of 64
Intracellular traffic in polarity
and development
Gwyn W. Gould
Henry Wellcome Laboratory of Cell Biology
Davidson Building.
You have seen already from Gareth Jenkins that
the acquisition of assymetry - polarity - is a key step in early development as it leads to the
production of cells with different properties and
hence different developmental fates.
Today, I will describe how membrane trafficking
can contribute to polarity in a range of different
systems.
First, how are cells organised?
Cells have multiple
membrane-bound compartments.
These compartments serve to provide specialisation (e.g. mitochondria are specialised for energy production; lysosomes specialised for protein degradation).
This in turn provides grater efficiency to all cellular processes.
Serves to sequester certain activities away from others, e.g. lysosomes.
How are these compartments arranged?
Important later!
How do they communicate with each
other (and the outside world)?
Key steps in trafficking between organelles:
Cargo selection
Scission of transport vesicle from donor compartment
Movement to target compartment membrane
Tethering (aka: docking)
Fusion to deliver cargo.
NB cargo in this context can be stuff inside the vesicle lumen, or proteins/lipids within the
membrane of the vesicle.
So, what has this to do with polarity?
Polarisation of signals via membrane
trafficking in development/mitosis.
Think-Pair-Share #1
I pose a question, you think about it for one minute, spend two minutes talking with your neighbour sharing your thoughts, then I will randomly select people to share their thoughts with the class.
So: How might a cell equally distribute a signal during mitosis?
TGFb signalling in cell division
Extracellular signals, such as growth factors and hormones, are received by receptors on the surface of cells.
Signals generated on these receptors are transmitted to the nucleus by cascades of intracellular signalling molecules and result in changes in gene expression.
These signalling molecules need to be equally distributed between daughter cells during cell division.
This is particularly important in developing tissues, where morphogens can elicit concentration-dependent responses at very long ranges even small changes can have huge effects. During development, cells acquire positional information by reading the concentration of morphogens.
Drosophila wing development
In the developing fly wing, there is a sub-
set of intracellular vesicles (endosomes)
called Sara-endosomes, whose main
function seems to be equally distributing
components of the TGFb signalling
pathway during cell division. This makes
sure the signal is equally split between the
two daughter cells.
TGFb morphogen binds to two cell surface
receptors and induces their dimerisation.
This leads to the activation of the receptor
kinase domain which phosphorylates the
transcription factor R-Smad.
TGFb morphogen binds to two cell surface
receptors and induces their dimerisation.
This leads to the activation of the receptor
kinase domain which phosphorylates the
transcription factor R-Smad.
Sara (Smad anchor for receptor activation)
simultaneously binds the receptor complex
and the R-Smad.
Sara localises to the surface of endosomes.
Endosomes accumulate in the midbody of
cells during cytokinesis, meaning that as
TGFb binds its receptors, these are
internalised and trafficked into the central
region of the cell.
The result is an equal distribution of the
signal between the two daughter cells.
During mitosis, the Sara endosomes and the receptors therein associated with the spindle
machinery to segregate into the two daughter cells. Daughter cells thereby inherited equal
amounts of signalling molecules and thus retained the TGFb signalling levels of the mother cell
But what about endosomes in
polarised cell development?
Drosophila Sensory Organ Precursor cells
(SOP)s.
Still with fly development now looking at sensory organ precursors (SOPs).
SOP cell divides into two cells, called pIIaand pIIb.
These two cells communicate to set up a polarised pattern of cell growth and differentiation.
Will consider briefly the role of
endosomes in assymetric cell division
using this system.
A key facet of the subsequent development of the SOP
from pIIa and pIIb is that the pII cells are distinct.
pIIb presents on its surface a ligand called Delta.
Delta is in turn then recognised by a receptor (Notch) on
the surface of the pIIa cell.
The differential activation of the Notch pathway in pIIa is
a key facet of the subsequent differential development of
the SOP.
Clue: endosomes!
Delta and Notch
After SOP division, Delta (ligand) and Notch (receptor for delta) are present in both pIIa and pIIb.
Differential regulation of endocytic trafficking events ensures that Notch is active in the future pIIa cell, but not the pIIb cell.
Membrane trafficking thus plays a seminal role in the development of a polarised signal.
We shall look at the mechanism(s) which regualte this.
Lets meet the molecules involved:
Delta ligand for the Notch receptor
Notch receptor for delta
Numb endocytic protein that can bind Notch
Sandopo can bind Notch, Numb and is required for activation of Notch pathway in pIIa.
Notch signalling is activated when it binds its ligand, Delta. Ligand binding results in endocytosis of the Delta/Notch complex, whereupon a series of proteolytic cleavage events release a Notch fragment, which moves into the nucleus where it turns on a specific program of gene transcription.
So how do pIIa and pIIb exhibit
differential notch signalling?
Membrane traffic turns out to be key!
Thus, differential endocytosis contributes
To the development of polarity.
Early in SOP mitosis, Numb (red) accumulates
on the anterior face of the SOP.
In the subsequent pIIb cell, Numb and Sandopo
result in the trafficking of Notch away from the
cell surface in a degradative compartment
(lysosomes), so reducing Notch levels in pIIb.
Directional transport of internalized
Delta/Notch during asymmetric cell division.
Found that Delta (Dl) enters Sara-positive endosomes in SOP cells
at ~10 min after internalisation. Likewise, so does Notch (not shown).
But, strikingly, it was found that during the cell division from pI into pIIa and pIIb, the
Distribution of Sara-positive endosomes was remarkably polarised:
[Pon: protein which forms a crescent at the anterior end and set up polarity gradiant]
Endosome dynamics during asymmetric cell division.
Sara-positive endosomes
So what does this mean?
Thus, in the pIIa cell, there is an increased level of Sara-endosomes containing internalised Delta and Notch.
This has two effects: a decrease in levels of Delta (ligand) on cell surface, and an increase in levels of Notch in pIIa.
The Notch in these endosomes in cleaved by a protease, releasing active Notch to drive changes in gene expression in pIIa (but not pIIb), thereby establishing polarity in development.
Net result: Two trafficking steps play
similar roles
Differential ligand endocytosis after division of SOP into pIIa and pIIb contributes to polarised development (Numb).
Differential endosome sorting in assymetric cell division does likewise by driving directional transport of Delta/Notch into one of the daughter cells (Sara-endosomes).
Summary
Membrane traffic plays a key role in both symmetric cell division and assymetric cell
division.
It therefore also is a key player in the development of polarity and polarised cell
growth.
Multiple mechanisms at work, e.g. SOP development.
Intracellular reorganisation
in the cell cycle:
The fate of the Golgi
and making the final cut
Gwyn W. Gould
Henry Wellcome Laboratory of Cell Biology Davidson Building.
The issue at hand:
Successful cell reproduction requires faithful duplication and proper segregation of cellular contents, including not only the genome but also intracellular organelles.
The Golgi apparatus is an essential organelle of the secretory pathway, so its accurate inheritance is therefore of importance to sustain cellular function.
Regulation of Golgi division and its coordination with cell cycle progression involves a series of sequential events that are subjected to a precise spatiotemporal control.
The Golgi directs traffic operations.
Roles of the Golgi
Golgi is a membrane bound organelle, essential for glycosylation of proteins, lipid synthesis and
the control of membrane traffic into multiple
compartments.
In eukaryotes, there are multiple Golgi cisternae, arranged in flattened sacks, tethered together.
In most cells, this structure (shown on previous slide) resides near the centrosome in the middle
of the cell.
Think/Pair/Share #2
How is the Golgi equally inherited by the
two daughter cells after mitosis?
Early thoughts and observations
The Golgi apparatus grows in interphase and is divided into the daughter cells in mitosis.
During cell division, the single continuous Golgi ribbon in mammalian cells is disassembled in early mitosis and reformed upon partitioning in both daughter cells.
The nuclear envelope is also dissolved at the onset of mitosis to allow chromosome segregation. For the purpose of partitioning, the nuclear membranes are first absorbed into the ER and re-emerge out of the ER at the end of mitosis to assemble a nuclear envelope around the de-condensing chromosomes.
Two different mechanisms have been proposed for the partitioning of the Golgi. In one view, Golgi membranes are absorbed into and partitioned with the ER. The second view argues that the Golgi remains distinct from the ER and that the two compartments are inherited independently.
Movie.
In this figure, the
distribution of two Golgi
markers, Sphingosine-1-
phosphatase (A) and
GM130 (B) are revealed
by immunofluorescence
staining.
During prometaphase, the
Golgi cisternae unstacked
and fragmented into clusters
of tubules and vesicles
In the course of M-phase, the
Golgi membranes and S1P
were seen to be separated into
similar amounts on each side
of the equatorial plate.
Equal inheritance
Figure 1. Mitotic Golgi membranes accumulate at the spindle poles
Wei, Seemann J. Cell Biol. 2009:184:391-397
2009 Wei et.al.
What controls this?
Mitotic Golgi membranes concentrate around the spindle poles, suggesting that
the mitotic spindle may control Golgi
partitioning.
A really cool experiment
To test whether the spindle is required for
Golgi partitioning, Joachim Seeman and
his colleagues established a system in
which mitotic cells were induced to divide
asymmetrically into two daughter cells, but
only one of which received the entire
spindle.
If you do this, what happens to the Golgi?
Dont worry about what these are
After division, the karyoplasts received the chromosomes (Fig. 2, E and I), centrosomes (unpublished data), and microtubules (Fig. 2, D and H), whereas the cytoplasts lacked all of these.
Intriguingly, Golgi markers, including NAGT IGFP and GM130, were detected in both cells (Fig. 2, C and G), suggesting that parts of the Golgi were partitioned independently of the spindle (n = 29 for Mad1 injection; and n > 50 for Cdk1 inhibition).
However, the organization of the Golgi in the two daughter cells was very different. They analyzed the Golgi distribution in each daughter cell, where the Golgi was determined as a ribbon if 90% of the fluorescence resided in no more than three continuous structures (Puthenveedu et al., 2006). The Golgi in the karyoplasts localized to the perinuclear region and exhibited the characteristic ribbon structure (Fig. 2, C and G). In contrast, the Golgi in the cytoplasts was spread throughout the cytoplasm and failed to reform a ribbon (Fig. 2, C and G). This indicates that the spindle has a direct role in inheritance of the Golgi ribbon.
So why go to all this bother?
The appearance of the Golgi ribbon in higher organisms during evolution reflects a different mechanism for polarized secretion.
In mammalian cells, cargo is post-translationally sorted from the centrally located Golgi ribbon.
To establish cell polarity, the entire Golgi ribbon is reoriented toward the site of secretion which is essential for a variety of cellular processes such as the outgrowth of dendrites in neurons or fibroblast migration during wound healing.
Because the ribbon is particularly important for more advanced functions, tight coupling of ribbon determinants with the spindle ensures that each daughter cell receives the information to assemble it.
This might be the reason that mammalian cells use the highly regulated spindle apparatus to partition these factors instead of a stochastic ER-dependent process.
Some other aspects of membrane
traffic in mitosis.
A lot of cytokinesis:
100 x 1012 cells in each of us.
5 litres of blood, corresponds to 3 trillion cells with a life span of 120 days.
Do the maths: 290,000 cell divisions per second to regenerate blood.
Recent studies suggest that the formation of binucleate cells as a result of cytokinesis failure is an early event in tumour formation and underlies the subsequent development of genomic instability.
Many key cytokinesis genes are proto-oncogenes (e.g. ECT2, anillin) and loss of tumour suppressors may promote carcinogenesis by disrupting cytokinesis.
Evidence for requirement of
membrane traffic
The composition of the PM at the cleavage furrow is distinct from the rest of the PM, both in terms of lipids (PE,
Cholesterol) and proteins, e.g. SNAREs are specifically
localised at the furrow and function in furrowing and
abscission.
Cellularisation studies in flies and worms: small GTPases involved (Rab11, ADP-ribosylation factors (Arfs)) suggests
a role for internal membrane delivery to the PM.
Need to accommodate increased surface area, but may also reflect specialised cargo delivery of key proteins into
the furrow and midbody which may regulate (e.g.) shape
change, actin dynamics and abscission.
Rab familyOver 60 Rab genes in human genomeDistributed within distinct cellular compartments Involved in vesicle formation, motility, membrane fusion, etc.
Rab11Localises to TGN & recycling endosomeMediates vesicles traffic from RE and TGN to plasma membrane
Rab proteins
Rab proteins and their binding partners move into the
midbody during cytokinesis
Neto and Gould, Journal of Cell Science, 2011Click here to repeat first sound bite Click here for next sound bite
Barr and Gruneberg, 2007
Why might we need membrane traffic
into the furrow?
Barr and Gruneberg, 2007
Why might this happen?
So the model/hypothesis is that membrane vesicles (both endosomes and secretory vesicles) enter the midbody and become anchored there.
This provides a source of membrane (for fusion cf previous slide) but also a platform for the assembly of the abscission machinery.
Endosomes accumulate
Signalling complexAssembles on
or near endosomesAurora A, Plk1
Recruited by Cep55
Summary:
Membrane traffic is important for many facets of mitosis: here we have briefly considered the Golgi partitioning and endosomes in abscission. There are numerous other examples.
Membrane traffic can be controlled in space and time.
Membrane traffic is selective and specific. Membranes can be used to deliver
cargo/material (e.g. Golgi enzymes to two daughter cells) or as a platform onto which specific machines can be assembled (e.g. cytokinesis/abscission).
Some useful reading:
SARA-endosomes and Delta/Notch:
Bokel C, Schwabedissen A, Entchev E, Renaud O, and Gonzalez-Gaitan M, "Sara endosomes and the maintenance of Dpp signaling levels in mitosis". Science. 2008 314: 1135-1139.
Knobilch JA, "SARA splits the signal". Science. 2006 314: 1096.
Emery G and Knobilch JA, "Endosome dynamics during development". Curr. Opin. Cell Biol. 2006 18: 407-415.
Endosomes as machines/abscission:
Gould GW and Lippincott-Schwartz J, "New roles for endosomes: from vesicular carriers to multipurpose platforms." Nature Rev. Mol. Cell Biol. 2009 10: 287-292.
