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

eLife ◽  
2018 ◽  
Vol 7 ◽  
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.

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

2018 ◽  
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.

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

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.

scholarly journals Acute inhibition of heterotrimeric kinesin-2 function reveals mechanisms of intraflagellar transport in mammalian cilia

2018 ◽  
Author(s):  
Martin F. Engelke ◽  
Bridget Waas ◽  
Sarah E. Kearns ◽  
Ayana Suber ◽  
Allison Boss ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Genetic Approach ◽  
Ciliated Cells ◽  
Loss Of Function ◽  
Double Knockout ◽  
Chemical Genetic ◽  
Species Specific ◽  
And Function

ABSTRACTThe trafficking of components within cilia, called intraflagellar transport (IFT), is powered by kinesin-2 and dynein-2 motors. Loss of function in any subunit of the heterotrimeric KIF3A/KIF3B/KAP kinesin-2 motor prevents ciliogenesis in mammalian cells and has hindered an understanding of how kinesin-2 motors function in IFT. We used a chemical-genetic approach to engineer an inhibitable KIF3A/KIF3B (i3A/i3B) kinesin-2 motor that is capable of rescuing WT motor function in Kif3a/Kif3b double-knockout cells. Inhibitor addition blocks ciliogenesis or, if added to ciliated cells, blocks IFT within two minutes, which leads to a complete loss of primary cilia within six hours. The kinesin-2 family members KIF3A/KIF3C and KIF17 cannot rescue ciliogenesis in Kif3a/Kif3b double-knockout cells nor delay the disassembly of full-formed cilia upon i3A/i3B inhibition. These data suggest that KIF3A/KIF3B/KAP is the sole and essential motor for cilia assembly and function in mammalian cells, indicating a species-specific adaptation of kinesin-2 motors for IFT function.

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

2017 ◽  
Vol 216 (6) ◽  
pp. 1659-1671 ◽  
Author(s):  
Daniel Serwas ◽  
Tiffany Y. Su ◽  
Max Roessler ◽  
Shaohe Wang ◽  
Alexander Dammermann
Keyword(s):  
Fluorescence Microscopy ◽  
Transition Zone ◽  
Electron Tomography ◽  
Intraflagellar Transport ◽  
Basal Bodies ◽  
C Elegans ◽  
Cilia Formation ◽  
Syndrome Protein

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.

scholarly journals CEP19 cooperates with FOP and CEP350 to drive early steps in the ciliogenesis programme

Open Biology ◽  
2017 ◽  
Vol 7 (6) ◽  
pp. 170114 ◽  
Author(s):  
Bahareh A. Mojarad ◽  
Gagan D. Gupta ◽  
Monica Hasegan ◽  
Oumou Goudiam ◽  
Renata Basto ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Three Dimensional ◽  
Structured Illumination ◽  
Loss Of Function ◽  
Structured Illumination Microscopy ◽  
C Terminus ◽  
Interaction Partners ◽  
Cilia Formation ◽  
Mother Centriole ◽  
Section Electron

Primary cilia are microtubule-based sensory organelles necessary for efficient transduction of extracellular cues. To initiate cilia formation, ciliary vesicles (CVs) are transported to the vicinity of the centrosome where they dock to the distal end of the mother centriole and fuse to initiate cilium assembly. However, to this date, the early steps in cilia formation remain incompletely understood. Here, we demonstrate functional interplay between CEP19, FOP and CEP350 in ciliogenesis. Using three-dimensional structured-illumination microscopy (3D-SIM) imaging, we mapped the relative spatial distribution of these proteins at the distal end of the mother centriole and show that CEP350/FOP act upstream of CEP19 in their recruitment hierarchy. We demonstrate that CEP19 CRISPR KO cells are severely impaired in their ability to form cilia, analogous to the loss of function of CEP19 binding partners FOP and CEP350. Notably, in the absence of CEP19 microtubule anchoring at centromes is similar in manner to its interaction partners FOP and CEP350. Using GFP-tagged deletion constructs of CEP19, we show that the C-terminus of CEP19 is required for both its localization to centrioles and for its function in ciliogenesis. Critically, this region also mediates the interaction between CEP19 and FOP/CEP350. Interestingly, a morbid-obesity-associated R82* truncated mutant of CEP19 cannot ciliate nor interact with FOP and CEP350, indicative of a putative role for CEP19 in ciliopathies. Finally, analysis of CEP19 KO cells using thin-section electron microscopy revealed marked defects in the docking of CVs to the distal end of the mother centrioles. Together, these data demonstrate a role for the CEP19, FOP and CEP350 module in ciliogenesis and the possible effect of disrupting their functions in ciliopathies.

scholarly journals Prominins control ciliary length throughout the animal kingdom: New lessons from human prominin-1 and zebrafish prominin-3

2020 ◽  
Vol 295 (18) ◽  
pp. 6007-6022 ◽  
Author(s):  
József Jászai ◽  
Kristina Thamm ◽  
Jana Karbanová ◽  
Peggy Janich ◽  
Christine A. Fargeas ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Photoreceptor Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Loss Of Function ◽  
Animal Kingdom ◽  
Left Right Asymmetry ◽  
And Function ◽  
The Impact

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

scholarly journals NRF2-dependent gene expression promotes ciliogenesis and Hedgehog signaling

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ana Martin-Hurtado ◽  
Raquel Martin-Morales ◽  
Natalia Robledinos-Antón ◽  
Ruth Blanco ◽  
Ines Palacios-Blanco ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Embryonic Stem ◽  
Mtor Inhibitors ◽  
Gene Promoters ◽  
Protein Levels ◽  
Functional Connections ◽  
Differentiated Cells ◽  
Cilia Formation ◽  
And Function

Abstract The transcription factor NRF2 is a master regulator of cellular antioxidant and detoxification responses, but it also regulates other processes such as autophagy and pluripotency. In human embryonic stem cells (hESCs), NRF2 antagonizes neuroectoderm differentiation, which only occurs after NRF2 is repressed via a Primary Cilia-Autophagy-NRF2 (PAN) axis. However, the functional connections between NRF2 and primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae, remain poorly understood. For instance, nothing is known about whether NRF2 affects cilia, or whether cilia regulation of NRF2 extends beyond hESCs. Here, we show that NRF2 and primary cilia reciprocally regulate each other. First, we demonstrate that fibroblasts lacking primary cilia have higher NRF2 activity, which is rescued by autophagy-activating mTOR inhibitors, indicating that the PAN axis also operates in differentiated cells. Furthermore, NRF2 controls cilia formation and function. NRF2-null cells grow fewer and shorter cilia and display impaired Hedgehog signaling, a cilia-dependent pathway. These defects are not due to increased oxidative stress or ciliophagy, but rather to NRF2 promoting expression of multiple ciliogenic and Hedgehog pathway genes. Among these, we focused on GLI2 and GLI3, the transcription factors controlling Hh pathway output. Both their mRNA and protein levels are reduced in NRF2-null cells, consistent with their gene promoters containing consensus ARE sequences predicted to bind NRF2. Moreover, GLI2 and GLI3 fail to accumulate at the ciliary tip of NRF2-null cells upon Hh pathway activation. Given the importance of NRF2 and ciliary signaling in human disease, our data may have important biomedical implications.

scholarly journals Signaling compartment at the ciliary tip is formed and maintained by intraflagellar transport and functions as sensitive salt detector

2019 ◽  
Author(s):  
Servaas N. van der Burght ◽  
Suzanne Rademakers ◽  
Jacque-Lynne Johnson ◽  
Chunmei Li ◽  
Gert-Jan Kremers ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Physiological Function ◽  
Intraflagellar Transport ◽  
Distal Region ◽  
Central Question ◽  
Membrane Diffusion ◽  
High Concentration ◽  
C Elegans ◽  
Cgmp Signaling ◽  
And Function

AbstractPrimary cilia are ubiquitous antenna-like organelles that mediate cellular signaling and represent hotspots for human diseases termed ciliopathies. How signaling subcompartments are established within the microtubule-based organelle, and for example support Hedgehog or cGMP signal transduction pathways, remains a central question. Here we show that a C. elegans salt-sensing receptor type guanylate cyclase, GCY-22, accumulates at a high concentration within the distal region of the cilium. This receptor uses DAF-25 (Ankmy2 in mammals) to cross the transition zone (TZ) membrane diffusion barrier in the proximal-most region of the ciliary axoneme. Targeting of GCY-22 to the ciliary tip is dynamic, requiring the cargo-mobilizing intraflagellar transport (IFT) system. Disruption of transit across the TZ barrier or IFT trafficking causes GCY-22 protein mislocalization and defects in the formation, maintenance, and function of the ciliary tip compartment required for chemotaxis to low NaCl concentrations. Together, our findings reveal how a previously undescribed cilium tip cGMP signaling compartment is established and contributes to the physiological function of a primary cilium.

Sign in / Sign up

Export Citation Format

Share Document