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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 (A and B) and  an average of 16 similar images (C) reveal additional details of  central pair structure that are summarized diagramatically (D).  These include four densities (1a, 1b, 1c, and 1d) attached to the  C1 microtubule, three densities (2a, 2b, and 2c) attached to the  C2 microtubule, a bipartite bridge and a diagonal link spanning  the gap between the microtubules, and sheath material that  forms an arc from 1c to the tip of 1a, and from 1d to the tip of 1b.  Individual cross-sections through cpc1pf14 axonemes (E and F)  show that the 1b projection is invariably missing in cpc1 axonemes, whereas the 2b projection is sometimes retained (filled  arrowhead in E). An average of 12 images (G) reveals the presence of a densely staining region at the site where 1b attaches to  the C1 microtubule in wild-type axonemes. A difference image  (H) made by subtracting G from C produces a dark image of the  missing structures and a bright region where rotation of C1 relative to C2 has produced an apparent overlap of the 1a and 2a projections (asterisk). Cross-sections through pf6pf14 axonemes (I and  J), an average of 25 images (K), and a difference image (L) show  that only projection 1a and its associated sheath material are affected by the pf6 mutation. Bars, 100 nm in A and 20 nm in C.
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Figure 4: Cross-sections through pf14 axonemes (A and B) and an average of 16 similar images (C) reveal additional details of central pair structure that are summarized diagramatically (D). These include four densities (1a, 1b, 1c, and 1d) attached to the C1 microtubule, three densities (2a, 2b, and 2c) attached to the C2 microtubule, a bipartite bridge and a diagonal link spanning the gap between the microtubules, and sheath material that forms an arc from 1c to the tip of 1a, and from 1d to the tip of 1b. Individual cross-sections through cpc1pf14 axonemes (E and F) show that the 1b projection is invariably missing in cpc1 axonemes, whereas the 2b projection is sometimes retained (filled arrowhead in E). An average of 12 images (G) reveals the presence of a densely staining region at the site where 1b attaches to the C1 microtubule in wild-type axonemes. A difference image (H) made by subtracting G from C produces a dark image of the missing structures and a bright region where rotation of C1 relative to C2 has produced an apparent overlap of the 1a and 2a projections (asterisk). Cross-sections through pf6pf14 axonemes (I and J), an average of 25 images (K), and a difference image (L) show that only projection 1a and its associated sheath material are affected by the pf6 mutation. Bars, 100 nm in A and 20 nm in C.

Mentions: Further attempts to clarify central pair–associated structures in both wild-type and mutant axonemes were hampered by the close proximity of radial spoke heads to the central pair. To avoid this problem, axonemes were isolated from radial spoke assembly mutant pf14 and from double mutants between pf14, and three mutations that alter central pair structure, cpc1, pf6, and pf16. Two individual pf14 axonemes, a composite average of 16 similar images, and a cartoon summarizing densities associated with pf14 central pair microtubules are shown in Fig. 4. In addition to the four projections labeled in Fig. 3 A, the averaged image reveals densely stained material at ∼11 o'clock (1c) and 8 o'clock (1d) on C1, at 2 o'clock (2c) on C2, and in a broad zone between the two microtubules. Based on the image average and on additional information from double mutants (see following sections), this mid-zone has been further divided into a bipartite intermicrotubule bridge and a diagonal linker between projection 1b and the C2 microtubule. Although there is an electron lucent gap between the tip of the 1a and 2a projections, the tips of 1b and 2b always appear to be joined by a continuous thin arc extending from the 1d density on C1 through 180° to the lateral margin of C2. This arc of sheath material and the 2b projection together form a triangle of nearly uniform density at the bottom of C2, whereas the sheath generally appears separated by a less dense region from the 1a and 1b projections. Careful examination of favorable images of wild-type axonemes confirm that all of these structural details are also present in wild-type axonemes such as those in Fig. 3 A and are not artifacts created by the loss of radial spokes.


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 (A and B) and  an average of 16 similar images (C) reveal additional details of  central pair structure that are summarized diagramatically (D).  These include four densities (1a, 1b, 1c, and 1d) attached to the  C1 microtubule, three densities (2a, 2b, and 2c) attached to the  C2 microtubule, a bipartite bridge and a diagonal link spanning  the gap between the microtubules, and sheath material that  forms an arc from 1c to the tip of 1a, and from 1d to the tip of 1b.  Individual cross-sections through cpc1pf14 axonemes (E and F)  show that the 1b projection is invariably missing in cpc1 axonemes, whereas the 2b projection is sometimes retained (filled  arrowhead in E). An average of 12 images (G) reveals the presence of a densely staining region at the site where 1b attaches to  the C1 microtubule in wild-type axonemes. A difference image  (H) made by subtracting G from C produces a dark image of the  missing structures and a bright region where rotation of C1 relative to C2 has produced an apparent overlap of the 1a and 2a projections (asterisk). Cross-sections through pf6pf14 axonemes (I and  J), an average of 25 images (K), and a difference image (L) show  that only projection 1a and its associated sheath material are affected by the pf6 mutation. Bars, 100 nm in A and 20 nm in C.
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Figure 4: Cross-sections through pf14 axonemes (A and B) and an average of 16 similar images (C) reveal additional details of central pair structure that are summarized diagramatically (D). These include four densities (1a, 1b, 1c, and 1d) attached to the C1 microtubule, three densities (2a, 2b, and 2c) attached to the C2 microtubule, a bipartite bridge and a diagonal link spanning the gap between the microtubules, and sheath material that forms an arc from 1c to the tip of 1a, and from 1d to the tip of 1b. Individual cross-sections through cpc1pf14 axonemes (E and F) show that the 1b projection is invariably missing in cpc1 axonemes, whereas the 2b projection is sometimes retained (filled arrowhead in E). An average of 12 images (G) reveals the presence of a densely staining region at the site where 1b attaches to the C1 microtubule in wild-type axonemes. A difference image (H) made by subtracting G from C produces a dark image of the missing structures and a bright region where rotation of C1 relative to C2 has produced an apparent overlap of the 1a and 2a projections (asterisk). Cross-sections through pf6pf14 axonemes (I and J), an average of 25 images (K), and a difference image (L) show that only projection 1a and its associated sheath material are affected by the pf6 mutation. Bars, 100 nm in A and 20 nm in C.
Mentions: Further attempts to clarify central pair–associated structures in both wild-type and mutant axonemes were hampered by the close proximity of radial spoke heads to the central pair. To avoid this problem, axonemes were isolated from radial spoke assembly mutant pf14 and from double mutants between pf14, and three mutations that alter central pair structure, cpc1, pf6, and pf16. Two individual pf14 axonemes, a composite average of 16 similar images, and a cartoon summarizing densities associated with pf14 central pair microtubules are shown in Fig. 4. In addition to the four projections labeled in Fig. 3 A, the averaged image reveals densely stained material at ∼11 o'clock (1c) and 8 o'clock (1d) on C1, at 2 o'clock (2c) on C2, and in a broad zone between the two microtubules. Based on the image average and on additional information from double mutants (see following sections), this mid-zone has been further divided into a bipartite intermicrotubule bridge and a diagonal linker between projection 1b and the C2 microtubule. Although there is an electron lucent gap between the tip of the 1a and 2a projections, the tips of 1b and 2b always appear to be joined by a continuous thin arc extending from the 1d density on C1 through 180° to the lateral margin of C2. This arc of sheath material and the 2b projection together form a triangle of nearly uniform density at the bottom of C2, whereas the sheath generally appears separated by a less dense region from the 1a and 1b projections. Careful examination of favorable images of wild-type axonemes confirm that all of these structural details are also present in wild-type axonemes such as those in Fig. 3 A and are not artifacts created by the loss of radial spokes.

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