Journal of Cell Science, Vol 20, Issue 2 405-439, Copyright © 1976 by Company of Biologists

JOURNAL ARTICLES

Flagellar doublet microtubules: fractionation of minor components and alpha-tubulin from specific regions of the A-tubule

RW Linck

Proteins occurring minor amounts with purified sperm flagellar doublet microtubules were identified and studied by SDS-gel electrophoresis. Methods were developed to solubilize selectively these minor components; electron microscopy (EM) of the fractionated products revealed possible locations of these proteins in the tubule. Doublet microtubules were prepared from sea-urchin (Echinus esculentus and Stronglyocentrotus droebachiensis) and scallop (Pecten maximus) sperm by dialysing flagellar axonemes against 2 mM Tris-0-2 mM EDTA-0-5 mM DTT. EM indicates that these doublet tubule preparations retain at least 70% of their radial spokes; cross-sections show a globule or fibre applied to the inside wall of the A-tubule, across from the inner B-tubule junction. On SDS-gels these preparations separate into at least 10 minor bands, accounting for 20-30% of the total protein; the remaining 75 +/- 4% migrates as tubulin. For E. esculentus the molecular weights and relative amounts of these components are: Component Ee 8 (150000 Daltons; 1%), 11 (114000; 2-5%), 15 (89000; 2%), 16 (80000; 2-5%), 17 (74000; 2%), 18 (69000; 2%), 19 (66000; 2%), 21 (48000; 4-5%), 22 (45000; 3%) and 23 (44500; 3%). Treatment of sea-urchin tubules with 0-1-0-5% sarkosyl, 0-1-0-3 M KSCN or 0-3-0-6 M KI results in the selective solubilization of: first, component 8 and some B-subfibre tubulin; second, components 11 and 23 and the remaining B-subfire tubulin; third, most of the A-subfire tubulin and components 17, 18 and 19. Thermal fractionation extracts none of these components, suggesting they are principally associated with the A-tubule. Finally 25-35% of the original protein is resistant to solubilization, and appears in the EM as ribbons of 3 protofilaments with 16-nm axial repeats. The resistant ribbons contain components 15, 16, 21 and 22 (plus component 20 in S. droebachiensis) in addition to 25 +/- 4% of the total tubulin. The data support the existence of two stable moieties in each doublet tubule: (1) a ribbon of 3 protofilaments and (2) either a second ribbon of 3 protofilaments or an equivalent amount of tubulin in some other form. EM images suggest that one ribbon forms the lateral side of the A-tubule (e.g. protofilaments A1,2,3 or A13,1,2 in the model) and that the globule applied to A13 may be a multisubunit complex of remaining minor components. Treatment of scallop tubules with 0-3 M KSCN preferentially extracts alpha-tubulin, yielding ribbons 1-4 protofilaments wide. The significance of this finding is discussed.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				T. M. Durcan, E. S. Halpin, T. Rao, N. S. Collins, E. K. Tribble, J. E. Hornick, and E. H. Hinchcliffe Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis J. Cell Biol., October 17, 2008; 181(4): 595 - 603. [Abstract] [Full Text] [PDF]

				H.-a. Yanagisawa and R. Kamiya A Tektin Homologue Is Decreased in Chlamydomonas Mutants Lacking an Axonemal Inner-Arm Dynein Mol. Biol. Cell, May 1, 2004; 15(5): 2105 - 2115. [Abstract] [Full Text] [PDF]

				P. Yang, D. R. Diener, J. L. Rosenbaum, and W. S. Sale Localization of Calmodulin and Dynein Light Chain Lc8 in Flagellar Radial Spokes J. Cell Biol., June 11, 2001; 153(6): 1315 - 1326. [Abstract] [Full Text] [PDF]

				E. Hinchcliffe and R. Linck Two proteins isolated from sea urchin sperm flagella: structural components common to the stable microtubules of axonemes and centrioles J. Cell Sci., January 3, 1998; 111(5): 585 - 595. [Abstract] [PDF]

				J. Norrander, R. Linck, and R. Stephens Transcriptional control of tektin A mRNA correlates with cilia development and length determination during sea urchin embryogenesis Development, January 6, 1995; 121(6): 1615 - 1623. [Abstract] [PDF]

				R. Stephens Tubulin and tektin in sea urchin embryonic cilia: pathways of protein incorporation during turnover and regeneration J. Cell Sci., January 2, 1994; 107(2): 683 - 692. [Abstract] [PDF]

				R Chen, C. Perrone, L. Amos, and R. Linck Tektin B1 from ciliary microtubules: primary structure as deduced from the cDNA sequence and comparison with tektin A1 J. Cell Sci., January 11, 1993; 106(3): 909 - 918. [Abstract] [PDF]