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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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SDS-PAGE analysis of the cpc1 defect. Demembranated flagellar axonemes (Axoneme lanes) from wild-type cells  (WT) or a central pair assembly mutant (pf18) show that eight  bands are missing when the central pair is absent (arrowheads;  estimated Mr shown along the right margin). Three of these proteins are depleted or missing from cpc1 axonemes (open circles).  The HSP lanes show insoluble high salt pellet fractions produced  by extracting axonemes with 0.6 M NaCl. The WT HSP sample  retains the three proteins missing from cpc1 axonemes. Extraction of cpc1 axonemes removes two additional bands of 630 and  165 kD that are not removed from wild-type axonemes by high  salt treatment (cpc1 HSP, open circles). Gel was stained with  Coomassie blue. Molecular mass standards (kD) are indicated  along the left margin.
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Figure 8: SDS-PAGE analysis of the cpc1 defect. Demembranated flagellar axonemes (Axoneme lanes) from wild-type cells (WT) or a central pair assembly mutant (pf18) show that eight bands are missing when the central pair is absent (arrowheads; estimated Mr shown along the right margin). Three of these proteins are depleted or missing from cpc1 axonemes (open circles). The HSP lanes show insoluble high salt pellet fractions produced by extracting axonemes with 0.6 M NaCl. The WT HSP sample retains the three proteins missing from cpc1 axonemes. Extraction of cpc1 axonemes removes two additional bands of 630 and 165 kD that are not removed from wild-type axonemes by high salt treatment (cpc1 HSP, open circles). Gel was stained with Coomassie blue. Molecular mass standards (kD) are indicated along the left margin.

Mentions: To find out which central pair proteins were missing from cpc1, SDS-PAGE was used to compare axonemes isolated from wild-type, central pair assembly mutant pf18, and cpc1 cells. As shown in Fig. 8, eight bands with estimated sizes ranging from 630 to 79 kD could be recognized as depleted in pf18 axonemes using this 1-d gel system (marked with arrowheads), including four bands >200 kD that apparently correspond to the four high molecular weight proteins previously identified as central pair components on 1-d gels (Witman et al., 1978; Adams et al., 1981) and designated CP1–CP4 (Dutcher et al., 1984). Three of the eight proteins with apparent sizes of 350 (CP3), 265 (CP4), and 79 kD were greatly reduced or missing from cpc1 axonemes (open circles). Two others, 630 kD (CP1), and 380 kD (CP2), were slightly reduced in amount. CP1, CP2, and CP4 have been characterized as C1-associated proteins based on their loss from pf16 axonemes and resistance to high salt extraction from wild-type axonemes, whereas CP3 has been similarly identified as a C2-associated protein (Dutcher et al., 1984). Under our conditions, extraction of wild-type axonemes with 0.6 M NaCl to create a high salt pellet (HSP) fraction (Fig. 8, HSP WT lane) depleted only one of the eight identifiable central pair– associated bands (175 kD), but did not remove the 350-kD band. Either this band is not the same as CP3, or our extraction conditions differ in an unknown way from those of Dutcher et al. (1984). We favor the second explanation and have kept the designation CP3 for this band. Seven central pair proteins were retained in our experiments and thus reside in structures associated with C1 following salt extraction (Fig. 5). Similar extraction of cpc1 axonemes removed CP1 (630 kD) and a 165-kD band (open circles in Fig. 8, HSP cpc1 lane) in addition to the 175-kD band that was extracted from wild-type axonemes.


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

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

SDS-PAGE analysis of the cpc1 defect. Demembranated flagellar axonemes (Axoneme lanes) from wild-type cells  (WT) or a central pair assembly mutant (pf18) show that eight  bands are missing when the central pair is absent (arrowheads;  estimated Mr shown along the right margin). Three of these proteins are depleted or missing from cpc1 axonemes (open circles).  The HSP lanes show insoluble high salt pellet fractions produced  by extracting axonemes with 0.6 M NaCl. The WT HSP sample  retains the three proteins missing from cpc1 axonemes. Extraction of cpc1 axonemes removes two additional bands of 630 and  165 kD that are not removed from wild-type axonemes by high  salt treatment (cpc1 HSP, open circles). Gel was stained with  Coomassie blue. Molecular mass standards (kD) are indicated  along the left margin.
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Figure 8: SDS-PAGE analysis of the cpc1 defect. Demembranated flagellar axonemes (Axoneme lanes) from wild-type cells (WT) or a central pair assembly mutant (pf18) show that eight bands are missing when the central pair is absent (arrowheads; estimated Mr shown along the right margin). Three of these proteins are depleted or missing from cpc1 axonemes (open circles). The HSP lanes show insoluble high salt pellet fractions produced by extracting axonemes with 0.6 M NaCl. The WT HSP sample retains the three proteins missing from cpc1 axonemes. Extraction of cpc1 axonemes removes two additional bands of 630 and 165 kD that are not removed from wild-type axonemes by high salt treatment (cpc1 HSP, open circles). Gel was stained with Coomassie blue. Molecular mass standards (kD) are indicated along the left margin.
Mentions: To find out which central pair proteins were missing from cpc1, SDS-PAGE was used to compare axonemes isolated from wild-type, central pair assembly mutant pf18, and cpc1 cells. As shown in Fig. 8, eight bands with estimated sizes ranging from 630 to 79 kD could be recognized as depleted in pf18 axonemes using this 1-d gel system (marked with arrowheads), including four bands >200 kD that apparently correspond to the four high molecular weight proteins previously identified as central pair components on 1-d gels (Witman et al., 1978; Adams et al., 1981) and designated CP1–CP4 (Dutcher et al., 1984). Three of the eight proteins with apparent sizes of 350 (CP3), 265 (CP4), and 79 kD were greatly reduced or missing from cpc1 axonemes (open circles). Two others, 630 kD (CP1), and 380 kD (CP2), were slightly reduced in amount. CP1, CP2, and CP4 have been characterized as C1-associated proteins based on their loss from pf16 axonemes and resistance to high salt extraction from wild-type axonemes, whereas CP3 has been similarly identified as a C2-associated protein (Dutcher et al., 1984). Under our conditions, extraction of wild-type axonemes with 0.6 M NaCl to create a high salt pellet (HSP) fraction (Fig. 8, HSP WT lane) depleted only one of the eight identifiable central pair– associated bands (175 kD), but did not remove the 350-kD band. Either this band is not the same as CP3, or our extraction conditions differ in an unknown way from those of Dutcher et al. (1984). We favor the second explanation and have kept the designation CP3 for this band. Seven central pair proteins were retained in our experiments and thus reside in structures associated with C1 following salt extraction (Fig. 5). Similar extraction of cpc1 axonemes removed CP1 (630 kD) and a 165-kD band (open circles in Fig. 8, HSP cpc1 lane) in addition to the 175-kD band that was extracted from wild-type axonemes.

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