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Part III. Cell Motility
Part I I I .
Cell Motility
The first chapter in this section is by Fechheimer and Taylor and deals with use of controlled sonication to introduce exogenous molecules into the cytoplasm of Dictyostelium discoideum. They describe utilization of this technique to localize a 30,000-Da actin-binding protein which they previously purified and characterized from this organism. Their chapter emphasizes the generality and possible widespread usage of this technique, and they point out its use not only in normalized immunofluorescence microscopy, but also in the measurement of intracellular pH. Condeelis and his co-workers then describe electron microscope immunolabeling of cytoskeletal proteins, which are concentrated in a cortical actin matrix. They describe procedures for fixation of amoebae, suggestions on preparation of antibodies, preparation of colloidal gold probes, optimum staining procedures, and use of myosin fragments for localizing actin filaments. The next chapter, by A. Spudich, provides one method for the isolation of the cortical actin matrix using Triton X-100 to lyse the cells. The effects of calcium ion on the integrity of the isolated matrix is described, as well as a rapid method for obtaining an actin preparation from the cells in reasonable yield. A totally different actin isolation scheme was described by Uyemura et al. (J. Biol. Chem. 253, 9088-9096, 1978) and was used for the basis of studies being carried out by Stone et al. and described in Chapter 12. Neutron diffraction can provide critical information about the structure and function of protein complexes, and organisms that can provide highly deuterated, purified proteins are greatly needed. Stone et al. give the details of how to obtain such deuterated proteins from D. discoideum, using actin as an example. In Chapter 13, Rubenstein et al. describe procedures for preparing t3%]methionine-labeled actin containing acetylated methionine at its amino terminus, as well as methods for studying the processing of that amino terminus. The final two chapters of this part are concerned with tubulin structure and function. White and Katz describe biochemical and genetic approaches to microtubule function. Included in their chapter are methods for analyzing tubulin on two-dimensional gels, immunofluorescent staining of amoebae, a buffer condition using Nonidet P-40isolation of microtubule-containing cytoskeletons, and methods for obtaining potential microtubule mutants. The last chapter of this section by Roos concerns approaches to study mitosis. Suitable strains and maintenance of cultures are described, as are methods for live observations of mitosis and staining of fixed cells.
Cell Motility
The first chapter in this section is by Fechheimer and Taylor and deals with use of controlled sonication to introduce exogenous molecules into the cytoplasm of Dictyostelium discoideum. They describe utilization of this technique to localize a 30,000-Da actin-binding protein which they previously purified and characterized from this organism. Their chapter emphasizes the generality and possible widespread usage of this technique, and they point out its use not only in normalized immunofluorescence microscopy, but also in the measurement of intracellular pH. Condeelis and his co-workers then describe electron microscope immunolabeling of cytoskeletal proteins, which are concentrated in a cortical actin matrix. They describe procedures for fixation of amoebae, suggestions on preparation of antibodies, preparation of colloidal gold probes, optimum staining procedures, and use of myosin fragments for localizing actin filaments. The next chapter, by A. Spudich, provides one method for the isolation of the cortical actin matrix using Triton X-100 to lyse the cells. The effects of calcium ion on the integrity of the isolated matrix is described, as well as a rapid method for obtaining an actin preparation from the cells in reasonable yield. A totally different actin isolation scheme was described by Uyemura et al. (J. Biol. Chem. 253, 9088-9096, 1978) and was used for the basis of studies being carried out by Stone et al. and described in Chapter 12. Neutron diffraction can provide critical information about the structure and function of protein complexes, and organisms that can provide highly deuterated, purified proteins are greatly needed. Stone et al. give the details of how to obtain such deuterated proteins from D. discoideum, using actin as an example. In Chapter 13, Rubenstein et al. describe procedures for preparing t3%]methionine-labeled actin containing acetylated methionine at its amino terminus, as well as methods for studying the processing of that amino terminus. The final two chapters of this part are concerned with tubulin structure and function. White and Katz describe biochemical and genetic approaches to microtubule function. Included in their chapter are methods for analyzing tubulin on two-dimensional gels, immunofluorescent staining of amoebae, a buffer condition using Nonidet P-40isolation of microtubule-containing cytoskeletons, and methods for obtaining potential microtubule mutants. The last chapter of this section by Roos concerns approaches to study mitosis. Suitable strains and maintenance of cultures are described, as are methods for live observations of mitosis and staining of fixed cells.