scholarly journals Ciliary membrane proteins traffic through the Golgi via a Rabep1/GGA1/Arl3-dependent mechanism

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Hyunho Kim ◽  
Hangxue Xu ◽  
Qin Yao ◽  
Weizhe Li ◽  
Qiong Huang ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Ciliary Membrane ◽  
Dependent Mechanism

scholarly journals Trafficking to the primary cilium membrane

2017 ◽  
Vol 28 (2) ◽  
pp. 233-239 ◽  
Author(s):  
Saikat Mukhopadhyay ◽  
Hemant B. Badgandi ◽  
Sun-hee Hwang ◽  
Bandarigoda Somatilaka ◽  
Issei S. Shimada ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Signaling Pathways ◽  
Primary Cilium ◽  
Hedgehog Signaling ◽  
Diffusion Barriers ◽  
Disease Pathogenesis ◽  
Ciliary Membrane ◽  
Multiple Organs ◽  
Sensory Reception

The primary cilium has been found to be associated with a number of cellular signaling pathways, such as vertebrate hedgehog signaling, and implicated in the pathogenesis of diseases affecting multiple organs, including the neural tube, kidney, and brain. The primary cilium is the site where a subset of the cell's membrane proteins is enriched. However, pathways that target and concentrate membrane proteins in cilia are not well understood. Processes determining the level of proteins in the ciliary membrane include entry into the compartment, removal, and retention by diffusion barriers such as the transition zone. Proteins that are concentrated in the ciliary membrane are also localized to other cellular sites. Thus it is critical to determine the particular role for ciliary compartmentalization in sensory reception and signaling pathways. Here we provide a brief overview of our current understanding of compartmentalization of proteins in the ciliary membrane and the dynamics of trafficking into and out of the cilium. We also discuss major unanswered questions regarding the role that defects in ciliary compartmentalization might play in disease pathogenesis. Understanding the trafficking mechanisms that underlie the role of ciliary compartmentalization in signaling might provide unique approaches for intervention in progressive ciliopathies.

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 Evidence for a tubulin-containing lipid-protein structural complex in ciliary membranes.

1985 ◽  
Vol 100 (4) ◽  
pp. 1082-1090 ◽  
Author(s):  
R E Stephens
Keyword(s):  
Membrane Proteins ◽  
Critical Micelle Concentration ◽  
Analytical Ultracentrifugation ◽  
Lipid Fraction ◽  
Moderate Increase ◽  
Or Efficiency ◽  
Lipid Complex ◽  
Ciliary Membrane ◽  
Freeze Thaw ◽  
Membrane Tubulin

The proteins and lipids of the scallop gill ciliary membrane may be reassociated through several cycles of detergent solubilization, detergent removal, and freeze-thaw, without significant change in overall protein composition. Membrane proteins and lipids reassociate to form vesicles of uniform, discrete density classes under a variety of reassociation conditions involving detergent removal and concentration. Freed of the solubilizing detergent during equilibrium centrifugation, a protein-lipid complex equilibrates to a position on a sucrose density gradient characteristic of the original membrane density. When axonemal tubulin is solubilized by dialysis, mixed with 2:1 lecithin/cholesterol dissolved in Nonidet P-40, freed of detergent, and reconstituted by freeze-thaw, vesicles of a density essentially equal to pure lipid result. If the lipid fraction is derived through chloroform-methanol extraction of natural ciliary membranes, a moderate increase in density occurs upon reconstitution, but the protein is adsorbed and most is removed by a simple low ionic strength wash, in contrast to vesicles reconstituted from membrane proteins where even high salt extraction causes no loss of protein. The proteins of the ciliary membrane dissolve with constant composition, regardless of the type, concentration, or efficiency of detergent. Analytical ultracentrifugation demonstrates that monodisperse mixed micelles form at high detergent concentrations, but that membranes are dispersed to large sedimentable aggregates by Nonidet P-40 even at several times the critical micelle concentration, which suggests reasons for the efficacy of certain detergent for the production of ATP-reactivatable cell models. In extracts freed of detergent, structured polydisperse particles, but not membrane vesicles, are seen in negative staining; vesicles form upon concentration of the extract. Membrane tubulin is not in a form that will freely undergo electrophoresis, even in the presence of detergent above the critical micelle concentration. All chromatographic attempts to separate membrane tubulin from other membrane proteins have failed; lipid and protein are excluded together by gel filtration in the presence of high concentrations of detergent. These observations support the idea that a relatively stable lipid-protein complex exists in the ciliary membrane and that in this complex membrane tubulin is tightly associated with lipids and with a number of other proteins.

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 Formation of the B9-domain protein complex MKS1–B9D2–B9D1 is essential as a diffusion barrier for ciliary membrane proteins

2020 ◽  
Vol 31 (20) ◽  
pp. 2259-2268
Author(s):  
Misato Okazaki ◽  
Takuya Kobayashi ◽  
Shuhei Chiba ◽  
Ryota Takei ◽  
Luxiaoxue Liang ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Transition Zone ◽  
Diffusion Barrier ◽  
Protein Complex ◽  
Ciliary Membrane ◽  
Meckel Syndrome ◽  
Interaction Mode ◽  
Domain Protein

Meckel syndrome (MKS)1, B9 domain (B9D)1, and B9D2 are soluble transition zone (TZ) proteins and share a B9D. We demonstrate the interaction mode of these B9D proteins to be MKS1-B9D2-B9D1 and their interdependent localization to the TZ. We also show that formation of the B9D protein complex is crucial for creating a diffusion barrier for ciliary membrane proteins.

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 Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins.

1983 ◽  
Vol 97 (5) ◽  
pp. 1412-1420 ◽  
Author(s):  
L Eisenbach ◽  
R Ramanathan ◽  
D L Nelson
Keyword(s):  
Membrane Proteins ◽  
Cell Immobilization ◽  
Excitable Membrane ◽  
Cross Linking ◽  
Paramecium Tetraurelia ◽  
Electron Microscopic ◽  
Ciliary Membrane ◽  
Electrophysiological Properties ◽  
Immobilization Antigen ◽  
Major Antigen

The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. We produced rabbit antisera against isolated ciliary membranes and against partially purified immobilization antigens derived from three serotypes (A, B, and H), and used these antisera as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross-reactivities of the antisera were examined. We identified the antigens recognized by these sera using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), approximately 250,000 mol wt. Several secondary antigens, including a family of polypeptides of 42,000-45,000 mol wt, are common to the membranes of serotypes A, B, and H, and antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for our studies on the effects of the antibodies on electrophysiological properties of cells and electron microscopic localization studies, which are reported in the accompanying paper. We have also used these antibodies to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i-antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. We conclude that antigen-antibody interaction per se is not sufficient for immobilization, and that antibody bivalency, which allows antigen cross-linking, is essential.

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.

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