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  • Cofilin is an additional actin severing protein

    2024-05-14

    Cofilin is an additional ceramide kinase severing protein present in sperm cells that undergo phosphorylation/inactivation on serine 3 by Lim kinases (LIMK) and by Tes kinases (TESK) [48]. LIMK can be activated by several pathways, including one through the Rho/ROCK/LIMK cascade [49], [50], [51]. Another way of activating LIMK is its dimerization and transphosphorylation by Hsp90 [52]. It is possible that cofilin phosphorylation is modulated by PKA activation, but there are conflicting reports about its impact. It was shown that PKA inhibits ROCK phosphorylation and activation by phosphorylation/inhibition of RhoA on serine-188 [53]. Other works support this idea by showing that elevated cAMP levels may indirectly lead to cofilin dephosphorylation [54], [55]. In contrast, another study showed that LIMK is directly activated by PKA through phosphorylation on serine-323 and -596 [56]. Another pathway of cofilin regulation is phosphorylation on tyrosine 68 by v-Src, leading to cofilin ubiquitination and degradation by the proteasome [57]. Supporting that, we found that inhibition of PKA leads to cofilin dephosphorylation [58]. In a recent study, we demonstrated that like gelsolin, activation of sperm cofilin leads to actin depolymerization, inhibition of hyper-activated motility and the induction of the acrosomal exocytosis [58]. However, the kinetics of inactivation during capacitation is different between cofilin and gelsolin. Cofilin is highly phosphorylated/inactivated at the beginning of the capacitation process, whereas gelsolin shows high phosphorylation/inactivation towards the mid-end of the ceramide kinase capacitation process. In addition, the level of PIP2 does not affect cofilin phosphorylation, as it does in the case of gelsolin [18], [58]. Thus, for the sperm to achieve high levels of F-actin along the capacitation process both gelsolin and cofilin must be inactive at different times during the capacitation period and both are activated prior to the acrosomal exocytosis. It is important to mention that for its activity gelsolin needs the actin-depolymerizing-factor (ADF)/cofilin to depolymerize F-actin [59]. In conclusion, our data suggest the following model (see Fig. 1): The relatively small increase in [Ca2+]i during capacitation leads to conformational changes in gelsolin revealing the F-actin binding site. This change and the increase in F-actin and PIP2 in the sperm head, result in gelsolin translocation to the head. Nevertheless, the elevation of PIP2 levels and PKA/Src activation, maintain gelsolin in a phosphorylated/inactivated state and actin polymerization occurs. Activation of Src during capacitation lead to protein-phosphatase1γ2 (PP1γ2) inhibition resulting in activation of the cascade CaMKII-Pyk2-PI3K that mediates actin polymerization [19], [47], [60] as described in Fig. 1 here. The increase in F-actin in the tail leads to the development of hyper-activated motility as part of the capacitation process. The model for cofilin phosphorylation/inactivation during capacitation is seen in Fig. 1. HCO3− activates the soluble-adenylyl cyclase (sAC) to produce cAMP, which activates PKA leading to LIMK activation and cofilin phosphorylation. It is possible that the phosphatase SSH1L which dephosphorylates p-cofilin is inhibited during capacitation by CaMKII, keeping the cofilin in its phosphorylated/inactivated form. This suggestion should be confirmed in the future.
    Activation of gelsolin and cofilin prior to acrosomal exocytosis Gelsolin activation is regulated by calcium ions, phosphoinositides [61], [62] and by Src-dependent phosphorylation on tyr-438 [63]. Low concentration of calcium ions cause conformational changes in the C- terminus of gelsolin, which exposed its F-actin binding site and higher calcium caused a second conformational change exposing the catalytic site [62]. In human sperm, activation of gelsolin by enhancing intracellular calcium concentration or by using the peptide PBP10 causes fast depolymerization of F-actin and induction of the acrosomal exocytosis in capacitated sperm [41]. In Sertoli cells, the hydrolysis of PIP2 by PLC resulted in the release the bound gelsolin and its activation [64]. Releasing of gelsolin from binding to PIP2 due to its hydrolysis by phospholipase C (PLC) induced reverse translocation of gelsolin from the head to the tail [41]. It is well known that there is a high increase in intracellular Ca2+ concentrations as a result of the interaction between capacitated sperm and the egg zona pellucida [65]. This increase in calcium is essential for the occurrence of the acrosomal exocytosis which is known to be mediated by PLC activity [60]. Interestingly, activation of gelsolin by PBP10 in capacitated sperm, which induces PLC-independent acrosomal exocytosis, occurs under conditions by which intracellular Ca2+ concentration is relatively low, further indicating that the increase in intracellular Ca2+ is essential for the activation of PLC leading to gelsolin activation and F-actin dispersion an essential step for acrosomal exocytosis to occur. Activation of cofilin by 17-allylaminogelanamycin (17-AAG) resulted in F-actin breakdown, inhibition of hyper-activated motility and significant increase in acrosomal exocytosis [58]. 17-AAG inhibits Hsp90/LIMK and cofilin phosphorylation resulting in p-cofilin dephosphorylation/activation. Moreover, activation of cofilin during the entire capacitation period prevents the cells from undergoing proper capacitation. Thus, cofilin regulates sperm capacitation by controlling the cellular F-actin levels. This conclusion is further supported by the translocation of cofilin from the tail to the head during capacitation [58]. The decrease of cofilin in the tail during the capacitation allows the retention of a high level of F-actin in the tail, which is important for the development of hyperactivated motility, while its increase in the head is important for F-actin breakdown prior to the acrosomal exocytosis. It should be mentioned that inhibition of Hsp90 by 17 AAG results in Akt inactivation [66], [67], and that Akt is related to the regulation of human sperm motility and hyper-activated motility [68]; thus, it is possible that 17-AAG also inhibits sperm motility via this mechanism.