The UNI1 and UNI2 genes function in the transition of triplet to doublet microtubules between the centriole and cilium in Chlamydomonas

Abstract

One fundamental role of the centriole in eukaryotic cells is to nucleate the growth of cilia. The unicellular alga Chlamydomonas reinhardtii provides a simple genetic system to study the role of the centriole in ciliogenesis. Wild-type cells are biflagellate, but "uni" mutations result in failure of some centrioles (basal bodies) to assemble cilia (flagella). Serial transverse sections through basal bodies in uni1 and uni2 single and double mutant cells revealed a previously undescribed defect in the transition of triplet microtubules to doublet microtubules, a defect correlated with failure to assemble flagella. Phosphorylation of the Uni2 protein is reduced in uni1 mutant cells. Immunogold electron microscopy showed that the Uni2 protein localizes at the distal end of the basal body where microtubule transition occurs. These results provide the first mechanistic insights into the function of UNI1 and UNI2 genes in the pathway mediating assembly of doublet microtubules in the axoneme from triplet microtubules in the basal body template.

Figure 1.

Median longitudinal sections of basal bodies from uni1-1 uni2-3 double mutant cells (CC-4201) reveal TZ and docking defects. (A) The TZ in WT cells (white bracket) is composed of distal (d) and proximal (p) cylinders separated by an electron dense transitional plate (tp). The basal body docks with the plasma membrane (pm) allowing the TZ and axoneme to extend through an opening in the cell wall (cw). Projections extend from triplet microtubules into the lumen of the basal body. The transition from triplet to doublet microtubules occurs near the distal end of the basal body (black arrow). (B and C) Mutant uni1 uni2 (CC-4201) cells have highly elongated or multiplied and stacked TZ regions. The basal body in B is docked at the plasma membrane; the basal body in C is undocked. The distal striated fiber (df, dark arrow head) typically links the two basal bodies together and appears to be properly connected to each of the mutant basal bodies.

Figure 2.

(A) Immunoblot analysis of uniflagellar mutants expressing the HA-tagged Uni2 protein. Protein extracts from an untransformed WT strain (CC-124) and single and double mutant strains expressing the HA-tagged Uni2 protein (CC-4163, CC-4204, CC-4205) were separated by SDS-PAGE and transferred to a PVDF membrane. Top, a high-affinity anti-HA antibody was used to identify the HA-tagged Uni2 protein; bottom, an antibody against β-tubulin was used as a loading control. (B) Expression of the two distinct Uni2 protein isoforms was compared using densitometry. The ratio of the lower to the higher molecular-weight isoform was calculated and the fold change of this ratio as compared with the uni2-3 strain rescued to WT (lane 2) was determined.

Figure 3.

Indirect immunofluorescence microscopy of the HA-tagged Uni2 protein expressed in single and double mutant strains (A) uni2, strain CC-4163; (B) uni1 uni2, strain CC-4204; (C) uni2 uni3, strain CC-4205. Fixed whole cells were doubly stained with rat anti-HA and rabbit anti-α-tubulin antibodies. Secondary anti-rat Alexa Fluor 488 (green) and anti-rabbit Texas Red (red) antibodies were used to detect primary antibodies. Basal body regions were enlarged two times.

Figure 4.

Transverse serial sections through two individual basal bodies and axonemal regions in uni2-3 mutant cells (CC-4162) reveal that for some basal bodies, a normal transition of triplet to doublet microtubules occurs before assembly of a TZ and flagellum (A–L), whereas other basal bodies fail to transition all triplet microtubules to doublet microtubules at the distal end of the transitional fibers (M–X). Triplet numbers (labeled 1, 3, and 8 in A) can be determined based on the location of the distal striated fiber (df, solid arrowhead) or the proximal fiber (pf, solid arrow). Open arrowheads demarcate the transition from triplet to doublet microtubules, which typically occurs at the distal end of transitional fibers (tf, open arrow) in WT cells. The ciliary necklace (cn, asterisk) links the doublet microtubules at the base of the axoneme to the flagellar membrane (fm). Roman numerals in the bottom left of some panels represent the number of omitted sections between that panel and the previous panel.

Figure 5.

Transverse serial sections through a single basal body and axonemal region in A–L the uni1-1 mutant (CC-1926) and in M–X the uni1-1 uni2-3 double mutant (CC-4201) reveal an incomplete transition of triplet to doublet microtubules and flagellar assembly failure. The basal body microtubule triplets were oriented based on the location of the proximal fiber (pf, solid arrow), which is in congruence with a normal localization of the distal striated fiber (df, solid arrowhead) in series A–L, but not in series M–X. Open arrowheads demarcate the transition from triplet to doublet microtubules in all series, which typically occurs at the distal end of transitional fibers (tf, open arrow) in WT cells. The ciliary necklace (cn, asterisk) links the microtubules of the axoneme to the flagellar membrane (fm). Roman numerals in the bottom left of some panels represent the number of omitted sections between that panel and the previous panel.

Figure 6.

Postembedment immunogold electron microscopy of whole cells reveals the localization of the Uni2 protein at the distal end of basal bodies. (A and B) Thin sections of uni2-3 mutant cells rescued to WT with an HA-tagged UNI2 gene (CC-4163) were labeled with rat anti-HA antibodies and then labeled with secondary goat anti-rat antibodies conjugated with 12-nm gold particles. Gold particles are indicated by open arrowheads. The distal striated fiber (df, dark arrowhead) attaches to one side of the basal body. (C) The transitional plate (tp) provided a reference point for quantifying the localization of gold particles. The position of each gold particle with respect to the position of the transitional plate was determined (right bracket and bar graph). The position of the distal end of the triplet microtubules with respect to the transitional plate (left bracket) was determined from six basal body images similar to the one in Figure 1A.