Rapid assembly of new (ATP-rich) actin filaments and coordinated disassembly of aged (ADP-rich) filaments are processes that are essential for remodeling of cell shape during cell division, cell motility and cell morphogenesis. The actin-depolymerizing factor (ADF)/cofilins is a family of essential actin binding proteins that accelerate actin turnover by recognizing and severing aged actin filaments and thus increasing a number of actin filament ends available for depolymerization. In the cell, ADF/cofilins are assisted by other proteins among which Aip1 and cyclase associated proteins 1 and 2 (CAP1,2) are the most functionally important. Srv2, a yeast orthologue of mammalian CAPs, is the most studied member of the family and has been characterized initially as a monomeric actin sequestering protein. Later, the N-terminal half of Srv2 has been shown to promote actin filament dynamics by enhancing ADF/cofilin mediated actin severing; whereas the C-terminal half has been proposed to accelerate the nucleotide exchange from ADP to ATP and protein exchange from cofilin to profilin on newly depolymerized G-actin. In contrast to Srv2 in the yeast, human CAP1 and especially CAP2 are poorly characterized.<br/>We analyzed the ability of human N-terminal fragments of human CAP1 and CAP2 to affect actin dynamics potentiated by human isoforms of “muscle” (CFLN2) and “non-muscle” (CFLN1) cofilins. By conducting bulk actin depolymerization assays and monitoring single filament severing by TIRF microscopy, we found that N-terminal domains of both isoforms enhance cofilin-mediated severing and depolymerization at similar rates. Since Srv2 was proposed to form hexamers via oligomerization of its N-terminal coiled-coil region, we have tested whether the oligomerization contributes to the functional activity of CAP1 and 2. Surprisingly, our preliminary analytical sedimentation data suggested that the N-terminal fragments of both human CAP isoforms form trimers. Furthermore, by replacing the native oligomerization domain of CAPs by artificial coiled-coil sequence of known oligomerization pattern, we found that the activity of the proteins is directly proportional to the stoichiometry of their oligomerization: i.e., trimers are more potent that dimers, which are more effective than monomers. The work is in progress to check whether the observed formation of trimers rather than hexamers is the result of the N-terminally located 6His tags or the fundamental property of human CAPs.

Oligomerization affects the ability of human cyclase associated proteins 1 and 2 (CAP1 and CAP2) to promote actin severing by cofilins

2019 Spring National Meeting

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