Dynamics and mechanics of the actin cytoskeleton ex vivo
- Mark Akira Tsuchida.
- June 2012.
- Physical description
- online resource (xi, 52 pages) : illustrations (some color)
- Tsuchida, Mark Akira.
- Dunn, Alexander Robert thesis advisor.
- Herschlag, Daniel thesis advisor.
- Spudich, James A. thesis advisor.
- Theriot, Julie thesis advisor (primary).
- Stanford University. Department of Biochemistry.
- Stanford University. Committee on Graduate Studies. degree grantor.
- Includes bibliographical references (p. 43-52). 66 refs.
- In actin-based crawling motility, cells continuously build, reorganize, and disassemble an actin network in a process driven jointly by biochemical reactions and mechanical work. Herein, we use isolated actin cytoskeletons from fish epithelial keratocytes to study two aspects of this highly coordinated process. For the continuous actin network of a motile cell to drive translocation, net assembly of the actin network at the leading edge (which drives protrusion) must be balanced by net disassembly at the trailing edge. Although proteins such as ADF/cofilin and gelsolin are known to locally depolymerize actin filaments, it is not clear how such activity could be coordinated with assembly over the distance scale of the whole cell. Here we present experiments showing that activation of nonmuscle myosin II embedded in isolated cytoskeletons results in partial disassembly of the actin network. Taken together with prior work on the effect of myosin II inhibition in live cells, these results establish that myosin II contributes to actin network disassembly in the rear of motile cells, and suggest that gradual binding of myosin to a maturing actin network could serve as a mechanism to coordinate actin network assembly and disassembly over long distances. A thorough understanding of how forces produced by actin and myosin contribute to whole-cell movement will require detailed knowledge of the material properties of the cytoskeletal network at the relevant spatial and temporal scales. Measurements of mechanical properties have largely been limited to microscopic strains, whole-cell bulk measurements, or reconstituted gels that do not fully capture the cellular cytoskeletal organization. We have therefore sought to characterize the deformation of actin networks derived from motile cells under large applied strains. Strain could be applied to the lamellipodial actin network by displacing the cell body using a glass microneedle. The network behaves as a coherent material sheet that is mechanically well-coupled to the cell body and stretches to approximately 400% of its original dimensions. At smaller strains and time scales ranging from a fraction of a second to tens of seconds, the network behaves almost exclusively elastically, suggesting that the cytoskeletal network is capable of integrating and transmitting forces over large distances within the cell.
- Publication date
- Submitted to the Department of Biochemistry and the Committee on Graduate Studies of Stanford University.
- Thesis (Ph.D.)--Stanford University, 2012.