The FLA3 KAP subunit is required for localization of kinesin-2 to the site of flagellar assembly and processive anterograde intraflagellar transport

Abstract

Intraflagellar transport (IFT) is a bidirectional process required for assembly and maintenance of cilia and flagella. Kinesin-2 is the anterograde IFT motor, and Dhc1b/Dhc2 drives retrograde IFT. To understand how either motor interacts with the IFT particle or how their activities might be coordinated, we characterized a ts mutation in the Chlamydomonas gene encoding KAP, the nonmotor subunit of Kinesin-2. The fla3-1 mutation is an amino acid substitution in a conserved C-terminal domain. fla3-1 strains assemble flagella at 21 degrees C, but cannot maintain them at 33 degrees C. Although the Kinesin-2 complex is present at both 21 and 33 degrees C, the fla3-1 Kinesin-2 complex is not efficiently targeted to or retained in the basal body region or flagella. Video-enhanced DIC microscopy of fla3-1 cells shows that the frequency of anterograde IFT particles is significantly reduced. Anterograde particles move at near wild-type velocities, but appear larger and pause more frequently in fla3-1. Transformation with an epitope-tagged KAP gene rescues all of the fla3-1 defects and results in preferential incorporation of tagged KAP complexes into flagella. KAP is therefore required for the localization of Kinesin-2 at the site of flagellar assembly and the efficient transport of anterograde IFT particles within flagella.

Figure 1.

Expression of the Chlamydomonas KAP gene in response to deflagellation. (A) A Southern blot of wild-type (R) and S1D2 (S) genomic DNA was hybridized with the ∼3.4-kb insert from the KAP cDNA clone CL70g01. A single EcoRI/XhoI restriction fragment was seen in wild-type, and a polymorphic fragment was seen in S1D2. (B) A Northern blot of total RNA isolated from wild-type cells before (0) and 45 min after deflagellation (45) was hybridized with the KAP cDNA (top panel). Expression of the ∼3.4-kb KAP transcript was enhanced by deflagellation, as compared with a probe (Cry1) for a ribosomal protein subunit used as a loading control (bottom panel).

Figure 2.

Chlamydomonas KAP is similar to other members of the KAP family. A Clustal W alignment of the predicted amino acid sequence of the Chlamydomonas KAP polypeptide (CrKAP) with orthologues from humans (Hs), mouse (Mm), sea urchin (Sp), flies (Dm), and worms (Ce) is shown here. The regions containing the conserved armadillo repeats (residues 229–270, 274–314, 360–400, 401–441, 442–480, 481–523, 525–561, 562–604, 605–648, 649–690, 691–733) are underlined. The conserved phenylalanine residue that is mutated in fla3 is indicated by an asterisk.

Figure 3.

Linkage of the KAP gene to flagellar assembly mutations and diagram of constructs used for rescue by transformation. (A) RFLP mapping procedures were used to place the KAP gene on the right arm linkage group X, based on linkage to the genetic marker nic13 (∼20 cM) and the molecular marker Tcr1-A (∼7.7 cM), ∼12.5 cM from the centromere (see Materials and Methods for details). These data placed the KAP gene in the vicinity of two flagellar assembly mutations, fla3 and fla4 (Adams et al., 1982). (B) Schematic diagram of the intron-exon structure of the KAP transcription unit used in transformation experiments. The KAP gene is contained within an ∼8.6-kb BamHI fragment. The open boxes represent exons and the solid lines represent introns. The positions of predicted Tub boxes, TATA boxes, translation start and stop sites, and the site of polyadenylation (Poly A) are also indicated. The site of insertion of either the HA or GFP epitope tag into the last exon is also shown here.

Figure 4.

Identification of a KAP mutation in fla3-1. (A) Sequence analysis of the KAP gene in fla3-1 revealed a point mutation at nucleotide 6853 in exon 17 that changes amino acid residue 753 from a phenylalanine to a leucine. (B) Diagram of the KAP polypeptide showing the position of fla3-1 mutation relative to the armadillo repeats. (C) The fla3-1 mutation does not alter the level of the KAP subunit in whole cells. Western blots of wild-type, fla3, and fla10 cells grown at 21°C or overnight at 33°C were probed with antibodies against KAP, FLA10, and tubulin. Each lane was loaded with extracts prepared from ∼5 × 10⁶ cells. The relative intensities of the KAP and FLA10 signals were normalized to the tubulin signals to control for small variations in protein loading. No significant changes in the levels of the KAP subunit were observed in the fla3-1 mutant at either the permissive or restrictive temperature.

Figure 5.

The fla3 mutation disrupts the localization of the Kinesin-2 complex in the basal body region. Wild-type and mutant strains were fixed at both 21 and 33°C, stained with a specific antibody (indicated on the right), and then imaged using both DIC and fluorescence microscopy. The top row shows fla3 cells stained with an antibody to tubulin. The cytoplasmic microtubule array is wild-type, but flagellar microtubules are absent at the restrictive temperature. The second row shows wild-type cells stained with an antibody to the FLA10 motor subunit, which concentrates in the basal body region and in a punctate pattern along the length of the two flagella. The third row shows fla3 cells stained with the FLA10 antibody; note how FLA10 staining is dispersed at both temperatures. The fourth row shows fla10-1 cells strained with the FLA10 antibody. Although FLA10 is reduced in the fla10-1 mutant, basal body localization can still be observed at 21°C. The fifth row shows wild-type cells stained with an antibody to KAP; note that KAP is concentrated in the basal body region and in a punctate pattern in the flagella, similar to FLA10 above. The sixth row shows fla3 cells stained with the KAP antibody; note how KAP staining is dispersed in the mutant strain. The seventh and eighth rows are fla3 cells stained with antibodies against other IFT components, the dynein LIC and the IFT particle subunit p139. The basal body and flagellar localization of these components appears to be relatively wild-type at 21°C. These components remain concentrated in the basal body region of the aflagellate cells at 33°C.

Figure 6.

Epitope-tagged KAPs are incorporated into the Kinesin-2 complex. (A) A Western blot of flagella isolated from wild-type, fla3, and four rescued fla3 strains (A1, A6, B5, 1D1, obtained by transformation with the KAP-HA construct) was probed with an antibody against the HA epitope. The KAP-HA subunit contains 892 amino acids and migrates at ∼100 kDa. (B) A Western blot showing the sequential extraction of isolated flagella (F) prepared from the KAP-GFP–rescued strain was probed with antibodies against GFP and FLA10. (M+M) is the membrane plus matrix fraction obtained by extraction of isolated flagella with 1.0% Nonidet-P-40, and A is the resulting pellet of axonemes. Most of the Kinesin-2 complex is released by extraction of axonemes with 10 mM MgATP (ATP-S); a small amount remains in the ATP extracted pellet (ATP-P). Treatment with 0.6 M NaCl releases the remainder into a high salt extract (HS-S) and high salt pellet (HS-P). Note the cofractionation of the KAP-GFP subunit with the FLA10 subunit. The KAP-GFP contains 1089 amino acids and migrates at ∼122 kDa. (C) The 10 mM MgATP extract shown in B was fractionated by sucrose density gradient centrifugation. A Western blot of the resulting fractions was probed with antibodies against GFP and FLA10. Note the cosedimentation of KAP-GFP with FLA10 at ∼10S in fractions 13 and 14; the top of the gradient is on the right. Reprobing of the blot with the KAP antibody failed to detect any of the endogenous fla3 KAP subunit in the 10S complex. (D) A Western blot of isolated axonemes prepared from wild-type (wt), fla3 (fla3), KAP-HA–rescued (HA), and KAP-GFP–rescued (GFP) cells at 21 and 33°C was probed with an antibody against KAP. Note the preferential assembly of the larger, epitope-tagged KAP subunits in axonemes of the rescued strains at both temperatures. (E) A Western blot of whole cell extracts prepared from wild-type, fla3, KAP-HA–rescued, and KAP-GFP–rescued strains was probed with an antibody against KAP. Note that the epitope-tagged KAP subunits are expressed at levels similar to the endogenous fla3 KAP subunit in the rescued cells.

Figure 7.

Epitope-tagged KAPs restore localization of the FLA10 Kinesin-2 complex to the basal body region and flagella. fla3 and fla3 rescued cells were fixed at both 21 and 33°C, stained with the antibodies as indicated on the right, and then imaged using both DIC and fluorescence microscopy. The top row shows fla3 cells stained with an antibody against the HA epitope; only background staining is observed. The second row shows KAP-HA–rescued cells stained with the HA-antibody, note the concentration of KAP-HA in the basal body region and along the length of the flagella at both temperatures, similar to wild-type pattern observed with the FLA10 and KAP antibodies in Figure 5. KAP-HA–rescued cells (third row) and KAP-GFP–rescued cells (bottom row) were also stained with the FLA10 antibody. Note that the FLA10 motor subunit is now concentrated in the basal body region and the flagella of the rescued strains.

Figure 8.

Anterograde IFT is altered in fla3 cells at 21°C. Representative kymograms illustrating the movements of individual IFT particles within a single flagellum are shown here. Individual IFT particles are seen as diagonal tracks. Time elapsed is plotted along the lower horizontal axis of the kymogram with time zero starting at the left. The length of the flagellum analyzed is depicted along the vertical axis of the kymograms. Anterograde particles are larger and move from the bottom left (proximal region) to the top right (distal region) of the kymograms (see black arrows in top panel). Retrograde particles are smaller and move from the top left to the bottom right (see white arrows in top panel). Note that many of the anterograde IFT particles in fla3 are larger than anterograde particles in either wild-type or the KAP-GFP–rescued strain. Anterograde particles also pause more frequently in fla3 flagella, as indicated by the white arrowheads.

Figure 9.

Flagellar disassembly in fla3 occurs by active excision. Wild-type (triangles), fla3 (squares), and fla3::KAP-GFP rescued cells (circles) were shifted from 21 to 33°C at the indicated times. The lengths of flagella on at least 100 cells were measured for each time point. (A) The average flagellar lengths including the contribution of bald and aflagellate cells. (B) The same samples as in A, but the average flagellar lengths excluding the contribution of zero-length flagella. (C) Pie diagrams illustrating the percent of aflagellate (black), uniflagellate (white), and biflagellate cells at each time point.