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

Single-particle imaging reveals intraflagellar transport–independent transport and accumulation of EB1 in \u3cem\u3eChlamydomonas\u3c/em\u3e flagella

The microtubule (MT) plus-end tracking protein EB1 is present at the tips of cilia and flagella; end-binding protein 1 (EB1) remains at the tip during flagellar shortening and in the absence of intraflagellar transport (IFT), the predominant protein transport system in flagella. To investigate how EB1 accumulates at the flagellar tip, we used in vivo imaging of fluorescent protein–tagged EB1 (EB1-FP) in Chlamydomonas reinhardtii. After photobleaching, the EB1 signal at the flagellar tip recovered within minutes, indicating an exchange with unbleached EB1 entering the flagella from the cell body. EB1 moved independent of IFT trains, and EB1-FP recovery did not require the IFT pathway. Single-particle imaging showed that EB1-FP is highly mobile along the flagellar shaft and displays a markedly reduced mobility near the flagellar tip. Individual EB1-FP particles dwelled for several seconds near the flagellar tip, suggesting the presence of stable EB1 binding sites. In simulations, the two distinct phases of EB1 mobility are sufficient to explain its accumulation at the tip. We propose that proteins uniformly distributed throughout the cytoplasm like EB1 accumulate locally by diffusion and capture; IFT, in contrast, might be required to transport proteins against cellular concentration gradients into or out of cilia

H\u3csup\u3e+\u3c/sup\u3e- and Na\u3csup\u3e+\u3c/sup\u3e- elicited rapid changes of the microtubule cytoskeleton in the biflagellated green alga \u3cem\u3eChlamydomonas\u3c/em\u3e

Although microtubules are known for dynamic instability, the dynamicity is considered to be tightly controlled to support a variety of cellular processes. Yet diverse evidence suggests that this is not applicable to Chlamydomonas, a biflagellate fresh water green alga, but intense autofluorescence from photosynthesis pigments has hindered the investigation. By expressing a bright fluorescent reporter protein at the endogenous level, we demonstrate in real time discreet sweeping changes in algal microtubules elicited by rises of intracellular H+ and Na+. These results from this model organism with characteristics of animal and plant cells provide novel explanations regarding how pH may drive cellular processes; how plants may respond to, and perhaps sense stresses; and how organisms with a similar sensitive cytoskeleton may be susceptible to environmental changes

An age of enlightenment for cilia: The FASEB summer research conference on the “Biology of Cilia and Flagella”

AbstractFrom July 19–24, 2015, 169 clinicians and basic scientists gathered in the vertiginous heights of Snowmass, Colorado (2502m) for the fourth FASEB summer research conference on the ‘Biology of Cilia and Flagella’. Organizers Maureen Barr (Rutgers University), Iain Drummond (Massachusetts General Hospital/Harvard Medical School), and Jagesh Shah (Brigham and Women's Hospital/Harvard Medical School) assembled a program filled with new data and forward-thinking ideas documenting the ongoing growth of the field. Sixty oral presentations and 77 posters covered novel aspects of cilia structure, ciliogenesis, cilia motility, cilia-mediated signaling, and cilia-related disease. In this report, we summarize the meeting, highlight exciting developments and discuss open questions

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

Mutations in Hydin impair ciliary motility in mice

Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility, and mice with Hydin defects develop lethal hydrocephalus. To determine if defects in Hydin cause hydrocephalus through a mechanism involving cilia, we compared the morphology, ultrastructure, and activity of cilia in wild-type and hydin mutant mice strains. The length and density of cilia in the brains of mutant animals is normal. The ciliary axoneme is normal with respect to the 9 + 2 microtubules, dynein arms, and radial spokes but one of the two central microtubules lacks a specific projection. The hydin mutant cilia are unable to bend normally, ciliary beat frequency is reduced, and the cilia tend to stall. As a result, these cilia are incapable of generating fluid flow. Similar defects are observed for cilia in trachea. We conclude that hydrocephalus in hydin mutants is caused by a central pair defect impairing ciliary motility and fluid transport in the brain

Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin

The assembly and maintenance of most cilia and flagella rely on intraflagellar transport (IFT). Recent in vitro studies have suggested that, together, the calponin-homology domain within the IFT81 N-terminus and the highly basic N-terminus of IFT74 form a module for IFT of tubulin. By using Chlamydomonas mutants for IFT81 and IFT74, we tested this hypothesis in vivo Modification of the predicted tubulin-binding residues in IFT81 did not significantly affect basic anterograde IFT and length of steady-state flagella but slowed down flagellar regeneration, a phenotype similar to that seen in a strain that lacks the IFT74 N-terminus. In both mutants, the frequency of tubulin transport by IFT was greatly reduced. A double mutant that combined the modifications to IFT81 and IFT74 was able to form only very short flagella. These results indicate that, together, the IFT81 and IFT74 N-termini are crucial for flagellar assembly, and are likely to function as the main module for IFT of tubulin

Diffusion rather than intraflagellar transport likely provides most of the tubulin required for axonemal assembly in Chlamydomonas

Tubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using Chlamydomonas, we attenuated IFT-based tubulin transport of GFP-beta-tubulin by altering the IFT74N-IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP-beta-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by approximately 90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP-beta-tubulin in the axoneme was only moderately reduced. In vivo imaging showed more GFP-beta-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth

The IDA3 adapter, required for intraflagellar transport of I1 dynein, is regulated by ciliary length

Axonemal dyneins, including inner dynein arm I1, assemble in the cytoplasm prior to transport into cilia by intraflagellar transport (IFT). How I1 dynein interacts with IFT is not understood. We take advantage of the Chlamydomonas reinhardtii ida3 mutant, which assembles the inner arm I1 dynein complex in the cytoplasm but fails to transport I1 into the cilium, resulting in I1 dynein-deficient axonemes with abnormal motility. The IDA3 gene encodes an ∼115-kDa coiled-coil protein that primarily enters the cilium during ciliary growth but is not an axonemal protein. During growth, IDA3, along with I1 dynein, is transported by anterograde IFT to the tip of the cilium. At the tip, IDA3 uncouples from IFT and diffuses within the cilium. IFT transport of IDA3 decreases as cilia lengthen and subsides once full length is achieved. IDA3 is the first example of an essential and selective IFT adapter that is regulated by ciliary length. </jats:p

Cycling of the signaling protein phospholipase D through cilia requires the BBSome only for the export phase

The BBSome is a complex of seven proteins, including BBS4, that is cycled through cilia by intraflagellar transport (IFT). Previous work has shown that the membrane-associated signaling protein phospholipase D (PLD) accumulates abnormally in cilia of Chlamydomonas reinhardtii bbs mutants. Here we show that PLD is a component of wild-type cilia but is enriched approximately 150-fold in bbs4 cilia; this accumulation occurs progressively over time and results in altered ciliary lipid composition. When wild-type BBSomes were introduced into bbs cells, PLD was rapidly removed from the mutant cilia, indicating the presence of an efficient BBSome-dependent mechanism for exporting ciliary PLD. This export requires retrograde IFT. Importantly, entry of PLD into cilia is BBSome and IFT independent. Therefore, the BBSome is required only for the export phase of a process that continuously cycles PLD through cilia. Another protein, carbonic anhydrase 6, is initially imported normally into bbs4 cilia but lost with time, suggesting that its loss is a secondary effect of BBSome deficiency