scholarly journals Trafficking of ciliary membrane proteins by the intraflagellar transport/BBSome machinery

2018 ◽  
Vol 62 (6) ◽  
pp. 753-763 ◽  
Author(s):  
Jenna L. Wingfield ◽  
Karl-Ferdinand Lechtreck ◽  
Esben Lorentzen
Keyword(s):  
Membrane Proteins ◽  
Molecular Motors ◽  
Intraflagellar Transport ◽  
Inherited Disease ◽  
Cell Organelles ◽  
Bardet Biedl Syndrome ◽  
Ciliary Membrane ◽  
Peripheral Membrane Proteins ◽  
Abnormal Accumulation ◽  
And Function

Bardet–Biedl syndrome (BBS) is a rare inherited disease caused by defects in the BBSome, an octameric complex of BBS proteins. The BBSome is conserved in most organisms with cilia, which are microtubule (MT)-based cell organelles that protrude from the cell surface and function in motility and sensing. Cilia assembly, maintenance, and function require intraflagellar transport (IFT), a bidirectional motility of multi-megadalton IFT trains propelled by molecular motors along the ciliary MTs. IFT has been shown to transport structural proteins, including tubulin, into growing cilia. The BBSome is an adapter for the transport of ciliary membrane proteins and cycles through cilia via IFT. While both the loss and the abnormal accumulation of ciliary membrane proteins have been observed in bbs mutants, recent data converge on a model where the BBSome mainly functions as a cargo adapter for the removal of certain transmembrane and peripheral membrane proteins from cilia. Here, we review recent data on the ultrastructure of the BBSome and how the BBSome recognizes its cargoes and mediates their removal from cilia.

scholarly journals A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Tooba Quidwai ◽  
Jiaolong Wang ◽  
Emma A Hall ◽  
Narcis A Petriman ◽  
Weihua Leng ◽  
...  
Keyword(s):  
Coat Protein ◽  
Membrane Proteins ◽  
Sequence Homology ◽  
Protein Complex ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Ciliary Membrane ◽  
Mouse Mutants ◽  
Core Components

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits, and demonstrate an accumulation of 'coat-less' vesicles which fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in-situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.

scholarly journals Caenorhabditis elegans DYF-2, an Orthologue of Human WDR19, Is a Component of the Intraflagellar Transport Machinery in Sensory Cilia

2006 ◽  
Vol 17 (11) ◽  
pp. 4801-4811 ◽  
Author(s):  
Evgeni Efimenko ◽  
Oliver E. Blacque ◽  
Guangshuo Ou ◽  
Courtney J. Haycraft ◽  
Bradley K. Yoder ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Aspartic Acid ◽  
Critical Role ◽  
Intraflagellar Transport ◽  
Bardet Biedl Syndrome ◽  
Evolutionarily Conserved ◽  
Sensory Cilia ◽  
Mutant Phenotypes ◽  
And Function

The intraflagellar transport (IFT) machinery required to build functional cilia consists of a multisubunit complex whose molecular composition, organization, and function are poorly understood. Here, we describe a novel tryptophan-aspartic acid (WD) repeat (WDR) containing IFT protein from Caenorhabditis elegans, DYF-2, that plays a critical role in maintaining the structural and functional integrity of the IFT machinery. We determined the identity of the dyf-2 gene by transgenic rescue of mutant phenotypes and by sequencing of mutant alleles. Loss of DYF-2 function selectively affects the assembly and motility of different IFT components and leads to defects in cilia structure and chemosensation in the nematode. Based on these observations, and the analysis of DYF-2 movement in a Bardet–Biedl syndrome mutant with partially disrupted IFT particles, we conclude that DYF-2 can associate with IFT particle complex B. At the same time, mutations in dyf-2 can interfere with the function of complex A components, suggesting an important role of this protein in the assembly of the IFT particle as a whole. Importantly, the mouse orthologue of DYF-2, WDR19, also localizes to cilia, pointing to an important evolutionarily conserved role for this WDR protein in cilia development and function.

scholarly journals Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei

2015 ◽  
Vol 113 (3) ◽  
pp. 632-637 ◽  
Author(s):  
Gerasimos Langousis ◽  
Michelle M. Shimogawa ◽  
Edwin A. Saada ◽  
Ajay A. Vashisht ◽  
Roberto Spreafico ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Trypanosoma Brucei ◽  
Molecular Mechanisms ◽  
Protozoan Parasite ◽  
Infection Model ◽  
Protein Distribution ◽  
Bardet Biedl Syndrome ◽  
Ciliary Membrane ◽  
Mouse Infection Model ◽  
Endocytic Sorting

Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium’s capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet–Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet–Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host–parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host–pathogen interface and offering insights into BBSome molecular mechanisms.

scholarly journals Flagella Ca2+ elevations regulate pausing of retrograde intraflagellar transport trains in adherent Chlamydomonas flagella

2020 ◽  
Author(s):  
Cecile Fort ◽  
Peter Collingridge ◽  
Colin Brownlee ◽  
Glen Wheeler
Keyword(s):  
Membrane Proteins ◽  
Green Alga ◽  
High Frequency ◽  
Intraflagellar Transport ◽  
Gliding Motility ◽  
Membrane Glycoproteins ◽  
Ciliary Membrane ◽  
Process Uncertainty ◽  
Temporal Properties ◽  
Transient Interactions

AbstractThe movement of ciliary membrane proteins is directed by transient interactions with intraflagellar transport (IFT) trains. The green alga Chlamydomonas has adapted this process for gliding motility, using IFT to move adhesive glycoproteins (FMG-1B) in the flagella membrane. Although Ca2+ signalling contributes directly to the gliding process, uncertainty remains over the mechanisms through which Ca2+ acts to influence the movement of IFT trains. Here we show that flagella Ca2+ elevations regulate IFT primarily by initiating the movement of paused retrograde IFT trains. Flagella Ca2+ elevations exhibit complex spatial and temporal properties, including high frequency repetitive Ca2+ elevations that prevent the accumulation of paused retrograde IFT trains. We show that flagella Ca2+ elevations disrupt the IFT-dependent movement of microspheres along the flagella membrane. The results suggest that flagella Ca2+ elevations directly disrupt the interaction between retrograde IFT particles and flagella membrane glycoproteins to modulate gliding motility and the adhesion of the flagellum to a surface.

scholarly journals Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Fan Ye ◽  
David K Breslow ◽  
Elena F Koslover ◽  
Andrew J Spakowitz ◽  
W James Nelson ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Active Transport ◽  
Single Molecule ◽  
Primary Cilia ◽  
Somatostatin Receptor ◽  
Intraflagellar Transport ◽  
Signal Propagation ◽  
Protein Diffusion ◽  
Ciliary Membrane ◽  
Signaling Receptors

The dynamic organization of signaling cascades inside primary cilia is key to signal propagation. Yet little is known about the dynamics of ciliary membrane proteins besides a possible role for motor-driven Intraflagellar Transport (IFT). To characterize these dynamics, we imaged single molecules of Somatostatin Receptor 3 (SSTR3, a GPCR) and Smoothened (Smo, a Hedgehog signal transducer) in the ciliary membrane. While IFT trains moved processively from one end of the cilium to the other, single SSTR3 and Smo underwent mostly diffusive behavior interspersed with short periods of directional movements. Statistical subtraction of instant velocities revealed that SSTR3 and Smo spent less than a third of their time undergoing active transport. Finally, SSTR3 and IFT movements could be uncoupled by perturbing either membrane protein diffusion or active transport. Thus ciliary membrane proteins move predominantly by diffusion, and attachment to IFT trains is transient and stochastic rather than processive or spatially determined.

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

2018 ◽  
Vol 115 (5) ◽  
pp. E934-E943 ◽  
Author(s):  
Peiwei Liu ◽  
Karl F. Lechtreck
Keyword(s):  
Protein Complex ◽  
Intraflagellar Transport ◽  
Negative Regulator ◽  
Membrane Composition ◽  
Bardet Biedl Syndrome ◽  
Ciliary Membrane ◽  
Phototactic Behavior ◽  
Dependent Transport ◽  
Particle Imaging ◽  
Single Particle Imaging

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. Chlamydomonas bbs mutants lack phototaxis and accumulate phospholipase D (PLD) in the ciliary membrane. Single particle imaging revealed that PLD comigrates with BBS4 by intraflagellar transport (IFT) while IFT of PLD is abolished in bbs mutants. BBSome deficiency did not alter the rate of PLD entry into cilia. Membrane association and the N-terminal 58 residues of PLD are sufficient and necessary for BBSome-dependent transport and ciliary export. The replacement of PLD’s ciliary export sequence (CES) caused PLD to accumulate in cilia of cells with intact BBSomes and IFT. The buildup of PLD inside cilia impaired phototaxis, revealing that PLD is a negative regulator of phototactic behavior. We conclude that the BBSome is a cargo adapter ensuring ciliary export of PLD on IFT trains to regulate phototaxis.

scholarly journals A WDR35-dependent coatomer transports ciliary membrane proteins from the Golgi to the cilia

2020 ◽  
Author(s):  
Tooba Quidwai ◽  
Emma A. Hall ◽  
Margaret A. Keighren ◽  
Weihua Leng ◽  
Petra Kiesel ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Diffusion Barrier ◽  
Sequence Homology ◽  
Intraflagellar Transport ◽  
Structural Similarity ◽  
Retrograde Transport ◽  
Ciliary Membrane ◽  
Mouse Mutants ◽  
Core Components

AbstractIntraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia and across the diffusion barrier is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the IFT-A peripheral components are degraded and core components accumulate at the transition zone. We reveal deep sequence homology and structural similarity of WDR35 and other IFT-As to the coatomer COPI proteins α and β′, and demonstrate an accumulation of ‘coat-less’ vesicles which fail to fuse with Wdr35 mutant cilia. Our data provides the first in situ evidence of a novel coatomer function for WDR35 likely with other IFT-A proteins in delivering ciliary membrane cargo from the Golgi necessary for cilia elongation.

scholarly journals Ca2+ elevations disrupt interactions between intraflagellar transport and the flagella membrane in Chlamydomonas

2021 ◽  
Vol 134 (3) ◽  
pp. jcs253492
Author(s):  
Cecile Fort ◽  
Peter Collingridge ◽  
Colin Brownlee ◽  
Glen Wheeler
Keyword(s):  
Membrane Proteins ◽  
Green Alga ◽  
High Frequency ◽  
Traction Force ◽  
Intraflagellar Transport ◽  
Gliding Motility ◽  
Membrane Glycoproteins ◽  
Ciliary Membrane ◽  
Precise Control ◽  
Transient Interactions

ABSTRACTThe movement of ciliary membrane proteins is directed by transient interactions with intraflagellar transport (IFT) trains. The green alga Chlamydomonas has adapted this process for gliding motility, using retrograde IFT motors to move adhesive glycoproteins in the flagella membrane. Ca2+ signalling contributes directly to the gliding process, although uncertainty remains over the mechanism through which it acts. Here, we show that flagella Ca2+ elevations initiate the movement of paused retrograde IFT trains, which accumulate at the distal end of adherent flagella, but do not influence other IFT processes. On highly adherent surfaces, flagella exhibit high-frequency Ca2+ elevations that prevent the accumulation of paused retrograde IFT trains. Flagella Ca2+ elevations disrupt the IFT-dependent movement of microspheres along the flagella membrane, suggesting that Ca2+ acts by directly disrupting an interaction between retrograde IFT trains and flagella membrane glycoproteins. By regulating the extent to which glycoproteins on the flagella surface interact with IFT motor proteins on the axoneme, this signalling mechanism allows precise control of traction force and gliding motility in adherent flagella.

scholarly journals Transient accumulation and bidirectional movement of KIF13B in primary cilia

2021 ◽  
Author(s):  
Alice Dupont Juhl ◽  
Zeinab Anvarian ◽  
Julia Berges ◽  
Daniel Wustner ◽  
Lotte B Pedersen
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Primary Cilia ◽  
Cell Imaging ◽  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Ciliary Membrane ◽  
Bidirectional Movement ◽  
And Function ◽  
Male Specific

Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein 2 motors. Nematodes additionally employ a male-specific kinesin-3 motor, KLP-6, which regulates ciliary content and function by promoting release of bioactive extracellular vesicles (EVs) from cilia. Here we show by live cell imaging that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured mammalian cells at 0.64 +/- 0.07 μm/s in the anterograde direction and at 0.39 +/- 0.06 μm/s in the retrograde direction, reminiscent of conventional IFT. In addition, we found that KIF13B undergoes EV-like release from the ciliary tip whereas a ciliary membrane marker, SMO-tRFP, remains stably associated with cilia during such EV release. Our results suggest that KIF13B, similar to KLP-6, regulates ciliary membrane content by promoting ciliary EV release, possibly in coordination with conventional IFT.

scholarly journals Fractionation of the Epithelial Apical Junctional Complex: Reassessment of Protein Distributions in Different Substructures

2005 ◽  
Vol 16 (2) ◽  
pp. 701-716 ◽  
Author(s):  
Roger Vogelmann ◽  
W. James Nelson
Keyword(s):  
Membrane Proteins ◽  
Protein Interactions ◽  
Cell Structure ◽  
Specific Protein ◽  
Junctional Complex ◽  
Protein Protein Interactions ◽  
Peripheral Membrane Proteins ◽  
Apical Junctional Complex ◽  
Important Regulator ◽  
And Function

The epithelial apical junctional complex (AJC) is an important regulator of cell structure and function. The AJC is compartmentalized into substructures comprising the tight and adherens junctions, and other membrane complexes containing the membrane proteins nectin, junctional adhesion molecule, and crumbs. In addition, many peripheral membrane proteins localize to the AJC. Studies of isolated proteins indicate a complex map of potential binding partners in which there is extensive overlap in the interactions between proteins in different AJC substructures. As an alternative to a direct search for specific protein-protein interactions, we sought to separate membrane substructures of the AJC in iodixanol density gradients and define their protein constituents. Results show that the AJC can be fractured into membrane substructures that contain specific membrane and peripheral membrane proteins. The composition of each substructure reveals a more limited overlap in common proteins than predicted from the inventory of potential interactions; some of the overlapping proteins may be involved in stepwise recruitment and assembly of AJC substructures.

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