The actin-targeting toxins have not only proven to be invaluable tools in studies of actin cytoskeleton structure and function but they also served as a foundation for a new class of anticancer drugs. Here, we describe that amphidinolide H (AmpH) targets actin cytoskeleton. AmpH induced multinucleated cells by disrupting actin organization in the cells, and the hyperpolymerization of purified actin into filaments of apparently normal morphology in vitro. AmpH covalently binds on actin, and the AmpH binding site is determined as Tyr200 of actin subdomain 4 by mass spectrometry and halo assay using the yeast harboring site-directed mutagenized actins. Time-lapse analyses showed that AmpH stimulated the formation of small actin-patches, followed by F-actin rearrangement into aggregates via the retraction of actin fibers. These results indicate that AmpH is a novel actin inhibitor that covalently binds on actin.
Actin filaments are organised into sub-compartments of meshwork and bundles in lamellipodia. Localisation of fascin, the LIM and SH3 domain protein 1 (lasp-1), and lasp-2 to the bundles suggest their involvement in that organisation; however, their contributions remain unclear. We have compared the turnover of these proteins with actin at the bundle. After photobleaching, EGFP-actin recovered inwards from the bundle tip, consistent with the retrograde flow by treadmilling. In contrast, the recovery of EGFP-fascin, -lasp-1 and -lasp-2 occurred from the anterograde direction. These results suggest that these molecules would participate in the stabilisation of bundles but not in initiation.
The nebulin family of actin-binding proteins plays an important role in actin filament dynamics in a variety of cells including striated muscle. We report here the identification of a new striated muscle Z-disc associated protein: lasp-2 (LIM and SH3 domain protein-2). Lasp-2 is the most recently identified member of the nebulin family. To evaluate the role of lasp-2 in striated muscle, lasp-2 gene expression and localization were studied in chick and mouse tissue, as well as in primary cultures of chick cardiac and skeletal myocytes. Lasp-2 mRNA was detected as early as chick embryonic stage 25 and lasp-2 protein was associated with developing premyofibril structures, Z-discs of mature myofibrils, focal adhesions, and intercalated discs of cultured cardiomyocytes. Expression of GFP-tagged lasp-2 deletion constructs showed that the C-terminal region of lasp-2 is important for its localization in striated muscle cells. Lasp-2 organizes actin filaments into bundles and interacts directly with the Z-disc protein alpha-actinin. These results are consistent with a function of lasp-2 as a scaffolding and actin filament organizing protein within striated muscle Z-discs.
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