Flipping a Phosphate Switch on Kinesin-II to Turn IFT Around

Cilia and flagella are assembled and maintained by the motor-driven, bidirectional traffic of large protein complexes in a process termed intraflagellar transport (IFT). In this issue of Developmental Cell, Liang et al. (2014) report that IFT is regulated in part by the phosphorylation status of the kinesin-II subunit FLA8/KIF3B

The N-terminus of IFT46 mediates intraflagellar transport of outer arm dynein and its cargo-adaptor ODA16

Cilia are assembled via intraflagellar transport (IFT). The IFT machinery is composed of motors and multi-subunit particles, termed IFT-A and IFT-B, that carry cargo into the cilium. Knowledge of how the IFT subunits interact with their cargo is of critical importance for understanding how the unique ciliary domain is established. We previously reported a Chlamydomonas mutant, ift46-1, that fails to express the IFT-B protein IFT46, has greatly reduced levels of other IFT-B proteins, and assembles only very short flagella. A spontaneous suppression of ift46-1 restored IFT-B levels and enabled growth of longer flagella, but the flagella lacked outer dynein arms. Here, we show that the suppression is due to insertion of the transposon MRC1 into the ift46-1 allele, causing the expression of a fusion protein including the IFT46 C-terminal 240 amino acids. The IFT46 C-terminus can assemble into and stabilize IFT-B, but does not support transport of outer arm dynein into flagella. ODA16, a cargo adaptor specific for outer arm dynein, also fails to be imported into the flagella in the absence of the IFT46 N-terminus. We conclude that IFT46, ODA16, and the outer dynein arm interact for IFT of the latter

Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility

Mutations in Hydin cause hydrocephalus in mice, and HYDIN is a strong candidate for causing hydrocephalus in humans. The gene is conserved in ciliated species, including Chlamydomonas reinhardtii. An antibody raised against C. reinhardtii hydin was specific for an ∼540-kD flagellar protein that is missing from axonemes of strains that lack the central pair (CP). The antibody specifically decorated the C2 microtubule of the CP apparatus. An 80% knock down of hydin resulted in short flagella lacking the C2b projection of the C2 microtubule; the flagella were arrested at the switch points between the effective and recovery strokes. Biochemical analyses revealed that hydin interacts with the CP proteins CPC1 and kinesin-like protein 1 (KLP1). In conclusion, C. reinhardtii hydin is a CP protein required for flagellar motility and probably involved in the CP–radial spoke control pathway that regulates dynein arm activity. Hydrocephalus caused by mutations in hydin likely involves the malfunctioning of cilia because of a defect in the CP

The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly

Dyneins are microtubule-based molecular motors involved in many different types of cell movement. Most dynein heavy chains (DHCs) clearly group into cytoplasmic or axonemal isoforms. However, DHC1b has been enigmatic. To learn more about this isoform, we isolated Chlamydomonas cDNA clones encoding a portion of DHC1b, and used these clones to identify a Chlamydomonas cell line with a deletion mutation in DHC1b. The mutant grows normally and appears to have a normal Golgi apparatus, but has very short flagella. The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R. Diener, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993-1008) to the flagella. Rafts are particles that normally move up and down the flagella in a process known as intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523), which is essential for assembly and maintenance of flagella. The redistribution of raft subunits apparently occurs due to a defect in the retrograde component of IFT, suggesting that DHC1b is the motor for retrograde IFT. Consistent with this, Western blots indicate that DHC1b is present in the flagellum, predominantly in the detergent- and ATP-soluble fractions. These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is the motor for retrograde IFT

Flagellar central pair assembly in Chlamydomonas reinhardtii

BACKGROUND: Most motile cilia and flagella have nine outer doublet and two central pair (CP) microtubules. Outer doublet microtubules are continuous with the triplet microtubules of the basal body, are templated by the basal body microtubules, and grow by addition of new subunits to their distal ( plus ) ends. In contrast, CP microtubules are not continuous with basal body microtubules, raising the question of how these microtubules are assembled and how their polarity is established. METHODS: CP assembly in Chlamydomonas reinhardtii was analyzed by electron microscopy and wide-field and super-resolution immunofluorescence microscopy. To analyze CP assembly independently from flagellar assembly, the CP-deficient katanin mutants pf15 or pf19 were mated to wild-type cells. HA-tagged tubulin and the CP-specific protein hydin were used as markers to analyze de novo CP assembly inside the formerly mutant flagella. RESULTS: In regenerating flagella, the CP and its projections assemble near the transition zone soon after the onset of outer doublet elongation. During de novo CP assembly in full-length flagella, the nascent CP was first apparent in a subdistal region of the flagellum. The developing CP replaces a fibrous core that fills the axonemal lumen of CP-deficient flagella. The fibrous core contains proteins normally associated with the C1 CP microtubule and proteins involved in intraflagellar transport (IFT). In flagella of the radial spoke-deficient mutant pf14, two pairs of CPs are frequently present with identical correct polarities. CONCLUSIONS: The temporal separation of flagellar and CP assembly in dikaryons formed by mating CP-deficient gametes to wild-type gametes revealed that the formation of the CP does not require proximity to the basal body or transition zone, or to the flagellar tip. The observations on pf14 provide further support that the CP self-assembles without a template and eliminate the possibility that CP polarity is established by interaction with axonemal radial spokes. Polarity of the developing CP may be determined by the proximal-to-distal gradient of precursor molecules. IFT proteins accumulate in flagella of CP mutants; the abnormal distribution of IFT proteins may explain why these flagella are often shorter than normal

The unique catalytic subunit of sperm cAMP-dependent protein kinase is the product of an alternative Calpha mRNA expressed specifically in spermatogenic cells

cAMP-dependent protein kinase has a central role in the control of mammalian sperm capacitation and motility. Previous protein biochemical studies indicated that the only cAMP-dependent protein kinase catalytic subunit (C) in ovine sperm is an unusual isoform, termed C(s), whose amino terminus differs from those of published C isoforms of other species. Isolation and sequencing of cDNA clones encoding ovine C(s) and Calpha1 (the predominant somatic isoform) now reveal that C(s) is the product of an alternative transcript of the Calpha gene. C(s) cDNA clones from murine and human testes also were isolated and sequenced, indicating that C(s) is of ancient origin and widespread in mammals. In the mouse, C(s) transcripts were detected only in testis and not in any other tissue examined, including ciliated tissues and ovaries. Finally, immunohistochemistry of the testis shows that C(s) first appears in pachytene spermatocytes. This is the first demonstration of a cell type-specific expression for any C isoform. The conservation of C(s) throughout mammalian evolution suggests that the unique structure of C(s) is important in the subunit\u27s localization or function within the sperm

Proteomic analysis of a eukaryotic cilium

Cilia and flagella are widespread cell organelles that have been highly conserved throughout evolution and play important roles in motility, sensory perception, and the life cycles of eukaryotes ranging from protists to humans. Despite the ubiquity and importance of these organelles, their composition is not well known. Here we use mass spectrometry to identify proteins in purified flagella from the green alga Chlamydomonas reinhardtii. 360 proteins were identified with high confidence, and 292 more with moderate confidence. 97 out of 101 previously known flagellar proteins were found, indicating that this is a very complete dataset. The flagellar proteome is rich in motor and signal transduction components, and contains numerous proteins with homologues associated with diseases such as cystic kidney disease, male sterility, and hydrocephalus in humans and model vertebrates. The flagellum also contains many proteins that are conserved in humans but have not been previously characterized in any organism. The results indicate that flagella are far more complex than previously estimated

TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport

The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions

Intraflagellar transport is essential for mammalian spermiogenesis but is absent in mature sperm

Drosophila sperm are unusual in that they do not require the intraflagellar transport (IFT) system for assembly of their flagella. In the mouse, the IFT proteins are very abundant in testis, but we here show that mature sperm are completely devoid of them, making the importance of IFT to mammalian sperm development unclear. To address this question, we characterized spermiogenesis and fertility in the Ift88(Tg737Rpw) mouse. This mouse has a hypomorphic mutation in the gene encoding the IFT88 subunit of the IFT particle. This mutation is highly disruptive to ciliary assembly in other organs. Ift88(-/-) mice are completely sterile. They produce approximately 350-fold fewer sperm than wild-type mice, and the remaining sperm completely lack or have very short flagella. The short flagella rarely have axonemes but assemble ectopic microtubules and outer dense fibers and accumulate improperly assembled fibrous sheath proteins. Thus IFT is essential for the formation but not the maintenance of mammalian sperm flagella

Reduced tubulin polyglutamylation suppresses flagellar shortness in Chlamydomonas

Ciliary length control is an incompletely understood process essential for normal ciliary function. The flagella of Chlamydomonas mutants lacking multiple axonemal dyneins are shorter than normal; previously it was shown that this shortness can be suppressed by the mutation suppressor of shortness 1 (ssh1) via an unknown mechanism. To elucidate this mechanism, we carried out genetic analysis of ssh1 and found that it is a new allele of TPG2 (hereafter tpg2-3), which encodes FAP234 functioning in tubulin polyglutamylation in the axoneme. Similar to the polyglutamylation-deficient mutants tpg1 and tpg2-1, tpg2-3 axonemal tubulin has a greatly reduced level of long polyglutamate side chains. We found that tpg1 and tpg2-1 mutations also promote flagellar elongation in short-flagella mutants, consistent with a polyglutamylation-dependent mechanism of suppression. Double mutants of tpg1 or tpg2-1 and fla10-1, a temperature-sensitive mutant of intraflagellar transport, underwent slower flagellar shortening than fla10-1 at restrictive temperatures, indicating that the rate of tubulin disassembly is decreased in the polyglutamylation-deficient flagella. Moreover, alpha-tubulin incorporation into the flagellar tips in temporary dikaryons was retarded in polyglutamylation-deficient flagella. These results show that polyglutamylation deficiency stabilizes axonemal microtubules, decelerating axonemal disassembly at the flagellar tip and shifting the axonemal assembly/disassembly balance toward assembly