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Welcome to this third part of the iBioSeminar on rafts
in cell membranes. I'm Satyajit Mayor from the
National Centre for Biological Sciences, working in
close collaboration with my physicist colleague,
Madan Rao, trying to make sense of what we see in
living cell membranes. So if you remember, or in case
you haven't seen the previous part, what we had
observed was that many different lipid-anchored
proteins are organized as small nanoclusters in
membranes of living cells in a manner that seems
consistent with their being formed by some active
process, because they exhibit a concentration-
independent clustering phenomena in living cell
membranes. We had also proposed that these
nanoclusters of lipid-anchored molecules may come
together and form regions in cell membranes that we
believe are functional domains, which can perform
sorting and signaling functions under regulatory
control of the cell's machinery. Well, the questions
that we were left with at the end of the last part were
questions about what makes these nanoclusters of
these different lipid-anchored molecules in cell
membranes, and what induces their forming
functional domains. And I think if we think can
address those two issues, we would be well on our
way to understanding functional domains in
membranes of living cells. As you will appreciate,
coming to this point has required a development of
new technology, new tools, to image processes in
living cells at the scales at which they demand.
We've had to develop new types of microscopy to
be able to study and elucidate the structure of these
little nanoclusters in living cell membranes. And in
fact, understanding and beginning to observe the
formation of these functional, larger-scale domains
also has required new developments in microscopy.
So what we have been able to do is image the
signals that we had observed from these nanoscale
clusters, the FRET signals, the fluorescence or
Förster's resonance energy transfer signals, from
these nanoscale structures, we've begun to image
them at much higher resolution, using a high-
resolution FRET imaging technique, where we're
able to map the FRET, using this "***-FRET"
technique that we had developed in the previous
iBioSeminar segment, at much higher spatial
resolution. So the spatial resolution that we have
now achieved is about 300 nanometer resolution,
and at this resolution, if we begin to image the FRET
signals coming from these lipid-anchored proteins,
specifically GPI-linked proteins at the surface of
living cells, we see the following. So in living cell
membranes, here is the fluorescence intensity
distribution of these GPI-linked molecules at the
surface of a mammalian cell (a Chinese hamster
ovary cell, in this case), and if you notice, the
distribution in most parts looks quite uniform and flat.
There are some regions which correspond to
microvilli that have slightly higher intensity than the
surroundings, and there are other regions which
correspond to edges of cells which look rather
undifferentiated. But if you now switch our gaze to
the FRET signal, and here the code is red for very
low FRET and blue for very high FRET
(corresponding to high level of anisotropy and low level of
anisotropy), the low level of anisotropy, which is the
blue signals, indicate regions where there is
enormous amount of FRET going on, and the high
level of anisotropy indicates where very low levels of
FRET is taking place. And what you immediately see
is that there are different shades of FRET distributed
throughout the cell surface. There is a region of low
FRET and a region of high FRET, which correspond
to regions where these clusters of these GPI-
anchored proteins are enriched or depleted. If you
look more closely, for example at the microvilli, we
find that the microvilli are regions where the clusters
of the GPI-anchored protein seem to be specifically
depleted. The same seems to be the case at tips of
lamellipodia, so tips of lamellipodia we find that these
GPI-anchored proteins also look quite heavily
depleted. So these are edges of cells where the cells
begin to extend, protruding actin-driven membranes,
where at the very tips you see a very high anisotropy
that corresponds to the exclusion of these
nanoclusters. In fact, in living cells, when we
observe cells performing their natural ruffling
functions, we find that the cells seem to be
dynamically excluding the clusters from the edges
that are ruffling. In fact, ruffling cells, if you look at
the arrows in the movie, you find that the arrows
indicate moments in time when, in ruffling cells, the
ruffles have extended out, and the GPI-anchored
protein clusters as detected by the FRET signals are
completely depleted from these ruffling edges. Other
regions in the cells are where the nanoclusters of
these molecules are enriched, and they seem to be
enriched specifically in the flat regions of cells, or in
regions just behind the tips of lamellipodia, the
lamellum, where the distribution of actin is likely to be
very different from the tip of the lamellipodia, or in
fact even microvilli. So it appears from these sorts of
observations that the GPI-anchored protein
nanoclusters are, in many cellular environments,
correlating with the underlying cytoskeletal matrix. If
you just look at the flatscape of a cell, where the
membrane is relatively flat at least to the eye of the
microscope, we find that there are a patchwork of
domains where GPI-anchored proteins are extremely
enriched, these very deep blue regions, and GPI-
anchored proteins are not as enriched in these
slightly yellow regions. But these patchworks are
regions which are showing a kind of variegated
pattern, which when analyzed by some statistical
means, shows a completely nonrandom segregation
of clusters. So in other words, it appears that the
membrane is not a homogenous, well-mixed
membrane where clusters are present, sprinkled
uniformly over the surface of the membrane, but in
fact, there are regions where clusters are selectively
concentrated, and they're selectively concentrated
or excluded from regions of membrane that seem to
have distinct underlying cytoskeleton. Experiments
that we have begun to do suggest that the
localization of these nanoclusters correlate with
specific types of the cytoskeletal organization,
specifically the actin organization. In fact, perturbing
the actin cytoskeleton using drugs that can impair
the polymerization or prevent the depolymerization of
actin dramatically affect the distribution of these
nanoclusters in cell membranes. So it seems that we
may have something that motivates the formation of
these regions in cell membranes where nanoclusters
are concentrated. In fact, the regions where
nanoclusters are concentrated correlate with regions
of actin where the actin cytoskeleton is likely to be
arranged in a horizontal fashion beneath the plasma
membrane. And regions where these clusters are
excluded seem to be correlating with regions where
the cytoskeleton is effectively organized vertical to
the plane of the membrane. And this may be in some
ways a mechanism that the cell uses to regulate the
local composition of these clusters in different
regions of the cell membrane. And then it is quite
likely that the sorting and signaling functions that we
have ascribed to different types of membrane
domains could be realized through this kind of
organization of membrane components in the cell.
The experiments that I've just shown you are of
course extremely new and perhaps almost "hot off
the bench," but they are not unsupported by other
literature that has been accruing over the past
several years, specifically from work from Ken
Jacobson's group, Mike Sheetz's group, and Aki
Kusumi's group in Japan, where they have argued
now that the membrane of a living cell is in fact
supported by, it's sitting on top of, a cytoskeleton.
And the cytoskeleton in fact not only, as I indicated
to you, influences the local distribution of these
nanoclusters in the membrane, it also influences the
mobility of molecules on the cell surface. So when
Aki Kusumi looks at the movement of a single
particle tagged to a fluorophore on the cell
membrane, or tagged to a gold particle, if he looks at
a lipid or if he looks at a membrane protein, they
exhibit sort of a rather characteristic pattern of
diffusion when observed at a relatively slow frame
rate. When Aki Kusumi looks at the membrane at an
extremely fast frame rate, they picture that they
observe is quite different. It seems that the particles
that move spend times in small confined regions in
the cell membrane and then jump to other regions in
the cell membrane, as if they were exhibiting some
sort of a hop diffusion mechanism. And this type of
characteristic is only observed when the frame rate
for observation is in fact 40,000 times per second,
achievements that are, at the moment, at the edge
of technology today. These regions can be mapped
out, and when they are identified by computer
programs that can detect breaks in the diffusional
characteristics within different regions, or can identify
these little domains, they show a fairly interesting
pattern of adjacent regions that are organized in
some fashion. And when, again, Aki Kusumi's group
looks at the underside of the plasma membrane, very
close to the lipid bilayer, they find that the
cytoskeleton seems to be also arranged in a pattern
that corresponds to these domains in which these
lipids seem to be confined or these proteins seem to
be confined during the diffusional process. So,
putting these different observations together, it
seems as if the membrane of a living cell is not simply
the lipid bilayer situated on its own, but it's in fact a
bilayer that's in close conversation with the
underlying cytoskeleton, and that conversation
seems to be generating new structures, rafts, if you
want to call them that, or structures where molecules
can be confined by the scaffolding of this
cytoskeleton for periods of time necessary for
engaging signaling output. And we believe that
those kinds of organizational forms can also
generate budding pits at the cell surface. Of course,
these are speculations, and I think it's important to
be able to clearly state what we know and what is a
proposal. So what we know is that we can see small
clusters of these lipid-anchored proteins in
membranes whose distribution and whose existence,
as well, seems to be very dependent on the
presence of the specific organization of the
underlying cortical actin, and that it appears a two-
way conversation between the lipids in the bilayer
and the cortical cytoskeleton. And this obviously
implies that the membrane of a living cell is not a
passive fluid mosaic model, but in fact it's an
extremely active system, which is, I guess, a walking,
talking composite between the bilayer and the
underlying cortical cytoskeleton which it is
associated with, and there are multiple things going
on in the membrane that could influence the local
organization of components. There's trans-bilayer flip-
flop mechanisms, which can change the local
composition of lipids. There are protein-protein
interactions whose aggregation can again change
the nature of the lipids surrounding these protein-
protein aggregates. There could be synapses in the
membrane, which could bring other cells close to the
membrane, which again can influence the local lipid
composition. There is, as I think, determining
cytoskeletal interactions, cytoplasmic proteins like
negatively charged molecules like MARCKS proteins
which also can influence the local lipid composition.
And finally, the well-characterized clathrin- and
caveolae-coated regions of the membrane, I think,
represent one example of how proteins in fact, and
their dynamics, can influence the local structure and
composition in cell membranes. So I think together
putting many of the pieces of this puzzle, where we
have a bilayer consisting of a variety of different
membrane components, it seems that the cell is
using special organizing principles from these active
polymers of the cytoskeleton to locally control the
nature, composition properties, of the plasma
membrane in a way that I think is going to keep us
busy in trying to understand for many decades to
come. But I think this is just the beginning of a new
way of looking at the cell membrane, and for all the
young researchers out there, I think this is a very
important area of research because, although there
has been work in this area for such a long time, I
think we know very little about how the local
composition of the cell membrane is regulated. And
of course, what I've described to you today is in fact
a proposal, and like many, these proposals are open
for debate, challenge, and investigation. And I think
this investigation has to be a multidisciplinary one,
where we are using tools from every resource
possible, we're using tools of physics, of the latest
physics, of the physics of active systems; chemistry,
where we try to understand how the chemistry of the
membrane is coupling to the activity of components
in the cell; and also exploring all the tools that the
biological system allows us, the tools of genetics and
imaging, to bring together and to bring to bear on this
fundamental problem of how membranes of living
cells are organized. And I'd like to invite all the young
researchers out there to this open arena, which I
think is just the start of a new adventure in cell
biology. Thank you.