Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells

摘要

Purpose: To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed enda??capping protein. Methods: We investigated F-actin and F-actina??binding protein localization in interdigitations of Tmod1+/+ and Tmod1a??/a?? single mature lens fibers. Results: F-actina??rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1a??/a?? mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, ?22-spectrin, and ?±-actinin are localized in large puncta in valleys between paddles; but in Tmod1a??/a?? mature fibers, ?22-spectrin was dispersed while ?±-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1a??/a?? mature fibers. Conclusions: These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a ?22-spectrina??actin network stabilized by Tmod1. ?±-Actinina??crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actina??associated proteins required for the formation of paddles between lens fibers.