3

Welcome to the joint meeting of the Spanish Network of Excellence in Mechanobiology and the European Innovate Training Network BIOPOLBy factoring the role not only of biochemical but also of mechanical signals, the emerging discipline of Mechanobiology is reshaping our understanding of molecules, cells, and tissues. This meeting brings together two networks dedicated to this blooming topic: the Spanish Network of Excellence in Mechanobiology, and the European Training Network BIOPOL. The meeting will feature talks and poster presentations from both senior and junior members of both networks, and will address fundamental and applied questions in mechanobiology from the perspectives of physics, biology, and engineering. The meeting is also open for attendance to anyone interested in the topic.

Enjoy the meeting!

4

Information for participantsInformation DeskThe conference registration and information desk will be located in the main reception hall of the Antoni Caparrós Auditorium. It will be staffed from 08:15 to 18:00 on Thursday 6th October and from 8:45 to 13:30 on Friday 7th October.

BadgesEach registered participant will receive a name badge. For security reasons, the badge must be clearly exhibited in order to access the congress area during all scientific and social events. Replacements for lost badges will be available from the registration desk.

Speakers/Oral presentations All presentations should be upload during registration time (from 8:15 to 8:45) before the Opening Ceremony or during the coffee breaks.

Poster sessionsPosters should be hung during registration between 8:15 and 8:45 on Thursday 6th October. Please refer to the information board in the registration area or this book to check which board number has been allocated to you.

Posters can remain on display throughout the conference and should be removed between 13:30 and 14:30 on Friday 7th October. Any posters remaining after the indicated time will be removed by the organizers, who accept no responsibility for loss or damage.

Poster sessions will take place during the coffee and lunch breaks.

Certificate of attendanceIf you wish to have a Certificate of Attendance, you can request one from the Secretariat at ibecevents@ibecbarcelona.eu

Wi-Fi Conne ctionNetwork: PCBGuest

User: ibec-guestPassword: 2tz7aZWR

5

Programme

08:15 - 08:45 Registration

08:45 - 09:00 Welcome

Cell and tissue mechanics -Chair: Daniel Navajas-

09:00 - 09:45 Jochen Guck Mechanosensing in the central nervous system

09:45 - 10:05 Ignasi Jorba Effect of decellularization in heart scaffold mechanics

10:05 - 10:25 Léo ValonOptogenetic control of cell forces and mechanotransduction

10:25 - 10:45 Clara ValeroMechanical evaluation of collagen-based gels for in-vitro experiments

10:45 - 11:15 Coffee break

Mechanosensing and mechanoresponse (I) -Chair: Miguel Ángel del Pozo-

11:15 - 12:00Christophe Lamaze

Caveolae mechanotransduction and cancer : role of the EHD2 ATPase

12:00 - 12:20Fidel-Nicolás Lolo

Identifying mechanosensing and mechanotransducer elements within caveolae

12:20 - 12:40 Roger OriaForce loading explains how substrate rigidity and ligand nano-distribution regulate cell response

12:40 - 13:00 Arnaud Nicolas3D networks of iPSC-derived neurons for high-throughput neurotoxicity screening

13:00 - 14:00 Lunch and posters session

Mechanosensing and mechanoresponse (II) -Chair: José Manuel García Aznar-

14:00 - 14:45 Aurelien RouxBuckling of an epithelium growing under spherical con-finement

14:45 - 15:05Alejandro Torres

Modeling and simulation of the cell surface

15:05 - 15:25Roberto Moreno

Caveolin1 in Hippo pathway regulation by mechanical cues

15:25 - 15:45Alberto Elosegui

Force application to the nucleus is sufficient to trigger YAP nuclear entry

15:45 - 16:15 Coffee Break and posters session

Thursday October 6th

6

Polarity and morphogenesis (II) -Chair: Enrique Martín Blanco-

09:00 - 9:45 Stephan Grill Controlling contractile instabilities in the actomyosin cortex

09:45 - 10:05 Angughali SumiRole of contractile actomyosin flows in junctional homeo-stasis of constricting cells

10:05 - 10:25 Carlos PérezE-cadherin regulates epithelial contractility and active dewetting

10:25 - 10:45 Katerina KarkaliThe modular architecture of the Ventral Nerve Cord reflects the level of activity of the JNK signaling

10:45 - 11:15 Coffee break

Migration -Chair: Xavier Trepat-

11:15 - 12:00 Pietro Cicuta Collective dynamics in motile cilia: waves in the airways

12:00 - 12:20Antonio Quílez-Álvares

High Content Screening cell based assays for deciphering mechanotransduction-driven mechanisms of tumour invasion

12:20 - 12:40 Adrian MoureComputational modeling of amoeboid motion: chemotaxis and free movement in different environments

Single molecule mechanics -Chair: Pere Roca-Cusachs-

12:40 - 13:25Jorge Alegre-Cebollada

Mechanical biochemistry and the physiology of the heart muscle

13:25 - 13:30 Closing ceremony

13:30 - 14:30 Lunch

Friday October 7th

Polarity and morphogenesis (I) - Chair: Kai Erdmann -

16:15 - 17:00 Matthieu PielEffect of confinement on shape, volume, migration and survival of individual cells

17:00 - 17:20Petra Stockinger

Chromatin organisation in Drosophila melanogaster

17:20 - 17:40Guillermo Vilanova

Growth, regression, and regrowth in tumor-induced angiogenesis

17:40 - 18:00 Carlos BorauImage processing tool to quantify angiogenic sprouting in a microfluidic platform under different growth factor conditions

18:00 -19:00 Posters session

7

Keynote Lectures and oral presentations

8

Prof. Jochen GuckBiotechnology Center TU Dresden (BIOTEC), Germany

Thursday, 6th October 9:00

Mechanosensing in the central nervous systemIt is increasingly being recognized that cells measure and respond to the mechanics of their environment. We are especially interested in the influence of mechanics during CNS development and pathologies. Using quantitative scanning force microscopy we have shown that various neural tissues are very compliant (shear modulus < 1 kPa) and mechanically heterogeneous. We have recreated compliant polyacrylamide (PAA) gel substrate with shear moduli between 0.1 and 30 kPa to match and exceed those of CNS tissue. Various primary neurons and glial cells have been cultured on these gels and their reaction studied. Both primary rat microglia and astrocytes responded to increasing substrate stiffness by changes in morphology and upregulation of inflammatory genes and proteins.

Upon implantation of composite hydrogel stripes into rat brains, foreign body reactions were significantly enhanced around their stiff portions in vivo. It appears that the mechanical mismatch between a neural implant and native tissue might be at the root of foreign body reactions. Investigations into the molecular mechanisms are underway. Also oligodendrocytes, another type of glial cells, are mechanosensitive as their survival, proliferation, migration, and differentiation capacity in vitro depend on the mechanical stiffness of polymer hydrogel substrata. This finding might be linked to the failure of remyelination in chronic demyelinating diseases such as multiple sclerosis.

And finally, we have also shown retinal ganglion axon pathfinding in the early embryonic Xenopus brain development to be instructed by stiffness gradients. We could even identify a specific molecular mechanism involving piezo1, a stretch-activated ion channel. These results form the basis for further investigations into the mechanobiology of cell function in the CNS. Ultimately, this research could help treating previously incurable neuropathologies such as spinal cord injuries and neurodegenerative disorders.

9

Oral presentation 1 Ignasi Jorba

Effect of decellularization in heart scaffold mechanicsIgnasi Jorba1,*, Isaac Perea-Gil5,*, Cristina Prat-Vidal5, Ramon Farré2,3,4, Antoni Bayés-Genís5,6, Daniel Navajas1,2,4

1Institute for Bioengineering of Catalonia, Barcelona, Spain2Facultat de Medicina i Ciències de la Salud. Universitat de Barcelona, Spain3Institut d’Investigacions Biomèdiques August Pi I Sunyer, Barcelona, Spain4CIBER de Enfermedades Respiratorias, Madrid, Spain5ICREC Research Lab. Health Sciences Research Institute Germans Trias I Pujol, Badalona, Spain6Cardiology Service. Hospital Universitari Germans Trias I Pujol, Badalona, Spain

*Equal contributors

After myocardial infarction, effective treatments are needed to reduce scar formation, enhance cardiac regeneration and improve ventricular remodeling. Advances in cardiac tissue engineering have enabled the development of myocardial bioprostheses, based on seeding cells onto natural or synthetic three-dimensional (3D) matrices or scaffolds. In this context, scaffold biomaterial choice is a crucial step, as the scaffold must be able to provide biochemical and biomechanical microenvironments resembling the native structural organization. Decellularized myocardial extracellular matrix (ECM) can accomplish these premises. However, it is necessary to characterize the mechanical properties of ECM after decellularization process and after storage in freezing conditions in order to assess whether these procedures preserve the mechanical properties of the native ECM.

The aim of the study was to measure micro- and macromechanical properties of porcine cardiac tissue in native, decellularized and frozen/thawed decellularized conditions. Hearts (n=5) were obtained from healthy slaughterhouse pigs and blocks of 15x5x5 mm were cut from the left ventricle mid-myocardium and maintained with Kreb’s solution at 4 ºC. A custom-built atomic force microscope (AFM) was used for micromechanical measurements of 50 µm thick tissue slices obtained with a vibratome. The Young’s modulus (E) was computed from force-indentation curves recorded with a spherical tip 4.5 µm in diameter (cantilever spring constant of 0.03 N/m). After measurements, the slices were decellularized with SDS 1%, Triton X-100 1% and 3 washes of PBS (total time of 40 min) and measured again. Finally, the decellulariazed slices were measured after frozen in liquid nitrogen for one day and thawed at room temperature. E of native, decellularized and frozen/thawed decellularized slices was 13.75 ± 2.43

10

kPa (mean ± SE), 15.35 ± 1.07 kPa and 19.18 ± 2.00 kPa respectively with no significant differences between groups (ANOVA p > 0.1). For macromechanical measurements, tensile tests were performed (300C-LR, Aurora Scientific) on strips of 10x2x2 mm obtained from the heart blocks. The strips were decellularized, frozen/thawed and measured at each step. Stress-strain (σ-ε) curves were fitted with the Fung’s model (E = α·σ). α of native, decellularized and frozen/thawed decellularized strips was 14.06 ± 0.92, 15.27 ± 0. 72 and 14.11 ± 0.45 respectively, (ANOVA p > 0.4). These results show that micromechanical and macromechanical properties of the heart extracellular matrix are well preserved during decellularization and freezing/thawing processes, offering an ideal scaffold for cardiac regeneration.

11

Oral presentation 2 Léo Valon

Optogenetic control of cell forces and Mechanotransduction Léo Valon, Ariadna Marín, Xavier Trepat

Institute for Bioengineering of Catalonia, Barcelona, Spain

The daily life of most eukaryotic cells is critically influenced by contractile forces. At the subcellular level, these forces enable transport, polarization, protrusion, division, and mechanosensing. At longer scales, contractile forces are central for morphogenesis, wound healing, and cancer cell invasion. In each of these biological processes, contractile forces are tightly controlled in space and time. Yet current experimental tools to perturb forces, such as small molecule inhibitors or siRNA, target global cell populations in a uniform manner and are severely restricted in time resolution. This impedes their use to determine how local upregulation or downregulation of contractility could lead to cellular or multicellular shape changes.

Here we used optogenetics to control contractile forces with high spatiotemporal resolution. We report two tools that upregulate or downregulate cellular contractility over a four-fold dynamic range by controlling the subcellular localization of RhoA activator. Changes in contractility are rapid, local, fully reversible, and do not compromise cell viability. Our work is the first to demonstrate by direct measurement that optogenetics can be used to control cell-cell and cell-matrix forces. Besides this novelty, we also provide the first evidence that optogenetic control of physical forces drives rapid changes in key mechanostransduction pathways.

Acknowledgement: Mathieu Coppey, institut Curie, France

12

Oral presentation 3 Clara Valero

Mechanical evaluation of collagen-based gels for in-vitro experimentsC. Valero1,*, H. Amaveda2, M. Mora2, JM. García-Aznar1

1 Multiscale in Mechanical and Biological Engineering (M2BE), Aragon Institute of Engineering Research (I3A), Betancourt Building, Campus Rio Ebro, University of Zaragoza, Spain

2 Instituto de Ciencia de Materiales de Aragón (CSIC-Universidad de Zaragoza), Depto. de Ciencia y Tecnología de Materiales y Fluidos, C/María de Luna 3, 50018, Zaragoza (Spain)

* Correspondence: claraval@unizar.es, Universidad de Zaragoza, Campus Rio Ebro, Edif. Betancourt, 50018, Zaragoza (Spain)

Hydrogels are designed to mimic living tissues. Collagen hydrogels are one the most used for in-vitro experiments. Mechanical properties of collagen hydrogels vary with the collagen concentration and the incorporation of other substances. Cells respond differently depending on the resistance that the matrix creates to be deformed, which depends on the matrix mechanical properties. Thus, having an accurate mechanical characterization of hydrogels is crucial to understand its effect on the cellular behavior.

In this work, we have performed rheological measurements of collagen hydrogels with various collagen concentrations. We have analyzed the behavior of the hydrogels for a range of deformations. The results show that the shear modulus increases when the collagen concentration is higher. It has been observed that hydrogels present a constant shear modulus for low deformations and strain stiffening when deformations are higher, independently on the collagen concentration.

Finally, we have developed a computational model to evaluate the mechanical response of collagen-based gels depending on the collagen concentration. This model allows us to predict the shear modulus and the stiffening curve of each hydrogel and reproduces the gel behavior more accurately than existing material models.the transference of PrLD-containing proteins via this exosomal transport. Our objective is to unbalance the proteostasis of Plasmodium as a new antimalarial strategy, either by the introduction of pre-formed prionic nuclei into the parasite or by inhibiting its chaperone machinery.

13

Caveolae mechanotransduction and cancer: role of the EHD2 ATPase

Cells perceive their microenvironment not only through signaling receptors, but also through physical and mechanical cues, such as extracellular matrix stiffness, confined adhesiveness and shear pressure of blood. We recently established that caveolae are dynamic mechanosensors that buffer cell membrane tension variations and protect membrane integrity. Here, we demonstrate that upon mechanical stress, the EHD2 ATPase is released from the neck of caveolae, SUMOylated, and translocated to the nucleus where it controls caveolae constituents gene transcription. Caveolin1, the main component of caveolae, is often deregulated in cancers. We found that triple negative breast cancer cells presented low EHD2 RNA and protein levels, resulting in lack of gene regulation and loss of caveolae at the plasma membrane. Accordingly, we showed that EHD2 was required to reconstitute a stable reservoir of caveolae after mechanical stress release. RNA and immunohistochemistry analyses in human biopsies revealed that EHD2 expression was strongly decreased in basal-like breast cancers, melanoma and colon cancers. Our findings therefore define EHD2 as a new key player in caveolae-mediated mechanotransduction and cancer aggressiveness.

Christophe LamazeInstitut Curie, France

Thursday, 6th October 11:15

14

Identifying mechanosensing and mechanotransducer elements within caveolaeFidel-Nicolás Lolo1,2, Dácil María Pavón1, Alberto Elósegui2, Dobryna Zalvidea2, Moreno Zamai1, Geo Cojoc4, Asier Echarri1, Juan José Uriarte3, Daniel Navajas2,3, Jochen Guck4, Pere Roca Cusachs2 and Miguel Ángel del Pozo1

1 Vascular Biology and Inflammation Department. Spanish National Cardiovascular Centre (CNIC), Madrid, Spain

2 IBEC, Baldiri Reixac, Barcelona, Spain3 Unitat Biofísica i Bioenginyeria, Facultad de Medicina, Universitad de Barcelona, Spain4 Biotechnologisches Zentrum (Biotec), Dresden, Germany

Mechanosensing and mechanotransduction have emerged as important fields at the interface between physical cues and cellular signaling. Caveolae, small omega-shaped invaginations of the plasma membrane, have been revealed as key elements in sensing and transducing mechanical forces [1]. Previous studies described how this process is both ATP and actin independent [1]; however, work in our lab showed that caveolae can also flatten in a process that requires actin polymerization [2], suggesting that different types of forces can affect caveolae organization. Two main proteins, namely caveolin-1 (Cav1) and cavin-1 (also known as PTRF) have been shown to be required for both caveolae structure and function [3-5]. However, despite extensive research in caveolae biology nothing is known about the specific contribution of the caveolar protein components alone in cellular adaptation to mechanical forces. To this end we developed genetically modified fibroblast lines that express caveolae components alone or the whole structure.

To study cellular response to membrane deformation we took advantage of the magnetic tweezers technique that has been extensively used in the field of mechanobiology [6]; coating magnetic beads with Concanavalin A, that specifically binds to sugars, we were able to study forces that affect plasma membrane.

Results have shown that cells expressing Cav1 alone adapt to plasma membrane deformation to the same extent as cells with caveolae, indicating that Cav1 itself can buffer membrane tension. Studies on cholesterol distribution by fluorescence lifetime microscopy and membrane order by Laurdan staining indicated that this role of Cav1 is probably due to an increased in cholesterol-enriched areas that makes plasma membrane more flexible. Optical stretching and atomic force microscopy studies also confirmed these results by showing that cells expressing

Oral presentation 4 Lolo Fidel-Nicolás

15

Cav1 alone are as soft as cells with caveolae. Altogether these results show for the first time that Cav1 alone can modify the mechanical properties of the plasma membrane in a caveolae-independent manner.

References:

[1] Sinha, B. et al. Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 144, 402-413, doi:10.1016/j.cell.2010.12.031 (2011)

[2] Echarri, A. et al. Caveolar domain organization and trafficking is regulated by Abl kinases and mDia1. J Cell Sci 125, 3097-3113, doi:10.1242/jcs.090134 (2012)

[3] Rothberg, K. G. et al. Caveolin, a protein component of caveolae membrane coats. Cell 68, 673-682 (1992)

[4] Hill, M. M. et al. PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function. Cell 132, 113-124, doi:10.1016/j.cell.2007.11.042 (2008)

[5] Navarro, A., Anand-Apte, B. & Parat, M. O. A role for caveolae in cell migration. Faseb J 18, 1801-1811, doi:10.1096/fj.04-2516rev (2004)

[6] Tanase, M., Biais, N. & Sheetz, M. Magnetic tweezers in cell biology. Methods Cell Biol 83, 473-493, doi:10.1016/S0091-679X(07)83020-2 (2007)

16

Force loading explains how substrate rigidity and ligand nano-distribution regulate cell responseRoger Oria1,2, Tina Wiegand3,4, Jorge Escribano5, Alberto Elosegui-Artola1, Juan Jose Uriarte2, Daniel Navajas1,2, Xavier Trepat1,2,6 José Manuel García-Aznar5, Elisabetta Ada Cavalcanti-Adam3,4, and Pere Roca-Cusachs1,2

1 Institute for Bioengineering of Catalonia, Barcelona, Spain2 University of Barcelona, Spain3 Max-Planck-Institute for Medical Research, Heidelberg, Germany4 University of Heidelberg, Heidelberg, Germany5 Multiscale in Mechanical and Biological Engineering (M2BE), University of Zaragoza, Spain6 Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain

Processes in development, cancer, and wound healing are determined by the rigidity and ligand density of the extracellular matrix (ECM). ECM rigidity and ligand density are first probed and detected via integrins, transmembrane proteins that link the ECM to the actin cytoskeleton. Current understanding establishes an upper limit of 70nm spacing between integrins bound to ligands on glass surfaces for appropriate clustering and subsequent formation of focal adhesions (FAs). However, the mechanism behind this limit, and its regulation by rigidity, remain largely unknown.

Here, we developed a tunable rigidity substrate with controllable ligand spacing and distribution at the nanometer scale. In response to rigidity, we counterintuitively found that FA growth in breast myoepithelial cells was favored as ligand spacing increased from 50 to 100nm. In addition, disordering the distribution of ligands while keeping their density and average spacing constant triggered FA growth at lower rigidities and drastically increased their length. Further, we found that FAs collapse by decreasing their length above a rigidity threshold. Combined with measurements of traction forces and actin flows, these results match qualitatively with a molecular clutch model. This model predicts that substrate rigidity and ligand density affect adhesion formation by regulating integrin-ECM bond force loading, which in turn controls ensuing mechanosensing events. Taken together, our findings suggest a force-dependent mechanism which explains FA formation, growth and possibly collapse in response to rigidity and ligand density.

Oral presentation 5 Roger Oria

17

3D networks of iPSC-derived neurons for high-throughput neurotoxicity screeningArnaud Nicolas, Nienke R. Wevers, Karlijn J. Wilschut, Remko van Vught, Henriëtte L. Lanz, Sebastiaan J. Trietsch, Jos Joore, Paul Vulto

Although traditional 2D culture of human cells has increased our knowledge on many biological processes, they fail to mimic the complexity of in vivo tissues [1–4]. Over the last decades, great advances have been made in the field of 3D cell culture, where ECM supported cultures replace stiff plastic substrates. It has been observed that neurons grown in 3D culture systems show longer neurite outgrowth, better cell survival, and different patterns of differentiation as compared to 2D monolayers [5–7]. In current 3D culture systems compound transport is often diffusion limited. A lack of close contact between medium and the extracellular matrix containing the cells can cause nutrient deprivation or build-up of toxic waste products. Moreover, many 3D cell culture systems offer low throughput and cannot recapitulate structural and cellular tissue heterogeneity.

The OrganoPlate® (Fig. 1) is a microfluidic platform for 3D cell culture based on a 384-well microtiter plate [8]. A microfluidic network connects four neighboring wells of the plate to form one culture chip, resulting in up to 96 data points per plate. Neural stem cells are mixed with an extracellular matrix (ECM) and pipetted into the gel channel. The gel is patterned using a Phaseguide™; a small ridge on the channel bottom that acts as a capillary pressure barrier and prevents the gel from flowing into an adjacent channel [9]. After gelation, medium is added to the adjacent channel to enable a membrane-free culture system in which the neurons can differentiate.

The OrganoPlate® supports both long-term differentiation of neural stem cells and precursor cells (Axol neural stem cells, HIP™ neurons) as well as short-term culture of already terminally differentiated neurons, optionally in co-culture with astrocytes (Dopa.4U™ neurons, iCell® neurons®, iCell® astrocytes). Immunocytochemical stainings show three-dimensional networks of astrocytes (GFAP, S100β) and neurons (β3-tubulin, MAP2) (Fig. 2), including GABAergic and glutamatergic phenotypes. The neuronal-glial cultures in the OrganoPlate® were shown to be compatible with traditional assays for cyto- and neurotoxicity, including assays for cell viability and neurite outgrowth. Electrophysiological activity of the neurons, both spontaneous and pharmacologically modulated, was shown using calcium imaging with Fluo-4 AM dye (Fig. 3). Imaging was performed using a Molecular Devices ImageXpress Micro XLS-C high content microscope (imaged

Oral presentation 6 Arnaud Nicolas

18

Figure 1. Picture of an OrganoPlate viewed from the bottom with a representation of the Microfluidic culture compartment liking four adjacent wells together.Figure 2. iPSC-derived iCell® neurons (green) and astrocytes (magenta) in the OrganoPlate®, day 14 Figure 3. Schematic description of the image analysis process of calcium imaging data.Figure 3. Single frame captures of a calcium imaging time series showing active HIP neurons (A). Red Color represents the accumulation of Ca2+ around electrophysiologically active neuron soma (B and C). Figure 4 Neuron extraction algorithmFigure 5. Extracted Firing trace of a HIP neuron over a minute. Grey areas show regions of spiking activity, resulting in an increase of Calcium concentration

at 50Hz). We developed a method for automatically extracting and analyzing the firing trace of each individual neuron in the acquired time series (Fig.4). Neurons cultured in the OrganoPlate™ showed a more in vivo like firing pattern (Fig.5). The combination of iPSC-derived neuronal-glial cell culture, a high throughput microfluidic 3D cell culture platform, high content imaging and automated image analysis provides a valuable tool for assessing the effects of drug candidates on neuronal firing, including detection of seizurogenic effects.

References:

[1] Hoffman, R. M. To do tissue culture in two or three dimensions; that is the question. Stem Cells 11, 105–111 (1993).

[2] Cukierman, E., Pankov, R., Stevens, D. R. & Yamada, K. M. Taking cell-matrix adhesions to the third dimension. Science 294, 1708–12 (2001).

[3] Pampaloni, F., Reynaud, E. G. & Stelzer, E. H. K. The third dimension bridges the gap between cell culture and live tissue. Nat. Rev. Mol. Cell Biol. 8, 839–45 (2007).

[4] Ravi, M., Paramesh, V., Kaviya, S. R., Anuradha, E. & Solomon, F. D. P. 3D cell culture systems: advantages and applications. J. Cell. Physiol. 230, 16–26 (2015).

[5] Peretz, H., Talpalar, A. E., Vago, R. & Baranes, D. Superior Survival and Durability of Neurons and Astrocytes on 3-Dimensional Aragonite Biomatrices. Tissue Eng. 13, 461–472 (2007).

[6] 26th Southern Biomedical Engineering Conference SBEC 2010, April 30 - May 2, 2010, College Park, Maryland, USA. 32, (Springer Berlin Heidelberg, 2010).

[7] Zare-Mehrjardi, N. et al. Differentiation of embryonic stem cells into neural cells on 3D poly (D, L-lactic acid) scaffolds versus 2D cultures. Int. J. Artif. Organs 34, 1012–23 (2011).8.

[8] Trietsch, S. J., Israëls, G. D., Joore, J., Hankemeier, T. & Vulto, P. Microfluidic titer plate for stratified 3D cell culture. Lab Chip 13, 3548–54 (2013).

[9] Vulto, P. et al. Phaseguides: a paradigm shift in microfluidic priming and emptying. Lab Chip 11, 1596–602 (2011)

19

Buckling of an epithelium growing under spherical confinementIn embryonic development, many organs are formed by folding of an initially flat epithelium. Many genetic factors and secreted morphogens are important for this process, however the origin of the forces required for such deformation are still obscure. I will present a technique to encapsulate epithelial cells in hollow spheres of alginate. By proliferating within the capsules, the cells form a single epithelium that changes shape while growing. The cell aspect ratio increases while cells are accumuating lateral compression, and after a week, invaginations are seen. We further test the possibility that these invaginations are caused by the buckling of the epithelium under compression.

Thursday, 6th October 14:00

Aurélien RouxUniversity of Geneva, Switzerland

20

Modeling and simulation of the cell surfaceAlejandro Torres-Sánchez1, Daniel Millán1,2, Marino Arroyo1

1LaCàN, Universitat Politècnica de Catalunya-BarcelonaTech, Barcelona, Spain2CONICET, Facultad de Ciencias Aplicadas a la Industria, Universidad Nacional de Cuyo, Mendoza, Argentina

marino.arroyo@upc.edu

Many interesting cell components exhibit much larger lateral dimensions than thickness, and hence can be well-described mathematically by surface models. Surface models have been successfully applied to describe lipid monolayers and bilayers [1,4,6], or the cell cortex [2,8]. However previous works have mostly focused on the equilibrium behavior of these systems, or have examined problems with axisymmetry. However, many important problems in cell mechanobiology involve dynamics and general shapes. Furthermore, often mechanics closely interacts with chemistry, such as in adhesion through molecular binders or in shape generation by membrane proteins.

Here we present a general theoretical framework to study the dissipative dynamics of surface models of the cell surface. Our framework is similar to the approach in [6], and is based on identifying the gradient flow structure of the problem [5]. This requires finding appropriate dissipation and energy potentials from which the dynamics follows from a variational principle [3]. We then realize this model in a computer code based on subdivision surfaces [7] and on algorithms exploiting the variational structure of the dynamics.

We apply our framework to the simulation of lipid bilayers, where in particular we examine the rheology of membrane inclusions that generate intrinsic curvature in the membrane, and the cell cortex, where we study cytokinesis in asymmetric division.

Oral presentation 7 Alejandro Torres Sánchez

21

Figure 1: (A) Lipid flow around a protein inducing spontaneous curvature in the lipid bilayer (B)Cortex density profile in asymmetric cell division.

References:

[1] M. Arroyo and A. DeSimone. Dynamics of fluid membranes. Phys. Rev. E, 79:031915, 2009.

[2] H. Berthoumieux, J.-L. Matre, C.-P. Heisenberg, E. K. Paluch, F. Jlicher, and G. Salbreux. Active elastic thin shell theory for cellular deformations. New Journal of Physics, 16(6):065005, 2014.

[3] M. Doi. Onsager’s variational principle in soft matter. Journal of Physics: Condensed Matter, 23(28):284118, 2011.

[4] H. Noguchi and G. Gompper. Shape transitions of fluid vesicles and red blood cells in capillary flows. Proceedings of the National Academy of Sciences of the United States of America, 102(40):14159–14164, 2005.

[5] M. Peletier. Variational Modelling : Energies , gradient flows , and large deviationss. arXiv, 1402.1990, 2013.

[6] M. Rahimi and M. Arroyo. Shape dynamics, lipid hydrodynamics, and the complex viscoelasticity of bilayer membranes. Physical Review E, 86(1):011932, jul 2012.

[7] J. Stam. Evaluation of Loop subdivision surfaces. In SIGGRAPH’99 Course notes, Los Angeles, CA, 1999.

[8] H. Turlier, B. Audoly, J. Prost, and J. F. Joanny. Furrow constriction in animal cell cytokinesis. Biophysical Journal, 106(1):114–123, 2014.

22

Caveolin1 in Hippo pathway regulation by mechanical cuesRoberto Moreno Vicente

Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain

(e-mail: rmorenov@cnic.es)

The downstream component of the Hippo pathway, YAP, and its homolog, TAZ, are important elements in mechanotransduction, mediating many aspects of cell function in response to changes in ECM stiffness or cell shape. In this context, YAP regulates extracellular matrix (ECM) remodeling and is responsible for the increase in tumoral stroma stiffness which indeed enhances tumor invasion and metastasis. Given that Cav1 is also involved in mechanotransduction and ECM remodeling, we propose Cav1 as the unidentified upstream regulator of YAP in response to mechanical cues. Using specific assays, such as Flexcell® stretching device, substrates with tunable stiffness and CYTOO® micropatterns we expect to decipher the role of Cav1 in Hippo pathway modulation by substrate stiffness, mechanical tension and cell shape.

Oral presentation 8 Roberto Moreno Vicente

23

Force application to the nucleus is sufficient to trigger YAP nuclear entryAlberto Elosegui-Artola, Ion Andreu, Ainhoa Lezamiz, Marina Uroz, Anita Kosmalska, Roger Oria, , Xavier Trepat, Daniel Navajas, Sergi Garcia-Manyes, and Pere Roca-Cusachs

Institute for Bioengineering of Catalonia, Barcelona, Spain

Tissue rigidity plays a critical role during development, cancer and wound healing. Cells sense the rigidity of the extracellular matrix (ECM) through a dynamic molecular clutch which applies forces to the ECM, and then transduces them into biochemical signals leading to transcriptional regulation in the nucleus. One such transcriptional regulator is yes associated-protein (YAP), which regulates organ development and homeostasis in health and disease by controlling cell death, proliferation and differentiation. YAP is mechanosensitive since it translocates to the nucleus above a certain extracellular matrix rigidity, but the mechanism involved remains unknown. Here we show that YAP translocation in response to mechanical signals is directly triggered by mechanical force application to the nucleus. We demonstrate that the nucleus is mechanically connected to the ECM only in cells plated on stiff matrices, and that this connection triggers YAP nuclear entry. Further, force transmission to the nucleus is sufficient for YAP translocation independently of focal adhesions, the actin cytoskeleton, and YAP phosphorylation. Our results demonstrate a robust and reversible mechanosensitive mechanism directly mediated by the nucleus, where cell-ECM adhesions serve merely as a force relay checkpoint.

Oral presentation 9 Alberto Elosegui-Artola

24

Wednesday, 29th June 14:15

Effect of confinement on shape, volume, migration and survival of individual cellsIn tissues, cells have their physical space constrained by neighboring cells and extracellular matrix. In the recent years, we have developed simple and versatile devices to precisely and dynamically control this confinement parameter in cultured cells.

In the context of cell migration, confinement is important to allow cells to move at high speed without focal adhesions, by increasing friction with the substrate. I will present results on how confinement affects the switch to fast migration by inducing a sustained increase in contractility due to deformation of intracellular organelles and I will propose hypothesis for how friction is generated. I will also discuss our recent finding on rupture of the nuclear envelope due to nuclear deformation during cell migration under confinement.

Confinement might also affect cell volume and cell growth. I will show recent results on how cells regulate their volume during cycles of growth and division and on effects of confinement on volume regulation

Matthieu Piel Institut Curie, France

25

Drosophila chromatin organization: To scale or not to scale…? Petra Stockinger

Centre for Genomic Regulation (CRG), Barcelona, Spain

Chromatin undergoes dramatic reorganization throughout the cell cycle. Once cells enter mitosis, interphase compartmentalization is lost and chromatin is condensed in order to form compact mitotic structures. How cells achieve sufficient compaction in order to be able to correctly segregate their genomic content during anaphase is not known. We therefore investigated the sizes of mitotic chromosomes in Drosophila melanogaster developing neural system. During development, neuronal precursors divide asymmetrically yielding two daughter cells of different sizes: a large neuroblast (NB) and a small ganglion mother cell (GMC). We identified that mitotic chromosome length is highly correlated with anaphase spindle size, within the developing nervous system as well as during other developmental stages.

How cells scale mitotic chromosome size with spindle length is not known. Therefore, we analyze chromatin compaction prior to anaphase, using a combination of different imaging approaches with statistical/biophysical descriptions of compaction throughout the cell cycle. We are currently aiming at combining these analyses of wild type and perturbed chromatin states

Oral presentation10 Petra Stockinger

26

Growth, regression, and regrowth in tumor-induced angiogenesisGuillermo Vilanova, Ignasi Colominas, and Hector Gomez

Departamento de Metodos Matematicos e de Representacion, Universidade da Coruña, Spain

(gvilanovac@udc.es)

Angiogenesis is the growth of new capillaries from pre-existing ones. This complex biological phenomenon plays a critical role in the development of cancer, as tumors gain the ability to promote angiogenesis. The new capillaries provide the tumor the necessary nourishment for its fast growth as well as they are used by the cancer cells to metastasize. For this reason, impeding angiogenesis has become a promising cancer therapy [1]. However, many aspects of the physics and biology of angiogenesis are still unknown and researches from multiple disciplines study this phenomenon under different perspectives.

One of the most salient features of tumor-induced vascular networks is their instability: they are stimulus-dependent and may undergo alternating stages of growth, regression, and regrowth [2]. Thus, tumor angiogenesis is a highly dynamic phenomenon in which the new vasculature is a sequence of patterns that are continuously shaping to tumor angiogenic factor distribution to better nourish and oxygenate the cells. Previous efforts to model tumor angiogenesis [3-5], have chiefly focused on the initial growth of blood vessels, often using models which are fundamentally unable to predict the natural regression and regrowth observed in experiments. In this work, following a phase-field methodology, we present a new model of tumor angiogenesis that reproduces the aforementioned features and highlights the importance of vascular regression and regrowth. The model also includes a conceptualization of tip endothelial cell filopodia (the cellular protrusions that aid TECs in their migration) that plays a key role in regrowth and loop formation. The predictions of our model are in quantitative agreement with in vivo experiments and may prove useful for the design of antiangiogenic therapies.

Oral presentation 11 Guillermo Vilanova

27

References:

[1] Folkman, J. 1971. Tumor angiogenesis: Therapeutic implications. New England Journal of Medicine, vol. 285(21), pp. 1182{1186.

[2] Mancuso, M.R., Davis, R., Norberg, S.M., O'Brien, S., Sennino, B., Nakahara, T., Yao, V.J., Inai, T., Brooks, P., Freimark, B., Shalinsky, D.R., Hu-Lowe, D.D., and McDonald, D.M. 2006. Rapid vascular regrowth in tumors after reversal of VEGF inhibition. Journal of Clinical Investigation, vol. 116(10), pp. 2610{2621.

[3] Travasso, R.D.M., Corvera Poire, E., Castro, M., Rodrguez-Manzaneque, J.C., and Hernandez-Machado, A. 2011. Tumor angiogenesis and vascular patterning: A mathematical model. PLoS ONE, vol. 6(5), p. e19989.

[4] Vilanova, G., Colominas, I., and Gomez, H. 2013. Capillary networks in tumor angiogenesis: From discrete endothelial ells to phase-eld averaged descriptions via isogeometric analysis. International Journal for Numerical Methods in Biomedical Engineering, vol. 29(10), pp. 1015{1037.

[5] Vilanova, G., Colominas, I., and Gomez, H. 2014. Coupling of discrete random walks and continuous modeling for three-dimensional tumor-induced angiogenesis. Computational Mechanics, vol. 53(3), pp. 449{464.

28

Image processing tool to quantify angiogenic sprouting in a microfluidic platform under different growth factor conditionsCristina Del Amo*1, Carlos Borau*1, Raquel Gutiérrez1, Jesús Asín2, José Manuel García-Aznar1

1 Multiscale in Mechanical and Biological Engineering (M2BE). Aragón Institute of Engineering Research (I3A), Department of Mechanical Engineering, University of Zaragoza, Spain

2 Department of Statistical Methods, University of Zaragoza, Spain* Both authors contributed equally to this work

The regeneration of the global vascular network and its connection with the tissue is one of the first steps to restore the functionality when some injury is produced. When the tissue is damaged, the vascularity is interrupted, triggering the angiogenic process and the release of different growth factors (GFs). The collective migration of endothelial cells (ECs), is the main dynamic process that culminates in sprout formation from existing vessels. The main aim of this work is to quantify the effect of specific GFs during the initial stage of sprout formation, namely: VEGF, PDGF-BB, TGFβ and BMP-2, all of them involved in regenerative processes. For this purpose, we developed an image processing code (Matlab) that analyses z-stacks over time and builds a 3D volume to quantify the advance of the EC monolayer and the sprout structure. Our results show that VEGF is the main regulatory GF on angiogenesis processes, producing longer sprouts with higher frequency. However, the chemoattractant effect of VEGF depends on its concentration and its spatiotemporal location, having a critical impact on the sprout time evolution. PDGF-BB has a global negative effect on both the number and length of sprouts. TGFβ enhances sprout length at earlier times, although its effect gradually disappears over time. Finally, BMP-2 produces, overall, less number and shorter sprouts, but was the only GF with a positive evolution at longer times, producing fewer but longer sprouts.

Oral presentation 12 Carlos Borau

29

Friday, 7th October 09:00

Controlling contractile instabilities in the actomyosin cortexThe actomyosin cell cortex is an active contractile material for driving cell- and tissue morphogenesis. The cortex has a tendency to form a pattern of myosin foci, which is a signature of potentially unstable behavior. How a system that is prone to such instabilities can reliably drive morphogenesis remains an outstanding question. Here we report that in the Caenorhabditis elegans zygote, feedback between active RhoA and myosin induces a contractile instability in the cortex. We discover that an independent RhoA pacemaking oscillator controls this instability, generating a pulsatory pattern of myosin foci and preventing the collapse of cortical material into a few dynamic contracting regions. Our work reveals how contractile instabilities that are natural to occur in mechanically active media can be biochemically controlled in order to robustly drive morphogenetic events.

Stephan W GrillBiotechnology Center TU Dresden (BIOTEC), Germany

30

Role of contractile actomyosin flows in junctional homeostasis of constricting cellsA. Sumi1, D. Hayes1, J. Solon1

1 Centre for Genomic Regulation, PRBB, Barcelona, Spain

Epithelial tissues undergo extensive remodeling during embryonic development. Recent studies have revealed that, in a number of developmental processes, epithelial remodeling is associated with pulsations of individual cell surface areas and cortical actomyosin flows [Lecuit et al., 2010, Solon et al., 2009, Martin et al., 2009].

During Drosophila dorsal closure, the amnioserosa (AS), a contractile tissue covering the dorsal side of the embryo, shows contractile pulsations and regular actomyosin flows during the reduction of its apical surface area. The role of these pulsations in epithelial contraction and dorsal closure still remains unknown. We are investigating, here, the cellular role for these pulsations in tissue remodeling. By applying mechanical stretch on the AS tissue, we are able to arrest the pulsatile contractions and the actomyosin flows during DC. We show that this arrest is associated with a relocalizatin of actin and myosin from the medial area of the cells towards the adherens junctions to reduce AS cell surface area and maintain junction integrity upon stretch. This relocalisation of myosin directly correlates with the junctional strain and do not occur in cells having excess of membrane when endocytosis is blocked. In that later case, cells continue pulsating and seem “insensitive” to stretch.

After stretch release, myosin relocalizes to the medial area of the cells and pulsations are recovered, indicating that cells can switch between two states depending on tension: pulsatile contraction and actomyosin flows at low stretch and junctional reinforcement of actomyosin at high stretch.

In addition, with our mechanical perturbations, we showed that the stretch applied to cell junctions directly affect the rate of junction removal necessary for apical cell surface reduction during epithelial contraction. Upon stretch, junctional length remains constant and no removal is observed. After stretch release, the junctions are floppy and they strongly reduce in length in few minutes. The removal of this excess junctional material correlates with flows of myosin to the junction.

During dorsal closure, the AS cells constantly reduce their areas and junctional

Oral presentation 13 Angughali Sumi

31

References:

[1] Thomas Lecuit, Pierre-François Lenne, and Edwin Munro. Force Generation, Transmission, and Integration During Cell and Tissue Morphogenesis. Annual review of cell and developmental biology, 27(June):157–84, nov 2010. ISSN 1530-8995. doi: 10.1146/annurev-cellbio-100109-104027.

URL http://www.ncbi.nlm.nih.gov/pubmed/21740231.

[2] Adam C Martin, Matthias Kaschube, and Eric F Wieschaus. Pulsed contractions of an actin-myosin network drive apical constriction. Nature, 457 (7228):495–9, jan 2009. ISSN 1476-4687. doi: 10.1038/nature07522.

URL http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2822715&tool=pmcentrez&

[3] Jerome Solon, Aynur Kaya-Copur, Julien Colombelli, and Damian Brunner. Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure. Cell, 137(7):1331–1342, 2009.

URL http://www.ncbi.nlm.nih.gov/pubmed/19563762.

length at a similar rate. Our results indicate that this reduction in junctional length is controlled by the junctional tension and is promoted by the contractile actomyosin waves by coupling with the endocytic machinery.

32

E-cadherin regulates epithelial contractility and active de-wettingCarlos Pérez-González, Ricard Alert, Jaume Casademunt, Xavier Trepat

Adherens junctions are highly complex signal transducers that can be activated by force. However, how E-cadherin expression regulates force generation and downstream tissue organization remains unclear. Here we use MDA-MB-231, a non-cohesive cell line lacking classical cadherins, as a model to study the role of E-cadherin in the regulation of tissue mechanics. Taking advantage of an inducible promoter, we finely tune the amount of E-cadherin expressed by these cells. In our experimental setup, we confine a group of cells on a round micropattern of collagen. After allowing cells to adhere and reach subconfluence, we start the induction of E-cadherin expression and measure cell-matrix and cell-cell forces using Traction Force Microscopy and Monolayer Stress Microscopy, respectively. We observe that traction forces and intercellular tension increase proportionally to the expression levels of E-cadherin. Force buildup is paralleled by an increase in PMLC, despite the absence of cell spreading or migration. Tension keeps increasing after more than one day of induction, reaching a point in which cell-substrate adhesions suddenly break. This is paralleled by dewetting of the monolayer and the formation of a 3D aggregate. When we inhibit contractility, build-up of tension slows down and de-wetting delays; on the other hand, a reduced adhesion to the substrate speeds up this process.

These results suggest that the 2D-3D transition is driven by the high intercellular tension overcoming cell-substrate adhesion. In order to understand this phenomenon, we adapted wetting mechanics theory to tissues by taking into account their active properties. The model predicts that tissue dewetting critically depends on substrate maximum adhesion and, unexpectedly, on tissue radius. These predictions were observed experimentally, confirming that tissue size dependent wetting is a unique property of active systems. In conclusion, we generalized wetting mechanics to biological systems, finding unprecedented behaviors from the interplay between intercellular and cell-substrate forces. Furthermore, we found that all these forces are strongly regulated by E-cadherin expression.

Oral presentation 14 Carlos Pérez González

33

The modular architecture of the Ventral Nerve Cord reflects the level of activity of the JNK signalingKaterina Karkali1, Ignasi Jorba2, Timothy E. Saunders3, Daniel Navajas2, George Panayotou4 and Enrique Martin-Blanco1

1 Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), Barcelona, Spain2 Unitat de Biofísica i Bioenginyeria, Universitat de Barcelona, Spain

Institute for Bioengineering of Catalonia, Barcelona, Spain3 Mechanobiology Institute, National University of Singapore4 BSRC Alexander Fleming, Athens, Greece

In all higher metazoans most behavioral and cognitive functions depend on the integration capacity of the CNS. The CNS exhibits a complex architecture based on different organization levels each of which can be considered as an independent unit or a module. Recent studies have shown that CNS modularity is key to its function.

Here we aim to unravel the mechanisms underlying CNS modularity and architectural stability during CNS development. For this purpose we have focused on the arthropod VNC as a model system. Employing pan-axonal analysis of both Triops (a primitive branchyopod) as well as the Drosophila embryonic VNC we uncovered an iterated and conserved pattern of structural robustness, which responds to architectural needs. Further, we also found that the Drosophila CNS was extremely soft and that the most central regions of the embryonic VNC, where most axons bundle, were more rigid than lateral domains, where most of the somata are found. This suggests a role for the fasciculated axons acting as a suspension cable network providing structural support to the developing nervous system. Using these observations as entry points, we have further explored the molecular mechanisms that could be involved in accomplishing or supporting this modular architectural balance. We determined that a delicate equilibrium on the level of activity of the JNK signaling cascade in specific neurons was essential for proper structural patterning. Finally, we found that the JNK signaling seems to govern the mechanical balance within the VNC by non-autonomously controlling axonal adhesion.

Oral presentation 15 Katerina Karkali

34

Friday, 7th October 11:15

Collective dynamics in motile cilia: waves in the airways.Motile cilia are cell organelles able to exert a net force onto a liquid; they are highly conserved across eukaryotes, and enable a variety of functions from the motility of single cell organisms to flow that carries nutrients to our brains. A fascinating process takes place in mammalian airways: a carpet of motile cilia maintains the cell surface free of pathogens and particles by continuously refreshing and clearing a barrier of mucus. In order for this `muco-ciliary clearance' to be effective, cilia motion needs to be phase-locked across significant distances, in the form of a travelling wave, and it is not known how this is achieved.

Our lab is currently approaching this question from two directions: recently we have begun imaging ciliated cell carpets, quantifying the spatial and temporal coherence in the dynamics, and perturbing the system; we aim to match the understanding gained at that level with our previous work on model systems, which informed us of the importance of hydrodynamic coupling between driven oscillators, as a mechanism sufficient to establish collective large-scale dynamical patterns.

Pietro CicutaUniversity of Cambridge, UK

35

High Content Screening cell based assays for deciphering mechanotransduction-driven mechanisms of tumour invasionAntonio Quílez Álvarez

PhD Fellow ITN BIOPOL @ CNIC

Background: Tumour microenvironment is composed of tumour-associated cells and molecules that reciprocally signal for enabling invasion. During cancer progression, extracellular matrix (ECM) remodelling and homeostasis is shifted to favour cell migration. Aberrant ECM proteins deposition and fibre alignment occurs to allow malignant cells to metastasise. Tissues become stiffer during invasion due to ECM deposition and alignment. That is why, recently, cell mechanics have arisen as a promising field to better understand the forces involved in cell-cell and cell-ECM signalling driving disease. Cell contractility, migration capacity and intercellular adhesion together with ECM microstructure, composition and mechanical properties cannot be completely understood without knowing the force-dependent processes involved.

The aim of this project is to develop High-Content Screening (HCS) cell-based assays using siRNA technology to decipher the underlying pathways driving mechanotransduction. The development of a siRNA library for mechanotransduction will allow us to systematically knock down specific genes providing cellular features that can be quantified in vitro.

Methodology: We have developed a 2D assay for siRNA screening to study ECM organization, fibre alignment and stroma mediated remodelling in fibroblast-driven cancer progression. The use of FITC-labelled Fibronectin (FN) allows us to image ECM remodelling by Cancer-associated Fibroblasts (CAFs). Human CAFs are seeded onto FITC-FN and stained for actin cytoskeleton and nuclei. Matrix organization or chaotic structures can be quantified by High-Content image analysis methods developed in MATLAB by the Cellomics Unit. Controls for chaotic and organized phenotypes will be used.

To further investigate the invasive ability of tumour cells depending on fibroblast-driven ECM remodelling, a 3D assay will be optimised mimicking the tumour microenvironment of breast carcinoma. Mechanotransduction siRNA library will be tested in this 3D invasion assay. CAFs will be embedded in Collagen-I gels to

Oral presentation 16 Antonio Quílez Álvarez

36

remodel the matrix. A monolayer of breast carcinoma cells will be seeded and allowed to invade into the collagen matrix before fixation and staining for nuclei. The co-culture of CAFs and tumour cells (TC) will be analysed in a multiparametric way to measure proliferation, cell morphology (polarized phenotype), cell migration and ECM-remodelling. Cell features can be related to cell migration (measured by confocal Z-stacks of ·3D images and analysed by a software developed at Cellomics Unit) and to ECM-microstructure (imaged by Second-Harmonic Generation).

Hits obtained from the 2D and 3D assays will be validated by mechanical tests. Gel Compression tests, optical and magnetic tweezers, Real-Time Deformability Citometry (RT-DC) and AFM cell indentation will help to better understand how mechanotransduction occurs during tumour invasion and the gene networks involved in it.

37

Computational Modeling of Amoeboid Motion: Chemotaxis and Free Movement in Different EnvironmentsAdrian Moure, Hector Gomez

Department of Applied Mathematics - School of Civil Engineering, Universidade da Coruña, Spain

One of the features of eukaryotic cells is their ability to move. This motility emerges in several phenomena such as, for example, cellular nourishment, wound healing, tissue growth, pathogen removal or cancer metastasis. Here we focus on Dictyostelium Discoideum, a paradigm of amoeboid migration, which is a highly-deformable cell that translocates via rapidly alternating cycles of morphological expansion and contraction, thanks to the extension and retraction of actin-rich protrusions, called “pseudopods” [1].

Here, we propose a mathematical model for single cell migration, that couples both membrane dynamics and cytosolic mechanics. The cell is tracked by a phase-field which moves according to the actin filament network velocity [2]. The actin filament network is treated as a viscous fluid, whose behavior is governed by a Stokes-type equation that includes the forces acting into the cell, namely, surface tension, cell-substrate adhesion, myosin contraction, actin filament protrusion, and those arising from cell-obstacle contacts. The model introduces a novel description for actin filament and globular actin subunit dynamics inside the cell, as well as for an activator naturally located at the membrane. This activator controls pseudopod formation through probability distributions taken from experiments [3].

The high-order partial differential equation system is solved by using isogeometric analysis [4]. We perform simulations for free and confined environments, subject and not subject to chemotaxis. Examples of all these movements are compared to experiments, resulting in good agreement.

Keywords: Cell Motility, Amoeboid Migration, Phase-field Modeling, Isogeometric Analysis.

Oral presentation 17 Adrian Moure

38

References:

[1] R.H. Insall. Understanding eukaryotic chemotaxis: a pseudopod-centred view, Nat. Rev. Mol. Cell Biol. 11, 453-458, 2010.

[2] D. Shao, H. Levine and W.-J. Rappel. Coupling actin flow, adhesion, and morphology in a computational cell motility model, Proc. Natl. Acad. Sci. USA 109(18), 6851-6856, 2012.

[3] L. Bosgraaf and P.J.M. Van Haastert. The Ordered Extension of Pseudopodia by Amoeboid Cells in the Absence of External Cues, PLoS ONE 4(4), e5253, 2009.

[4] J.A. Cottrell, T.J.R. Hughes and Y. Bazilevs. Isogeometric Analysis: Toward Integration of CAD and FEA, Wiley, 2009.

39

Friday, 7th October 12:40

Mechanical biochemistry and the physiology of the heart muscleIn our laboratory, we investigate the mechanical properties of the myocardium and their regulation in health and disease. Mutations in cardiac proteins with mechanical roles cause different forms of life-threatening cardiomyopathy by mechanisms that remain unknown. We are currently testing the hypothesis that mutations in sarcomeric proteins can induce mechanical phenotypes that result in the development of disease. We also want to understand how the elasticity of the myocardium is tuned by posttranslational modifications of sarcomeric proteins. We are applying this fundamental knowledge to develop novel protein bio materials whose mechanical properties can be regulated by physiological cues. To bridge the gap between mechanics and biochemical regulation of sarcomeric proteins, we apply our double expertise in protein biochemistry and single-molecule force-clamp spectroscopy by atomic force microscopy.

Jorge Alegre-CebolladaCentro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Spain

Posters

41

Posters

Poster Name Title

1 Shada Abuhattum AFM-based microrheology for cell viscoelastic measurements and analysis

2 Rocio AguilarTyr phosphorylation of the RLC: A novel dominant mechanism of NMII activation

3 Ariadna ArasanzMuscle attachments mechanics in a Drosophila model for Congenital Muscle Dystrophies

4 Mar CóndorNumerical predictions of the chemical gradients generated in extracellular matrix networks

5 Henry De BellyPlasma Membrane and Cell Surface Mechanics in Embryonic Stem Cells

6 Ilaria Di Meglio Epithelium division under stress in 3D confinement

7 Asier EcharriA strain sensing mechanism protects the plasma membrane from increased tension

8 Joan-Carles EscolanoMolecular mechanisms of macrophage inflammatory mechanosensitivity

9 Giulio FulgoniInterplay between Rab8 and Caveolin-1 in membrane trafficking and mechano-transduction

10 Jose Manuel Garcia A hybrid approach to simulate collective cell durotaxis

11 Víctor González-TarragóBinding of ZO-1 to Œ±5Œ≤1 integrins regulates the mechanical properties of Œ±5Œ≤1-fibronectin bonds

12 Víctor JiménezCaveolin1 role as potential integrator of cellular mechanical and metabolic cues

42

13 Ignasi JorbaA Microfluidic Chip To Measure The Effect Of Oxygen On Cell Mechanics Probed With AFM

14 Ignasi JorbaMechanical properties of the Drosophila embryonic central nervous system

15 Dimitri Kaurin Modelling of cell adhesion at multiple scales

16 Hannes Maib Physical properties of single Clathrin Coated Pits

17 Giulia MencattelliMechanics in Central Nervous System Repair in Drosophila

18 Claire MurzeauRegulation of the Hippo pathway in polarized cells via multi-PDZ domain proteins

19 Alvaro OrtegaInducible expression of non-muscle myosin II-B controls T cell activation and shapes the immune synapse in a TCR- and integrin- dependent manner

20 Nicola PellicciottaInvestigating mechanical coupling between motile cilia in human airway epithelia

21 Thomas Pujol Folding for a segmented tissue

22 Carlos UreñaCaveolae mechanosignaling in multicelullar alginate spheroids

23 Larisa Venkova The role of acto-myosin cortex in cell volume regulation

24 Dobryna ZalvideaMeasuring forces during in vivo angiogenesis in the chorioallantoic membrane of the chicken embryo using a custom made multi-photon microscope

25 Joanna ZellStudy of glycosphingolipid- and lectin-dependant endocytosis with chemical biology

43

Poster 1 Shada Abuhattum

AFM-based microrheology for cell viscoelastic measurements and analysisShada Abuhattum1,2, Anna Taubenberger1, Paul Müller1, Jörg Barner2, Jochen Guck1 and Torsten Müller2

1Biotechnology Center,TU Dresden, Germany2JPK instruments AG, Berlin, Germany

Atomic force microscopy (AFM) has rapidly become one of the most used techniques to quantify the mechanics of biological samples ranging from single molecules to cells and tissues. The most conventional approach used in AFM quanties the elastic modulus of the sample from force indentation curves. However, the elastic modulus solely is not sufficient to characterize the mechanical properties of cells and tissues.

Cells and tissues behave neither solid-like (elastic) nor fluid-like (viscous), but rather a combination of both. This viscoelastic behavior plays an significant role in many biophysical and biological responses and thus the development of measurement and analysis methods is crucial to quantify such a behavior. This study aims to extend AFM data acquisition and quantification to a wider range of probe-based mechanical interrogation modes. To do so, we will develop and implement AFM-based microrheology method to determine the complex shear modulus from oscillatory measurements. These measurements will enable us to quantify the elastic and viscous responses of the sample and accordingly the dissipative energy over a wide range of frequencies.

44

Poster 2 Rocio Aguilar Cuenca

Tyr phosphorylation of the RLC: A novel dominant mechanism of NMII activationRocio Alguilar Cuenca

Hospital Universitario La Princesa

Non-muscle myosin II (NMII) is the main generator of mechanical forces inside cells. As such, it is a master regulator of several fundamental biological processes, e.g. cell division and migration.

Structurally, NMII is a hexamer made of two heavy chains, two essential light chains and two regulatory light chains. The heavy chain contains an ATPase domain together with an actinbinding domain. The essential light chain plays a structural role, whereas the regulatory light chain controls the activation state of NMII.

The mechanism of control of NMII activation by RLC depends of phosphorylation. To this point, many studies have focused on the role of Ser/Thr phosphorylation of the RLC in the control of the ATPase activity of the heavy chain and the conformational extension of NMII.

In this study, we reveal novel phosphorylation sites that regulate activation of NMII. Mass spectrometry and a custom-made phospho-specific antibody reveal robust phosphorylation in Tyr residues in a growth factor-dependent manner. Using mutagenesis, we have generated nonphosphorylatable (Y143F and Y156F) mutants. Whereas Y143F behaves largely like wild type RLC, Y156F mutation prevents RLC binding to the heavy chain and remains largely cytoplasmic and not in filaments.

Use of double mutants of the canonical Thr/ Ser residues and the Tyr residues demonstrate that Tyr phosphorylation supersedes the canonical T/S cluster that controls the conformational activation and ATPase activity of NMII. Together, these results indicate the existence of an additional level of conformational regulation of the NMII dependent of the incorporation of RLC to the hexamer in a Tyr156 phosphorylation-dependent manner.

45

Poster 3 Ariadna Arasanz

Muscle attachments mechanics in a Drosophila model for Congenital Muscle DystrophiesAriadna Arasanz, Adria Prado and Enrique Martin-Blanco

Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), Barcelona, Spain

Walker-Warburg syndrome (WWS) is an inherited Congenital Muscle Dystrophy (CMD) that affects the development of muscles early during infancy. WWS has been linked to the dysfunction of the O-mannosyltransferases 1 and 2 (POMT1 and POMT2), which are required for the glycosylation of the α-Dystroglycan. This is a component of the dystrophin glycoprotein complex (DGC) that connects the extracellular matrix with the intracellular cytoskeleton.

To explore the developmental defects associated to this congenital disorder, we are employing Drosophila as a model system. Her, we are first typifying in vivo the development of muscles and associated nerves (growth and positional orientation) as well as their attachments to the epidermis in wild type animals during metamorphosis.

To reveal the mechanistic details of the coordination process we are employing in vivo microscopy. These studies constitute an entry point for the mechanical analysis of the establishment muscle-epithelial attachments.

Same approaches are being employed for the characterization of mutant phenotypes. In Drosophila the POMT1 and POMT2 orthologs are rotated abdomen (rt) and twisted (tw), respectively. To obtain a reliable understanding of the problems underlying congenital muscular dystrophies, we aim to evaluate in rt and tw mutants the mechanical resistance of muscles and their attachments. Progress in these aspects will be presented at the meeting.

46

Poster 4 Mar Cóndor

Numerical predictions of the chemical gradients generated in extracellular matrix networksM. Cóndor1, J.M. García Aznar1

1 M2BE, Aragon Institute of Engineering Research (I3A), University of Zaragoza, Spain (mcondor@unizar.es, jmgaraz@unizar.es)

Microfluidic devices allow for the production of physiologically relevant cellular microenvironments by including biomimetic hydrogels and generating controlled chemical gradients. During transport, the biomolecules interact in distinct ways with the fibrillar networks: as purely diffusive factors in the soluble fluid or bound to the matrix proteins. These two main mechanisms may regulate distinct cell responses in order to guide their directional migration: caused by the substrate bound chemoattractant gradient (haptotaxis) or by the gradient established within the soluble fluid (chemotaxis) [1, 2].

In this work we present a numerical model based on a reaction-diffusion transport model, in combination with ELISA assays. This model allows us to predict the chemical gradients distribution within the microfluidic platforms of different growth factor as PDGF-BB and TGF-β1 across collagen and fibrin gels. In addition, the model yields an accurate prediction of the experimental results (Fig.1), confirming that diffusion and binding phenomena are established within the microdevice [3].

Moreover, we present a Web application for showing the numerical results provided by the model in order to characterize the chemical gradients generated within different microfluidic devices. This application allows the user to define online the geometrical parameters that characterize the microfluidic device (hydrogel height and width, among others); as well as the input parameters of the diffusion case: the growth factor (PDGF-BB and TGF-β1), the initial concentration, the type of matrix (collagen of fibrin) or the time to simulate.Acknowledgments: This study is supported by the European Research Council (ERC) through project

ERC-2012-StG 306751, the Spanish Ministry of Economy and Competitiveness (DPI2015-64221-C2-1-R)

and the Government of Aragon (C126/2015).

47

References:

[1] Aznavoorian, S., et al., Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells. J Cell Biol, 1990. 110(4): p. 1427-38.

[2] Daub, J.T. and R.M. Merks, A cell-based model of extracellular-matrix-guided endothelial cell migration during angiogenesis. Bull Math Biol, 2013. 75(8): p. 1377-99.

[3] Moreno-Arotzena, O., et al., Inducing chemotactic and haptotactic cues in microfluidic devices for threedimensional in vitro assays. Biomicrofluidics, 2014. 8(6).

Fig. 1. In-vitro and in-silico images of the diffusive gradient of dextran in collagen (a),(c) and fibrin (b),(d) hydrogels, after 4 h since addition. Dextran advance speed for collagen (e) and fibrin (f) hydrogel [3].

48

Poster 5 Henry De Belly

Plasma Membrane and Cell Surface Mechanics in Embryonic Stem Cellsde Belly, H.1,2 Chalut, K.J.3, and Paluch, E.K.1,2

1MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT, UK.2Institute for the Physics of Living Systems, UCL, London WC1E 6BT, UK.3Wellcome Trust/Medical Research Council, Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

Embryonic Stem Cells (ESCs) are able to generate any tissue in a given organism; this ability is called pluripotency. Understanding exit from pluripotency is key for the study of pluripotency itself but it is also important in order to control the exit for therapeutic approaches in regenerative medecine. Many molecular players such as LIFR (receptor of leukemia inhibitory factor, repressor of differentiation)and the kinase PIP3k involved in the exit from pluripotency are primarily localised at the plasma membrane. Furthermore, membrane mechanical properties, such as tension, have been shown to influence cell behaviour and differentiation in many systems. However, very little is known about plasma membrane dynamics and mechanics in ESCs. Here, using mouse ESCs as a model system, we investigate the link between the mechanical properties of the plasma membrane and the molecular factors that lead to exit from pluripotency. Preliminary results show a difference in expression and activation of membrane tension regulators between naïve cells and primed cells that are already exiting from pluripotency, suggesting a higher membrane tension in naïve cells. Furthermore, using electron microscopy, we show that membrane organisation changes during exit from pluripotency, with a strong increase in membrane reservoirs in primed cells. We also observe higher expression and differential localisation of caveolae components in primed cells compared to naïve cells. Caveolae have been suggested to play an important role in other stem cell type such as human mesenchymal stem cells and are sensitive to change in membrane tension. Together, our data strongly suggest that membrane organisation and tension changes during exit from naïve pluripotency in mouse ESCs. We are currently investigating the impact of these changes in cellular fate transitions in ESCs.

49

Poster 6 Ilaria Di Meglio

Epithelial division and growth under stress in 3D confinementIlaria Di Meglio

University of Geneva

Tight regulation of processes like cell growth and proliferation must take place during development to ensure the formation of organs with defined sizes and shapes. These cellular processes lead to global tissue changes due to accumulation of local stresses, that in turn, feed back onto these processes. The ability to sense and respond to the environment is therefore a key aspect of normal tissue development and morphogenesis. In order to quantitatively investigate this coupling, we propose to use a simple artificial 3D model to study epithelial growth within a confined spherical shell. We combine 3D printing and microfluidics to produce a co-extrusion device (Alessandri et al., 2016) that allows for production of alginate microcapsules in to which cells are encapsulated. The inner surface of these alginate shells is coated with Matrigel, to which cells adhere and form a monolayer. Because shell thickness and capsule size can be tuned, this allows us to study the effect of the substrate on epithelial cell proliferation. After establishing stable epithelial cell lines expressing the H2B (chromatin marker) and FUCCI (cell cycle indicator), we can assess the division rate of the monolayer within this 3D confinement. Using this model, we hope to gain further understanding of the coupling between physical constraints within a tissue, its curvature and rigidity, with cell growth and proliferation.

50

Poster 7 Asier Echarri

A strain sensing mechanism protects the plasma membrane from increased tensionAsier Echarri1, María García-García1, Dácil M. Pavón1, Enrique Calvo2, Nicholas Ariotti3, Juan José Uriarte4, Sara Sánchez, Daniel Navajas4, Robert G. Parton3, and Miguel A. Del Pozo1

1Integrin Signaling Laboratory and 2Proteomic Unit, Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain

3University of Queensland, Australia4Facultad de Medicina, Universidad de Barcelona, Spain

Cells adapt the plasma membrane to mechanical strain; however, how changes in plasma membrane tension are translated into biochemical signals that control the organization of the plasma membrane during the cell response to mechanical strain is poorly understood. Here, we show that plasma membrane adaptation to mechanical strain is controlled by a tension sensing pathway composed of c-Abl tyrosine kinase and FBP17, an F-BAR family member that bends the plasma membrane. FBP17 regulates the formation of mechanosensitive plasma membrane invaginations decorated with caveolae that unfold in response to increased tension. As a consequence, cells deficient for FBP17 are more sensitive to mechanical strain. Mechanistically, tension is transduced to the F-BAR domain of FBP17 in the form of a direct phosphorylation mediated by c-Abl. This modification inhibits the membrane bending activity and oligomerization of FBP17, favoring membrane adaptation to increased tension. The inhibitory action of FBP17 on stress fibers is also controlled by this tension sensing pathway, thus coordinating membrane curvature and stress fiber remodeling. Overall, these results shed light on the mechanisms used to adapt the plasma membrane and the actin cytoskeleton to tension changes in mammalian cells.

51

Poster 8 Joan-Carles Escolano

Molecular mechanisms of macrophage inflammatory mechanosensitivityJoan-Carles Escolano1, Gilbert Ng1,2, Andrew Ekpenyong1, Panagiotis Athanaspoulos3, Weiwei Shen4, Christophe Lamaze4, Clare Bryant3, Jochen Guck1

1Biotechnology Center, Technische Universität Dresden, Germany2Cavendish Laboratory, Department of Physics, University of Cambridge, UK3Department of Veterinary Medicine, University of Cambridge, UK4Institut Curie, Paris, France

Mechanical mismatch between compliant tissue and stiff materials hinders biointegration and limits the long-term success of implanted prostheses. This limitation is primarily due to the foreign body reaction, a proinflammatory response that damages surrounding tissue and engulfs the region in fibrotic tissue. We have previously shown that microglia, macrophage-like immunosensory cells in neural tissue, are mechanosensitive to inert polyacrylamide gels both in vitro and in vivo [1,2]. These cells, like macrophages, direct inflammatory reactions and the foreign body response. However, the mechanism for their inflammatory mechanosensitivity has remained elusive. Here, we utilize an engineered novel polymer with independently tunable mechanical properties, star-polyethylene-glycol (star-PEG) crosslinked to heparin, to investigate how bone marrow derived macrophages (BMDM) sense the stiffness of their substrate and trigger inflammatory reactions. Using this system, we also examine the potential role played by different membrane reservoir components and the activity of certain ion channels in macrophage mechanosensitivity. Our findings could contribute to better explain how engineered bioinert in vitro polymers can trigger robust inflammatory reactions in vivo. Furthermore, information of the underlying molecular and biophysical mechanism could aid in the rational design of biomaterials, enable pharmacological control against mechanosensitive pathways, and have further implications in amyloid-driven and chronic inflammatory disorders.

References:

[1] Moshayedi, P. et al. Mechanosensitivity of astrocytes on optimized polyacrylamide gels analyzed by quantitative morphometry. J. Phys. Condens. Matter 22, 194114 (2010).

[2] Moshayedi, P. et al. The relationship between glial cell mechanosensitivity and foreign body reactions in the central nervous system. Biomaterials 35, 3919–3925 (2014).

52

Poster 9 Giulio Fulgoni

Mechanobiology across networks abstract Title: Interplay between Rab8 and Caveolin-1 in membrane trafficking and mechano-transduction G. Fulgoni1,2, F. Lolo Romero1, M. Cordani2, Anita Joanna Kosmalska3, Pere Roca-Cusachs3, M.A. Del Pozo Barriuso1 and M. C. Montoya2

1 Integrin Signalling Lab., Cell & Developmental Biology Area. Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC). Madrid (Spain)

2 Cellomics Unit. Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC). Madrid (Spain)3 Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain

Rab8 is a small GTPase that has been classically involved in the control of exocytic traffic and membrane recycling. More recently, Rab8-traffic has been reported to control trafficking pathways that are tightly coupled with changes in PM tension. It promotes the formation of PM protrusion [1] and directional cell migration [2]. It controls a form of stretch-activated exocytosis [3] and promotes the formation of new PM areas during Drosophila embryo development [4]. Therefore Rab8 could act as a mechano-trasductor that would modulate PM tension by the simply addition or removal of membranes, or by the regulation of membrane signaling pathways in response to membrane tension.

Caveolin-1 (Cav1) is the main component of caveolae, PM invaginations involved in signal transduction and mechano-sensing [5]. Caveolae are very dynamic structures that can flatten in response to an increase in PM tension [6] or form complex structures in response to a lowering in PM tension [7]. Since Rab8 and Cav-1 have been shown to colocalize at tubular membranes that sense actomyosin tension [8] and are involved in complementary traffic pathways, we decided to explore their possible interplay in in mechano-sensing and mechano-transduction. Our preliminary data suggest that caveolae are required for proper Rab8 traffic, as the silencing of Cav1 lead to an accumulation of Rab8 in the endocytic recycling compartment. Conversely, silencing of Rab8 affects Cav1 recycling to the PM during cell adhesion and spreading. Although co-localization of these proteins has been described in some cell lines [8], our in-vivo microscopy imaging studies showed no clear co-localization, suggesting an indirect interaction between Rab8 and Cav1.

To test Rab8 involvement in mechano-transduction, cells were subjected to various mechanical stimuli. After hyper-osmotic shock, Rab8 accumulated in vesicular

53

structures that did not co-localize with any of the canonical endocytic markers. The same effect was obtained after a hypo-osmotic to isosmotic treatment. Using a custom designed cell-stretcher, we induced a strain that corresponds to 12% increase in cell area and we observed a redistribution of Rab8 from the endocytic compartment toward the PM. After strain relaxation, Rab8 localized to membrane structures that gradually disappeared within a few minutes. These structures showed the same morphology and dynamics of the “reservoir” tubules previously described [9]. Rab8-tubules also showed fast dynamism after stretching-relaxation, moving from the reservoirs toward the perinuclear compartment of some cells. Further experiments will elucidate the trafficking route of Rab8 during these processes, its role in mechano-sensing and mechano-transduction pathways and its interplay with caveolae dynamics.

References:

[1] Peranen J. Rab8 GTPase as a Regulator of Cell Shape. Cytoskeleton, 68:527–539 (2011)

[2] Bravo-Cordero J. Rab8 coordinates signaling of Rho GTPases and focal adhesion turnover to direct cell migration (2016)

[3] Khandelwal P. A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells. Molecular Biology of the Cell; E12-08-0568 (2013)

[4] Mavor LM. Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster. Development 10.1242/dev.128876 (2016)

[5] Parton R & Del Pozo MA. Caveolae as plasma membrane sensors, protectors and organizers. Nat Rev Mol Cell Biol. Feb; 14(2):98-112 (2013)

[6] Sinha B. Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 144, 402–413 (2011)

[7] Echarri A. Caveolar domain organization and trafficking is regulated by Abl kinases and mDia1 J. Cell Sci. 125, 3097–3113 (2012)

[8] Verma P. Caveolin-1 Induces Formation of Membrane Tubules That Sense Actomyosin Tension and Are Inhibited by Polymerase I and Transcript Release Factor/Cavin-1. Molecular Biology of the Cell Vol. 21, 2226–2240 (2010)

[9] Kosmalska AJ. Physical principles of membrane remodelling during cell mechanoadaptation. Nature Communications 6, 10.1038 (2015)

54

Poster 10 José Manuel García-Aznar

A hybrid approach to simulate collective cell durotaxisJorge Escribano1, Raimon Sunyer2, Pere Roca-Cusachs2,3, Xavier Trepat2,3 and José Manuel García-Aznar1

1 University of Zaragoza, Zaragoza, Spain2 Institute for Bioengineering of Catalonia, Barcelona, Spain3 University of Barcelona, Spain

Durotaxis is an important guidance mechanism in collective cell migration. Here we present a hybrid computational framework that combines a particle-based and a finite element-based approach, in order to reproduce a cell monolayer migrating over a flat substrate with a stiffness gradient. The model is based on the mechanical interactions between the cells and the substrate. These interactions are driven by dynamic adhesion complexes that engage and disengage constantly as a function of their failure mechanical properties. We explore the crucial role that different geometrical and mechanical conditions exert on collective cell migration: different substrate gradient slopes, different positions of the cluster in the gradient, size of the cell cluster, and different concentration of binding proteins. Results are compared with experimental data, showing that the model is able to predict qualitatively every phenomenon observed and quantitatively with relative accuracy the migration speed for the different cases.

55

Poster 11 Víctor González-Tarragó

Binding of ZO-1 to α5β1 integrins regulates the mechanical properties of α5β1-fibronectin bondsVíctor González-Tarragó, Alberto Elosegui-Artola, Elsa Bazellières, Roger Oria, Carlos Pérez-González, Xavier Trepat, Pere Roca-Cusachs

Fundamental processes in cell adhesion, motility, and rigidity adaptation are regulated by integrin-mediated adhesion to the extracellular matrix (ECM). The bond between the ECM component fibronectin (fn) and integrin α5β1 forms a complex with ZO-1 in cells at the edge of migrating monolayers, regulating cell migration. However, how this complex affects the α5β1-fn bond is unknown. Here we show that the α5β1/ZO-1 complex decreases α5β1-fn bond resistance to forces in cells at the edge of migrating monolayers, while also increasing α5β1 recruitment. Those changes can be explained by a ZO-1 mediated increase in both the binding an unbinding rates of α5β1-fn bonds. Consistently with a molecular clutch model of adhesion, this effect of ZO-1 leads to a decrease in the density and intensity of focal adhesions in cells at the edge of migrating monolayers. Taken together, our results unveil a new mode of integrin regulation through modification of the integrin-ECM binding dynamics, which may be harnessed by cells to control adhesion and migration.

56

Poster 12 Victor Jiménez-Jiménez

Caveolin1 role as potential integrator of cellular mechanical and metabolic cuesV Jiménez-Jiménez, M Sánchez-Álvarez, FN Lolo, G Cojoc, JC Escolano, Y Ge, S Ciucci, CV Cannistraci, J Guck, MA del Pozo.

Caveolin 1 (CAV1) participates of multiple processes in the cell, including plasma membrane organization (through structures termed caveolae), lipid metabolism and trafficking, signaling integration and regulation, and mechanotransduction, but we are far from fully understanding its contribution to cell behavior and disease. The main aim of our project is to explore the role of CAV1 as an integrator of metabolic and mechanical cues in the cell. Furthermore, we propose that CAV1 expression levels decreased through evolution in the immune system and the neural tissue, conferring those tissues with some of their differential characteristics: anchorage-independent growth, metabolic inflexibility and mechanical sensitivity- all of which are both required for the functional properties of these tissues, but also potentially expose them to disorders such as neoplasia.To explore these systems-level aspects of CAV1 biology, we are combining complementary approaches.

First, we are deploying comparative bioinformatics and functional network analysis of CAV1-related co-express