Characterization of a Chlamydomonas insertional mutant that disrupts flagellar central pair microtubule-associated structures - PubMed (original) (raw)

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

D R Mitchell et al. J Cell Biol. 1999.

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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Figures

Figure 1

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.

Figure 2

Figure 2

Flagellar beat frequencies were measured on freely swimming cells. The cpc1 defect reduces beat frequency by 38% from wild-type (WT) levels. Absence of outer row dyneins (pf28) reduces beat frequency by 60%, and the combination of cpc1 and absence of outer row dyneins (pf28cpc1) results in a further decrease to 17 Hz, or 30% below pf28 alone. Each bar shows mean and standard deviation (n = 31).

Figure 3

Figure 3

Electron micrographs of thin sections through demembranated wild-type axonemes (A) show several electron-dense projections from the two central pair microtubules. All micrographs are oriented as if viewed from inside the cell and rotated so that the C1 central microtubule is to the left. The longest central pair projections (1a, 1b, 2a, and 2b) have been labeled in the third panel. In similar micrographs of cpc1 axonemes (B) the central pair microtubules lack the 1b and 2b densities. Images of intact cpc1 flagella (C) show that the C1b projection is not lost during axoneme preparation, but rather fails to assemble in this mutant. Bar, 100 nm.

Figure 4

Figure 4

Cross-sections through pf14 axonemes (A and B) and an average of 16 similar images (C) reveal additional details of central pair structure that are summarized diagramatically (D). These include four densities (1a, 1b, 1c, and 1d) attached to the C1 microtubule, three densities (2a, 2b, and 2c) attached to the C2 microtubule, a bipartite bridge and a diagonal link spanning the gap between the microtubules, and sheath material that forms an arc from 1c to the tip of 1a, and from 1d to the tip of 1b. Individual cross-sections through cpc1pf14 axonemes (E and F) show that the 1b projection is invariably missing in cpc1 axonemes, whereas the 2b projection is sometimes retained (filled arrowhead in E). An average of 12 images (G) reveals the presence of a densely staining region at the site where 1b attaches to the C1 microtubule in wild-type axonemes. A difference image (H) made by subtracting G from C produces a dark image of the missing structures and a bright region where rotation of C1 relative to C2 has produced an apparent overlap of the 1a and 2a projections (asterisk). Cross-sections through pf6pf14 axonemes (I and J), an average of 25 images (K), and a difference image (L) show that only projection 1a and its associated sheath material are affected by the pf6 mutation. Bars, 100 nm in A and 20 nm in C.

Figure 5

Figure 5

Cross-sections through pf14 axonemes that were extracted with 0.6 M NaCl. (A) Survey view of extracted axonemes. (B–E) Cross-sections in which C2 has been solubilized. These C1 microtubules retain all of the electron densities identified as C1-associated in Fig. 4. Bars, 100 nm in both A and B.

Figure 6

Figure 6

Selected images of cross-sections through pf16pf14 axonemes which illustrate apparent progressive steps in the loss of C1 and its associated structures. In row A, the 1a projection and its associated sheath are missing, and little remains at the 1c position, but the 1b and 1d densities are retained. In row B, several protofilaments are missing from the wall of the C1 microtubules. In rows C and D, C1 is completely missing. The 1b projection can remain attached to the C2 microtubule through apparent connections at its base to the diagonal link and at its tip to 2b (seen most clearly in row C). Bar, 100 nm.

Figure 7

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.

Figure 8

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 M r 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.

Figure 9

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 M r 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.

Figure 10

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, M r 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.

Figure 11

Figure 11

Sucrose gradient fractionation of C1-associated proteins. The KIS fraction shown in Fig. 10 was centrifuged on a 5–20% sucrose gradient and collected from the bottom in 24 fractions. Equal volumes of fractions 2–19 were analyzed by SDS-PAGE and silver stained. All three proteins missing from cpc1 axonemes (filled circles) cosediment in a complex at 16S (fractions 7 and 8) along with three additional proteins (diamonds). Std, mass standards; L, material loaded onto the gradient. The mass of markers (kD) is indicated along the left margin.

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