Shape changes, membrane extension and the actin cytoskeleton

CeU shape and extent o f ceU-matrix adhesion can change in response to environmental signals (Zigmond, 1996; Ingber, 1997), an example being ECM-dependent shape control (Sims et al. 1992). Rearrangement of the actin cytoskeleton is a contributory factor to such change. During formation o f adhesions in migrating ceUs the actin cytoskeleton reorganises and stabiUses itself in order for movement to occur, ie: polarisation and membrane extension (Figure 6). The mechanism by which this is initiated is stiU unclear ie: through actin flow or via ceUular tensegrity (Ingber, 1993).

Figure 6. The reorganisation of actin during migration

F ilo p o d i» . tv n g a c tin bundle:»

i

Î

Cells must attain a polarised morphology (ie: clear distinction between the front and the rear of the cell) in order for forward cell translocation to occur. This can be initiated by a change in F-actin distribution. Actin filament binding proteins interact with the actin filaments and cause actin bundling and crosslinking. Such changes in actin filament mechanical properties are required to enable membrane extension during migration. Initially F-actin is arranged in long bundles and forms plasma membrane protrusions called filopodia. Next the actin transforms into a meshwork generating lamellipodia (ruffles) that resemble sheet-like protrusions. Finally, the F-actin forms actin stress fibre bundles that are coupled to focal adhesions, and it is these structures that provide a link between the internal cytoskeleton and the external ECM. The focal adhesions exert force against the substrate and in turn generate traction (Huttenlocher et al. 1995; Zigmond, 1996). Possible mechanisms responsible for such actin cytoskeletal reorganisation and shape changes are through actin polymerisation or cellular tensegrity.

Actin Flow Theory:

The actin flow theory suggests that upon cell-matrix attachment, the actin cytoskeleton is rearranged through the treadmilling effect o f actin polymerisation and depolymerisation (Lauffenburger and Horwitz, 1996; Welch et al. 1997). The cell cytoplasm maintains a high level o f globular actin monomers that can undergo rapid assembly and disassembly at specific sites. The ability o f actin polymerisation to drive membrane extension is dependent on the activity o f actin filament binding proteins. These proteins, eg: a-actinin and filamin, provide actin filaments with appropriate mechanical properties (ie: filament bundling or filament crosslinking). It is thought that together these induce a cellular shape change, ie: formation o f protrusion, necessary for migration. Such a phenomenon may be termed ‘protrusive force’ as opposed t o ‘contractile force’ generated by actin-myosin dynamics during motility (Lauffenburger and Horwitz, 1996) (Figure 7).

Cellular Tensegrity Theory:

The second theory, that o f tensional integrity (tensegrity model) (Ingber, 1993, 1997) suggests that shape changes and protrusion formation are dependent on cytoskeletal tension (generated through actin-myosin dynamics), and a cellular force balance between the cell and its underlying matrix (Sims et al. 1992; Chicurel et al. 1998). This theory is based on two concepts, mechanotransduction and tensegrity (Ingber, 1997). Mechanotransduction can be defined as the conversion o f a physical signal (such as mechanical stress) into a biological or chemical response (such as cellular signalling) (Ingber 1997). Where as tensegrity attempts to explain how cells stabilise their structure and shape in response to a mechanical signal. Ingber suggests that all cells possess an internal pre-stress or equilibrium, that if altered, is rapidly adjusted to reinstate a homeostatic environment (Ingber, 1993). Cells exert changes in their shape and motility by adjusting to the tension that they perceive (Ingber, 1997; Chicurel et al. 1998). It suggests that cells are constructed so as to immediately respond to any physical changes, and that these physical changes are transmitted over cell surface mechanoreceptors (eg: integrins), that physically join the cytoskeleton to the extracellular matrix through focal contact complexes (Ingber, 1997; Chicurel et al. 1998). This model, unlike the actin flow model attempts to give insight into the cellular mechanics occurring during migration. Figure 8 and 9 depict the basic tensegrity model.

Figure 7. Actin dynamics during migration retrograde flow dcpolymcrisation ■ ADP-actin A D P-Pi-actin protrusion nucleotide exchange ■ A1 P-aclin plasma membrane LEADING EDGE

Myosin induced retrograde flow

Transport o f depolymerised material

This is a schematic representation of actin dynamics occurring at the leading edge (protrusion) of a motile cell. Filamentous actin undergoes continuous polymerisation at the leading edge of the cell (right-hand side), which takes place at the inner surface of the

plasma membrane. Myosin induced retrograde flow transports newly polymerised actin

filaments away fi-om the plasma membrane, towards the inside of the cell. It is here that depolymerisation occurs (left-hand side), subunits are recycled by the ATP-hydrolysis driven exchange of ADP for ATP, and prepared for polymerisation once again. This cascade of events results in a protrusive force necessary for membrane extension.

(adapted from Welch et al. 1997).

Figure 8. Basic tensegrity model

Microfilament-like tensioned elements

Microtubule-like compression-resistant elements

Blue rigid compression-resistant elements push out against the inward pull of the black tensioned elements. A construction of this sort provides the structure, eg: a cell, with an internal tension or pre-stress.

(taken from Ingber and Jamieson, 1985).

Figure 9. Tensegrity cell model

unattached cell

substrate

flattened cell

rigid substrate overcom es the cell-m ediated tension

cell contracts the substrate through its internal tension

flexible substrate that is pulled in by the cell's internal tension

Unattached cells (A) appear round due to their internal tension or pre-stress. Upon attachment to a rigid substrate (eg: tissue culture plastic) the cell and its nucleus flattens (B), as the substrate can resist the cell-mediated tension. However, if the rigid substrate is made flexible (C), the cell pulls the substrate around it, ie: contracts the substrate, and returns to its round configuration. This phenomenon is again due to the cell generating its own internal tension. This can be seen on the classic experiment conducted by Harris et al. (1980) where cellular migration caused wrinkling of silicone sheets.

(taken fi-om Ingber and Jamieson, 1985).