Shaping Cells Using Modular Actin Filament Bundles

University Of Pennsylvania, Philadelphia PA

Investigators

Abstract

Actin is one of the key cytoskeletal elements in eukaryotic cells. Actin protein molecules (monomers) reversibly polymerize to form actin microfilaments, which can further assemble into bundles with the assistance of actin-associated bundling proteins. Microfilaments are dynamic in the living cell, and can shorten and elongate in a manner that is strictly regulated by a variety of other actin-associated proteins. Microfilaments serve many critical roles in the life of the cell, either as structural elements for cell shape or resiliency or as tracks along which the actin-based motor, myosin, translocates. This research is aimed at understanding how actin bundles are formed and maintained using the Drosophila bristle cell as a model. Cross-linked bundles of unipolar (parallel) actin filaments are common in eukaryotic cells and provide the scaffolding that maintains cell shape. Familiar examples include the brush border (microvilli) of intestinal epithelial cells and the stereocilia of the inner ear. In these and other systems there is an array of actin-binding proteins that serve to control the dynamics of actin polymerization, assemble actin filaments into bundles, and regulate bundle maintenance. The Drosophila bristle cell develops a gigantic cellular extension hundreds of microns long over a period of 16 hours and is an excellent example of how a cell uses its cytoskeleton to change and maintain its shape. This cellular extension is supported by a scaffold of long actin bundles constructed by the end-to-end assembly of short modules composed of actin filaments bundled together with cross-linkers. This system offers important experimental advantages for the study of actin dynamics, by allowing the application of genetic and molecular approaches to a fundamental cell biological feature that can be analyzed in the living cell by light microscopy and at high resolution by electron microscopy. Drs. Guild and Tilney plan to determine how the modular components of actin bundles are formed, maintained, and eventually removed from the cytoplasm. They plan to use elongating bristle cells as in vivo test tubes and to employ genetic and transgenic tools to perturb and modify bundle assembly. The cytoskeletal consequences of these changes will be assessed by examining the actin cytoskeleton in the context of living cells by confocal microscopy and by examining the detailed structure of the cytoskeleton by electron microscopy. The project has two aims. First, the investigators will determine whether actin-capping proteins play a role in module length regulation and, if so, they will test whether they can engineer module length with different capping proteins. Second, they will determine how modules break down and whether actin capping proteins play a role in this process. In addition, they will test whether retrograde flow of actin modules plays a role in cell extension and in bundle breakdown and whether myosin motors drive this flow. Modules of actin bundles are used in many specialized cells to support cellular extensions and in all cases module length is an issue. Thus, the principles that are uncovered in the Drosophila system should be generally applicable to many other systems.