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to the formation of localized focusing platforms called asters (Rao and Mayor, 2014). Any cell membrane component that can bind to these dynamic actin filaments (directly or indirectly) willmovealongthemovingactinfilamentsand end up into tiny, localized clusters at the centre of an aster (Gowrishankar, Ghosh et al., 2012).
‘What I cannot create, I do not understand.’ – Richard Feynman
The intrinsic complexity of the cell membrane makes it difficult to gain a systematic understanding of the system as such. To identify the underlying microscopic processes, we decided to adopt a bottom-up approach and learn by building an artificial cell membrane from its purified components. This powerful method, called in vitro reconstitution biology, allows researchers to study a complex biological system in a simplified and controlled manner. One can test theoretical predictions (hypotheses) as well as
complement cell- and animal- based studies.
Our in vitro reconstitution
system consists of a synthetic
lipid bilayer connected to an
actomyosin network via linker
proteins. We can fine-tune
different parameters such as
the density of the linker proteins,
length of actin filaments and
relative number of actin and
myosin molecules. We extract
these proteins in their functional
form from chicken muscles or
purify them from large bacterial
cultures by exploiting their unique size and amino acid composition. Purified components are then labelled with fluorescent tags, and the whole preparation is visualized under a high-resolution microscope with the help of advanced detection systems.
Prior to myosin addition, actin filaments show a scattered distribution on the lipid bilayer. Upon myosin addition, actin filaments undergo a dramatic reorganization due to myosin contractility and transform into localized platforms (asters). These asters concomitantly drive the linker proteins into localized, transient clusters beneath the asters. This clustering depends on ATP-fuelled myosin activity: as soon as ATP is depleted, these clusters disperse into individual linker proteins. Thus, using this novel system, we were able to recapitulate the clustering and sorting behaviour of cell membrane proteins as described by our theoretical model (Gowrishankar, Ghosh et al., Cell 2012). These results were published in 2016 (Koester et al., PNAS 2016).
This mechanism of active (energy- fuelled) clustering might seem very general at the molecular level since it only requires an actin-binding capacity of cell membrane proteins. However, different membrane proteins bind actin filaments with different strengths and are present in different relative quantities in the membrane. Therefore, our question was what happened when this diversity of actin- binding proteins was coupled with the active clustering mechanism, that is, whether it would give rise to molecular patterning and ensure clusters
of consistent composition.
We generated a set of linker proteins with measured affinities ranging from very strong to very weak. Again, we used the same reconstitution system, but now, we used different linker proteins in various combinations and relative quantities. We found that when
Mr. Abrar Bhat || 465
   The cell membrane is draped on a specialized actin network called actin cortex. Actin is a small, round protein and can exist as individual units or filaments of variable length. The composition and architecture of the actin cortex are regulated by actin modulators and myosin motors.
  










































































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