Mastigoneme structure reveals insights into the O-linked glycosylation code of native hydroxyproline-rich helices.

Hydroxyproline-rich glycoproteins (HRGPs) are a ubiquitous class of protein in the extracellular matrices and cell walls of plants and algae, yet little is known of their native structures or interactions. Here, we used electron cryomicroscopy (cryo-EM) to determine the structure of the hydroxyproline-rich mastigoneme, an extracellular filament isolated from the cilia of the alga Chlamydomonas reinhardtii. The structure demonstrates that mastigonemes are formed from two HRGPs (a filament of MST1 wrapped around a single copy of MST3) that both have hyperglycosylated poly(hydroxyproline) helices.

Distribution and bulk flow analyses of the intraflagellar transport (IFT) motor kinesin-2 support an "on-demand" model for Chlamydomonas ciliary length control.

Most cells tightly control the length of their cilia. The regulation likely involves intraflagellar transport (IFT), a bidirectional motility of multi-subunit particles organized into trains that deliver building blocks into the organelle. In Chlamydomonas, the anterograde IFT motor kinesin-2 consists of the motor subunits FLA8 and FLA10 and the nonmotor subunit KAP.

Assembly of FAP93 at the proximal axoneme in Chlamydomonas cilia.

To identify proteins specific to the proximal ciliary axoneme, we used iTRAQ to compare short (~2 μm) and full-length (~11 μm) axonemes of Chlamydomonas. Known components of the proximal axoneme such as minor dynein heavy chains and LF5 kinase as well as the ciliary tip proteins FAP256 (CEP104) and EB1 were enriched in short axonemes whereas proteins present along the length of the axoneme were of similar abundance in both samples. The iTRAQ analysis revealed that FAP93, a protein of unknown function, and protein phosphatase 2A (PP2A) are enriched in the short axonemes.

The Small Interactor of PKD2 protein promotes the assembly and ciliary entry of the Chlamydomonas PKD2-mastigoneme complexes.

In Chlamydomonas, the channel polycystin 2 (PKD2) is primarily present in the distal region of cilia, where it is attached to the axoneme and mastigonemes, extracellular polymers of MST1. In a smaller proximal ciliary region that lacks mastigonemes, PKD2 is more mobile. We show that the PKD2 regions are established early during ciliogenesis and increase proportionally in length as cilia elongate.

Global and local functions of the Fused kinase ortholog CdaH in intracellular patterning in Tetrahymena.

Ciliates assemble numerous microtubular structures into complex cortical patterns. During ciliate division, the pattern is duplicated by intracellular segmentation that produces a tandem of daughter cells. In Tetrahymena thermophila, the induction and positioning of the division boundary involves two mutually antagonistic factors: posterior CdaA (cyclin E) and anterior CdaI (Hippo kinase).

Small Interactor of PKD2 (SIP), a novel PKD2-related single-pass transmembrane protein, is required for proteolytic processing and ciliary import of PKD2.

In cilia, the ciliopathy-relevant TRP channel PKD2 is spatially compartmentalized into a distal region, in which PKD2 binds the axoneme and extracellular mastigonemes, and a smaller proximal region, in which PKD2 is more mobile and lacks mastigonemes. Here, we show that the two PKD2 regions are established early during cilia regeneration and increase in length as cilia elongate. In abnormally long cilia, only the distal region elongated whereas both regions adjusted in length during cilia shortening.

Wbm0076, a candidate effector protein of the Wolbachia endosymbiont of Brugia malayi, disrupts eukaryotic actin dynamics.

Brugia malayi, a parasitic roundworm of humans, is colonized by the obligate intracellular bacterium, Wolbachia pipientis. The symbiosis between this nematode and bacterium is essential for nematode reproduction and long-term survival in a human host. Therefore, identifying molecular mechanisms required by Wolbachia to persist in and colonize B.

ARMC2/PF27 is an obligate cargo adapter for intraflagellar transport of radial spokes.

Intraflagellar transport (IFT) carries proteins into flagella but how IFT trains interact with the large number of diverse proteins required to assemble flagella remains largely unknown. Here, we show that IFT of radial spokes in requires ARMC2/PF27, a conserved armadillo repeat protein associated with male infertility and reduced lung function. ARMC2 was highly enriched in growing flagella and tagged ARMC2 and the spoke protein RSP3 co-migrated on anterograde trains.

Bardet-Biedl syndrome 3 protein promotes ciliary exit of the signaling protein phospholipase D via the BBSome.

Certain ciliary signaling proteins couple with the BBSome, a conserved complex of Bardet-Biedl syndrome (BBS) proteins, to load onto retrograde intraflagellar transport (IFT) trains for their removal out of cilia in . Here, we show that loss of the Arf-like 6 (ARL6) GTPase BBS3 causes the signaling protein phospholipase D (PLD) to accumulate in cilia. Upon targeting to the basal body, BBSomes enter and cycle through cilia via IFT, while BBS3 in a GTP-bound state separates from BBSomes, associates with the membrane, and translocates from the basal body to cilia by diffusion.

The BBSome restricts entry of tagged carbonic anhydrase 6 into the cis-flagellum of Chlamydomonas reinhardtii.

The two flagella of Chlamydomonas reinhardtii are of the same size and structure but display functional differences, which are critical for flagellar steering movements. However, biochemical differences between the two flagella have not been identified. Here, we show that fluorescence protein-tagged carbonic anhydrase 6 (CAH6-mNG) preferentially localizes to the trans-flagellum, which is organized by the older of the two flagella-bearing basal bodies.

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

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 , we attenuated IFT-based tubulin transport of GFP-β-tubulin by altering the IFT74N-IFT81N tubulin-binding module and the C-terminal E-hook of tubulin.

Intraflagellar transport protein RABL5/IFT22 recruits the BBSome to the basal body through the GTPase ARL6/BBS3.

Bardet-Biedl syndrome (BBS) is a ciliopathy caused by defects in the assembly or distribution of the BBSome, a conserved protein complex. The BBSome cycles via intraflagellar transport (IFT) through cilia to transport signaling proteins. How the BBSome is recruited to the basal body for binding to IFT trains for ciliary entry remains unknown.

In vivo analysis of outer arm dynein transport reveals cargo-specific intraflagellar transport properties.

Outer dynein arms (ODAs) are multiprotein complexes that drive flagellar beating. Based on genetic and biochemical analyses, ODAs preassemble in the cell body and then move into the flagellum by intraflagellar transport (IFT). To study ODA transport in vivo, we expressed the essential intermediate chain 2 tagged with mNeonGreen (IC2-NG) to rescue the corresponding Chlamydomonas reinhardtii mutant oda6.

In vivo analyses of radial spoke transport, assembly, repair and maintenance.

Radial spokes (RSs) are multiprotein complexes that regulate dynein activity. In the cell body, RS proteins (RSPs) are present in a 12S precursor, which enters the flagella and converts into the axoneme-bound 20S spokes consisting of a head and stalk. To study RS dynamics in vivo, we expressed fluorescent protein (FP)-tagged versions of the head protein RSP4 and the stalk protein RSP3 to rescue the corresponding Chlamydomonas mutants pf1, lacking spoke heads, and pf14, lacking RSs entirely.

The Bardet-Biedl syndrome protein complex is an adapter expanding the cargo range of intraflagellar transport trains for ciliary export.

Bardet-Biedl syndrome (BBS) is a ciliopathy resulting from defects in the BBSome, a conserved protein complex. BBSome mutations affect ciliary membrane composition, impairing cilia-based signaling. The mechanism by which the BBSome regulates ciliary membrane content remains unknown.

H- and Na- elicited rapid changes of the microtubule cytoskeleton in the biflagellated green alga .

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 , 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.

Protein transport in growing and steady-state cilia.

Cilia and eukaryotic flagella are threadlike cell extensions with motile and sensory functions. Their assembly requires intraflagellar transport (IFT), a bidirectional motor-driven transport of protein carriers along the axonemal microtubules. IFT moves ample amounts of structural proteins including tubulin into growing cilia likely explaining its critical role for assembly.

Methods for Studying Movement of Molecules Within Cilia.

The assembly of cilia and eukaryotic flagella (interchangeable terms) requires the import of numerous proteins from the cell body into the growing organelle. Proteins move into and inside cilia by diffusion and by motor-based intraflagellar transport (IFT). Many aspects of ciliary protein transport such as the distribution of unloading sites and the frequency of transport can be analyzed using direct in vivo imaging of fluorescently tagged proteins.

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.

Single-particle imaging reveals intraflagellar transport-independent transport and accumulation of EB1 in Chlamydomonas 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.

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

From July 19-24, 2015, 169 clinicians and basic scientists gathered in the vertiginous heights of Snowmass, Colorado (2502 m) 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.

IFT-Cargo Interactions and Protein Transport in Cilia.

The motile and sensory functions of cilia and flagella are indispensable for human health. Cilia assembly requires a dedicated protein shuttle, intraflagellar transport (IFT), a bidirectional motility of multi-megadalton protein arrays along ciliary microtubules. IFT functions as a protein carrier delivering hundreds of distinct proteins into growing cilia.

Tubulin transport by IFT is upregulated during ciliary growth by a cilium-autonomous mechanism.

The assembly of the axoneme, the structural scaffold of cilia and flagella, requires translocation of a vast quantity of tubulin into the growing cilium, but the mechanisms that regulate the targeting, quantity, and timing of tubulin transport are largely unknown. In Chlamydomonas, GFP-tagged α-tubulin enters cilia as an intraflagellar transport (IFT) cargo and by diffusion. IFT-based transport of GFP-tubulin is elevated in growing cilia and IFT trains carry more tubulin.

Kinesin-13 regulates the quantity and quality of tubulin inside cilia.

Kinesin-13, an end depolymerizer of cytoplasmic and spindle microtubules, also affects the length of cilia. However, in different models, depletion of kinesin-13 either lengthens or shortens cilia, and therefore the exact function of kinesin-13 in cilia remains unclear. We generated null mutations of all kinesin-13 paralogues in the ciliate Tetrahymena.

NPHP4 controls ciliary trafficking of membrane proteins and large soluble proteins at the transition zone.

The protein nephrocystin-4 (NPHP4) is widespread in ciliated organisms, and defects in NPHP4 cause nephronophthisis and blindness in humans. To learn more about the function of NPHP4, we have studied it in Chlamydomonas reinhardtii. NPHP4 is stably incorporated into the distal part of the flagellar transition zone, close to the membrane and distal to CEP290, another transition zone protein.