ESCRT subunit CHMP4B localizes to primary cilia and is required for the structural integrity of the ciliary membrane

2019 ◽  
Vol 34 (1) ◽  
pp. 1331-1344 ◽  
Author(s):  
Eunji Jung ◽  
Tae‐Ik Choi ◽  
Ji‐Eun Lee ◽  
Cheol‐Hee Kim ◽  
Joon Kim
Keyword(s):  
Primary Cilia ◽  
Structural Integrity ◽  
Ciliary Membrane

scholarly journals A Model for Primary Cilium Biogenesis by Polarized Epithelial Cells: Role of the Midbody Remnant and Associated Specialized Membranes

2021 ◽  
Vol 8 ◽  
Author(s):  
Leticia Labat-de-Hoz ◽  
Armando Rubio-Ramos ◽  
Javier Casares-Arias ◽  
Miguel Bernabé-Rubio ◽  
Isabel Correas ◽  
...  
Keyword(s):  
Cell Surface ◽  
Primary Cilium ◽  
Primary Cilia ◽  
Control Cell ◽  
Future Research ◽  
Alternative Route ◽  
Ciliary Membrane ◽  
Cilium Formation ◽  
Membrane Compartmentalization

Primary cilia are solitary, microtubule-based protrusions surrounded by a ciliary membrane equipped with selected receptors that orchestrate important signaling pathways that control cell growth, differentiation, development and homeostasis. Depending on the cell type, primary cilium assembly takes place intracellularly or at the cell surface. The intracellular route has been the focus of research on primary cilium biogenesis, whereas the route that occurs at the cell surface, which we call the “alternative” route, has been much less thoroughly characterized. In this review, based on recent experimental evidence, we present a model of primary ciliogenesis by the alternative route in which the remnant of the midbody generated upon cytokinesis acquires compact membranes, that are involved in compartmentalization of biological membranes. The midbody remnant delivers part of those membranes to the centrosome in order to assemble the ciliary membrane, thereby licensing primary cilium formation. The midbody remnant's involvement in primary cilium formation, the regulation of its inheritance by the ESCRT machinery, and the assembly of the ciliary membrane from the membranes originally associated with the remnant are discussed in the context of the literature concerning the ciliary membrane, the emerging roles of the midbody remnant, the regulation of cytokinesis, and the role of membrane compartmentalization. We also present a model of cilium emergence during evolution, and summarize the directions for future research.

scholarly journals FBW7 couples structural integrity with functional output of primary cilia

2020 ◽  
Author(s):  
Eleni Petsouki ◽  
Vasileios Gerakopoulos ◽  
Nicholas Szeto ◽  
Wenhan Chang ◽  
Mary Beth Humphrey ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Structural Integrity ◽  
Stem Cell Differentiation ◽  
Bone Architecture ◽  
Structural Defects ◽  
Independent Manner ◽  
Mesenchymal Stem Cell Differentiation ◽  
Cilia Formation ◽  
Length Changes ◽  
Msc Differentiation

AbstractStructural defects in cilia have robust effects in diverse tissues and systems. However, how ciliary length changes influence signaling output are unknown. Here, we examined the functional role of a ciliary length control mechanism whereby FBW7-mediated destruction of NDE1 positively regulated ciliary length, in mesenchymal stem cell differentiation. We show that FBW7 functions as a master regulator of both negative (NDE1) and positive (TALPID3) regulators of ciliogenesis, with an overall positive net effect on cilia formation, MSC differentiation, and bone architecture. Deletion of Fbxw7 suppresses ciliation, Hedgehog activity, and differentiation, which are rescued in Fbxw7/Nde1-null cells. However, despite formation of abnormally long cilia in Nde1-null cells, MSC differentiation is suppressed. NDE1 promotes MSC differentiation by increasing the activity of the Hedgehog pathway by direct binding and enhancing GLI2 activity in a cilia-independent manner. We propose that ciliary structure-function coupling is determined by intricate interactions of structural and functional proteins.

scholarly journals Defining the Layers of a Sensory Cilium with STORM and Cryo-Electron Nanoscopies

2017 ◽  
Author(s):  
Michael A. Robichaux ◽  
Valencia L. Potter ◽  
Zhixian Zhang ◽  
Feng He ◽  
Michael F. Schmid ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Electron Tomography ◽  
Super Resolution ◽  
Intraflagellar Transport ◽  
Structural Features ◽  
Animal Cells ◽  
Ciliary Membrane ◽  
Acetylated Tubulin ◽  
Sensory Cilium ◽  
Syntaxin 3

ABSTRACTPrimary cilia are cylindrical organelles extending from the surface of most animal cells that have been implicated in a host of signaling and sensory functions. Genetic defects in their component molecules, known as “ciliopathies” give rise to devastating symptoms, ranging from defective development, to kidney disease, to progressive blindness. The detailed structures of these organelles and the true functions of proteins encoded by ciliopathy genes are poorly understood because of the small size of cilia and the limitations of conventional microscopic techniques. We describe the combination of cryo-electron tomography, enhanced by sub-tomogram averaging, with super-resolution stochastic reconstruction microscopy (STORM) to define substructures and subdomains within the light-sensing rod sensory cilium of the mammalian retina. Longitudinal and radial domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of these compartments, including Ca2+-binding protein centrin-2 in the lumen of the axoneme, acetylated tubulin forming the axoneme, the glycocalyx extending outward from the surface of the plasma membrane, and molecular residents of the space between axoneme and ciliary membrane, including Arl13B, intraflagellar transport proteins, BBS5, and syntaxin-3. Within this framework we document that deficiencies in the ciliopathy proteins BBS2, BBS7 and BBS9 lead to inappropriate accumulation of proteins in rod outer segments while largely preserving their sub-domain localization within the connecting cilium region, but alter the distribution of syntaxin-3 clusters.

scholarly journals Lateral transport of Smoothened from the plasma membrane to the membrane of the cilium

2009 ◽  
Vol 187 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Ljiljana Milenkovic ◽  
Matthew P. Scott ◽  
Rajat Rohatgi
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Primary Cilia ◽  
Protein Transport ◽  
Transmembrane Protein ◽  
Signaling Proteins ◽  
Lateral Transport ◽  
Transport Pathway ◽  
Ciliary Membrane ◽  
Specific Proteins

The function of primary cilia depends critically on the localization of specific proteins in the ciliary membrane. A major challenge in the field is to understand protein trafficking to cilia. The Hedgehog (Hh) pathway protein Smoothened (Smo), a 7-pass transmembrane protein, moves to cilia when a ligand is received. Using microscopy-based pulse-chase analysis, we find that Smo moves through a lateral transport pathway from the plasma membrane to the ciliary membrane. Lateral movement, either via diffusion or active transport, is quite distinct from currently studied pathways of ciliary protein transport in mammals, which emphasize directed trafficking of Golgi-derived vesicles to the base of the cilium. We anticipate that this alternative route will be used by other signaling proteins that function at cilia. The path taken by Smo may allow novel strategies for modulation of Hh signaling in cancer and regeneration.

scholarly journals BLOC-1 is required for selective membrane protein trafficking from endosomes to primary cilia

2017 ◽  
Vol 216 (7) ◽  
pp. 2131-2150 ◽  
Author(s):  
William J. Monis ◽  
Victor Faundez ◽  
Gregory J. Pazour
Keyword(s):  
Protein Trafficking ◽  
Primary Cilia ◽  
Recycling Endosome ◽  
Ciliary Membrane ◽  
Membrane Protein Trafficking ◽  
Selective Membrane ◽  
Specific Proteins ◽  
Lysosome Related Organelles ◽  
Extracellular Environment

Primary cilia perceive the extracellular environment through receptors localized in the ciliary membrane, but mechanisms directing specific proteins to this domain are poorly understood. To address this question, we knocked down proteins potentially important for ciliary membrane targeting and determined how this affects the ciliary trafficking of fibrocystin, polycystin-2, and smoothened. Our analysis showed that fibrocystin and polycystin-2 are dependent on IFT20, GMAP210, and the exocyst complex, while smoothened delivery is largely independent of these components. In addition, we found that polycystin-2, but not smoothened or fibrocystin, requires the biogenesis of lysosome-related organelles complex-1 (BLOC-1) for ciliary delivery. Consistent with the role of BLOC-1 in sorting from the endosome, we find that disrupting the recycling endosome reduces ciliary polycystin-2 and causes its accumulation in the recycling endosome. This is the first demonstration of a role for BLOC-1 in ciliary assembly and highlights the complexity of pathways taken to the cilium.

scholarly journals Exposure to 16 Hz Pulsed Electromagnetic Fields Protect the Structural Integrity of Primary Cilia and Associated TGF-β Signaling in Osteoprogenitor Cells Harmed by Cigarette Smoke

2021 ◽  
Vol 22 (13) ◽  
pp. 7036
Author(s):  
Yangmengfan Chen ◽  
Romina H. Aspera-Werz ◽  
Maximilian M. Menger ◽  
Karsten Falldorf ◽  
Michael Ronniger ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cigarette Smoke ◽  
Electromagnetic Fields ◽  
Primary Cilia ◽  
Structural Integrity ◽  
Immunofluorescent Staining ◽  
Pulsed Electromagnetic Fields ◽  
Migration Assay ◽  
Daily Exposure ◽  
Pulsed Electromagnetic

Cigarette smoking (CS) is one of the main factors related to avoidable diseases and death across the world. Cigarette smoke consists of numerous toxic compounds that contribute to the development of osteoporosis and fracture nonunion. Exposure to pulsed electromagnetic fields (PEMF) was proven to be a safe and effective therapy to support bone fracture healing. The aims of this study were to investigate if extremely low frequency (ELF-) PEMFs may be beneficial to treat CS-related bone disease, and which effect the duration of the exposure has. In this study, immortalized human mesenchymal stem cells (SCP-1 cells) impaired by 5% cigarette smoke extract (CSE) were exposed to ELF-PEMFs (16 Hz) with daily exposure ranging from 7 min to 90 min. Cell viability, adhesion, and spreading were evaluated by Sulforhodamine B, Calcein-AM staining, and Phalloidin-TRITC/Hoechst 33342 staining. A migration assay kit was used to determine cell migration. Changes in TGF-β signaling were evaluated with an adenoviral Smad2/3 reporter assay, RT-PCR, and Western blot. The structure and distribution of primary cilia were analyzed with immunofluorescent staining. Our data indicate that 30 min daily exposure to a specific ELF-PEMF most effectively promoted cell viability, enhanced cell adhesion and spreading, accelerated migration, and protected TGF-β signaling from CSE-induced harm. In summary, the current results provide evidence that ELF-PEMF can be used to support early bone healing in patients who smoke.

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 FBW7 couples structural integrity with functional output of primary cilia

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Eleni Petsouki ◽  
Vasileios Gerakopoulos ◽  
Nicholas Szeto ◽  
Wenhan Chang ◽  
Mary Beth Humphrey ◽  
...  
Keyword(s):  
Stem Cell ◽  
Primary Cilia ◽  
Structural Integrity ◽  
Interaction Network ◽  
Stem Cell Differentiation ◽  
Bone Architecture ◽  
Structural Defects ◽  
Independent Manner ◽  
And Function ◽  
Msc Differentiation

AbstractStructural defects in primary cilia have robust effects in diverse tissues and systems. However, how disorders of ciliary length lead to functional outcomes are unknown. We examined the functional role of a ciliary length control mechanism of FBW7-mediated destruction of NDE1, in mesenchymal stem cell (MSC) differentiation. We show that FBW7 functions as a master regulator of both negative (NDE1) and positive (TALPID3) regulators of ciliogenesis, with an overall positive net effect on primary cilia formation, MSC differentiation to osteoblasts, and bone architecture. Deletion of Fbxw7 suppresses ciliation, Hedgehog activity, and differentiation, which are partially rescued in Fbxw7/Nde1-null cells. We also show that NDE1, despite suppressing ciliogenesis, promotes MSC differentiation by increasing the activity of the Hedgehog pathway by direct binding and enhancing GLI2 activity in a cilia-independent manner. We propose that FBW7 controls a protein-protein interaction network coupling ciliary structure and function, which is essential for stem cell differentiation.

scholarly journals Dopamine receptors reveal an essential role of IFT-B, KIF17, and Rab23 in delivering specific receptors to primary cilia

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Alison Leaf ◽  
Mark Von Zastrow
Keyword(s):  
Dopamine Receptors ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Dopaminergic Receptors ◽  
Guanine Nucleotide Binding Protein ◽  
Ciliary Membrane ◽  
Specific Receptors ◽  
Guanine Nucleotide Binding ◽  
Transport Complex

Appropriate physiological signaling by primary cilia depends on the specific targeting of particular receptors to the ciliary membrane, but how this occurs remains poorly understood. In this study, we show that D1-type dopaminergic receptors are delivered to cilia from the extra-ciliary plasma membrane by a mechanism requiring the receptor cytoplasmic tail, the intraflagellar transport complex-B (IFT-B), and ciliary kinesin KIF17. This targeting mechanism critically depends on Rab23, a small guanine nucleotide binding protein that has important effects on physiological signaling from cilia but was not known previously to be essential for ciliary delivery of any cargo. Depleting Rab23 prevents dopamine receptors from accessing the ciliary membrane. Conversely, fusion of Rab23 to a non-ciliary receptor is sufficient to drive robust, nucleotide-dependent mis-localization to the ciliary membrane. Dopamine receptors thus reveal a previously unrecognized mechanism of ciliary receptor targeting and functional role of Rab23 in promoting this process.

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.

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