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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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Coomassie blue–stained gel of pf18 axonemes and  various fractions of wild-type axonemes demonstrating selective  extraction of C1-associated proteins. Ax, unextracted axonemes;  HSP, pellet following extraction with high salt (0.6 M NaCl);  KIP, pellet following extraction of the HSP fraction with 0.2 M  KI; KIS, supernatant from extraction with 0.2 M KI; Std, Mr standards. All seven central pair proteins identified as C1-associated  based on their retention in the HSP fraction, indicated by diamonds, are also present in the KIS fraction. Molecular mass standards (kD) are indicated along the right margin.
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Figure 10: Coomassie blue–stained gel of pf18 axonemes and various fractions of wild-type axonemes demonstrating selective extraction of C1-associated proteins. Ax, unextracted axonemes; HSP, pellet following extraction with high salt (0.6 M NaCl); KIP, pellet following extraction of the HSP fraction with 0.2 M KI; KIS, supernatant from extraction with 0.2 M KI; Std, Mr standards. All seven central pair proteins identified as C1-associated based on their retention in the HSP fraction, indicated by diamonds, are also present in the KIS fraction. Molecular mass standards (kD) are indicated along the right margin.

Mentions: To determine whether the three proteins missing from Coomassie blue–stained gels of cpc1 axonemes represent subunits of a single structure, C1-associated proteins were solubilized and fractionated by centrifugation on a sucrose gradient. Wild-type axonemes were extracted with 0.6 M NaCl to remove dyneins and most C2-associated proteins, and the resulting high salt pellet (HSP) was further extracted with 0.2 M KI and centrifuged to generate pellet (KIP) and supernatant (KIS) fractions (Fig. 10). All seven C1 proteins identifiable with this gel system are quantitatively solubilized by 0.2 M KI treatment (Fig. 10, KIS lane). Electron microscopic examination of the KIP fraction confirmed that C1 microtubules and associated structures were removed by this treatment (i.e., no central pair material remained). After dialysis to remove salt, the KI soluble material was centrifuged through a sucrose gradient and fractions were examined by SDS-PAGE (Fig. 11). The three proteins missing in cpc1 (CP3, CP4, and 79 kD, marked with circles) consistently cosedimented along with three additional proteins of 135, 125, and 56 kD (marked with diamonds), and reached a peak at approximately 16S (fractions 7 and 8). When 16S peak fractions from two separate experiments were analyzed on 12% acrylamide gels, no additional cosedimenting proteins of lower Mr were observed. A second and much smaller peak of 350- and 265-kD bands, without any smaller subunits, cosedimented at 9S (fraction 16). These bands may represent another smaller complex containing only CP3 and CP4, or a complex of unrelated proteins of similar apparent size to CP3 and CP4. The remaining C1-associated bands identified on our 1-d gels sedimented elsewhere in the gradient.


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

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

Coomassie blue–stained gel of pf18 axonemes and  various fractions of wild-type axonemes demonstrating selective  extraction of C1-associated proteins. Ax, unextracted axonemes;  HSP, pellet following extraction with high salt (0.6 M NaCl);  KIP, pellet following extraction of the HSP fraction with 0.2 M  KI; KIS, supernatant from extraction with 0.2 M KI; Std, Mr standards. All seven central pair proteins identified as C1-associated  based on their retention in the HSP fraction, indicated by diamonds, are also present in the KIS fraction. Molecular mass standards (kD) are indicated along the right margin.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132896&req=5

Figure 10: Coomassie blue–stained gel of pf18 axonemes and various fractions of wild-type axonemes demonstrating selective extraction of C1-associated proteins. Ax, unextracted axonemes; HSP, pellet following extraction with high salt (0.6 M NaCl); KIP, pellet following extraction of the HSP fraction with 0.2 M KI; KIS, supernatant from extraction with 0.2 M KI; Std, Mr standards. All seven central pair proteins identified as C1-associated based on their retention in the HSP fraction, indicated by diamonds, are also present in the KIS fraction. Molecular mass standards (kD) are indicated along the right margin.
Mentions: To determine whether the three proteins missing from Coomassie blue–stained gels of cpc1 axonemes represent subunits of a single structure, C1-associated proteins were solubilized and fractionated by centrifugation on a sucrose gradient. Wild-type axonemes were extracted with 0.6 M NaCl to remove dyneins and most C2-associated proteins, and the resulting high salt pellet (HSP) was further extracted with 0.2 M KI and centrifuged to generate pellet (KIP) and supernatant (KIS) fractions (Fig. 10). All seven C1 proteins identifiable with this gel system are quantitatively solubilized by 0.2 M KI treatment (Fig. 10, KIS lane). Electron microscopic examination of the KIP fraction confirmed that C1 microtubules and associated structures were removed by this treatment (i.e., no central pair material remained). After dialysis to remove salt, the KI soluble material was centrifuged through a sucrose gradient and fractions were examined by SDS-PAGE (Fig. 11). The three proteins missing in cpc1 (CP3, CP4, and 79 kD, marked with circles) consistently cosedimented along with three additional proteins of 135, 125, and 56 kD (marked with diamonds), and reached a peak at approximately 16S (fractions 7 and 8). When 16S peak fractions from two separate experiments were analyzed on 12% acrylamide gels, no additional cosedimenting proteins of lower Mr were observed. A second and much smaller peak of 350- and 265-kD bands, without any smaller subunits, cosedimented at 9S (fraction 16). These bands may represent another smaller complex containing only CP3 and CP4, or a complex of unrelated proteins of similar apparent size to CP3 and CP4. The remaining C1-associated bands identified on our 1-d gels sedimented elsewhere in the gradient.

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