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Characterization of a Chlamydomonas insertional mutant that disrupts flagellar central pair microtubule-associated structures.

Mitchell DR, Sale WS - J. Cell Biol. (1999)

Bottom Line: These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells.By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins.Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, State University of New York Health Science Center, Syracuse, New York 13210, USA. mitchrld@vax.cs.hscsyr.edu

ABSTRACT
Two alleles at a new locus, central pair-associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke-defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.

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Selected images of cross-sections through pf16pf14 axonemes which illustrate apparent progressive steps in the loss of  C1 and its associated structures. In row A, the 1a projection and  its associated sheath are missing, and little remains at the 1c position, but the 1b and 1d densities are retained. In row B, several  protofilaments are missing from the wall of the C1 microtubules.  In rows C and D, C1 is completely missing. The 1b projection can  remain attached to the C2 microtubule through apparent connections at its base to the diagonal link and at its tip to 2b (seen most  clearly in row C). Bar, 100 nm.
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Figure 6: Selected images of cross-sections through pf16pf14 axonemes which illustrate apparent progressive steps in the loss of C1 and its associated structures. In row A, the 1a projection and its associated sheath are missing, and little remains at the 1c position, but the 1b and 1d densities are retained. In row B, several protofilaments are missing from the wall of the C1 microtubules. In rows C and D, C1 is completely missing. The 1b projection can remain attached to the C2 microtubule through apparent connections at its base to the diagonal link and at its tip to 2b (seen most clearly in row C). Bar, 100 nm.

Mentions: To create axonemes lacking C1 but retaining C2, samples were prepared from a pf16pf14 double mutant strain. Although intact pf16 flagella retain both central pair microtubules, the C1 tubule is reportedly unstable under the detergent extraction conditions used to prepare axonemes (Dutcher et al., 1984). To our surprise, >90% of pf16 axonemes prepared by our standard isolation procedure retained both central pair tubules. However, when pf16 or pf16pf14 flagella were demembranated in a buffer containing sodium chloride (Smith and Lefebvre, 1996) rather than potassium acetate, central pair complexes lost variable amounts of C1-associated structures. Both central pair tubules were intact in only 10% of pf16pf14 axonemal cross-sections compared with 17% in which part of C1 was missing, 38% that contain only C2, and 35% that lacked both central pair tubules (n = 230). The most complete structures observed (under conditions used here for axoneme preparation) lacked only the 1a projection and its associated sheath, and showed a reduced interdoublet bridge density (Fig. 6 A). Fig. 6, B–D, illustrates increasingly disrupted C1 structures and shows that the 1b projection and its associated sheath remain attached to C2, even when no C1 microtubule remains in the section plane, through connections at both the base and tip of 1b (Fig. 6 C). In some images (e.g., the last panel of Fig. 6 C), 2b has been lost and the only remaining connection between 1b and the C2 tubule is the diagonal link at the base of the 1b projection. When no C1-associated structures were evident (Fig. 6 D), most C2-associated structures were also absent and proper orientation of C2 could not be accurately determined. Densities similar to projection 2a were sometimes present, but densities with the characteristic triangular shape of 2b were never observed. Because we never observed an identifiable 2b density unless 1b was also visible, the pf16pf14 data favor the hypothesis that the tips of 1b and 2b do not just overlap, but physically interact.


Characterization of a Chlamydomonas insertional mutant that disrupts flagellar central pair microtubule-associated structures.

Mitchell DR, Sale WS - J. Cell Biol. (1999)

Selected images of cross-sections through pf16pf14 axonemes which illustrate apparent progressive steps in the loss of  C1 and its associated structures. In row A, the 1a projection and  its associated sheath are missing, and little remains at the 1c position, but the 1b and 1d densities are retained. In row B, several  protofilaments are missing from the wall of the C1 microtubules.  In rows C and D, C1 is completely missing. The 1b projection can  remain attached to the C2 microtubule through apparent connections at its base to the diagonal link and at its tip to 2b (seen most  clearly in row C). Bar, 100 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132896&req=5

Figure 6: Selected images of cross-sections through pf16pf14 axonemes which illustrate apparent progressive steps in the loss of C1 and its associated structures. In row A, the 1a projection and its associated sheath are missing, and little remains at the 1c position, but the 1b and 1d densities are retained. In row B, several protofilaments are missing from the wall of the C1 microtubules. In rows C and D, C1 is completely missing. The 1b projection can remain attached to the C2 microtubule through apparent connections at its base to the diagonal link and at its tip to 2b (seen most clearly in row C). Bar, 100 nm.
Mentions: To create axonemes lacking C1 but retaining C2, samples were prepared from a pf16pf14 double mutant strain. Although intact pf16 flagella retain both central pair microtubules, the C1 tubule is reportedly unstable under the detergent extraction conditions used to prepare axonemes (Dutcher et al., 1984). To our surprise, >90% of pf16 axonemes prepared by our standard isolation procedure retained both central pair tubules. However, when pf16 or pf16pf14 flagella were demembranated in a buffer containing sodium chloride (Smith and Lefebvre, 1996) rather than potassium acetate, central pair complexes lost variable amounts of C1-associated structures. Both central pair tubules were intact in only 10% of pf16pf14 axonemal cross-sections compared with 17% in which part of C1 was missing, 38% that contain only C2, and 35% that lacked both central pair tubules (n = 230). The most complete structures observed (under conditions used here for axoneme preparation) lacked only the 1a projection and its associated sheath, and showed a reduced interdoublet bridge density (Fig. 6 A). Fig. 6, B–D, illustrates increasingly disrupted C1 structures and shows that the 1b projection and its associated sheath remain attached to C2, even when no C1 microtubule remains in the section plane, through connections at both the base and tip of 1b (Fig. 6 C). In some images (e.g., the last panel of Fig. 6 C), 2b has been lost and the only remaining connection between 1b and the C2 tubule is the diagonal link at the base of the 1b projection. When no C1-associated structures were evident (Fig. 6 D), most C2-associated structures were also absent and proper orientation of C2 could not be accurately determined. Densities similar to projection 2a were sometimes present, but densities with the characteristic triangular shape of 2b were never observed. Because we never observed an identifiable 2b density unless 1b was also visible, the pf16pf14 data favor the hypothesis that the tips of 1b and 2b do not just overlap, but physically interact.

Bottom Line: These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells.By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins.Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, State University of New York Health Science Center, Syracuse, New York 13210, USA. mitchrld@vax.cs.hscsyr.edu

ABSTRACT
Two alleles at a new locus, central pair-associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke-defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.

Show MeSH