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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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Analysis of central pair structure in longitudinal thin  sections. The top of each panel indicates the plane of section and  direction of view for the longitudinal image in the bottom. Cross-sections are printed looking from base to tip of the axoneme, longitudinal sections are printed with the base of the axoneme toward the bottom of the page. (A–G) pf14 axonemes; (H)  cpc1pf14 axonemes. In A, arrows point to two rows of rectangular densities (corresponding to 1d and 1c in Fig. 4 D) that repeat  every 32 nm (indicated by parallel lines); arrowheads emphasize  the 96-nm periodicity of inner row dyneins. In B, the section  plane contains microtubule C1, with projections that correspond  to densities 1a and 1b along the right-hand and left-hand edges.  In C, diagonal lines indicate material with a 16-nm repeat periodicity in the intermicrotubule bridge region, superimposed on an  image of the C2 microtubule. D includes the C2 microtubule and  projections 2a and 2b (brackets), as well as material in the bridge  region. The longitudinal section in E passes tangentially through  the sheath and reveals pairing of sheath fibers. In F, the 1c or 1d  densities project with a 32-nm period from the left edge of C1,  but material projecting from C2 does not show a distinct periodicity. The oblique section in G passes through the lumen of C1  and C2 (top of longitudinal image) out to the tips of 1a and 2a  (bottom of image). In H, absence of the 1b/sheath complex  changes the outline of 1-d densities to a saw-tooth (compare with  the similar section through a wild-type central pair in A). Bar in  A, 100 nm.
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Figure 7: Analysis of central pair structure in longitudinal thin sections. The top of each panel indicates the plane of section and direction of view for the longitudinal image in the bottom. Cross-sections are printed looking from base to tip of the axoneme, longitudinal sections are printed with the base of the axoneme toward the bottom of the page. (A–G) pf14 axonemes; (H) cpc1pf14 axonemes. In A, arrows point to two rows of rectangular densities (corresponding to 1d and 1c in Fig. 4 D) that repeat every 32 nm (indicated by parallel lines); arrowheads emphasize the 96-nm periodicity of inner row dyneins. In B, the section plane contains microtubule C1, with projections that correspond to densities 1a and 1b along the right-hand and left-hand edges. In C, diagonal lines indicate material with a 16-nm repeat periodicity in the intermicrotubule bridge region, superimposed on an image of the C2 microtubule. D includes the C2 microtubule and projections 2a and 2b (brackets), as well as material in the bridge region. The longitudinal section in E passes tangentially through the sheath and reveals pairing of sheath fibers. In F, the 1c or 1d densities project with a 32-nm period from the left edge of C1, but material projecting from C2 does not show a distinct periodicity. The oblique section in G passes through the lumen of C1 and C2 (top of longitudinal image) out to the tips of 1a and 2a (bottom of image). In H, absence of the 1b/sheath complex changes the outline of 1-d densities to a saw-tooth (compare with the similar section through a wild-type central pair in A). Bar in A, 100 nm.

Mentions: Longitudinal images were used to determine the periodicity and angular orientation of the 1b projection and the other densities defined by cross-section analysis, and to see whether the 1b and 2b densities are longitudinally aligned. Longitudinal sections that graze the lateral surface of C1 show that projection 1a and its associated sheath fibers (extending from the right-hand edge of C1 in the longitudinal image of Fig. 7 A) have a 16-nm repeat period and tilt toward the tip of the axoneme at ∼24°. Also visible in Fig. 7 A are two rows of rectangular or rhomboidal densities, corresponding to densities 1c and 1d in cross-sectional images, which overlie the walls of C1, and project out from its left-hand margin, respectively. They are indicated in the longitudinal image by arrows and by parallel lines marking their 32-nm repeat period. When the section plane included both 1a and 1b projections (Fig. 7 B), their identical periodicity was revealed. Sections passing perpendicular to the central pair microtubule axes and including the intermicrotubule bridge (Fig. 7, C and D) show the presence of closely spaced rope-like material with a 16-nm repeat overlying the central pair microtubule image in the bridge region (diagonal lines in Fig. 7 C). This material, which may include both the diagonal link and the intermicrotubule bridge, appears to follow a left-handed helix with a pitch matching that of the underlying dimer lattice of the C2 microtubule (Linck et al., 1981). Both the 2a and 2b projections repeat at a 16-nm period, as seen in the lower half of the longitudinal image in Fig. 7 D.


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

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

Analysis of central pair structure in longitudinal thin  sections. The top of each panel indicates the plane of section and  direction of view for the longitudinal image in the bottom. Cross-sections are printed looking from base to tip of the axoneme, longitudinal sections are printed with the base of the axoneme toward the bottom of the page. (A–G) pf14 axonemes; (H)  cpc1pf14 axonemes. In A, arrows point to two rows of rectangular densities (corresponding to 1d and 1c in Fig. 4 D) that repeat  every 32 nm (indicated by parallel lines); arrowheads emphasize  the 96-nm periodicity of inner row dyneins. In B, the section  plane contains microtubule C1, with projections that correspond  to densities 1a and 1b along the right-hand and left-hand edges.  In C, diagonal lines indicate material with a 16-nm repeat periodicity in the intermicrotubule bridge region, superimposed on an  image of the C2 microtubule. D includes the C2 microtubule and  projections 2a and 2b (brackets), as well as material in the bridge  region. The longitudinal section in E passes tangentially through  the sheath and reveals pairing of sheath fibers. In F, the 1c or 1d  densities project with a 32-nm period from the left edge of C1,  but material projecting from C2 does not show a distinct periodicity. The oblique section in G passes through the lumen of C1  and C2 (top of longitudinal image) out to the tips of 1a and 2a  (bottom of image). In H, absence of the 1b/sheath complex  changes the outline of 1-d densities to a saw-tooth (compare with  the similar section through a wild-type central pair in A). Bar in  A, 100 nm.
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Figure 7: Analysis of central pair structure in longitudinal thin sections. The top of each panel indicates the plane of section and direction of view for the longitudinal image in the bottom. Cross-sections are printed looking from base to tip of the axoneme, longitudinal sections are printed with the base of the axoneme toward the bottom of the page. (A–G) pf14 axonemes; (H) cpc1pf14 axonemes. In A, arrows point to two rows of rectangular densities (corresponding to 1d and 1c in Fig. 4 D) that repeat every 32 nm (indicated by parallel lines); arrowheads emphasize the 96-nm periodicity of inner row dyneins. In B, the section plane contains microtubule C1, with projections that correspond to densities 1a and 1b along the right-hand and left-hand edges. In C, diagonal lines indicate material with a 16-nm repeat periodicity in the intermicrotubule bridge region, superimposed on an image of the C2 microtubule. D includes the C2 microtubule and projections 2a and 2b (brackets), as well as material in the bridge region. The longitudinal section in E passes tangentially through the sheath and reveals pairing of sheath fibers. In F, the 1c or 1d densities project with a 32-nm period from the left edge of C1, but material projecting from C2 does not show a distinct periodicity. The oblique section in G passes through the lumen of C1 and C2 (top of longitudinal image) out to the tips of 1a and 2a (bottom of image). In H, absence of the 1b/sheath complex changes the outline of 1-d densities to a saw-tooth (compare with the similar section through a wild-type central pair in A). Bar in A, 100 nm.
Mentions: Longitudinal images were used to determine the periodicity and angular orientation of the 1b projection and the other densities defined by cross-section analysis, and to see whether the 1b and 2b densities are longitudinally aligned. Longitudinal sections that graze the lateral surface of C1 show that projection 1a and its associated sheath fibers (extending from the right-hand edge of C1 in the longitudinal image of Fig. 7 A) have a 16-nm repeat period and tilt toward the tip of the axoneme at ∼24°. Also visible in Fig. 7 A are two rows of rectangular or rhomboidal densities, corresponding to densities 1c and 1d in cross-sectional images, which overlie the walls of C1, and project out from its left-hand margin, respectively. They are indicated in the longitudinal image by arrows and by parallel lines marking their 32-nm repeat period. When the section plane included both 1a and 1b projections (Fig. 7 B), their identical periodicity was revealed. Sections passing perpendicular to the central pair microtubule axes and including the intermicrotubule bridge (Fig. 7, C and D) show the presence of closely spaced rope-like material with a 16-nm repeat overlying the central pair microtubule image in the bridge region (diagonal lines in Fig. 7 C). This material, which may include both the diagonal link and the intermicrotubule bridge, appears to follow a left-handed helix with a pitch matching that of the underlying dimer lattice of the C2 microtubule (Linck et al., 1981). Both the 2a and 2b projections repeat at a 16-nm period, as seen in the lower half of the longitudinal image in Fig. 7 D.

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
Related in: MedlinePlus