH).
High-Content Imaging
Both MDA-MB-231 and MCF-10A cells were seeded on 384-well plates and then
fixed at approximately 95% confluence. The nuclei were stained with same
concentration of H33342 dye and imaged with an Operetta CLS system (PerkinElmer,
Waltham, MA) with the same intensity of UV light. The images were analyzed by Cell
Profiler software (Carpenter lab, Boston, MA) to acquire data of nuclear morphology and
signal intensity.
Results
The Deforming Cell Shape Amplifies Cancer Nuclear Abnormalities
To gain insight into how irregular cancer nuclear shapes are established, we
quantitatively tracked nuclear irregularities during cell spreading by imaging GFP-NLS
expressing nuclei in MCF-10A and MDA-MB-231 cells (Figure 3-1A). We observed
instances of transient nuclear membrane rupture (indicated by a loss and later recovery
of GFP-NLS signal in the nucleus) during spreading in MDA-MB-231 cells but not in
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MCF-10A cells (Figure 3-1A). The occurrence of membrane rupture indicates the
presence of mechanical stresses on the nuclear membranes during spreading. To
calculate irregularities while the nucleus deforms significantly in three dimensions, we fit
a convex hull to the three-dimensional nuclear shape constructed from confocal stacks,
and calculated the fractional deviation of nuclear volume from the fitted hull. The 3-D
abnormality increased in cancer nuclei dynamically during spreading of cancer cells, but
not in MCF-10A cells (Figure 3-1B). These results show that dynamic deformation of the
cell during spreading amplifies nuclear irregularities in cancer cells, but not in non-
malignant cells. Consistent with our previous results, cancer nuclear abnormality
increases during spreading even in the absence of actomyosin contraction, although the
increase is attenuated (Figure 3-1C). We therefore cannot rule out a contribution of
actomyosin stresses in establishing abnormal nuclear shapes.
Disrupting Either the LINC Complex or the Cytoskeleton Dampens Cancer Nuclear Abnormality
Cytoskeletal forces are transmitted to the nuclear lamina and associated proteins
through nuclear envelope proteins that form the Linker of the Nucleus to Cytoskeleton
(LINC) complex [34, 80, 81]. We therefore hypothesized that LINC complex disruption in
MDA-MB-231 cells normalizes the nuclear shape. We disrupted the LINC complex with
two approaches: overexpression of GFP-KASH4 and GFP-SUN1L-KDEL. KASH4 is the
binding domain of nesprin4 interacting with SUN1/2 in the inner nuclear membrane, and
overexpression of GFP-KASH4 competitively prevents the binding of endogenous
nepsrin proteins with SUN1/2, which consequently disrupts the LINC complex. GFP-
SUN1L-KDEL is negative domain protein disabled to bind with KASH domain, which
can disconnect nuclear membrane and cytoskeleton by competing with their
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endogenous counterparts after overexpression. LINC complex disruption with either
KASH4 or SUN1L-KDEL over-expression indeed reduced the three-dimensional
abnormality of the nucleus without change of cell spreading area (Figure3-2B and C).
Consistent with this data, disrupting cytoskeletal forces also reduced the 3D abnormality
of the nucleus (Figure 3-3A and B) with little change of cell spreading area (Figure 3-3
C), either through myosin inhibition by blebbistatin or disrupting microtubules with
nocodazole treatment. Conversely, overexpressing laminB1 or laminA/C both increased
3D nuclear abnormality (Figure 3-2B).
The Reduction of Nuclear Abnormality by LINC Complex Disruption Impairs Cellular Motility
As shown above, LINC disruption ‘normalized’ the abnormal cancer nuclear
shape. Based on previous results from Alam et al [37] which showed that transmission
of forces to the nucleus is necessary for persistent cell migration, we hypothesized that
LINC disruption in cancer cells reduces cancer cell migration. The LINC complex was
again disrupted with GFP-KASH or GFP-SUN1L-KDEL expression as above. Compared
with control groups (GFP and GFP-KDEL), migration is significantly reduced in LINC-
disrupted cells (GFP-KASH4 and GFP-SUN1L-KDEL, Figure 3-4). This is reflected in
over 50% drop in the mean squared displacement (MSD) (Figure 3-5). This drop in
migration is likely due to the fact that the nucleus does not transmit forces efficiently,
because recent data from the Lele lab suggests that cytoskeletal signaling pathways
that control migration are unaltered in LINC disrupted cells (data not shown).
Abnormal Morphology of Cancer Nucleus do NOT Necessarily Reflect Chromatin Content
There is some evidence that chromatin instabilities could cause abnormal
nuclear shapes in cancer [74, 76]. We showed above that nuclear abnormalities
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increase during cancer cell spreading (as opposed to decreasing in normal cell
spreading). Here we asked if the ploidy (chromosomal content) in cancer nuclei
correlates with nuclear shape abnormalities. To do this, we imaged a large number of
nuclei stained with H33342 with a high content imaging technique. We observed a
bimodal distribution of the total amount of DNA content in both MCF-10A and MDA-MB-
231 cells, consistent with cells being in different phases of the cell cycle (Figure 3-6A).
MDA-MB-231 cells were observed to have a long tail in the distribution, suggestive of
aneuploidy (Figure 3-6A). MDA-MB-231 cells had higher average DNA content as
evident from the right-shifted distribution of MDA-MB-231 cells compared with MCF-10A
cells (Figure 3-6B). We found only minor difference in the dependence of 2-D nuclear
abnormality on DNA content (Figure 3-6C) between MDA-MB-231 with MCF-10A cells.
DNA content also did not correlate with 3-D nuclear abnormality (Figure 3-6D). Taken
together, these results suggest that abnormal nuclear morphology is not necessarily
reflective of chromatin content.
Nuclear Abnormality can be Inherited by Offspring
We next examined if cells containing abnormal nuclear shapes have defects in
proliferation. MDA-MB-231 cells with either normal or abnormal nucleus were imaged
up to 18 hr. In this time period, cells with abnormally shaped nuclei were half as likely to
divide as cells that contained a normal nucleus (Figure 3-7A). We also tracked nuclear
shapes in daughter cells. Mother cells with regular nuclei tended to divide into cells with
regular nuclei, while cells with abnormal nuclei (Figure 3-7B) tended to divide into cells
with abnormal nuclei. We also found that over half of the cells with highly abnormal
nuclei (nuclei with two or more lobed structures) were able to remain alive up to 18hr
imaging and the fraction of cell with successful division is similar with cells with regularly
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abnormal nuclei (Figure 3-7C). These results indicate that the abnormal shape of the
nucleus attenuates but does not eliminate proliferation in breast cancer cells.
Discussion
Nuclear Abnormalities in Cancer.
We quantified the degree of abnormality in the cancer nuclear shape during
dynamic deformations in cell shape. The abnormality increased in MDA-MB-231 cells,
but not in MCF-10A cells during the process of cell spreading. Coupled with the
irreversibility of the shape deformations upon cell microdissection and the observed
reduction of cancer nuclear irregularities upon LINC disruption, we interpret these
results to suggest that mechanical stress on the cancer nucleus generated by the
dynamic process of spreading amplifies cancer nuclear irregularities. Furthermore,
treatment with Y27632 which inhibits myosin but promotes actin polymerization, did not
prevent the increase in the nuclear abnormalities during cell spreading. This is similar to
our observations in Chapter 2 where myosin activity was not required for nuclear
shaping during cell spreading.
Recent literature suggests that many LINC complex components are
downregulated, both in patient breast tumors and breast cancer cell lines [14]. SUN1
and SUN2 levels are downregulated in breast cancer tumors relative to surrounding
non-cancer tissue, and genes encoding nesprin-1 and nesprin-2 are mutated in breast
cancer tissue [82, 83]. Our result that LINC disruption reduces cancer nuclear
irregularities suggests that the LINC complex is still mechanically functional in cancer
cells despite such changes to its protein components, and contributes to nuclear
irregularities.
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It has been reported that lamins in cancer nuclei are spatially inhomogeneously
distributed in cancer nuclei resulting in a mechanically ‘patchy’ nuclear surface [77].
Thus, certain regions of the heterogeneous nuclear surface may be more compliant in
response to mechanical stresses, resulting in the observed irregular nuclear shapes.
We note here that knockdown of nuclear proteins other than the nuclear lamins [84] can
also result in irregularities- these include the chromatin remodeling protein BRG1 [85]
and endosome regulator protein Wash [86].
We have previously shown that cell spreading is necessary and sufficient to drive
nuclear flattening in fibroblasts under a wide range of conditions [78], including in the
absence of microtubules, vimentin intermediate filaments and myosin activity.
Therefore, we speculate that here it is primarily the F-actin network that transmits stress
from the moving membrane to the cancer nucleus. However, once F-actin is completely
disrupted with pharmacological agents, any further cell protrusion and spreading are not
possible [78], which makes it difficult to test the role of F-actin in mediating nuclear
response to deformations in cell shape over the required time scale, at least several
minutes. Alternatively, it is possible that any of the three cytoskeletal filaments can
transmit stresses to the nucleus, and disruption of one of these initiates other filaments
to engage in the force transmission. If there is such redundancy, then identifying the
molecular structures that cause an increase in cancer nuclear abnormalities is likely to
remain a fundamental challenge.
The shape of the nucleus has been commonly assumed to store elastic energy.
The prediction of this model is that nucleus should restore the original rounded shape
once removing cytoplasmic forces. Similarly, nuclear lobes and invaginations,
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characteristic of cancer nuclei, are thought to be elastically deformed in response to
cytoskeletal forces. These results are contradicted, however, by recent results from the
Lele lab [87] that isolation of the cancer nucleus from the cell by micro-dissection does
not cause a relaxation of the nuclear shape. The lack of elastic relaxation upon removal
of the surrounding cell implies that the deformed nuclear shape in cancer cells stores no
elastic energy and that the deformation is not an elastic response to the instantaneous
cell shape-dependent cytoskeletal forces on the nucleus.
Because gene expression and protein synthesis potentially depend on the
physical properties of the nucleus [17, 55, 88], reversible deformations of the cancer
nucleus may trigger expression of genes during cell migration by modulating chromatin
compaction [89, 90]. Therefore, knowing how dynamic cell shape information is
transmitted to the cancer nucleus will be key to a complete understanding of the
relationship between cell shape and cell function.
Mechanical Stress and Cancer Cell Migration.
Our observation that LINC disruption normalizes the nucleus and reduces cancer
cell motility is surprising considering that the LINC complex components are
downregulated in MDA-MB-231 cells [14]. It appears that the LINC complex continues
to be functional in these cells despite low levels of these proteins. Unpublished results
from the Lele lab have shown that LINC disruption does not alter Rho/Rac/Cdc42
signaling pathways in these cells. Wu et al. [57] and Alam et al. [37] have suggested
that the nucleus can act as an intracellular scaffold, transmitting forces from one end of
the cell to the other. This mechanical integration allows the cell to migrate efficiently.
Thus, we interpret these results to suggest that an intact LINC complex transmits forces
in breast cancer cells.
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The existence of aneuploidy or an abnormal number of chromosomes has been
shown in most cancer types and is known to promote the progression of cancer [76, 91,
92]. As expected, our results show that MDA-MB-231 cells have more DNA than
MCF10A cells. Coupled with the fact that MDA-MB-231 cells have abnormal nuclei, one
might conclude that abnormal shapes of nuclei correlate with DNA content. However,
further examination shows that there is little correlation between DNA content and
nuclear shape abnormalities. Therefore, we conclude that nuclear shape abnormalities
in cultured cancer cells are not explained by DNA content alone. We have also shown
that the abnormality of nuclear shapes is heritable. The mechanism for this is unclear
and will need to be probed further in future studies.
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Figure 3-1. The abnormality of nucleus amplifies during cell spreading. A) Shown is an
example of nuclear rupture evident from the loss of GFP-NLS from the nucleus of a spreading MDA-MB-231 cell. An MCF-10A cell during spreading is shown for comparison. The change of NLS signal intensity normalized with its initial intensity is shown in line plots at right of the corresponding cell type. Scale bar is 10µm B) Schematic shows the calculation of 3D abnormality defined here as the difference in volume between the nucleus and a convex hull fit to the nucleus divided by nuclear volume. Plot shows 3D abnormality plotted against time of spreading in MCF-10A and MDA-MB-231 cells. C) The abnormality of nuclear shape increases during cell spreading even in the absence of myosin contraction. (n>30 at each time point, data are shown as Mean ± SEM, p value of ANOVA analysis is 0.02, 0.04 and 0.83 for DMSO, Y27 and Blebbistatin, respectively)
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Figure 3-2. Disrupting the LINC complex reduces nuclear abnormality. A). Images of nuclei stained with H33342 (blue) are shown in cells transfected with GFP (control), GFP-KASH4, GFP-KDEL (control), GFP-SUN1L-KDEL, GFP-Lamin A/C or GFP-lamin B1. The 3-D nuclear abnormality B) and cell spreading area C) for populations of cells quantified. (Three biological replicates with n=59, 62, 90, 86, 93 and 72 for 3D nuclear abnormality of GFP, GFP-KASH4, GFP-KDEL, GFP-SUN1L-KDEL, GFP-Lamin A, GFP-Lamin B1, respectively; and n>30 for cell spreading area of all conditions. Data are shown as Mean ± SEM, * indicates p<0.05 by t-test)
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Figure 3-3. Disrupting cytoskeletal elements reduces nuclear abnormality. A) Images of cells treated with latrunculin A (Lat A), blebbistatin (Blebb), or nocodazole (Noco) are shown stained for microtubules (green), actin (red) and DNA (blue). The 3-D nuclear abnormality B) and cell spreading area C) for populations of cells quantified.(Three biological replicates with n=39, 142, 107 and 75 of nuclear 3D abnormality for wild type, nocodazole, blebbistatin, and latrunculin A, respectively; and n>30 for cell spreading area of all conditions. Data are shown as Mean ± SEM, * indicates p<0.05 by t-test).
87
Figure 3-4. Trajectory maps of MDA-MB-231 cells with or without LINC disruption.
Each color line in the trajectory maps represent one cell (More than Three biological replicates with n=42, 40, 40 and 39 for GFP, GFP-KASH4, GFP-KDEL and GFP-SUN1L-KDEL, respectively). The inserted images at top right corner of trajectory map are the representative images of transfected cell for each condition.
88
Figure 3-5. LINC complex disruption impairs cellular motility. Shown is the MSD of individual cell (A) and plot of average MSD (B) in all four conditions. (More than Three biological replicates with n=42, 40, 40 and 39 for GFP, GFP-KASH4, GFP-KDEL and GFP-SUN1L-KDEL, respectively. Data are shown as Mean ± SEM, * indicates p<0.05 by Mann-Whitney Test).
89
Figure 3-6. The abnormal shape of nucleus does not correlate with DNA content. A) Shown is the distribution of total DNA content (proportional to the total signal intensity of H33342) of MCF-10A and MDA-MB-231 cells (Three biological replicates with n = 9995 and 7970 for MCF-10A and MDA-MB-231, respectively). B) Shown is the distribution of average DNA content (defined as total H33342 intensity over total pixel number) for MCF-10A and MDA-MB-231 cells. C) Nuclei of MCF-10A and MDA-MB-231 cells were binned according to total DNA content and the average nuclear 2D abnormality (defined as the difference in nuclear area between the nucleus and a convex hull fit to the nucleus divided by nuclear area) is plotted for each bin. Error bars represent SEM for n = 625, 7157, 1633, 494, 81 (MCF-10A) and 0, 232, 2839, 2970, 1865 (MDA-MB-231) nuclei for each bin, respectively. D) Scatter plot of total DNA content with respect to nuclear 3D abnormality. (Data are shown as Mean ± SEM, * indicates p<0.05 by t-test).
90
91
Figure 3-7 The abnormality of nuclear shape is heritable. A) MDA-MB-231 cells were manually classified as having a regular or irregular nucleus (More than Three biological replicates with n = 225 and 281, respectively) and tracked for 18 hours with DIC microscopy. As shown, 14% of cells with regular nuclei and 6% of cells with irregular nuclei underwent mitosis during this period. B) Of the cells that divided, the resulting daughter nuclei were classified as regular and irregular (as before) and plotted in the shown pie charts (“unclear” indicates that the nucleus could not be classified due to changing focal plane or migrating out of the field of view). C) Shown is the pie plot about the fate of mother cell with highly irregular nuclei (“highly” is identified according to the number of lobules of nucleus, which is at least two).
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CHAPTER 4 CONCLUSIONS
Summary of Findings
This thesis has discovered a novel mechanism for nuclear shaping. We
discovered that the degree of nuclear flattening is closely tied to the degree of cell
spreading. For example, Figure 4.1 shows a plot of the x-z nuclear aspect ratio versus
cell spreading area. This data is pooled from fibroblast experiments across different
conditions- myosin inhibition, microtubule disruption, what else, etc. As seen, all data
seem to suggest a ‘master’ curve that captures the fact that nuclear flattening is
inversely correlated with the degree of spreading. As long as cells are able to spread,
the nucleus is flattened. This observation directly challenges the currently accepted
model in the literature in which myosin activity causes actomyosin bundles to compress
and thereby shape the nucleus [22, 38].
To explain the dependence of nuclear flattening on cell spreading, we have
proposed a model in which motion of the cell boundary transmits a frictional stress
through the cytoskeleton to the nuclear surface. Model calculations show that this
mechanism adequately explains how the vertical nuclear shape mimics vertical cell
shape. After publication of our paper, Alam et al. [37] reported that local protrusions
proximal to the nuclear surface can transiently deform the nucleus, and relaxation of the
protrusions caused relaxation of the deformed shape (Figure 4.2). Similarly, Neelam et
al. [93] have shown that vertical nuclear shapes mimic vertical cell shapes in confluent
epithelial cell monolayers. Our proposed mechanism in which the nuclear shape mimics
cell shape through local movement of cell boundaries can easily explain how the
nucleus takes on an hour-glass shape in cells migrating through small pores as
93
observed by Lammerding, Friedl and coworkers [94, 95]. In contrast, it is not clear how
actomyosin bundles could compress the nucleus into such hourglass shapes that mimic
cell shapes.
Our work also suggested that the nucleus reaches a steady-state shape once the
nuclear lamina becomes completely taut, beyond which is stiff to cellular forces. Our
concept has since received support from direct measurements of the stiffness of
isolated nuclei by Stephens et al. [96]. They showed that the nucleus is soft below
~30% strains and this stiffness is due to chromatin inside the nucleus. Beyond this
strain, the stiffness increases abruptly and the authors attribute this to the taut nuclear
lamina. Also, Neelam et al. [93] have since shown that the folds in the nuclear lamina
decrease with cell spreading in MCF10A cells.
During cell spreading, we showed that the nuclear volume remains constant. Our
results thus suggest a mechanistic picture in which nuclear shape changes in the cell at
constant volume and constant surface area. Lammerding and coworkers have also
shown that the nuclear volume does not change during the large deformations observed
of the nucleus as the cell migrates through narrow pores [97].
If nuclear volume and surface area remain constant during changes in nuclear
shape, then relieving stresses on the nuclear surface should produce no changes in
nuclear shape. This concept directly contradicts the notion that the nucleus is elastically
deformed in cells, an assumption made in all current models in the literature [98-101].
Our concept has since received support from experiments by Keith Christopher in which
he removed nucleus from cell body by micro-excision and showed that the nucleus
94
undergoes no change in its shape upon isolation from the cell [87]. No elastic energy is
stored in the nucleus inside cells, both for fibroblasts as well as cancer cells.
We also demonstrated that lobes and invaginations in the cancer nucleus are
amplified by the process of cell spreading, and this amplification again does not require
myosin activity. This is not to suggest that actomyosin bundles cannot indent the
nuclear surface or constrict it in cancer cells after the initial nuclear shape is established
[102]. However, the frictional stress transmission is responsible for establishing the
steady-state shapes during processes like cell spreading.
Future Work
We proposed that the cell exerts frictional or viscous stresses on the nuclear
surface based on the physical concept that the cellular cytoskeleton does not store
elastic stresses over the time scales of several minutes over which the nuclear and cell
shapes are established. However, direct evidence that the stresses are frictional in
nature has not been developed yet. Experiments that correlate the rate of motion of the
cell boundary with the rate of motion of the nuclear surface could offer stronger support
to this transmission mechanism.
A crucial open question for the mechanism proposed in this thesis is the identity
of the cytoskeletal elements that propagate the frictional stresses to the nuclear surface.
We have already shown that microtubules, intermediate filaments and myosin activity
are not required for this stress transmission mechanism. We therefore hypothesize that
F-actin structures transmit these frictional stresses to shape the nucleus. This
hypothesis is challenging to test because without changes in cell shape, the nucleus
cannot change shape, and disrupting F-actin filaments rounds up the cell.
95
One possible experiment is to test the hypothesis that proteins which cross-link
F-actin filaments to transmit stresses. Such cross-linking proteins include alpha-actinin
and filamin. Assuming that knockdown of these proteins does not prevent F-actin
polymerization, it is possible that the nucleus will remain ‘rounded’ during cell spreading
and therefore prevent cell spreading despite normal F-actin polymerization at the
leading edge.
It is also possible that all three cytoskeletal elements, as well as membranous
structures like the endoplasmic reticulum or the Golgi apparatus might transmit stresses
to the nuclear surface. Even though disruption of the microtubules or intermediate
filaments does not prevent nuclear flattening, it is possible that these structures act
redundantly to transmit stresses. If this is indeed the case, then it will remain difficult to
unequivocally identify the structures that participate in the shaping of the nucleus.
How the nucleus itself behaves during shaping is also of interest. Our data
suggests that inhibiting myosin activity reduces nuclear volume. The nuclear volume
resists changes presumably because of osmotic stresses exerted by macromolecules
that do not leave the nucleus. This property likely accounts for the resistance to volume
expansion or compression. Therefore it is possible that myosin inhibition decreases cell
volume, which causes osmotic flow out of the nucleus and thereby reduce its volume.
Whether this mechanism is indeed responsible for decrease in the nuclear volume
remains unknown. Experiments which measure cell volume simultaneously with myosin
inhibition can prove useful. However, cell volume is not easy to accurately measure in
spread cells with current methods including confocal microscopy. Other measurements
96
might involve quantifying the concentration of tracer molecules upon myosin inhibition in
the cytoplasm and the nucleus.
We have shown that cancer nuclear abnormalities increase during spreading.
The reasons for this are unclear. It is possible that the nucleus has a mechanically
patchy surface as has been suggested by others [77], but direct evidence is not
available for this. It may be possible that nuclear manipulation experiments developed
by Neelam et al. [103] might be used to reveal if cancer nuclei indeed have spatially
distributed mechanical properties in the same cancer nucleus.
Our assays have focused on in vitro model systems in which cells spread on 2-D
surfaces. Whether these principles apply in tissues inside organisms remains to be
established. Nuclei in tissues formed by epithelial cells do not have ‘flattened’ nuclei.
Cells in epithelia tend to be cuboidal with corresponding nuclear shapes. We predict
that the mechanism by which these nuclear shapes are established in these tissues is
similar to the mechanisms we have discovered with in vitro model systems. While
testing these mechanisms in vivo is highly challenging, we expect that progress will be
made with intermediate model systems, such as 3D mammary epithelial acinar
assemblies in matrigel.
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Figure 4-1 Data pool of x-z nuclear aspect ratio versus cell spreading area. (Three
biological replicates with n=40, 34, 43, 38, 39, 35, 30, 33 and 34 for Control, ML-7, Y27, Blebbistatin, Nocodazole0.83µM, Vim+/+, Vim-/- MEF, Nocodazole1.67µM and Blebbistatin 6hr, respectively).
98
Courtesy of Samer Alam [37] Figure 4-2 Local nuclear deformation in response to local protrusion and retraction of
cell memrbane. Shown is the formation and retraction of a lateral protrusion of cell membrane near the nucleus at various confocal planes (planes 1-4 in cartoon on the left), and the reversible nuclear deformation (in the xy-plane) accompanying the protrusion. YZ view of the nucleus at the mid-plane shows the deformation in the yz-plane. Vertical dashed lines indicate the position of the corresponding yz plane. Cell is labeled with RFP-LifeAct for F-actin and GFP-histone H1 for nucleus. Scale bar, 10μm. Photo credits Samer Alam.
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APPENDIX A COMPUTATIONAL MODEL FOR NUCLEAR DEFORMATION DURING CELL
SPREADING
Constitutive Model for Cytoskeletal Network Stress
The assumed constitutive equation for the stress tensor in the network phase of
the cytoplasm is
𝛔 = 2𝜇�̇� + (σ𝐜 + λ∇ ∙ 𝐯)𝐈 A1-1
where I is the unit dyadic, �̇� = 1
2(∇𝐯 + ∇𝐯𝑻) is the rate-of-strain tensor, and 𝜇 and λ are
viscosity parameters. Eq. A1-1 models the cytoskeletal network as a compressible
contractile network. Network density changes, which may affect these properties, are
assumed to equilibrate by local assembly/disassembly over the slow time-scale of cell
spreading; therefore no continuity equation for the network density is required. Since
network volume is not locally conserved, Eq. A1-1 reflects both shear and
expansion/compression strains. If the strains caused by both modes of deformation
have the equivalent resistances, then we can assume λ ~0, reducing Eq. A1-1 to a
single viscosity parameter 𝜇. (In linear elasticity, this is analogous to assuming
Poisson’s ratio is zero such that the Young’s modulus (a measure of longitudinal
stiffness) equates to twice the shear modulus (a measure of shear stiffness).
The longitudinal transmission of normal stress to a surface due to a distant
moving boundary is an important property of Eq. 1.1 that is relevant to our model for cell
spreading. To illustrate this, consider first a one-dimensional case of a contracting
network that is fixed at one end (𝑣𝑥(𝑥 = 0) = 0) and moving with velocity V at a distance
𝑥 = 𝐿 (i.e. 𝑣𝑥(𝑥 = 𝐿) = 𝑉)). The stress balance ∇ ∙ 𝛔 = 𝟎 in the x-direction is
𝑑𝜎𝑥𝑥
𝑑𝑥= 2𝜇
𝑑2𝑣𝑥
𝑑𝑥2 = 0 A1-2
100
Applying the boundary conditions yields the velocity field
𝑣𝑥(𝑥) = 𝑉𝑥/𝐿 A1-3
as well as the stress field
𝜎𝑥𝑥 = σ𝐜 + 2𝜇𝑉/𝐿 A1-4
which is uniform in this case. Hence, noting the second term in Eq. A1-4, translating the
one boundary at 𝑥 = 𝐿 at speed V transmits an additional stress 2𝜇𝑉/𝐿 to the surface at
𝑥 = 0 due to longitudinal friction, which is positive for expansion (𝑉 > 0), and negative
for compression (𝑉 < 0).
Now consider a spherical cell of radius R with a nucleus of radius 𝑅𝑛, under the
assumption of spherical symmetry, the stress balance in the cytoplasm is
𝑑𝜎𝑟𝑟
𝑑𝑟+
1
𝑟(2𝜎𝑟𝑟 − 𝜎𝜃𝜃 − 𝜎𝜙𝜙) = 0 A1-5
Where
𝜎𝑟𝑟 = 𝜎𝑐 + 2𝜇𝑑𝑣𝑟
𝑑𝑟
𝜎𝜃𝜃 = 𝜎𝑐 + 2𝜇𝑣𝑟
𝑟 A1-6
𝜎𝜙𝜙 = 𝜎𝑐 + 2𝜇𝑣𝑟
𝑟
Assume now that new network is assembled at the cell membrane and moves
centripetally with speed 𝑣𝑎, and allow the cell radius to expand at speed V (ignoring for
now any volume constraints). Substituting Eqs. 2-2 into Eq. 2-1 and applying the
boundary conditions, 𝑣𝑟(𝑟 = 𝑅𝑛) = 0 and 𝑣𝑟(𝑟 = 𝑅) = 𝑉 − 𝑣𝑎 yields the r-velocity field,
𝑣𝑟 = (𝑉 − 𝑣𝑎)𝑟
𝑅
(1−(𝑅𝑛
𝑟)
3)
(1−(𝑅𝑛𝑅
)3
) A1-7
Eq. A1-3 then provides the rr-component of the stress tensor:
101
𝜎𝑟𝑟 = 𝜎𝑐 + 2𝜇(𝑉 − 𝑣𝑎)1
𝑅
(1+2(𝑅𝑛
𝑟)
3)
(1−(𝑅𝑛𝑅
)3
) (A1-8)
Such that stress of the nucleus surface is:
𝜎𝑟𝑟(𝑟 = 𝑅𝑛) = 𝜎𝑐 + 2𝜇(𝑉 − 𝑣𝑎)1
𝑅
3
(1−(𝑅𝑛𝑅
)3
) A1-9
(In the small gap limit, 𝐿 = 𝑅 − 𝑅𝑛 ≪ 𝑅 , Eq. A1-7 and A1-8 become equivalent to
Eqs.A1-3 and A1-4.). Therefore, similar to the one-dimensional case, the net tensile
stress on the nucleus is increased by movement of the cell boundary to expand the
network, but is also reduced by assembly of network at the membrane and the resulting
centripetal flow, which causes compression of the intervening network. In this way, the
movement of the cell boundary and network assembly at the cell membrane can
modulate the stresses on the nuclear surface. In general, the nucleus will tend to distort
to follow the changes in cell shape and will follow the flow field generated by network
assembly at the membrane. This is the basis for our model for nuclear shape changes
during cell spreading.
Model for Cell Mechanics
We apply a simple mechanical model of the cell that takes into account (1) the
resistance of the nucleus to volume compression/expansion; (2) resistance of the
nuclear surface to area expansion; (3) tension of the cell membrane, and (4) friction due
to centripetal flow of network tangent to the adhesive substratum. The network normal
stress on the nuclear surface is balanced by the nuclear internal tension 𝜏𝑛𝑢𝑐 (or
pressure when 𝜏𝑛𝑢𝑐 < 0) due to its resistance to volume changes, and the nuclear
surface tension Τ𝑛𝑢𝑐, due to its resistance to surface area expansion. The internal
nuclear tension is modeled as
102
𝜏𝑛𝑢𝑐 = 𝐾ln(𝑉/𝑉0) A2-1
Where K is the bulk compressibility and 𝑉0 is the unstressed volume. The surface
tension Τ𝑛𝑢𝑐 of the nucleus is expected to depend on strained surface area of the
nuclear lamina A above the unstressed area 𝐴0. We note that surface area undulations
are evident in cross-sectional images of nuclei, indicating roughly 20%-40% excess
area. Therefore, to account for the energy associated with smoothing the nuclear
lamina, we estimated Τ𝑛𝑢𝑐 using the following equation, which is normally applied to
calculate vesicle surface tension accounting for thermal undulations [104]:
𝐴−𝐴0
𝐴0=
𝐸𝑠
8𝜋𝑘𝑐𝑙𝑛 (1 +
𝐴0
24𝜋𝑘𝑐𝑇𝑛𝑢𝑐) +
𝑇𝑛𝑢𝑐
𝜅 A2-2
for > 𝐴0 , where κ is the area extensional modulus of the nuclear lamina, 𝑘𝑐 is its
bending modulus of the lamina, and 𝐸𝑠 is a parameter that can be considered the
magnitude of the energy driving the undulations (equal to 𝑘𝐵𝑇 - Boltzmann’s constant
multiplied by temperature – for undulations driven by thermal energy). In this equation,
the first logarithmic term dominates at low area expansion (low lamina tension), while
the second term dominates at high area expansion. Assuming a value of 𝐸𝑠 ~100 𝑘𝐵𝑇
(Boltzmann’s constant multiplied by temperature) yields excess area in the observed
range, which is reasonable noting intracellular energy fluctuations tend to be on the
order of 100-fold larger that thermal fluctuations [105].
Except for the adhesive substratum, tangential traction stresses on cell and
nuclear membrane surfaces are assumed negligible (i.e., slip boundary conditions).
The normal stress exerted on the cell membrane is assumed to be balanced by the
cell’s internal hydrostatic pressure 𝑃ℎ (assumed uniform throughout the cell and
nucleus) and the stress due to membrane tension 𝑇𝑚𝑒𝑚. Due to the high cytoplasmic
103
osmolality, cells are resistant to volume changes under typical cellular stresses, hence
simulations were performed under the constraint of constant cell volume, maintained by
varying 𝑃ℎ.
For the boundary at the substratum, network flow at the substratum is assumed
to exert a tangential stress vector equal to 𝜂𝒗(𝑧 = 0), where 𝒗(𝑧 = 0) is the network
velocity tangential to the substratum. The limit 1/η→0 represents the case of perfect
adhesion, such that 𝒗(𝑧 = 0) = 0 (no-slip boundary condition). In either case, it is
assumed there is no network flow in the direction normal to substratum.
To account for cortical actin assembly at the cell membrane, the net boundary
velocity is increased by the actin assembly speed 𝑣𝑎 directed normal to the surface,
except near the substratum contact boundary, where assembly occurs with speed 𝑣𝑎𝑐
directed tangential to the substratum. The net local velocity of the cell membrane is
therefore equal to the difference between the network assembly velocity and the
retrograde flow velocity.
Model Parameters
Parameter Estimates: A list of parameters used in the simulations is shown in
Table A-1. It should be emphasized that key qualitative conclusions from the model –
network flow-driven translation of the nucleus to the surface, nuclear flattening resulting
from cell spreading rather than network tension -- do not strongly depend on several
parameter values, as noted below. Values for nucleus area modulus 𝜅, nuclear bulk
modulus 𝐾 were obtained from measurements by Dahl et al. [11], with the latter
parameter value calculated from their measured osmotic resistance to volume
expansion. Values for membrane tension 𝑇𝑚𝑒𝑚 varies widely from 0.01 nN/μm to 0.3
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nN/μm [106, 107], so a mid-range value of 0.1 nN/μm was used (the quantitative
predictions depend only weakly on the value of this parameter). The network assembly
speed at the contact boundary 𝑣𝑎 = 𝑣𝑎𝑐 was estimated from the observed initial speed
of cell spreading (0.5 μm/min). The assembly speed of network at the cell cortex v_a is
not known, but we show results for two cases: 𝑣𝑎 = 𝑣𝑎𝑐 and 𝑣𝑎 = 0, to demonstrate that
cytoskeleton assembly and resulting flow (𝑣𝑎 > 0) is necessary for initial translation and
flattening of the nucleus against the surface. The value of network viscosity was
estimated from the literature [108]. The contractile stress 𝜎𝑐 could be estimated from
Eqs. A1-6 and A2-1, noting that volume was ~50% reduced upon myosin inhibition. If
𝜎𝑐 is assumed to be zero in this case, then 𝜎𝑐 for the control case can be estimated from
the volume difference. Under typical values of other parameters, the second term in Eq.
1.6 is relatively small, such that 𝜎𝑐 ≅ 𝐾 ln(𝑉𝑛 𝑉𝑛∗⁄ ), or 𝜎𝑐 ≅ 0.69𝐾 for a 50% volume
reduction. However, as in the main text, a key prediction is that shape changes during
spreading do not significantly on this background network tension.
Parametric Sensitivity: The key model predictions – translation of the nucleus to
the substratum and flattening of the nucleus during cell spreading – were found depend
to only on the following quantities. The speed of network assembly at the cortex relative
to the contact boundary assembly speed, 𝑣𝑎/𝑣𝑎𝑐 determines how fast the nucleus
translates to the surface while the cell spreads. The nuclear envelope stiffness relative
to viscous stresses (dimensionless ratio 𝜅/𝜇𝑣𝑎𝑐) as well as amount of excess nuclear
surface area (reflected in Eq. A2-2) determines the extent of nucleus flattening in a fully
spread cell. The substratum adhesivity relative to the viscous stresses (dimensionless
ratio 𝜂𝑅𝑛/𝜇) determines the steady-state cell spreading area (hence the amount of
105
flattening). Model simulations were found to not depend strongly on the bulk modulus of
the nucleus because nuclear shape changes during flattening can proceed without
requiring volume compression (i.e. at constant volume). The contractility parameter 𝜎𝑐
was unimportant since the tension this parameter quantifies was assumed to be uniform
throughout the cytoplasm hence it acts uniformly on all surfaces, and motion is driven
by the divergence of the stress tensor, ∇ ∙ 𝛔 (c.f. Eq A1-1), in which case the constant 𝜎𝑐
disappears. The assumed value of cell membrane tension relative to viscous stresses
(dimensionless ratio, 𝑇𝑚𝑒𝑚𝜇/𝑣𝑎𝑐) had a modest effect on the curvature of the cell
membrane of a spread cell, but had little effect the predicted cell spreading dynamics
and nuclear shape changes.
Methods for Simulating Cell Spreading
The resulting quasistatic stress balance ∇ ∙ 𝛔 = 0 based on Eq. A1-1 is
mathematically equivalent to the classic problem of elastostatic deformation of an
isotropic elastic medium. Therefore, axisymmetric velocity field 𝑣𝑗(𝐱′) (𝑗 = 𝑅, 𝑍) at
position 𝐱′ = [𝑅 𝑍]𝑇can be obtained from the following boundary integrals over the
nucleus, substratum, and cell membrane boundaries (represented by 𝛤):
𝑐𝑖𝑗𝑣𝑗(𝐱′) + 2π ∫ 𝑣𝑗(𝐱)𝑝𝑖𝑗(𝐱, 𝐱′)𝑅𝑑𝛤(𝐱) = 2π ∫ 𝑇𝑗(𝐱)𝑢𝑖𝑗(𝐱, 𝐱′)𝑅𝑑𝛤(𝐱)𝛤𝛤
4.1
where 𝑢𝑖𝑗(𝐱, 𝐱′) and 𝑝𝑖𝑗(𝐱, 𝐱′) are velocities and tractions, respectively, arising from a
concentrated point force located at position x, given by Kelvin’s fundamental solutions
for the axisymmetric case for linear elasticity (provided in reference [109]), but with the
shear modulus replaced with 𝜇 and the Poisson ratio set to zero. The tensor 𝑐𝑖𝑗 is equal
to the Kronecker delta (identity tensor) 𝛿𝑖𝑗, on the cytosplasmic domain and is a known
tensor on the surface 𝛤. The boundary element method was used to estimate the
106
instantaneous velocities and stresses at the boundaries [110], such that the evolution of
cell and nuclear shapes could be simulated by numerically integrating the boundary
positions over time. The cell surface was discretized into 100 axisymmetric quadratic
boundary elements and the nuclear surface into 50 elements. Integrals on the elements
were approximated with 10th order Gauss quadrature except strongly singular integrals,
which were obtained from analytical rigid body translation (z-direction) and plan strain
(r-direction) conditions [109]. At each time step, the surface velocities and stresses were
calculated under the constraint of constant volume, and time-stepping was performed
using the 4th-order Runge-Kutta method. The constraint of constant volume was
imposed by simultaneously solving for the hydrostatic pressure 𝑃ℎ at each time step that
keeps the net volume change equal to zero. The node spacing was reset at each time
step by interpolation using cubic Hermite interpolating polynomials (MATLAB function
pchip). To prevent close approach of the nuclear surface to the cell membrane (which
mathematically allowed by way of Eq. A1-1, but creates numerical issues due to surface
singularities in the boundary element method), a close-range repulsive pressure was
imposed for close separation distances 𝑧 of the form 𝑃(𝑧) = (𝑑 𝑧⁄ )3𝑒−𝑧 𝑑⁄ 𝑑 = 0.01 𝑅𝑛.
The initial condition was set to a nearly spherical cell with a small contact area of
approximately 0.5% of the total cell membrane surface area.
107
Table A-1. Parameters of Cell Spreading Model
Parameter Symbol Value Source
Contractile stress 𝜎𝑐 0.19
nN/m2
Estimated from myosin-induced
nuclear volume change
Nucleus bulk modulus 𝐾 0.25
nN/m2
Ref [11], isolated Xenopus oocyte
nuclei
Nucleus area modulus 𝜅 25 nN/m Ref [11], isolated Xenopus oocyte
nuclei
Membrane tension 𝑇𝑚𝑒𝑚 0.1 nN/m Ref [106], moving fish keratocytes;
Ref [107]
Network Viscosity
Parameter
𝜇 0.21 nN-
s/m2
Ref [108], adherent J774
macrophages
Nuclear lamina bending
stiffness
𝑘𝑐 3.5x10-4
nN-m
Ref [111], MEFs
Network assembly speed
at contact boundary with
substratum
𝑣𝑎𝑐 0.5 m/min Estimated from spreading speed
Network assembly speed
at cell cortex
𝑣𝑎 Varied
Energy parameter in area
expansion equation
𝐸𝑠 3.2x10-4
nN-m
Estimated from excess surface
area
Nuclear radius 𝑅𝑛 6.3 m Measured
Cell radius (rounded) 𝑅 8.3 m Measured
108
APPENDIX B THE INFLUENCE OF CELL GEOMETRY ON NUCLEAR VOLUME
The evidence that nuclear shape mimics the change of cell shape triggers our
interests on the change of nuclear volume with different cell geometry. Vincent Tocco, a
graduate in my lab, has shown that the nuclear volume decreases significantly in cells
cultured on 1D line comparing with 2D surface, which means that cell geometry is able
to regulate nuclear volume. Then will the nuclear volume increase if the cell geometry is
removed? To answer this question, alive cells with GFP-histone expression on 1D line
and 2D surface were detached from substratum with trypsinization and imaged until
them completely became rounded. For cells on 2D surface, no significant change of
nuclear volume was observed for individual cells along with the decreased cell
spreading area (Figure B-1A). Consistently, average change of nuclear volume does not
change significantly in statistics although there is a tendency of decay (~5% drop)
during the process of detachment (Figure B-1B). However, the nuclear volume of cells
on 1D line increases after detaching from substratum (Figure B-1C) and this increase is
statistically significant (Figure B-1D). Comparing the nuclear volume of cells on 1D line
with 2D surface at different stages of cell detachment, the significant difference of
nuclear volume disappears after removal of the cell geometry (Figure B-1E). Taken
together, the change of nuclear volume induced by cell geometry is reversible without
constrain of the certain cell geometry.
109
Figure B-1 The difference of nuclear volume induced by cell geometry vanishes after
the removal of geometry constrain. The nuclear volume of individual cell A) remains constant during the cell detachment from 2D substratum accompanying with the decreased spreading area of cell. B) The average nuclear volume (normalized by initial volume before detachment) does not have statistically significant difference among the time points in the whole process. C) The nuclear volume of individual cells continuously increases along with the detachment of cell from 1D line, which is further supported by the increasing average of normalized volume of nucleus in D). E) There is statistically significant difference of nuclear volume between cell on 1D line and 2D surface before and immediately after detachment. Yet, this significant difference continuously decreases until disappear with completely rounded cell. (n=20 and 15 for 2D surface and 1D line, respectively. Data are shown as Mean ± SEM.* indicates p<0.05)
110
111
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BIOGRAPHICAL SKETCH
Yuan Li was born in Baoji City, Shaanxi Province, China in 1988 to Yinchi Liu
and Heng Li. He finished his high school from Baoji Middle School, China in 2006. In
July of 2010, he received the Bachelor of Science degree in chemistry from College of
Science, Nanchang University, Jiangxi, China. He joined University of Florida in 2011
and conducted research on mechanics of cell and nucleus in Dr. Tanmay Lele’s lab in
the Department of Chemical Engineering. He earned his Master of Science degree in
chemical engineering in 2013. In 2014, he converted to PhD program in Dr. Tanmay
Lele’s group and continued his research on studying mechanisms of nuclear shaping in
fibroblasts, epithelial cells and breast cancer cells. He earned his Doctor of Philosophy
in chemical engineering from the University of Florida in 2017.
