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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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Cross-sections through pf14 axonemes that were extracted with 0.6 M NaCl. (A) Survey view of extracted axonemes.  (B–E) Cross-sections in which C2 has been solubilized. These C1  microtubules retain all of the electron densities identified as C1-associated in Fig. 4. Bars, 100 nm in both A and B.
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Figure 5: Cross-sections through pf14 axonemes that were extracted with 0.6 M NaCl. (A) Survey view of extracted axonemes. (B–E) Cross-sections in which C2 has been solubilized. These C1 microtubules retain all of the electron densities identified as C1-associated in Fig. 4. Bars, 100 nm in both A and B.

Mentions: Although projections 1b and 2b appear physically connected at their tips in wild-type axonemes, it is possible that the images of these structures overlap, but the structures themselves could be noninteracting. The disrupted assembly or reduced stability of 2b in cpc1 axonemes could result either because a normal interaction of 2b with the tip of 1b is absent, if these structures do interact, or because the cpc1 gene product is part of (or needed for the stability of) each structure alone. To test for the presence of direct interactions between these projections, we created axonemes in which only one or the other central pair microtubule had disassembled, and then looked for retention of 1b-2b connections, or for loss of one when the other was removed. Extraction of Chlamydomonas axonemes with 0.6 M NaCl has been shown to selectively destabilize C2, as well as to extract most of the inner and outer dynein arms, without removing C1 (Piperno and Luck, 1979). When this procedure was applied to pf14 axonemes, most axonemal cross-sections displayed either 0 (33%) or 1 (44%) central microtubule, and the one remaining tubule was invariably C1 (n = 208). In half of the remaining images (12%) a part of the C2 tubule and its associated projections was visible and 11% retained both central pair microtubules (Fig. 5 A). In the absence of C2, C1 appears to retain all of its projections along with material in the interdoublet bridge-diagonal link region, but does not retain material with the appearance of 2b (Fig. 5, B–E). Although we did not attempt to make image averages of these extracted axonemes due to variability in the extent of extraction, none of the structures that directly interact with C1 appear to have been removed by 0.6 M NaCl. C1 microtubules lacking the 1b projection were never seen, whereas the 2b projection was often missing even in cross-sections where other C2 structures remained.


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

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

Cross-sections through pf14 axonemes that were extracted with 0.6 M NaCl. (A) Survey view of extracted axonemes.  (B–E) Cross-sections in which C2 has been solubilized. These C1  microtubules retain all of the electron densities identified as C1-associated in Fig. 4. Bars, 100 nm in both A and B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132896&req=5

Figure 5: Cross-sections through pf14 axonemes that were extracted with 0.6 M NaCl. (A) Survey view of extracted axonemes. (B–E) Cross-sections in which C2 has been solubilized. These C1 microtubules retain all of the electron densities identified as C1-associated in Fig. 4. Bars, 100 nm in both A and B.
Mentions: Although projections 1b and 2b appear physically connected at their tips in wild-type axonemes, it is possible that the images of these structures overlap, but the structures themselves could be noninteracting. The disrupted assembly or reduced stability of 2b in cpc1 axonemes could result either because a normal interaction of 2b with the tip of 1b is absent, if these structures do interact, or because the cpc1 gene product is part of (or needed for the stability of) each structure alone. To test for the presence of direct interactions between these projections, we created axonemes in which only one or the other central pair microtubule had disassembled, and then looked for retention of 1b-2b connections, or for loss of one when the other was removed. Extraction of Chlamydomonas axonemes with 0.6 M NaCl has been shown to selectively destabilize C2, as well as to extract most of the inner and outer dynein arms, without removing C1 (Piperno and Luck, 1979). When this procedure was applied to pf14 axonemes, most axonemal cross-sections displayed either 0 (33%) or 1 (44%) central microtubule, and the one remaining tubule was invariably C1 (n = 208). In half of the remaining images (12%) a part of the C2 tubule and its associated projections was visible and 11% retained both central pair microtubules (Fig. 5 A). In the absence of C2, C1 appears to retain all of its projections along with material in the interdoublet bridge-diagonal link region, but does not retain material with the appearance of 2b (Fig. 5, B–E). Although we did not attempt to make image averages of these extracted axonemes due to variability in the extent of extraction, none of the structures that directly interact with C1 appear to have been removed by 0.6 M NaCl. C1 microtubules lacking the 1b projection were never seen, whereas the 2b projection was often missing even in cross-sections where other C2 structures remained.

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