Centrioles initiate cilia assembly but are dispensable for maturation and maintenance in C. elegans

Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance.

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Functional Redundancy of the B9 Proteins and Nephrocystins in Caenorhabditis elegans Ciliogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-10-1070 ◽

2008 ◽

Vol 19 (5) ◽

pp. 2154-2168 ◽

Cited By ~ 74

Author(s):

Corey L. Williams ◽

Marlene E. Winkelbauer ◽

Jenny C. Schafer ◽

Edward J. Michaud ◽

Bradley K. Yoder

Keyword(s):

Caenorhabditis Elegans ◽

Joubert Syndrome ◽

Cystic Kidney ◽

Basal Bodies ◽

The Novel ◽

C Elegans ◽

Human Genes ◽

Cilia Formation ◽

Strong Candidate ◽

Candidate Loci

Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Joubert syndrome (JBTS) are a group of heterogeneous cystic kidney disorders with partially overlapping loci. Many of the proteins associated with these diseases interact and localize to cilia and/or basal bodies. One of these proteins is MKS1, which is disrupted in some MKS patients and contains a B9 motif of unknown function that is found in two other mammalian proteins, B9D2 and B9D1. Caenorhabditis elegans also has three B9 proteins: XBX-7 (MKS1), TZA-1 (B9D2), and TZA-2 (B9D1). Herein, we report that the C. elegans B9 proteins form a complex that localizes to the base of cilia. Mutations in the B9 genes do not overtly affect cilia formation unless they are in combination with a mutation in nph-1 or nph-4, the homologues of human genes (NPHP1 and NPHP4, respectively) that are mutated in some NPHP patients. Our data indicate that the B9 proteins function redundantly with the nephrocystins to regulate the formation and/or maintenance of cilia and dendrites in the amphid and phasmid ciliated sensory neurons. Together, these data suggest that the human homologues of the novel B9 genes B9D2 and B9D1 will be strong candidate loci for pathologies in human MKS, NPHP, and JBTS.

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Dependency relationships between IFT-dependent flagellum elongation and cell morphogenesis in Leishmania

Open Biology ◽

10.1098/rsob.180124 ◽

2018 ◽

Vol 8 (11) ◽

pp. 180124 ◽

Cited By ~ 3

Author(s):

Jack Daniel Sunter ◽

Flavia Moreira-Leite ◽

Keith Gull

Keyword(s):

Fluorescence Microscopy ◽

Cell Shape ◽

Electron Tomography ◽

Intraflagellar Transport ◽

Flagellar Pocket ◽

Cell Morphogenesis ◽

Multiple Functions ◽

Cell Shape Changes ◽

Shape Changes

Flagella have multiple functions that are associated with different axonemal structures. Motile flagella typically have a 9 + 2 arrangement of microtubules, whereas sensory flagella normally have a 9 + 0 arrangement. Leishmania exhibits both of these flagellum forms and differentiation between these two flagellum forms is associated with cytoskeletal and cell shape changes. We disrupted flagellum elongation in Leishmania by deleting the intraflagellar transport (IFT) protein IFT140 and examined the effects on cell morphogenesis. Δift140 cells have no external flagellum, having only a very short flagellum within the flagellar pocket. This short flagellum had a collapsed 9 + 0 (9v) axoneme configuration reminiscent of that in the amastigote and was not attached to the pocket membrane. Although amastigote-like changes occurred in the flagellar cytoskeleton, the cytoskeletal structures of Δift140 cells retained their promastigote configurations, as examined by fluorescence microscopy of tagged proteins and serial electron tomography. Thus, Leishmania promastigote cell morphogenesis does not depend on the formation of a long flagellum attached at the neck. Furthermore, our data show that disruption of the IFT system is sufficient to produce a switch from the 9 + 2 to the collapsed 9 + 0 (9v) axonemal structure, echoing the process that occurs during the promastigote to amastigote differentiation.

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The Caenorhabditis elegans nephrocystins act as global modifiers of cilium structure

The Journal of Cell Biology ◽

10.1083/jcb.200707090 ◽

2008 ◽

Vol 180 (5) ◽

pp. 973-988 ◽

Cited By ~ 80

Author(s):

Andrew R. Jauregui ◽

Ken C.Q. Nguyen ◽

David H. Hall ◽

Maureen M. Barr

Keyword(s):

Caenorhabditis Elegans ◽

Intraflagellar Transport ◽

Basal Bodies ◽

Structural Components ◽

Transition Zones ◽

C Elegans ◽

Cell Type Specific ◽

Children And Young Adults ◽

Stage Renal Disease ◽

End Stage

Nephronophthisis (NPHP) is the most common genetic cause of end-stage renal disease in children and young adults. In Chlamydomonas reinhardtii, Caenorhabditis elegans, and mammals, the NPHP1 and NPHP4 gene products nephrocystin-1 and nephrocystin-4 localize to basal bodies or ciliary transition zones (TZs), but their function in this location remains unknown. We show here that loss of C. elegans NPHP-1 and NPHP-4 from TZs is tolerated in developing cilia but causes changes in localization of specific ciliary components and a broad range of subtle axonemal ultrastructural defects. In amphid channel cilia, nphp-4 mutations cause B tubule defects that further disrupt intraflagellar transport (IFT). We propose that NPHP-1 and NPHP-4 act globally at the TZ to regulate ciliary access of the IFT machinery, axonemal structural components, and signaling molecules, and that perturbing this balance results in cell type–specific phenotypes.

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STORM imaging reveals the spatial arrangement of transition zone components and IFT particles at the ciliary base in Tetrahymena

Scientific Reports ◽

10.1038/s41598-021-86909-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Khodor S. Hazime ◽

Zhu Zhou ◽

Ewa Joachimiak ◽

Natalia A. Bulgakova ◽

Dorota Wloga ◽

...

Keyword(s):

Transition Zone ◽

Spatial Organization ◽

Intraflagellar Transport ◽

Spatial Arrangement ◽

Interaction Sites ◽

Narrow Region ◽

Cilia Formation ◽

Axial Dimension ◽

Optical Reconstruction ◽

And Function

AbstractThe base of the cilium comprising the transition zone (TZ) and transition fibers (TF) acts as a selecting gate to regulate the intraflagellar transport (IFT)-dependent trafficking of proteins to and from cilia. Before entering the ciliary compartment, IFT complexes and transported cargoes accumulate at or near the base of the cilium. The spatial organization of IFT proteins at the cilia base is key for understanding cilia formation and function. Using stochastic optical reconstruction microscopy (STORM) and computational averaging, we show that seven TZ, nine IFT, three Bardet–Biedl syndrome (BBS), and one centrosomal protein, form 9-clustered rings at the cilium base of a ciliate Tetrahymena thermophila. In the axial dimension, analyzed TZ proteins localize to a narrow region of about 30 nm while IFT proteins dock approximately 80 nm proximal to TZ. Moreover, the IFT-A subcomplex is positioned peripheral to the IFT-B subcomplex and the investigated BBS proteins localize near the ciliary membrane. The positioning of the HA-tagged N- and C-termini of the selected proteins enabled the prediction of the spatial orientation of protein particles and likely cargo interaction sites. Based on the obtained data, we built a comprehensive 3D-model showing the arrangement of the investigated ciliary proteins.

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Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

10.1101/251694 ◽

2018 ◽

Cited By ~ 1

Author(s):

Laura Vuolo ◽

Nicola L. Stevenson ◽

Kate J. Heesom ◽

David J. Stephens

Keyword(s):

Transition Zone ◽

Cell Lines ◽

Primary Cilia ◽

Point Mutations ◽

Intraflagellar Transport ◽

Jeune Syndrome ◽

Cilia Formation ◽

Microtubule Motor ◽

And Function ◽

Intermediate Chains

AbstractThe dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 comprises a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilia function. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport, only WDR34 is essential for axoneme extension. In contrast, only WDR60 is essential for co-assembly of the other subunits. Furthermore, WDR60 cannot compensate for loss of WDR34 or vice versa. This work defines a functional asymmetry to match the subunit asymmetry within the dynein-2 motor. Analysis of causative point mutations in WDR34 and WDR60 can partially restore function to knockout cells. Our data show that Jeune syndrome is caused by defects in transition zone architecture as well as intraflagellar transport.SUMMARYHere, Vuolo and colleagues use engineered knockout human cell lines to define roles for dynein-2 intermediate chains. WDR34 is required for axoneme extension, while WDR60 is not. Both subunits are required for cilia transition zone organization and bidirectional intraflagellar transport.

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MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis

The Journal of Cell Biology ◽

10.1083/jcb.201012116 ◽

2011 ◽

Vol 192 (6) ◽

pp. 1023-1041 ◽

Cited By ~ 293

Author(s):

Corey L. Williams ◽

Chunmei Li ◽

Katarzyna Kida ◽

Peter N. Inglis ◽

Swetha Mohan ◽

...

Keyword(s):

Transition Zone ◽

Basal Body ◽

Intraflagellar Transport ◽

C Elegans ◽

Disease Spectrum ◽

Phenotypic Features ◽

Surrounding Membrane

Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and related ciliopathies present with overlapping phenotypes and display considerable allelism between at least twelve different genes of largely unexplained function. We demonstrate that the conserved C. elegans B9 domain (MKS-1, MKSR-1, and MKSR-2), MKS-3/TMEM67, MKS-5/RPGRIP1L, MKS-6/CC2D2A, NPHP-1, and NPHP-4 proteins exhibit essential, collective functions at the transition zone (TZ), an underappreciated region at the base of all cilia characterized by Y-shaped assemblages that link axoneme microtubules to surrounding membrane. These TZ proteins functionally interact as members of two distinct modules, which together contribute to an early ciliogenic event. Specifically, MKS/MKSR/NPHP proteins establish basal body/TZ membrane attachments before or coinciding with intraflagellar transport–dependent axoneme extension and subsequently restrict accumulation of nonciliary components within the ciliary compartment. Together, our findings uncover a unified role for eight TZ-localized proteins in basal body anchoring and establishing a ciliary gate during ciliogenesis, and suggest that disrupting ciliary gate function contributes to phenotypic features of the MKS/NPHP disease spectrum.

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Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

eLife ◽

10.7554/elife.39655 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 9

Author(s):

Laura Vuolo ◽

Nicola L Stevenson ◽

Kate J Heesom ◽

David J Stephens

Keyword(s):

Transition Zone ◽

Primary Cilia ◽

Complex Function ◽

Intraflagellar Transport ◽

Loss Of Function ◽

Jeune Syndrome ◽

Cilia Formation ◽

Microtubule Motor ◽

And Function ◽

Intermediate Chains

The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport.

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In situ architecture of the ciliary base reveals the stepwise assembly of IFT trains

10.1101/2021.10.17.464685 ◽

2021 ◽

Author(s):

Hugo van den Hoek ◽

Nikolai Klena ◽

Mareike A Jordan ◽

Gonzalo Alvarez Viar ◽

Miroslava Schaffer ◽

...

Keyword(s):

Transition Zone ◽

Electron Tomography ◽

Intraflagellar Transport ◽

Cellular Functions ◽

Cryo Electron Tomography ◽

Cargo Proteins ◽

Stepwise Assembly

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. Here, we use in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base. We combine this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which IFT-A, then dynein-1b, and finally kinesin-2 sequentially bind before entry into the cilium.

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