Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons

Abstract

We previously described a kinesin-dependent movement of particles in the flagella of Chlamydomonas reinhardtii called intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523). When IFT is inhibited by inactivation of a kinesin, FLA10, in the temperature-sensitive mutant, fla10, existing flagella resorb and new flagella cannot be assembled. We report here that: (a) the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin; (b) IFT particles are composed of 15 polypeptides comprising two large complexes; (c) the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies; and, (d) mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. In the accompanying report by Pazour, G.J., C.G. Wilkerson, and G.B. Witman (1998. J. Cell Biol. 141:979-992), a Chlamydomonas mutant (fla14) is described in which only the retrograde transport of IFT particles is disrupted, resulting in assembly-defective flagella filled with an excess of IFT particles. This microtubule- dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia.

Figure 1

Gel filtration profile of axonemal ATP eluate. Axonemes from wild-type cells were isolated in the presence of AMPPNP and then extracted with ATP. The resulting ATP eluate, shown in the first lane (L) was separated on an S400 sizing column. (A) Coomassie-stained gel (7.5%) of fractions 46–69 (left to right) shows the majority of eluted protein. The vertical arrowheads over lanes 56–58 indicate the peak fractions of three comigrating bands of M _r 100, 90, and 85 kD. Molecular weight markers are indicated on the right. (B) Corresponding immunoblot probed with anti-FLA10T reveals that the 90-kD FLA10 gene product coelutes with the three comigrating bands, primarily between fractions 55 and 60.

Figure 2

Sucrose density gradient profile of FLA10 kinesin-II and subsequent antibody analysis. The peak FLA10-containing fractions from S400 chromatography were pooled, concentrated, and further fractionated on a 5-ml, 5–20% sucrose gradient. (A) Coomassie-stained gel shows cosedimentation at 9.7 S of three polypeptides at 100, 90, and 85 kD with molar ratios of 0.8, 1.0, and 1.0, respectively. Only the portion of the gradient from 20% (left) to 12% (right) sucrose is shown. (B) Analysis of FLA10 kinesin-II subunits with antibodies. The left panel is a Coomassie-stained gel of sucrose density gradient–purified FLA10 kinesin-II. The right panel contains separate strips from corresponding immunoblots that have been probed with the antibodies listed above each strip. Both of the pan-kinesin peptide antibodies, LAGSE and HIPYR, reacted with both the 85- and 90-kD polypeptides, indicating a high probability that both subunits are kinesin-like proteins. Anti-FLA10T and K2.4, an mAb raised against the 85-kD subunit of sea urchin kinesin-II, reacted only with the 90-kD subunit (FLA10). The polyclonal anti-115k antisera, raised against the 115-kD nonmotor subunit of sea urchin kinesin-II, reacted only with the 100-kD subunit. The identities of the reactive bands were confirmed in lanes probed with mixtures of the antibodies. The mixture of K2.4 and anti-FLA10T reacted only with the 90-kD band, verifying that a monoclonal raised against the smaller of the two kinesin-like subunits (85 kD) of sea urchin kinesin-II recognizes the larger of the two kinesin-like subunits (90 kD) of Chlamydomonas kinesin-II.

Figure 3

Polypeptide analysis of flagellar extracts from wild-type and fla mutant cells. Flagella were isolated from cells incubated at either 23°C or the restrictive temperature of 33°C. Membrane plus matrix was isolated in the presence of 0.05% NP-40 and 10 mM ATP to optimize extraction of FLA10 kinesin-II. (A) Coomassie-stained 7.5% gels of membrane plus matrix. Arrowheads, prominent bands that are reduced after incubation of the fla10 and fla8 mutants at 33°C. • on the far right indicates a band, M _r 140 kD, that is reduced in fla3 after heating, indicating that only a subset of the polypeptides lost in fla8 and fla10 appear to be reduced in the flagella of fla3 incubated at 33°C. An asterisk indicates a band at ∼100 kD that increases in fla8 after incubation at 33°C. Molecular weight standards, dynein, and tubulin are indicated on the right. (B) Corresponding immunoblots probed with anti-FLA10T. The FLA10 protein is significantly depleted in the flagellar extracts of the fla10 and fla8 mutants after incubation at 33°C.

Figure 4

Sucrose density gradient profile of membrane plus matrix. The membrane plus matrix fraction from flagella of wild-type cells was fractionated on an 11-ml 5–20% sucrose gradient in 10 mM Hepes, pH 7.2. The Coomassie-stained gel of the gradient profile shows that the putative IFT particle polypeptides cosediment at 16 S (highlighted by both sets of arrowheads with their apparent mobilities listed on the left).

Figure 5

Loss of 16 S polypeptides in the flagellar extracts of fla1 mutant cells at the restrictive temperature. Membrane plus matrix from fla1 mutant cells incubated at the permissive and restrictive temperatures of 23° and 33°C was fractionated on 11 ml 5–20% sucrose gradients in HMDEK buffer. Only the portion of the gradient from 20% (left) to 10% (right) is shown here on Coomassie-stained 7.5% gels. At 23°C, the IFT particle polypeptides, at 15–16 S, are reduced relative to wild type (arrowheads). These polypeptides are further depleted after incubation at 33°C (arrowheads). Similar results were obtained with membrane plus matrix isolated from fla8 and fla10 cells incubated at 23° and 33°C. Note that in the upper gel the polypeptides at 140 and 122 kD peak in the fraction highlighted by the 16 S arrow, whereas the other highlighted bands are sedimenting slightly slower.

Figure 6

Two-dimensional gel of IFT particle polypeptides. The 15–16 S fraction from a sucrose density gradient of a wild-type flagellar extract was separated by two-dimensional gel electrophoresis and stained with Coomassie blue. Identifiable bands are labeled according to apparent molecular mobility in the second dimension. An asterisk may represent p74. p57/55 are resolved as two closely migrating bands. p172 and p122 frequently run as streaks. The pIs of two-dimensional gel standards are shown.

Figure 7

Resolution of 15 IFT particle polypeptides. Membrane plus matrix from fla2 cells incubated at 23°C was fractionated on an 11-ml 10–25% sucrose density gradient in HMDEK buffer + 50 mM NaCl. Only a portion of the gradient from fraction 7 (21% sucrose, left side) to fraction 18 (15% sucrose, right side) are shown here on 5–20% SDS-PAGE. (A) Silver-stained gel. A subset of the IFT particle polypeptides, p144, p140, p139, and p122 (complex A), peak at fraction 11. The wide band at ∼140 kD is a triplet of unresolved bands at 144, 140, and 139 kD. The p172 polypeptide peaks at fraction 14. The remaining 10 IFT particle polypeptides, p88, p81, p80, p74, p72, p57/55, p52, p46, p27, and p20 (complex B minus p172), peak between fractions 12 and 13. The bands at ∼80 and 74 kD are two sets of unresolved doublets at 81 and 80 kD, and 74 and 72 kD, respectively (these two sets of doublets were routinely resolved on 7.5% SDS-PAGE as seen in Fig. 4). Note that at this ionic strength, p172 is clearly sedimenting slower than the rest of complex B. (B) Corresponding immunoblot probed with mAbs 172.1, 139.1, 81.1, 57.1, raised against IFT particle polypeptides.

Figure 8

Specificity of anti-FLA10N antibody and anti-IFT particle polypeptide antibodies and immunoprecipitation of complex A. (A) Whole wild-type cells and flagella were separately boiled in SDS-PAGE sample buffer, clarified, and then fractionated on 7.5% gels. Corresponding immunoblots were probed with antibodies as labeled. (B) Immunoprecipitation of complex A with 139.1. The membrane plus matrix from wild-type flagella was fractionated on a sucrose density gradient and fractions containing the 16 S proteins were pooled. An immunoprecipitate with the 139.1 antibody from this pool was analyzed on a Coomassie blue–stained 7.5% gel. Lane 1, 16 S pool; lane 2, 16 S pool after incubation with protein A–agarose resin; lane 3, 16 S pool immunodepleted with 139.1; lane 4, 139.1 immunoprecipitate of 16 S pool in HMDEK buffer; lane 5, 139.1 immunoprecipitate of 16 S pool after 0.5 M NaCl wash.

Figure 9

Immunofluorescent localization of FLA10, p172, p139, and p81 in cw92 cells (cell wall deficient but otherwise wild type). (a–c) 172.1. (d–f) Anti-FLA10N. (g) a pool of mAbs to p81: 81.1, 81.2, 81.3, and 81.4. (h) 139.1. Note the bi- or tri-lobed staining at the base of the flagella in a–h, insets. (i) Polyclonal anti–α-tubulin. Bar, 5 μm (inset, 10 μm).

Figure 10

Immunofluorescent staining of p172 and FLA10 in bld2 cells. (a and c) 172.1 staining. (b and d) Anti-FLA10N staining. The background chloroplast labeling of anti-FLA10N is intentionally bright to indicate the cells' orientation and to demonstrate that FLA10 kinesin-II is not concentrated at what should be the basal body region of the cell (highlighted by arrows).

Figure 11

Immunogold electron micrographs of p172 and FLA10 in wild-type cells. All micrographs illustrate the basal body region. (a–c) 172.1 staining was limited to the basal bodies and surrounding area with occasional gold particles seen between the flagellar membrane and outer doublet MTs. (d and e) Anti-FLA10N staining is similar to 172.1 staining. (e) Occasional labeling of the rootlet MTs (Mt) was seen in the vicinity of the basal bodies (BB).

Figure 12

(A) Alignment of three tryptic peptide sequences derived from p52 of complex B with deduced amino acids sequences from C. elegans OSM-6 and mouse neuronal NGD5. (B) Alignment of endoproteinase Lys-C peptide sequence derived from p172 with deduced amino acid sequence from C. elegans OSM-1.

Figure 13

Model of IFT in a Chlamydomonas flagellum. Fla10 kinesin-II and IFT complexes A and B are highly concentrated around the basal body region of the cell. Multiple copies of complexes A and B associate to form IFT particles. FLA10 kinesin-II binds to the basal bodies and then IFT particles bind to FLA10 kinesin-II. IFT particles may also bind to the kinesin before docking to the basal bodies. After oligomerization of IFT particles to form rafts, FLA10 kinesin-II transports IFT particles toward the flagellar tip. At the tip, the rafts dissociate into smaller oligomers of IFT particles and are returned to the base of the flagellum by cytoplasmic dynein.