C3G localizes to the mother centriole in a cenexin-dependent manner and regulates centrosome duplication and primary cilium length

Primary cilium drives the left-right asymmetry process during embryonic development. Moreover, its dysregulation contributes to cancer progression by affecting various signaling pathways. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system modulates primary cilium length and plays a pivotal role in embryogenesis and tumor growth. Here, we investigated the impact of the natural FGF trap long-pentraxin 3 (PTX3) on the determination of primary cilium extension in zebrafish embryo and cancer cells. The results demonstrate that down modulation of the PTX3 orthologue ptx3b causes the shortening of primary cilium in zebrafish embryo in a FGF-dependent manner, leading to defects in the left-right asymmetry determination. Conversely, PTX3 upregulation causes the elongation of primary cilium in FGF-dependent cancer cells. Previous observations have identified the PTX3-derived small molecule NSC12 as an orally available FGF trap with anticancer effects on FGF-dependent tumors. In keeping with the non-redundant role of the FGF/FGR system in primary cilium length determination, NSC12 induces the elongation of primary cilium in FGF-dependent tumor cells, thus acting as a ciliogenic anticancer molecule in vitro and in vivo. Together, these findings demonstrate the ability of the natural FGF trap PTX3 to exert a modulatory effect on primary cilium in embryonic development and cancer. Moreover, they set the basis for the design of novel ciliogenic drugs with potential implications for the therapy of FGF-dependent tumors.

Download Full-text

Casein Kinase II is Required for Proper Cell Division and Acts as a Negative Regulator of Centrosome Duplication in C. elegans Embryos

10.1101/083378 ◽

2016 ◽

Author(s):

Jeffrey C. Medley ◽

Megan M. Kabara ◽

Michael D. Stubenvoll ◽

Lauren E. DeMeyer ◽

Mi Hye Song

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Cell Cycle Progression ◽

Casein Kinase Ii ◽

Casein Kinase ◽

Negative Regulator ◽

Centrosome Duplication ◽

Dependent Manner ◽

C Elegans ◽

Mother Centriole

Summary statementThe conserved protein kinase CK2 negatively regulates centrosome assembly and is required for proper cell cycle progression and cytokinesis in early C. elegans embryos.AbstractCentrosomes are the primary microtubule-organizing centers that orchestrate microtubule dynamics during the cell cycle. The correct number of centrosomes is pivotal for establishing bipolar mitotic spindles that ensure accurate segregation of chromosomes. Thus, centrioles must duplicate once per cell cycle, one daughter per mother centriole, the process of which requires highly coordinated actions among core factors and modulators. Protein phosphorylation is shown to regulate the stability, localization and activity of centrosome proteins. Here, we report the function of Casein Kinase II (CK2) in early C. elegans embryos. The catalytic subunit (KIN-3/CK2α) of CK2 localizes to nuclei, centrosomes and midbodies. Inactivating CK2 leads to cell division defects, including chromosome missegregation, cytokinesis failure and aberrant centrosome behavior. Furthermore, depletion or inhibiting kinase activity of CK2 results in elevated ZYG-1 levels at centrosomes, restoring centrosome duplication and embryonic viability to zyg-1 mutants. Our data suggest that CK2 functions in cell division and negatively regulates centrosome duplication in a kinase-dependent manner.

Download Full-text

The retromer complex regulates C. elegans development and mammalian ciliogenesis

10.1101/2021.09.24.461716 ◽

2021 ◽

Author(s):

Shuwei Xie ◽

Ellie Smith ◽

Carter Dierlam ◽

Danita Mathew ◽

Angelina Davis ◽

...

Keyword(s):

Potential Function ◽

Primary Cilium ◽

Mammalian Cells ◽

Primary Cilia ◽

Vulval Development ◽

Sorting Nexin ◽

Capping Protein ◽

C Elegans ◽

Retromer Complex ◽

Mother Centriole

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.

Download Full-text

GJA1 Depletion Causes Ciliary Defects by Affecting Rab11 Trafficking to the Ciliary Base

10.1101/2020.11.13.381764 ◽

2020 ◽

Author(s):

Dong Gil Jang ◽

Keun Yeong Kwon ◽

Yeong Cheon Kweon ◽

Byung-gyu Kim ◽

Kyungjae Myung ◽

...

Keyword(s):

Gap Junction ◽

Primary Cilium ◽

Transport Channel ◽

Junction Protein ◽

Complex Functions ◽

Mother Centriole ◽

Pericentriolar Material ◽

Gap Junction Protein ◽

And Function ◽

Distinct Distribution

AbstractThe gap junction complex functions as a transport channel across the membrane. Among gap junction subunits, gap junction protein alpha 1 (GJA1) is the most commonly expressed subunit. However, the roles of GJA1 in the formation and function of cilia remain unknown. Here, we examined GJA1 functions during ciliogenesis in vertebrates. GJA1 was localized to the motile ciliary axonemes or pericentriolar material (PCM) around the primary cilium. GJA1 depletion caused the severe malformation of both primary cilium and motile cilia. Interestingly, GJA1 depletion caused strong delocalization of BBS4 from the PCM and basal body and distinct distribution as cytosolic puncta. Further, CP110 removal from the mother centriole was significantly reduced by GJA1 depletion. Importantly, Rab11, key regulator during ciliogenesis, was immunoprecipitated with GJA1 and GJA1 knockdown caused the mis-localization and mis-accumulation of Rab11. These findings suggest that GJA1 is necessary for proper ciliogenesis by regulating the Rab11 pathway.

Download Full-text

Loss of CEP162 function at the primary cilium delays ciliogenesis and causes retinal ciliopathy in humans

10.1101/2021.11.23.469779 ◽

2021 ◽

Author(s):

Nafisa Nuzhat ◽

Kristof Van Schil ◽

Sandra Liakopoulos ◽

Miriam Bauwens ◽

Alfredo Dueñas Rey ◽

...

Keyword(s):

Transition Zone ◽

Primary Cilium ◽

Outer Segment ◽

Retinal Dystrophy ◽

Mrna Levels ◽

Loss Of Function ◽

Photoreceptor Outer Segment ◽

Zone Formation ◽

Mother Centriole

Ciliopathies often comprise retinal degeneration since the photoreceptor outer segment is an adapted primary cilium. CEP162 is a distal end centriolar protein required for proper transition zone assembly during ciliogenesis and whose loss causes ciliopathy in zebrafish. CEP162 has so far not been implicated in human disease. Here, we identified a homozygous CEP162 frameshift variant, c.1935dupA (p.(E646R*5)), in retinitis pigmentosa patients from two unrelated Moroccan families, likely representing a founder allele. We found that even though mRNA levels were reduced, the truncated CEP162-E646R*5 protein was expressed and localized to the mitotic spindle during mitosis, but not at the basal body of the cilium. In CEP162 knockdown cells, expression of the truncated CEP162-E646R*5 protein is unable to restore ciliation indicating its loss of function at the cilium. In patient fibroblasts, cilia overcome the absence of CEP162 from the primary cilium by delaying ciliogenesis through the persistence of CP110 at the mother centriole. The patient fibroblasts are ultimately able to extend some abnormally long cilia that are missing key transition zone components. Defective transition zone formation likely disproportionately affects the long-living ciliary outer segment of photoreceptors resulting in retinal dystrophy. CEP162 is expressed in human retina, and we show that wild-type CEP162, but not truncated CEP162-E646R*5, specifically localizes to the distal end of centrioles of mouse photoreceptor cilia. Together, our genetic, cell-based, and in vivo modeling establish that CEP162 deficiency causes retinal ciliopathy in humans.

Download Full-text

Reduction of oxidative stress during recovery accelerates normalization of primary cilia length that is altered after ischemic injury in murine kidneys

AJP Renal Physiology ◽

10.1152/ajprenal.00427.2012 ◽

2013 ◽

Vol 304 (10) ◽

pp. F1283-F1294 ◽

Cited By ~ 24

Author(s):

Jee In Kim ◽

Jinu Kim ◽

Hee-Seong Jang ◽

Mi Ra Noh ◽

Joshua H. Lipschutz ◽

...

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Primary Cilium ◽

Primary Cilia ◽

Mdck Cells ◽

Reactive Oxygen ◽

Renal Tubular Cell ◽

Oxygen Species ◽

Renal Tubular ◽

Cilium Length

The primary cilium is a microtubule-based nonmotile organelle that extends from the surface of cells, including renal tubular cells. Here, we investigated the alteration of primary cilium length during epithelial cell injury and repair, following ischemia/reperfusion (I/R) insult, and the role of reactive oxygen species in this alteration. Thirty minutes of bilateral renal ischemia induced severe renal tubular cell damage and an increase of plasma creatinine (PCr) concentration. Between 8 and 16 days following the ischemia, the increased PCr returned to normal range, although without complete histological restoration. Compared with the primary cilium length in normal kidney tubule cells, the length was shortened 4 h and 1 day following ischemia, increased over normal 8 days after ischemia, and then returned to near normal 16 days following ischemia. In the urine of I/R-subjected mice, acetylated tubulin was detected. The cilium length of proliferating cells was shorter than that in nonproliferating cells. Mature cells had shorter cilia than differentiating cells. Treatment with Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP), an antioxidant, during the recovery of damaged kidneys accelerated normalization of cilia length concomitant with a decrease of oxidative stress and morphological recovery in the kidney. In the Madin-Darby canine kidney (MDCK) cells, H2O2 treatment caused released ciliary fragment into medium, and MnTMPyP inhibited the deciliation. The ERK inhibitor U0126 inhibited elongation of cilia in normal and MDCK cells recovering from H2O2 stress. Taken together, our results suggest that primary cilia length reflects cell proliferation and the length of primary cilium is regulated, at least, in part, by reactive oxygen species through ERK.

Download Full-text

Faculty Opinions recommendation of CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-β/BMP Signaling at the Primary Cilium.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732811228.793582060 ◽

2021 ◽

Author(s):

Christoper J Westlake

Keyword(s):

Primary Cilium ◽

Bmp Signaling ◽

Mother Centriole

Download Full-text

Caveolin-1α regulates primary cilium length by controlling RhoA GTPase activity

Scientific Reports ◽

10.1038/s41598-018-38020-5 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 9

Author(s):

Laura Rangel ◽

Miguel Bernabé-Rubio ◽

Jaime Fernández-Barrera ◽

Javier Casares-Arias ◽

Jaime Millán ◽

...

Keyword(s):

Primary Cilium ◽

Gtpase Activity ◽

Rhoa Gtpase ◽

Cilium Length

Download Full-text

Transepithelial pressure pulses induce nucleotide release in polarized MDCK cells

AJP Renal Physiology ◽

10.1152/ajprenal.00238.2004 ◽

2005 ◽

Vol 288 (1) ◽

pp. F133-F141 ◽

Cited By ~ 53

Author(s):

H. A. Praetorius ◽

J. Frøkiær ◽

J. Leipziger

Keyword(s):

Epithelial Cells ◽

Laminar Flow ◽

Primary Cilium ◽

Mdck Cells ◽

Chamber Pressure ◽

Dependent Manner ◽

Nucleotide Release ◽

Pressure Pulses ◽

Kidney Epithelial Cells ◽

Pressure Changes

The release of nucleotides is involved in mechanosensation in various epithelial cells. Intriguingly, kidney epithelial cells are absolutely dependent on the primary cilium to sense changes in apical laminar flow. During fluid passage, the renal epithelial cells are subjected to various mechanical stimuli in addition to changes in the laminar flow rate. In the distal part of the collecting duct, the epithelial cells are exposed to pressure changes and possibly distension during papillary contractions. The aim of the present study was to determine whether nucleotide release contributes to mechanosensation in kidney epithelial cells, thereby establishing whether pressure changes are sufficient to produce nucleotide-mediated responses. Madin-Darby canine kidney (MDCK) cells grown on permeable supports were mounted in a closed double perfusion chamber on an inverted microscope. The intracellular Ca2+ concentration ([Ca2+]i) was monitored with the Ca2+-sensitive fluorescence probe fluo 4. Transepithelial pressure pulses of 30–80 mmHg produced a transient increase in [Ca2+]i of MDCK cells. This response is independent of the primary cilium, since it is readily observed in immature cells that do not yet express primary cilia. The amplitudes of the pressure-induced Ca2+ transients varied with the applied chamber pressure in a quantity-dependent manner. The ATPase apyrase and the P2Y antagonist suramin significantly reduced the pressure-induced Ca2+ transients. Applying apyrase or suramin to both sides of the preparation simultaneously nearly abolished the pressure-induced Ca2+ response. In conclusion, these observations suggest that rapid pressure changes induce both apical and basolateral nucleotide release that contribute to mechanosensation in kidney epithelial cells.

Download Full-text

Interaction between ROCK II and Nucleophosmin/B23 in the Regulation of Centrosome Duplication

Molecular and Cellular Biology ◽

10.1128/mcb.01383-06 ◽

2006 ◽

Vol 26 (23) ◽

pp. 9016-9034 ◽

Cited By ~ 66

Author(s):

Zhiyong Ma ◽

Masayuki Kanai ◽

Kenji Kawamura ◽

Kozo Kaibuchi ◽

Keqiang Ye ◽

...

Keyword(s):

Cyclin E ◽

Active Form ◽

Small Gtpase ◽

Centrosome Duplication ◽

Physical Interaction ◽

Dependent Manner ◽

Physical Separation ◽

Initial Event ◽

Activity Dependent

ABSTRACT Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr199 by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr199 remains at centrosomes. It has been shown that Thr199 phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr199. Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr199-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

Download Full-text