These studies demonstrate the Ca2+ and calpain dependence of cell spreading. Calpastatin overexpression and treatment with four distinct calpain inhibitors all inhibit spreading by suppressing the intimately related processes of early filopodial and lamellipodial protrusion formation. Calpain inhibition results in abnormal transient extensions containing a narrow rim of cortical F-actin. When spreading finally occurs in calpain-inhibited cells, the cells are significantly smaller than normal and have abnormal actin networks that lack true lamellipodia. In contrast, stress and retraction fiber formation do not appear to be affected by calpain inhibition. Kinetic analysis of spreading indicates that calpain is involved in spreading almost immediately after cell contact with the substratum. These results indicate that calpain plays a critical, rate-limiting role in the early stages of spreading via its effects on motility and actin network remodeling, perhaps through cleavage of actin-associated cytoskeletal proteins.
Our finding that calpain is involved in lamellipodial protrusion formation is consistent with the recent observation that cell migration is calpain dependent (Huttenlocher, et al., 1997). Although migration studies in CHO cells indicate that calpain inhibition stabilizes peripheral focal adhesions and decreases detachment rates at the rear of the cell, our results reveal the important role that calpain–cytoskeletal interactions play in forward protrusion formation as well. The CHO cell migration studies demonstrated no inhibition of lamellipodial projections and ruffling. In contrast, we find by multiple modalities of calpain inhibition that lamellipodial protrusion formation is a calpain-dependent process. In our hands, extended treatment of crawling NIH-3T3 cells with calpain inhibitors results in very small lamellae that would be difficult to quantitate by video microscopy because of their size and thinness. Thus, our studies point to the importance of calpain-dependent cytoskeletal remodeling, in both the polarized movement of crawling and the nonpolarized movement of spreading. It is therefore proposed that calpain activity is involved both in spreading and crawling, but in the latter case, both at the rear of the cell and the forward lamella. Calpain may thus coordinately regulate forward protrusion formation and rearward retraction, providing a proteolytic tool to couple Ca2+ fluxes and cytoskeletal remodeling in crawling.
The methods of calpain inhibition chosen for these studies were chosen on the basis of their specificity. Calpastatin, which was overexpressed at two different levels in five independent cell lines, inhibits calpain specifically. No other protease has been found to be inhibited by calpastatin, including other sulfhydryl proteases such as cathepsins (Nishiura et al., 1978
; Waxman and Krebs, 1978
; for reviews see Murachi et al., 1980
; Crawford, 1990
; Croall and DeMartino, 1991
). The high-affinity binding of calpain to calpastatin, having a dissociation constant of 3.1 × 10−9
M (Yang et al., 1994
), is Ca2+
dependent (for review see Goll et al., 1992
), adding a further level of specificity. Although artificial inhibitors also require Ca2+
to interact with calpain, they do not bind to the enzyme with high affinity. The high-affinity interaction between calpain and calpastatin can be used to affinity-purify calpain to homogeneity from crude cell fractions, again suggesting that its interaction with calpain is highly specific (Anagli et al., 1996
). Nonetheless, the inhibition of calpain by calpastatin overexpression is not an immediate process and thus peptidyl inhibitors were used to confirm a direct role for calpain in cell spreading.
The peptidyl inhibitors used in these studies, ZLLYCHN2
, MDL, calpeptin, and E64d, were chosen on the basis of their specificities. All of these inhibitors inhibit only cysteine proteases. Calpeptin and MDL have similar specificities and inhibit both calpain and cathepsin B (Mehdi, 1991
). Calpeptin has also been shown to inhibit cathepsin L (Angliker et al., 1992
). E64 inhibits calpain and cathepsins B and L (for review see Wang and Yuen, 1994
, in contrast, inhibits calpain and cathepsin L, but inhibits cathepsin B poorly (Shaw, 1994
, although not immediately cell penetrating, has been found to be the most selective inhibitor, since radioiodinated ZLLYCHN2
primarily labels two cellular proteins, calpain and to a lesser extent, cathepsin L (Anagli et al., 1991
). Together, these data strongly suggest that a cysteine protease is required for spreading. The corresponding NH4
Cl control experiment, demonstrating that lysosomal inhibition does not affect spreading, greatly decreases the likelihood that this cysteine protease is a cathepsin. Finally, we found that MDL is the most potent inhibitor of calpain under the conditions used in our studies. It is well established that E64 is the least potent calpain inhibitor of the above group, with a 50% inhibitory concentration of 1.09 μM for m-calpain by caseinolysis assay (Saito and Nixon, 1993
), ~20-fold higher than the corresponding 50% inhibitory concentration of calpeptin (Tsujinaka, 1988). The observation that MDL is the most potent inhibitor of spreading (minimal inhibitory concentration, 50 μM), among the immediately acting calpain inhibitors, whereas E64d is the least potent (minimal inhibitory concentration, 200 μM), indicates that the peptidyl calpain inhibitors inhibit spreading with minimal inhibitory concentrations corresponding to their potency of calpain inhibition. This observation strongly suggests that calpain is the biological target of the immediately acting inhibitors that is relevant to spreading.
These studies demonstrate that calpastatin regulates calpain levels and activity in vivo. The regulatory role of calpastatin on calpain has been studied extensively in vitro (for review see Maki et al., 1990
; Mellgren and Lane, 1990
; Croall and DeMartino, 1991
). The biological importance of the calpain/calpastatin ratio in cells has been demonstrated for myoblast (Barnoy et al., 1996
) and erythrocyte (Glaser and Kosower, 1986
) fusion. Here, it is found that the reduction of calpain activity in NIH-3T3 fibroblasts induced by calpastatin overexpression can be accounted for mainly by the reduction of calpain itself, as demonstrated in five calpastatin-overexpressing cell lines. This observation is contrary to the simplest model for regulation of calpain activity, in which elevated calpastatin levels inhibit an unaltered level of calpain protease. The finding that the calpastatin/calpain ratio controls the abundance of m-calpain mRNA is also contrary to the simplest model for regulation of calpain gene expression, in which calpastatin should play no role. Our observations suggest that there is a calpastatin-dependent mechanism for turnover of calpain, since calpain downregulation was found to be proportional to calpastatin overexpression. Our observations also suggest that the level of calpain and/or the degree of calpastatin overexpression is connected to m-calpain gene expression. Steady-state levels of m-calpain mRNA are significantly elevated in the low overexpressors, but decreased in the high overexpressors. These unexpected results suggest that there is a calpain-dependent feedback loop that regulates m-calpain gene expression, since modest repression of calpain activity results in compensatory increases in m-calpain mRNA, whereas high repression of calpain activity results not only in loss of the compensatory response, but in a decrease of calpain mRNA. The increased levels of ezrin mRNA in the low overexpressors, but not the high overexpressors, also raises the question of whether the calpain–calpastatin system regulates ezrin gene expression in a similar fashion. Calpastatin overexpressing NIH-3T3 fibroblasts will be ideal for testing the mechanisms by which the calpastatin/calpain ratio is regulated in cells, both at the levels of gene expression and protein turnover.
Calpain inhibition, both biological and pharmacological, results in an increase of the steady-state levels of the actin-associated protein ezrin. These results are consistent with the observation that ezrin is a biological substrate of calpain during cell movement in wounded monolayers (Shuster and Herman, 1995
). A role for calpain in the regulation of ezrin is also suggested by the increases in ezrin, in contrast to other ERM proteins, observed with both calpastatin overexpression and ZLLYCHN2
-treatment. The greater increase in ezrin protein levels in the low calpastatin overexpressors, compared to the high overexpressors, may be due to increased levels of ezrin mRNA. The mechanisms by which the calpain–calpastatin system regulates ezrin levels will be addressed in future studies.
Although it is possible that the spreading abnormalities of calpastatin-overexpressing cells could be related to increased ERM proteins, which might act in a dominant-negative fashion, this is unlikely. It is known that overexpression of wild-type ezrin, although enhancing adhesion, does not appear to affect cell morphology or spreading (Martin et al., 1995
; Crepaldi et al., 1997
). Similarly, overexpressed radixin does not appear to affect morphology or spreading in NIH-3T3 fibroblasts (Henry et al., 1995
). Another argument against a dominant-negative role for ezrin is the finding that the excess ERM proteins accumulating in the calpastatin-overexpressing cell lines are present primarily in soluble form in the cytosol, where they are likely to be self-associating and self-inhibitory, in terms of their interaction with either the plasma membrane or actin filaments (Gary and Bretscher, 1993
). Finally, immediate inhibition of cell spreading with MDL and calpeptin is unlikely to alter the levels of cytoskeletal proteins.
Until recently, models of Ca2+
function at the lamellipodial protrusion have mainly focused on gelsolin rather than calpain. The Ca2+
-dependent actin-severing function of gelsolin provides a Ca2+
-regulated mechanism for coordinated uncoupling of the actin cytoskeleton from the cell membrane and simultaneous exposure of barbed ends (Cunningham et al., 1991
; Stossel, 1993
; Witke et al., 1995
). The observations presented here now suggest a second pathway by which Ca2+
-transients can regulate the dynamics of the actin cytoskeleton and raises the question of whether some cell types have predominantly a gelsolin-mediated uncoupling and uncapping process whereas others have a predominantly calpain-dependent process. Since motility of NIH-3T3 cells and other fibroblasts is clearly gelsolin dependent (Cunningham et al., 1991
; Witke et al., 1995
) although NIH-3T3 cell spreading is calpain-dependent, the functions of calpain and gelsolin may be nonredundant. It is also possible that there are subtle differences in lamellipodial actin dynamics between spreading and crawling. The calpain and gelsolin systems may complement or supplement one another during spreading and/or crawling.
There are at least three mechanisms by which calpain could facilitate actin filament extension and thus cell spreading: (a
) cleavage of ezrin linkages between capped β-actin filaments and the plasma membrane; (b
) stabilization of phosphatidylinositols that uncap β-actin filaments; and (c
) activation of the uncapping function of PKC. Supporting the first mechanism, ezrin indirectly binds barbed ends of β-actin filaments, is cleaved by calpain in motile cells, and colocalizes with β-actin and Ca2+
transients at the leading lamella of crawling cells (Shuster and Herman, 1995
; Shuster et al., 1996
). Furthermore, calpain cleavage of ezrin specifically disrupts its linkage with capped β-actin filaments. Calpain could also expose barbed ends for actin polymerization in a coordinated Ca2+
-dependent fashion, suggested by the observation that both ezrin and the novel β-actin barbed-end capping protein βcap73 are displaced from β-actin filaments by cytochalasin D (Shuster et al., 1996
). A second mechanism by which calpain may function in spreading might be to elevate phosphatidylinositols (PIs), such as PI(4,5)P and PI(3,4)P, which can directly uncap β-actin filaments (Schafer et al., 1996). It has recently been demonstrated that PI-4-phosphatase is a calpain substrate, which can be inactivated by calpain cleavage (Norris et al., 1997
). Inactivation of PI-4-phosphatase by calpain could thus elevate PtdIns(3,4)P and PtdIns(4,5)P levels and facilitate uncapping. A third mechanism by which reduction of calpain activity could affect spreading is through inhibition of PKC-dependent pathways of cell shape change. Calpain can cleave PKC to the constitutively active and diacylglycerol/Ca2+
-independent product PKM. PKC facilitates cell spreading on fibronectin, although its specific function in spreading remains to be determined (Vuori and Ruoslahti, 1993
). One mechanism by which PKC could facilitate spreading is through its modest actin uncapping activity, which has been described in platelets (Hartwig et al., 1995
). Nonetheless, given the observation that PKC-α/-β levels vary over a fivefold range between the low and high calpastatin overexpressors with no difference in spreading rates between them, and given the observation that the high calpastatin overexpressors and the control line exhibit no significant difference in the levels of PKC-α/-β, it is unlikely that PKC levels are rate-limiting for spreading in this system.
The demonstration that calpain activity is integral to lamella protrusion formation suggests a functional role for calpain in the regulation of β-actin dynamics at the leading lamella. The β-actin isoform is specifically localized to the lamellae of spreading and motile cells (Hoock et al., 1991
; Herman, 1993
; Latham et al., 1994
; Shuster and Herman, 1995
; Bassell et al., 1998
). Furthermore, the localization of β-actin mRNA to the leading lamella is important for cell motility (Kislauskis et al., 1997
). The localization of β-actin to the leading lamella may be due to local synthesis, since β-actin mRNA is specifically localized to the leading lamella of crawling cells (Hoock et al., 1991
; Hill and Gunning, 1993
; Kislauskis et al., 1993
; Bassell et al., 1998
). Lamellar localization of β-actin may also be due to the specific binding of β-actin to ezrin (Herman and Shuster, 1995; Yao et al., 1995
) via βcap73 (Shuster et al., 1996
). Electron microscopy has revealed that β-actin at the plasma membrane exists as short oligomers, 7–10 monomers in length (Shujath. J., and I. Herman, manuscript in preparation). Tethering of these β-actin oligomers to the plasma membrane by ezrin may provide a rich source of actin nuclei for the extension of free barbed ends, providing force and structure for lamellipodial extension. Because the binding of β-actin to ezrin has been shown to be indirect, involving the novel β-actin–specific barbed end capping protein, βcap73, it is proposed that calpain proteolysis of substrates, as yet unknown, lowers the affinity of βcap73 for barbed ends of β-actin nuclei or disrupts this interaction. One candidate substrate is ezrin, which has been demonstrated to be cleaved by calpain in motile cells (Shuster and Herman, 1995
). The resulting β-actin nuclei would serve as sites for the elongation of barbed ends, facilitating the force pushing against the membrane, resulting in lamellipodial protrusion formation. Calpain proteolysis could thus be one of the mechanisms by which Ca2+
-transients that colocalize with ezrin and β-actin at the leading lamella uncap β-actin filaments to initiate lamellipodial protrusion formation (Shuster and Herman, 1995
). Such a model would be consistent with the spreading defects seen when calpain is inhibited. Under these conditions, βcap73 would sequester β-actin and thus inhibit monomer addition and spreading. Identification of the biologically relevant targets of calpain, as well as molecular dissection of the interaction between βcap73, ezrin, and cortical β-actin filaments will likely be of critical importance to the understanding of how cytoskeletal remodeling occurs during cell motility and forward protrusion formation and why the β-actin isoform is specifically involved in these processes.