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- Micromanipulation of Entamoeba histolytica -



Dynamical organization of the cytoskeletal cortex probed by micropipette aspiration
 Jan Brugués, Benoit Maugis, Jaume Casademunt, Pierre Nassoy, François Amblard & Pierre Sens
Submitted (Dec. 2009) 
preprint


Probing membrane-cortex adhesion by micropipette experiments
 Jan Brugués, Jaume Casademunt & Pierre Sens
In preparation
The dynamical properties of the actin cytoskeleton and its interaction with the plasma membrane control crucial aspects of cellular shape change and motility. Actin-based motility can result from the polarization of the cell, with actin polymerization at the leading edge, in a flat cellular extension called lamellidopium, and myosin contraction at the rear end of the cell. Renewed attention is currently being paid to an alternative form of actin-myosin based cell motility, where the contraction of the actomyosin cortex leads to cortex unbinding from the plasma membrane and the pressure-driven inflation of micron-size spherical membrane protrusions called "blebs". Blebs are often the sign of apoptotis, but are also used for motility by several cell types, including amoebae and possibly cancer cells. 


The life cycle of a bleb is a remarkable illustration of the highly dynamical interplay between the cytoskeleton and the plasma membrane. The local detachment of a patch of membrane from the actin cortex is followed by the inflation of a cytoskeleton-free membrane blister. A new actin cortex containing myosin  eventually repolymerizes under the bare membrane (while the membrane-free cortex depolymerizes), and myosin contraction brings the bleb and the cell body back together. Blebbing occurs in a stochastic fashion and may be harnessed for cell motility when coupled with adhesion on a substrate. This example illustrates the importance of the mechanical interaction between the cytoskeleton and the plasma membrane for the conversions of a contractile stress into forward cellular protrusion in bleb-based motility. More generally, it calls for a quantitative understanding of the role of membrane-cortex cohesion in dictating many of the cell morphological changes. 

In collaboration with the experimental groups of F. Amblard and P. Nassoy at institut Curie (Paris), we have combine theoretical modeling and micropipette manipulation of Entamoeba hystolytica to study the mechanical response of the cell to a controlled perturbation in  controlled geometry. Our model includes polymerization and depolymerization of actin, coupled to acto-myosin contraction within the cytoskeletal cortex, as well as the dynamical nature of the CSK-PM interactions. 


 
Sketch of the mechano-kinetic model for a cell inside a micropipette. a. A cell is drawn inside a micropipette by a difference of pressure. Inside the pipette, the cell forms a cylindrical tongue with an hemispherical cap. The plasma membrane is in green and the actin cortex in red.  b. The cortex is a gel of crosslinked actin filaments (red), and contractile myosin microfilaments (orange). c. The cortical actin layer is constantly being renewed by polymerization near the membrane and depolymerization, leading to treadmilling. An element of cortex is stretched by the extension of the tongue before being depolymerized. d. The cortex is attached to the PM by dynamical molecular linkers. e. Linkers have association and dissociation rates dependent on their energy landscape (black curve). When a force is applied on the bond, its energy landscape and associated rates are modified (red curve).

The dynamic nature of the membrane-cortex attachement, with mechano-sensitive binding and unbinding rate for each attachement complex, renders the cellular surface unstable to large mechanical stress, such as a strong micropipette suction pressure. Since the cortex is visco-elastic, and "looses" its mechanical memory after the time needed to recycle actin by polymerisation and depolymerisation (actin treadmilling), we have shown that membrane detachement from the cortex strongly depends on the rate at which the perturbation is imposed (by the experimentalist, or by the cortex itself).
The cytoskeleton cortex is a contractile meshwork of actin filaments and myosin motors, which exerts a normal force of the membrane linkers that is proportional to the interface curvature. For a given curvature there exist a cortex thickness hb for which this stress is sufficient to lead to membrane unbinding. Furthermore, in the context of micropipette suction, the cortical contractile stress manages to balance the pipette suction pressure (and drive cell retraction) for another critical thickness hs. Finally, the actual (stationnary) cortex thickness h is mostly fixed by a balance between actin polymerization and depolymerization. Depending on the relative values of these three particular cortex thicknesses, one can predict different cellular responses to a sustained micropipette suction, ranging from a steady cell engulfment inside the pipette, to remarkable sustained periodic oscillations.
 

 Entamoeba hystolytica is a cell whose motility proceeds exclusively through blebbing in the absence of chemotactic signals. The cortex is thus able to generate its detachement from the membrane even in the absence of external perturbation (h>hb), and the cell does not present a static equilibrium (such as the one corresponding to the retraction regime below). Al other regimes predicted by the theory have been observed by the experimental teams, and the actual motion of the cell in the pipette could be quantitatively reproduced by fitting our theory to the observations.

                      


Top-Left:Variation of the critical suction pressure for membrane-cytoskeleton unbinding with the loading rate of the perturbation, obtained by comparing the maximal cortical tension with the critical tension for linkers unbinding.
Top-Right: Predicted classes of cell motion inside a micropipette depending on the cortex thickness up to which the membrane linkers remain stable hb and the thickness needed to stop the membrane hs. , and the stationnary cortex thickness h. The blue line corresponds to the motion of the cell tip inside the pipette
Bottom: Motion of  Entamoeba hystolytica cells inside the pipette in the Saltatory (a, b) and Oscillatory (c, d) pipette suction regimes. (a) and (c) show snapshots of the movies (see below) and (b) and (d) shows a comparizon with theoretical predictions for the cell motion.


See the movies
 
Saltatory Regime (x5)  

Movie of the motion of an Entamoeba histolytica cell subjected to micropipette suction (pipette radius 5μm, pipette pressure ∆P=1.9kPa). This cell is initially in the saltatory regime, and two rupture events can be seen (white arrows), where the membrane detaches from the cytoskeleton cortex, jumps forward, and slows down due to cortex repolymerization. A transition to a steady flow regime is then observed (possibly due to the increasing pipette radius), where aborted attempts of cortex repolymerization only manage to transiently slow down part of the membrane tongue (yellow arrows).
 Oscillatory Regime (x20)
  
Movie of the motion of an Entamoeba histolytica cell subjected to micropipette suction (pipette radius 5μm, pipette pressure ∆P=1.2kPa). After a transient regime, this cell settles in the oscillatory regime, where events of membrane detachment and fast forward motion alternate with membrane retraction caused by cortex repolymerization and contraction. After 15 periodic oscillation, the cell also ends up being swallowed by the pipette.