CHLAMYDOMONAS FLAGELLA I. Isolation and Electrophoretic Analysis of Microtubules, Matrix, Membranes, and Mastigonemes G. B. WITMAN, K. CARLSON, J. BERLINER, and JOEL L. ROSENBAUM Prom the Department of Biology~Yale University, New Haven, Connecticut 065~0 ABSTRACT Methods were developed for the isolauon of Chlarnygomonas flagella and for their fractiona- tion into membrane, mastigoneme, "matrix," and axoneme components. Each com- ponent was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flageUar membrane m situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of ~-~ 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "'9 -1- 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 -1- 2 flagella of wild-type cells and of ':9 -t- 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubuhns 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively INTRODUCTION The biflagellate, unicellular alga Chlamydomonas has been shown to have several unique advantages for studies on flagellar formation and function: it can be grown to high densities in defined medium (Sager and Granick, 1953; Gorman and Levine, 1965), its division can be synchronized by use of alternating light and dark periods (Bernstein, 1960; Kates and Jones, 1964), the flagella can be detached and isolated in large quantitms, and tile deflagellated ceils will synchronously regenerate new flagella (Rosenbaum et al., 1969) There are, in addition, detailed ultrastructural analyses of the cell (Sager and Palade, 1957; Johnson and Porter, 1968) and its flagella (Ringo, 1967 a, 1967 b, Hopkins, 1970). Perhaps of even greater impor- tance, mutants with defective flagella can be iso~ ThE JOm~NAL OF CELL BIOLOGY"VOLUME 54, I97~- pages 507-589 507 Downloaded from http://rupress.org/jcb/article-pdf/54/3/507/1626444/507.pdf by guest on 25 July 2023