The retromer complex regulates C. elegans development and mammalian ciliogenesis

The mammalian retromer is comprised of subunits VPS26, VPS29 and VPS35, and a more loosely-associated sorting nexin (SNX) heterodimer. Despite known roles for the retromer in multiple trafficking events in yeast and mammalian cells, its role in development is poorly understood, and its potential function in primary ciliogenesis remains unknown. Using CRISPR-Cas9 editing, we demonstrated that vps-26 homozygous knockout C. elegans have reduced brood sizes and impaired vulval development, as well as decreased body length which has been linked to defects in primary ciliogenesis. Since many endocytic proteins are implicated in the generation of primary cilia, we addressed whether the retromer regulates ciliogenesis in mammalian cells. We observed VPS35 localized to the primary cilium, and depletion of VPS26, VPS35 or SNX1/SNX5 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the capping protein, CP110, and was required for its removal from the mother centriole. Herein, we characterize new roles for the retromer in C. elegans development and in the regulation of ciliogenesis in mammalian cells, and suggest a novel role for the retromer in CP110 removal from the mother centriole.

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Current understandings of the relationship between extracellular vesicles and cilia

The Journal of Biochemistry ◽

10.1093/jb/mvaa112 ◽

2020 ◽

Author(s):

Koji Ikegami ◽

Faryal Ijaz

Keyword(s):

Extracellular Vesicles ◽

Primary Cilium ◽

Mammalian Cells ◽

Primary Cilia ◽

Research Field ◽

Historical Background ◽

Unicellular Organism ◽

The Relationship

Abstract Mammalian cells have a tiny hair-like protrusion on their surface called a primary cilium. Primary cilia are thought to be the antennae for the cells, receiving signals from the environment. In some studies, extracellular vesicles (EVs) were found attached to the surface of the primary cilium. An idea for the phenomenon is that the primary cilium is the receptor for receiving the EVs. Meanwhile, a unicellular organism, Chlamydomonas, which has two long cilia, usually called flagella, release EVs termed ectosomes from the surface of the flagella. Accumulating evidence suggests that the primary cilium also functions as the ‘emitter’ of EVs. Physiological and pathological impacts are also elucidated for the release of EVs from primary cilia. However, the roles of released cilia-derived EVs remain to be clarified. This review introduces the historical background of the relationship between EVs and cilia, and recent progresses in the research field.

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The Ca2+-activated Cl− channel ANO1/TMEM16A regulates primary ciliogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.e13-10-0599 ◽

2014 ◽

Vol 25 (11) ◽

pp. 1793-1807 ◽

Cited By ~ 31

Author(s):

Chelsey Chandler Ruppersburg ◽

H. Criss Hartzell

Keyword(s):

Ion Channels ◽

Primary Cilium ◽

Primary Cilia ◽

Chloride Transport ◽

Small Gtpases ◽

Sensory Transduction ◽

Anoctamin 1 ◽

Hairpin Rna ◽

Cellular Morphogenesis ◽

Mother Centriole

Many cells possess a single, nonmotile, primary cilium highly enriched in receptors and sensory transduction machinery that plays crucial roles in cellular morphogenesis. Although sensory transduction requires ion channels, relatively little is known about ion channels in the primary cilium (with the exception of TRPP2). Here we show that the Ca2+-activated Cl− channel anoctamin-1 (ANO1/TMEM16A) is located in the primary cilium and that blocking its channel function pharmacologically or knocking it down with short hairpin RNA interferes with ciliogenesis. Before ciliogenesis, the channel becomes organized into a torus-shaped structure (“the nimbus”) enriched in proteins required for ciliogenesis, including the small GTPases Cdc42 and Arl13b and the exocyst complex component Sec6. The nimbus excludes F-actin and coincides with a ring of acetylated microtubules. The nimbus appears to form before, or independent of, apical docking of the mother centriole. Our data support a model in which the nimbus provides a scaffold for staging of ciliary components for assembly very early in ciliogenesis and chloride transport by ANO1/TMEM16A is required for the genesis or maintenance of primary cilia.

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The β-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signalling

10.1101/2021.08.16.456473 ◽

2021 ◽

Author(s):

Gonzalo Sanchez ◽

Tugce Ceren Incedal ◽

Juan Prada Salcedo ◽

Paul O'Callaghan ◽

Santiago Echeverry ◽

...

Keyword(s):

Primary Cilium ◽

Mammalian Cells ◽

Primary Cilia ◽

Β Cells ◽

Intact Mouse ◽

B1 Receptors ◽

Pancreatic Islets Of Langerhans ◽

Mouse Pancreatic Islets ◽

Concentration Changes ◽

Dependent Transport

The primary cilium is an organelle present in most adult mammalian cells and is thought of as an antenna for detection of a variety of signals. Here we use intact mouse pancreatic islets of Langerhans to investigate signalling properties of the primary cilium in β-cells. Using cilia-targeted Ca2+ indicators we find that the resting Ca2+ concentration in the cilium is lower than that of the cytosol, and we uncover a Ca2+ extrusion mechanism in the cilium that effectively insulates the cilium from changes in cytosolic Ca2+. Stimuli that give rise to pronounced cytosolic Ca2+ concentration increases, such as glucose- and depolarization-induced Ca2+ influx, and mobilization of Ca2+ from the ER, was accompanied by minor increases in cilia Ca2+ concentrations that were spatially restricted to a small compartment at the base. Conversely, we observe pronounced Ca2+ concentration changes in the primary cilia of islet β-cells that do not propagate into the cytosol and show that paracrine GABA signalling via cilia-localized GABA- B1-receptors is responsible for this Ca2+ signalling. Finally, we demonstrate that the cilia response to GABA involves ligand-dependent transport of GABA-B1 receptors into the cilium.

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xbx-4, a homolog of the Joubert syndrome gene FAM149B1, acts via the CCRK and MAK kinase cascade to regulate cilia morphology

10.1101/2021.05.09.443182 ◽

2021 ◽

Author(s):

Ashish K Maurya ◽

Piali Sengupta

Keyword(s):

Sensory Neuron ◽

Mammalian Cells ◽

Primary Cilia ◽

Cell Types ◽

Joubert Syndrome ◽

C Elegans ◽

Kinase Cascade ◽

Multiple Cell ◽

Kinase Pathway ◽

Sensory Functions

Primary cilia are microtubule (MT)-based organelles that mediate sensory functions in multiple cell types. Disruption of cilia structure or function leads to a diverse collection of diseases termed ciliopathies. Mutations in the DUF3719 domain-containing protein FAM149B1 have recently been shown to elongate cilia via unknown mechanisms and result in the ciliopathy Joubert syndrome. The highly conserved CCRK and MAK/RCK kinases negatively regulate cilia length and structure in Chlamydomonas, C. elegans, and mammalian cells. How the activity of this kinase cascade is tuned to precisely regulate cilia architecture is unclear. Here we identify XBX-4, a DUF3719 domain-containing protein related to human FAM149B1, as a novel regulator of the DYF-18 CCRK and DYF-5 MAK kinase pathway in C. elegans. As in dyf-18 and dyf-5 mutants, sensory neuron cilia are elongated in xbx-4 mutants and exhibit altered axonemal MT stability. XBX-4 promotes DYF-18 CCRK activity to regulate DYF-5 MAK function and localization. We find that Joubert syndrome-associated mutations in the XBX-4 DUF3719 domain also elongate cilia in C. elegans. Our results identify a new metazoan-specific regulator of this highly conserved kinase pathway, and suggest that FAM149B1 may similarly act via the CCRK/MAK kinase pathway to regulate ciliary homeostasis in humans.

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Genotoxic stress-activated DNA-PK-p53 cascade and autophagy cooperatively induce ciliogenesis to maintain the DNA damage response

Cell Death and Differentiation ◽

10.1038/s41418-020-00713-8 ◽

2021 ◽

Author(s):

Ting-Yu Chen ◽

Bu-Miin Huang ◽

Tang K. Tang ◽

Yu-Ying Chao ◽

Xiao-Yi Xiao ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Primary Cilium ◽

Primary Cilia ◽

Genotoxic Stress ◽

Dna Damage Signaling ◽

Damage Response ◽

Cilia Formation ◽

Mother Centriole ◽

Autophagy Inhibition

AbstractThe DNA-PK maintains cell survival when DNA damage occurs. In addition, aberrant activation of the DNA-PK induces centrosome amplification, suggesting additional roles for this kinase. Here, we showed that the DNA-PK-p53 cascade induced primary cilia formation (ciliogenesis), thus maintaining the DNA damage response under genotoxic stress. Treatment with genotoxic drugs (etoposide, neocarzinostatin, hydroxyurea, or cisplatin) led to ciliogenesis in human retina (RPE1), trophoblast (HTR8), lung (A459), and mouse Leydig progenitor (TM3) cell lines. Upon genotoxic stress, several DNA damage signaling were activated, but only the DNA-PK-p53 cascade contributed to ciliogenesis, as pharmacological inhibition or genetic depletion of this pathway decreased genotoxic stress-induced ciliogenesis. Interestingly, in addition to localizing to the nucleus, activated DNA-PK localized to the base of the primary cilium (mother centriole) and daughter centriole. Genotoxic stress also induced autophagy. Inhibition of autophagy initiation or lysosomal degradation or depletion of ATG7 decreased genotoxic stress-induced ciliogenesis. Besides, inhibition of ciliogenesis by depletion of IFT88 or CEP164 attenuated the genotoxic stress-induced DNA damage response. Thus, our study uncovered the interplay among genotoxic stress, the primary cilium, and the DNA damage response.

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The Primary Cilium of MDCK Cells is a Flow Sensor

Microscopy and Microanalysis ◽

10.1017/s143192760002609x ◽

2001 ◽

Vol 7 (S2) ◽

pp. 4-5

Author(s):

H. A. Praetorius ◽

K. R. Spring

Keyword(s):

Primary Cilium ◽

Mammalian Cells ◽

Primary Cilia ◽

Cultured Cells ◽

Mdck Cells ◽

Collecting Duct ◽

Renal Tubules ◽

Intercalated Cells ◽

Principal Cells ◽

Renal Tubular Cells

The primary cilium, a solitary non-motile structure projecting from the centriole of mammalian cells, has been a subject of interest of anatomists since 1898. Surprisingly, the function of the primary cilium in mammalian cells is completely unknown. Virtually all of the epithelial cells of the mammalian kidney, with the exception of the intercalated cells of the collecting duct, express a single primary cilium on their apical (lumenal) surface. These structures were ignored by physiologists until recently when the mechanical properties of the primary cilia of cultured cells of renal origin were measured and it was proposed that they could serve as flow sensors. The proposal stemmed from the observation that flow rates comparable to those observed in renal tubules resulted in a deflection of the cilium of a few microns and that the stiffness of the cilium was commensurate with mechanosensing.The primary cilia of renal tubular cells are generally about 2 - 3 μm long whereas those of cultured renal cells become much longer as the cells age and may extend 50 μm or more into the medium. MDCK cells, a cultured cell line derived from the collecting duct of the canine kidney, exhibit two cell types analogous to the principal and intercalated cells of the collecting duct. MDCK principal cells express a primary cilium that is visible by high-resolution DIC microscopy. About one week after splitting, the primary cilium of MDCK principal cells is about 8 μm long and is well suited for testing the hypothesis that the cilium serves as a mechanosensor of fluid flow.

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Interchangeable but Essential Functions of SNX1 and SNX2 in the Association of Retromer with Endosomes and the Trafficking of Mannose 6-Phosphate Receptors

Molecular and Cellular Biology ◽

10.1128/mcb.00156-06 ◽

2006 ◽

Vol 27 (3) ◽

pp. 1112-1124 ◽

Cited By ~ 140

Author(s):

Raul Rojas ◽

Satoshi Kametaka ◽

Carol R. Haft ◽

Juan S. Bonifacino

Keyword(s):

Mammalian Cells ◽

Human Cell Line ◽

Sorting Nexin ◽

Retromer Complex ◽

Cell Line Hela ◽

Endosomal Membranes ◽

Mannose 6 Phosphate ◽

And Function ◽

Golgi Network ◽

Functional Analyses

ABSTRACT The retromer is a cytosolic/peripheral membrane protein complex that mediates the retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network (TGN) in mammalian cells. Previous studies showed that the mammalian retromer comprises three proteins, named Vps26, Vps29, and Vps35, plus the sorting nexin, SNX1. There is conflicting evidence, however, as to whether a homologous sorting nexin, SNX2, is truly a component of the retromer. In addition, the nature of the subunit interactions and assembly of the mammalian retromer complex are poorly understood. We have addressed these issues by performing biochemical and functional analyses of endogenous retromers in the human cell line HeLa. We found that the mammalian retromer complex consists of two autonomously assembling subcomplexes, namely, a Vps26-Vps29-Vps35 obligate heterotrimer and a SNX1/2 alternative heterodimer or homodimer. The association of Vps26-Vps29-Vps35 with endosomes requires the presence of either SNX1 or SNX2, whereas SNX1/2 can be recruited to endosomes independently of Vps26-Vps29-Vps35. We also found that the presence of either SNX1 or SNX2 is essential for the retrieval of the cation-independent mannose 6-phosphate receptor to the TGN. These observations indicate that the mammalian retromer complex assembles by sequential association of SNX1/2 and Vps26-Vps29-Vps35 subcomplexes on endosomal membranes and that SNX1 and SNX2 play interchangeable but essential roles in retromer structure and function.

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STAM and Hrs Down-Regulate Ciliary TRP Receptors

Molecular Biology of the Cell ◽

10.1091/mbc.e07-03-0239 ◽

2007 ◽

Vol 18 (9) ◽

pp. 3277-3289 ◽

Cited By ~ 43

Author(s):

Jinghua Hu ◽

Samuel G. Wittekind ◽

Maureen M. Barr

Keyword(s):

Growth Factor ◽

Mammalian Cells ◽

Primary Cilia ◽

Membrane Receptors ◽

Sensory Transduction ◽

Kinase Substrate ◽

C Elegans ◽

Early Endosomes ◽

Transient Receptor ◽

And Function

Cilia are endowed with membrane receptors, channels, and signaling components whose localization and function must be tightly controlled. In primary cilia of mammalian kidney epithelia and sensory cilia of Caenorhabditis elegans neurons, polycystin-1 (PC1) and transient receptor polycystin-2 channel (TRPP2 or PC2), function together as a mechanosensory receptor-channel complex. Despite the importance of the polycystins in sensory transduction, the mechanisms that regulate polycystin activity and localization, or ciliary membrane receptors in general, remain poorly understood. We demonstrate that signal transduction adaptor molecule STAM-1A interacts with C. elegans LOV-1 (PC1), and that STAM functions with hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs) on early endosomes to direct the LOV-1-PKD-2 complex for lysosomal degradation. In a stam-1 mutant, both LOV-1 and PKD-2 improperly accumulate at the ciliary base. Conversely, overexpression of STAM or Hrs promotes the removal of PKD-2 from cilia, culminating in sensory behavioral defects. These data reveal that the STAM-Hrs complex, which down-regulates ligand-activated growth factor receptors from the cell surface of yeast and mammalian cells, also regulates the localization and signaling of a ciliary PC1 receptor-TRPP2 complex.

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Role of Primary Cilia in Odontogenesis

Journal of Dental Research ◽

10.1177/0022034517713688 ◽

2017 ◽

Vol 96 (9) ◽

pp. 965-974 ◽

Cited By ~ 6

Author(s):

M. Hampl ◽

P. Cela ◽

H.L. Szabo-Rogers ◽

M. Kunova Bosakova ◽

H. Dosedelova ◽

...

Keyword(s):

Signaling Pathways ◽

Primary Cilium ◽

Mammalian Cells ◽

Primary Cilia ◽

Tooth Development ◽

Critical Role ◽

Current Evidence ◽

Developmental Defects ◽

Dental Tissues ◽

The Impact

Primary cilium is a solitary organelle that emanates from the surface of most postmitotic mammalian cells and serves as a sensory organelle, transmitting the mechanical and chemical cues to the cell. Primary cilia are key coordinators of various signaling pathways during development and maintenance of tissue homeostasis. The emerging evidence implicates primary cilia function in tooth development. Primary cilia are located in the dental epithelium and mesenchyme at early stages of tooth development and later during cell differentiation and production of hard tissues. The cilia are present when interactions between both the epithelium and mesenchyme are required for normal morphogenesis. As the primary cilium coordinates several signaling pathways essential for odontogenesis, ciliary defects can interrupt the latter process. Genetic or experimental alterations of cilia function lead to various developmental defects, including supernumerary or missing teeth, enamel and dentin hypoplasia, or teeth crowding. Moreover, dental phenotypes are observed in ciliopathies, including Bardet-Biedl syndrome, Ellis-van Creveld syndrome, Weyers acrofacial dysostosis, cranioectodermal dysplasia, and oral-facial-digital syndrome, altogether demonstrating that primary cilia play a critical role in regulation of both the early odontogenesis and later differentiation of hard tissue–producing cells. Here, we summarize the current evidence for the localization of primary cilia in dental tissues and the impact of disrupted cilia signaling on tooth development in ciliopathies.

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Hook2 is involved in the morphogenesis of the primary cilium

Molecular Biology of the Cell ◽

10.1091/mbc.e11-05-0405 ◽

2011 ◽

Vol 22 (23) ◽

pp. 4549-4562 ◽

Cited By ~ 42

Author(s):

Carole L. Baron Gaillard ◽

Emilie Pallesi-Pocachard ◽

Dominique Massey-Harroche ◽

Fabrice Richard ◽

Jean-Pierre Arsanto ◽

...

Keyword(s):

Primary Cilium ◽

Primary Cilia ◽

Protein Transport ◽

Adaptor Protein ◽

Functional Interaction ◽

Mother Centriole ◽

Pericentriolar Material ◽

Underlying Mechanisms ◽

Interfering Rna ◽

Two Hybrid

Primary cilia originate from the centrosome and play essential roles in several cellular, developmental, and pathological processes, but the underlying mechanisms of ciliogenesis are not fully understood. Given the involvement of the adaptor protein Hook2 in centrosomal homeostasis and protein transport to pericentrosomal aggresomes, we explored its role in ciliogenesis. We found that in human retinal epithelial cells, Hook2 localizes at the Golgi apparatus and centrosome/basal body, a strategic partitioning for ciliogenesis. Of importance, Hook2 depletion disrupts ciliogenesis at a stage before the formation of the ciliary vesicle at the distal tip of the mother centriole. Using two hybrid and immunoprecipitation assays and a small interfering RNA strategy, we found that Hook2 interacts with and stabilizes pericentriolar material protein 1 (PCM1), which was reported to be essential for the recruitment of Rab8a, a GTPase that is believed to be crucial for membrane transport to the primary cilium. Of interest, GFP::Rab8a coimmunoprecipitates with endogenous Hook2 and PCM1. Finally, GFP::Rab8a can overcome Hook2 depletion, demonstrating a functional interaction between Hook2 and these two important regulators of ciliogenesis. The data indicate that Hook2 interacts with PCM1 in a complex that also contains Rab8a and regulates a limiting step required for further initiation of ciliogenesis after centriole maturation.

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