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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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Dark-field stroboscopic images of reactivated axonemes used for comparison of waveforms. Wild-type (A) and  cpc1 (B) axonemes reactivated at 10−8 M Ca2+ beat with highly  asymmetric waveforms. Both axonemes are freely swimming  near the coverslip. Wild-type (C) and cpc1 (D) axonemes reactivated at 10−4 M Ca2+ beat with symmetric waveforms. Both axonemes have adhered to the coverslip at their proximal ends. All  panels were photographed at a flash rate of 50 Hz. Bar in A, 10 μm.
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Figure 1: Dark-field stroboscopic images of reactivated axonemes used for comparison of waveforms. Wild-type (A) and cpc1 (B) axonemes reactivated at 10−8 M Ca2+ beat with highly asymmetric waveforms. Both axonemes are freely swimming near the coverslip. Wild-type (C) and cpc1 (D) axonemes reactivated at 10−4 M Ca2+ beat with symmetric waveforms. Both axonemes have adhered to the coverslip at their proximal ends. All panels were photographed at a flash rate of 50 Hz. Bar in A, 10 μm.

Mentions: Previous analysis of motility defects associated with inner and outer row dynein assembly mutations showed that only inner row dynein defects have a marked effect on asymmetric waveforms, but that both types of defect greatly reduce bend amplitude during symmetric beating (Brokaw and Kamiya, 1987). Images of wild-type and cpc1 axonemes reactivated at either 10−8 M or 10−4 M Ca2+ (Fig. 1) show that cpc1 axonemes beat asymmetrically at low Ca2+ concentrations and symmetrically at high Ca2+ concentrations, and display no radical differences from wild-type waveforms under either condition. The combination of reduced beat frequency with apparently normal waveform in cpc1 flagella is phenotypically similar to motility parameters of some outer row dynein defects such as sup1 and sup2 (Brokaw and Luck, 1985). To see whether cpc1 acts exclusively through a modulation of outer row dynein activity, we compared beat frequencies of strains carrying cpc1 alone, an outer row dynein assembly mutation alone (pf28), or both mutations (pf28cpc1). As illustrated in Fig. 2, cpc1 reduced beat frequency almost identically in a pf28 background (30% reduction) and a wild-type background (38% reduction), and thus does not act exclusively through effects on outer row dyneins.


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

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

Dark-field stroboscopic images of reactivated axonemes used for comparison of waveforms. Wild-type (A) and  cpc1 (B) axonemes reactivated at 10−8 M Ca2+ beat with highly  asymmetric waveforms. Both axonemes are freely swimming  near the coverslip. Wild-type (C) and cpc1 (D) axonemes reactivated at 10−4 M Ca2+ beat with symmetric waveforms. Both axonemes have adhered to the coverslip at their proximal ends. All  panels were photographed at a flash rate of 50 Hz. Bar in A, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132896&req=5

Figure 1: Dark-field stroboscopic images of reactivated axonemes used for comparison of waveforms. Wild-type (A) and cpc1 (B) axonemes reactivated at 10−8 M Ca2+ beat with highly asymmetric waveforms. Both axonemes are freely swimming near the coverslip. Wild-type (C) and cpc1 (D) axonemes reactivated at 10−4 M Ca2+ beat with symmetric waveforms. Both axonemes have adhered to the coverslip at their proximal ends. All panels were photographed at a flash rate of 50 Hz. Bar in A, 10 μm.
Mentions: Previous analysis of motility defects associated with inner and outer row dynein assembly mutations showed that only inner row dynein defects have a marked effect on asymmetric waveforms, but that both types of defect greatly reduce bend amplitude during symmetric beating (Brokaw and Kamiya, 1987). Images of wild-type and cpc1 axonemes reactivated at either 10−8 M or 10−4 M Ca2+ (Fig. 1) show that cpc1 axonemes beat asymmetrically at low Ca2+ concentrations and symmetrically at high Ca2+ concentrations, and display no radical differences from wild-type waveforms under either condition. The combination of reduced beat frequency with apparently normal waveform in cpc1 flagella is phenotypically similar to motility parameters of some outer row dynein defects such as sup1 and sup2 (Brokaw and Luck, 1985). To see whether cpc1 acts exclusively through a modulation of outer row dynein activity, we compared beat frequencies of strains carrying cpc1 alone, an outer row dynein assembly mutation alone (pf28), or both mutations (pf28cpc1). As illustrated in Fig. 2, cpc1 reduced beat frequency almost identically in a pf28 background (30% reduction) and a wild-type background (38% reduction), and thus does not act exclusively through effects on outer row dyneins.

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