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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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Electron micrographs of thin sections through demembranated wild-type axonemes (A) show several electron-dense  projections from the two central pair microtubules. All micrographs are oriented as if viewed from inside the cell and rotated  so that the C1 central microtubule is to the left. The longest central pair projections (1a, 1b, 2a, and 2b) have been labeled in the  third panel. In similar micrographs of cpc1 axonemes (B) the central pair microtubules lack the 1b and 2b densities. Images of intact cpc1 flagella (C) show that the C1b projection is not lost during axoneme preparation, but rather fails to assemble in this  mutant. Bar, 100 nm.
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Figure 3: Electron micrographs of thin sections through demembranated wild-type axonemes (A) show several electron-dense projections from the two central pair microtubules. All micrographs are oriented as if viewed from inside the cell and rotated so that the C1 central microtubule is to the left. The longest central pair projections (1a, 1b, 2a, and 2b) have been labeled in the third panel. In similar micrographs of cpc1 axonemes (B) the central pair microtubules lack the 1b and 2b densities. Images of intact cpc1 flagella (C) show that the C1b projection is not lost during axoneme preparation, but rather fails to assemble in this mutant. Bar, 100 nm.

Mentions: As seen in electron micrographs of wild-type axonemal cross-sections (Fig. 3 A), the two central pair microtubules can be differentiated by the presence of two long projections (1a and 1b) on the C1 tubule and two short projections (2a and 2b) on the C2 tubule. These and all other micrographs have been printed as if viewed from inside the cell (base to tip of the axoneme) with A tubules of each outer doublet pointing clockwise, and have been rotated so that the C1 central pair tubule is on the left. The two C1-associated projections make perpendicular connections to the C1 microtubule surface. Thinner arcs (previously described as a sheath around the central pair; Warner, 1976) apparently connect the tip of each projection to the lateral side of C1. The two long C1-associated projections can be distinguished from each other by a thickening at the tip of 1a that is not seen at the tip of 1b. Axonemes of cpc1 (Fig. 3 B) consistently lack the 1b projection and its associated arc, and frequently lack the 2b projection as well. To determine whether a similar structural defect is present in vivo, whole cells were fixed for electron microscopy and images of intact flagella were examined (Fig. 3 C). These images lack the contrast seen in axonemal images because tannic acid does not penetrate through cell membranes, but they nonetheless show that the 1b projection and its associated arc are missing from flagella as well as axonemes of this mutant. The presence or absence of the 2b projection in cpc1 flagella could not be determined from these preparations.


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

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

Electron micrographs of thin sections through demembranated wild-type axonemes (A) show several electron-dense  projections from the two central pair microtubules. All micrographs are oriented as if viewed from inside the cell and rotated  so that the C1 central microtubule is to the left. The longest central pair projections (1a, 1b, 2a, and 2b) have been labeled in the  third panel. In similar micrographs of cpc1 axonemes (B) the central pair microtubules lack the 1b and 2b densities. Images of intact cpc1 flagella (C) show that the C1b projection is not lost during axoneme preparation, but rather fails to assemble in this  mutant. Bar, 100 nm.
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Related In: Results  -  Collection

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Figure 3: Electron micrographs of thin sections through demembranated wild-type axonemes (A) show several electron-dense projections from the two central pair microtubules. All micrographs are oriented as if viewed from inside the cell and rotated so that the C1 central microtubule is to the left. The longest central pair projections (1a, 1b, 2a, and 2b) have been labeled in the third panel. In similar micrographs of cpc1 axonemes (B) the central pair microtubules lack the 1b and 2b densities. Images of intact cpc1 flagella (C) show that the C1b projection is not lost during axoneme preparation, but rather fails to assemble in this mutant. Bar, 100 nm.
Mentions: As seen in electron micrographs of wild-type axonemal cross-sections (Fig. 3 A), the two central pair microtubules can be differentiated by the presence of two long projections (1a and 1b) on the C1 tubule and two short projections (2a and 2b) on the C2 tubule. These and all other micrographs have been printed as if viewed from inside the cell (base to tip of the axoneme) with A tubules of each outer doublet pointing clockwise, and have been rotated so that the C1 central pair tubule is on the left. The two C1-associated projections make perpendicular connections to the C1 microtubule surface. Thinner arcs (previously described as a sheath around the central pair; Warner, 1976) apparently connect the tip of each projection to the lateral side of C1. The two long C1-associated projections can be distinguished from each other by a thickening at the tip of 1a that is not seen at the tip of 1b. Axonemes of cpc1 (Fig. 3 B) consistently lack the 1b projection and its associated arc, and frequently lack the 2b projection as well. To determine whether a similar structural defect is present in vivo, whole cells were fixed for electron microscopy and images of intact flagella were examined (Fig. 3 C). These images lack the contrast seen in axonemal images because tannic acid does not penetrate through cell membranes, but they nonetheless show that the 1b projection and its associated arc are missing from flagella as well as axonemes of this mutant. The presence or absence of the 2b projection in cpc1 flagella could not be determined from these preparations.

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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