During the execution phase of apoptosis, a numer of plasmamembrane changes occur, resulting in the recognition and subsequent phagocytosis of the apoptotic cell either by professional phagocytes or by a neighbouring cell. Cleavage of important cytoskeletal proteins, including actin, Gas2 and fodrin, during apoptosis may induce cell shrinkage and membrane blebbing, and alter cell survival signalling systems.

Alpha-fodrin, an abundant membrane-associated cytoskeletal protein, is cleaved rapidly and specifically during Fas- and TNF-induced apoptosis. Studies in cells treated with these apoptotic stimuli reveal that both fodrin and poly(ADP-ribose) polymerase proteolysis are inhibited by acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and CrmA, both specific inhibitors of caspases. This cleavage is mediated by a caspase distinct from the poly(ADP-ribose) polymerase protease caspase-3 (Cryns et al., 1996). The same kind of experiments showed that cleavage of alpha-fodrin (non-erythroid spectrin) accompanies apoptosis, induced by activation via the CD3/T cell receptor complex in a murine T cell hybridoma. Furthermore, inhibition of activation-induced apoptosis by pre-treatment of T hybridoma cells with antisense oligonucleotides directed against c-myc also inhibited fodrin proteolysis, confirming that this cleavage process is tightly coupled to apoptosis (Martin et al., 1995).

Purified fodrin is cleaved in vitro by caspase-3 (but not by caspase-) into fragments of the same size observed in vivo during apoptosis. Fodrin proteolysis can be distinguished from poly(ADP-ribose) polymerase (PARP) proteolysis by its relative insensitivity to DEVD-CHO, a selective inhibitor of a subset of caspases that includes caspase-3. DEVD-CHO protects cells from Fas- induced apoptosis but does not prevent fodrin proteolysis, indicating that cleavage of this protein can be uncoupled from apoptotic cell death. It suggests that some caspases (those responsible for the cleavage of fodrin) may be activated and yet apoptosis may still be prevented. The inhibitory characteristics of the caspase responsible for fodrin proteolysis, good inhibition by Ac-YVADcmk and relatively insensitive to Ac-DEVD.CHO are reminiscent of caspase-6.

Human leukemia U937 cells are induced to undergo apoptosis by several chemotherapeutic agents. An actin-cleavage activity (ACA) was found in the VP-16- treated U937 cytosolic fraction producing 15K- and 30K-actin fragments. This ACA was inhibited by caspase inhibitors Z-Asp-CH2-DCB, YVAD-CHO, TPCK, TLCK and iodoacetamide. Although caspase-1 can cleave actin in vitro, caspase-1 was not activated in the VP-16 treated U937 cells, since ACA could not process pro-IL1b to mature IL-1b . These results indicate that actin might be a potential substrate of caspases.

However it is shown that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and caspase-3 were capable of cleaving actin in vitro. Although actin contains cleavage sites for caspases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation (Song et al., 1997)

Gas2, a component of the microfilament system, belongs to the class of gas genes whose expression is induced at growth arrest. After serum or growth factor addition to quiescent NIH 3T3 cells, Gas2 is hyperphosphorylated and relocalized at the membrane ruffles. Overexpression of Gas2 deleted at its C-terminal region (delta 276-314 and delta 236-314), but not its wild-type form, induces dramatic changes in the actin cytoskeleton and cell morphology. These effects are not due to interference of the deleted forms with the endogenous Gas2wt function but could be ascribed to a gain of function. During apoptosis the C-terminal domain of Gas2 is removed by proteolytic cleavage, resulting in a protein that is similar in size to the described delta 276-314. Moreover, by using in vitro mutagenesis, it was demonstrates that the proteolytic processing of Gas2 during apoptosis is dependent on an aspartic acid residue at position 279 pointing indirectly in the direction of caspases. This provides this a mechanism linking apoptosis with the co-ordinated microfilament-dependent cell shape changes (Brancolini et al., 1995).