Limits...
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.

Show MeSH
Kinesin-like central pair proteins are not disrupted by  the cpc1 defect. Axoneme samples from wild-type (WT), pf18,  pf16, and cpc1 strains were separated by SDS-PAGE as in Fig. 8,  and either stained with Coomassie blue (CB lanes) or transferred  and probed with affinity-purified polyclonal anti-HIPYR antibody (HIPYR lanes). Bands missing from pf18 axonemes are indicated by diamonds, bands missing from cpc1 by circles. The apparent Mr of two bands recognized by anti-HIPYR and missing  from pf18 axonemes are shown along the right margin. The 105-kD  band is missing from pf16 axonemes, whereas neither band is  missing from cpc1 axonemes. Molecular mass standards (kD) are  indicated along the left margin.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132896&req=5

Figure 9: Kinesin-like central pair proteins are not disrupted by the cpc1 defect. Axoneme samples from wild-type (WT), pf18, pf16, and cpc1 strains were separated by SDS-PAGE as in Fig. 8, and either stained with Coomassie blue (CB lanes) or transferred and probed with affinity-purified polyclonal anti-HIPYR antibody (HIPYR lanes). Bands missing from pf18 axonemes are indicated by diamonds, bands missing from cpc1 by circles. The apparent Mr of two bands recognized by anti-HIPYR and missing from pf18 axonemes are shown along the right margin. The 105-kD band is missing from pf16 axonemes, whereas neither band is missing from cpc1 axonemes. Molecular mass standards (kD) are indicated along the left margin.

Mentions: Three kinesin-like proteins have been localized to the Chlamydomonas central pair apparatus by blot analysis of central pair mutants (Bernstein et al., 1994; Fox et al., 1994) and by immunoelectron microscopy (Bernstein et al., 1994; Johnson et al., 1994). Klp1 (83 kD) is missing from central pair assembly mutants such as pf15 and has been localized to the C2 microtubule both by virtue of its presence in pf16 axonemes and by immunoelectron microscopy (Bernstein et al., 1994). Blot analysis with affinity-purified anti-Klp1 showed that normal amounts of this antigen were present in cpc1 axonemes (not shown) and thus Klp1 is not associated with projection 2b. A 110-kD antigen recognized by a polyclonal antibody to Drosophila kinesin, αHD, has also been immunolocalized to a single central pair microtubule (Johnson et al., 1994), but remains associated with axonemes in all central pair assembly mutants tested. A second 110-kD antigen recognized by polyclonal sera raised against peptides spanning either of two highly conserved kinesin head domain sequences, HIPYR and LAGSE, is missing from axonemes of central pair mutants such as pf18 (Fox et al., 1994; Johnson et al., 1994) but has not been localized within the central pair structure. We tested for the presence of this kinesin-like protein in cpc1 axonemes by blot analysis using affinity-purified anti-HIPYR antibody. As seen in Fig. 9, this antibody detects multiple bands in wild-type axonemes, and two of these (105 and 85 kD) are depleted in pf18 axonemes. The 105-kD HIPYR antigen, which most likely corresponds to the 110-kD central pair antigen previously detected with this antibody, is missing from the pf16 sample and thus should be designated a C1-associated protein, but is present in cpc1 axonemes. The 85-kD band, which does not correspond to any previously reported kinesin-like proteins in Chlamydomonas flagella, was retained in both pf16 and cpc1 axonemes at wild-type levels and is, like Klp1, a C2-associated protein that is not part of projection 2b.


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

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

Kinesin-like central pair proteins are not disrupted by  the cpc1 defect. Axoneme samples from wild-type (WT), pf18,  pf16, and cpc1 strains were separated by SDS-PAGE as in Fig. 8,  and either stained with Coomassie blue (CB lanes) or transferred  and probed with affinity-purified polyclonal anti-HIPYR antibody (HIPYR lanes). Bands missing from pf18 axonemes are indicated by diamonds, bands missing from cpc1 by circles. The apparent Mr of two bands recognized by anti-HIPYR and missing  from pf18 axonemes are shown along the right margin. The 105-kD  band is missing from pf16 axonemes, whereas neither band is  missing from cpc1 axonemes. Molecular mass standards (kD) are  indicated along the left margin.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Kinesin-like central pair proteins are not disrupted by the cpc1 defect. Axoneme samples from wild-type (WT), pf18, pf16, and cpc1 strains were separated by SDS-PAGE as in Fig. 8, and either stained with Coomassie blue (CB lanes) or transferred and probed with affinity-purified polyclonal anti-HIPYR antibody (HIPYR lanes). Bands missing from pf18 axonemes are indicated by diamonds, bands missing from cpc1 by circles. The apparent Mr of two bands recognized by anti-HIPYR and missing from pf18 axonemes are shown along the right margin. The 105-kD band is missing from pf16 axonemes, whereas neither band is missing from cpc1 axonemes. Molecular mass standards (kD) are indicated along the left margin.
Mentions: Three kinesin-like proteins have been localized to the Chlamydomonas central pair apparatus by blot analysis of central pair mutants (Bernstein et al., 1994; Fox et al., 1994) and by immunoelectron microscopy (Bernstein et al., 1994; Johnson et al., 1994). Klp1 (83 kD) is missing from central pair assembly mutants such as pf15 and has been localized to the C2 microtubule both by virtue of its presence in pf16 axonemes and by immunoelectron microscopy (Bernstein et al., 1994). Blot analysis with affinity-purified anti-Klp1 showed that normal amounts of this antigen were present in cpc1 axonemes (not shown) and thus Klp1 is not associated with projection 2b. A 110-kD antigen recognized by a polyclonal antibody to Drosophila kinesin, αHD, has also been immunolocalized to a single central pair microtubule (Johnson et al., 1994), but remains associated with axonemes in all central pair assembly mutants tested. A second 110-kD antigen recognized by polyclonal sera raised against peptides spanning either of two highly conserved kinesin head domain sequences, HIPYR and LAGSE, is missing from axonemes of central pair mutants such as pf18 (Fox et al., 1994; Johnson et al., 1994) but has not been localized within the central pair structure. We tested for the presence of this kinesin-like protein in cpc1 axonemes by blot analysis using affinity-purified anti-HIPYR antibody. As seen in Fig. 9, this antibody detects multiple bands in wild-type axonemes, and two of these (105 and 85 kD) are depleted in pf18 axonemes. The 105-kD HIPYR antigen, which most likely corresponds to the 110-kD central pair antigen previously detected with this antibody, is missing from the pf16 sample and thus should be designated a C1-associated protein, but is present in cpc1 axonemes. The 85-kD band, which does not correspond to any previously reported kinesin-like proteins in Chlamydomonas flagella, was retained in both pf16 and cpc1 axonemes at wild-type levels and is, like Klp1, a C2-associated protein that is not part of projection 2b.

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