Primary Cilia Formation Research Articles

STIL/AURKA axis promotes cell proliferation by influencing primary cilia formation in bladder cancer

BackgroundThe primary cilia (PC) is a microtubule-based and nonmotile organelle which protrudes from the surface of almost all mammalian cells. At present, PC has been found to be a deficiency or loss in multiple cancers. Restoring PC could be a novel targeting therapy strategy. Our research showed that PC was reduced in human bladder cancer (BLCA) cells, and PC deficiency promotes cell proliferation. However, the concrete mechanisms remain unknown. SCL/TAL1 interrupting locus (STIL), a PC-related protein, was screened in our previous study and could influence the cell cycle by regulating PC in tumor cells. In this study, we aimed to elucidate the function of STIL for PC to explore the underlying mechanism of PC in BLCA.MethodsPublic database analysis, western blot, and enzyme-linked immunosorbent assay (ELISA) were used to screen genes and explore gene expression alteration. Immunofluorescence and western blot were utilized to investigate PC. Wound healing assay, clone formation assay, and CCK-8 assay were used to explore cell migration, growth, and proliferation. The co-immunoprecipitation and western blot were employed to reveal the interaction of STIL and AURKA.ResultsWe found that high STIL expression is correlated with poor outcomes of BLCA patients. Further analysis revealed that STIL overexpression could inhibit PC formation, activate SHH signaling pathways, and promote cell proliferation. In contrast, STIL-knockdown could promote PC formation, inactivate SHH signaling, and inhibit cell proliferation. Furthermore, we found that the regulatory functions of STIL for PC depend on AURKA. STIL could influence proteasome activity and maintain AURKA stabilization. AURKA-knockdown could reverse PC deficiency caused by STIL overexpression for PC in BLCA cells. We observed that co-knockdown in STIL and AURKA significantly enhanced PC assembly.ConclusionIn summary, our result provides a potential therapy target for BLCA based on the restoration of PC.

Protein quality control machinery supports primary ciliogenesis by eliminating GDP-bound Rab8-family GTPases

The small GTPase Rab8 plays a vital role in the vesicular trafficking of cargo proteins from the trans-Golgi network to target membranes. Upon reaching its target destination, Rab8 is released from the vesicular membrane into the cytoplasm via guanosine triphosphate (GTP) hydrolysis. The fate of GDP-bound Rab8 released from the destination membranes, however, has not been investigated adequately. In this study, we found that GDP-bound Rab8 subfamily proteins are targeted for immediate degradation, and the pre-emptive quality control machinery is responsible for eliminating these proteins in a nucleotide-specific manner. We provide evidence that components of this quality control machinery have a critical role in vesicular trafficking events, including the formation of primary cilia, a process regulated by the Rab8 subfamily. These results suggest that the protein degradation machinery plays a critical role in the integrity of membrane trafficking by limiting the excessive accumulation of GDP-bound Rab8 subfamily proteins.

Exome Analysis Reveals Novel Missense and Deletion Variants in the CC2D2A Gene as Causative of Joubert Syndrome.

The CC2D2A gene is essential for primary cilia formation, and its disruption has been associated with Joubert Syndrome-9 (JBTS9), a ciliopathy with typical neurodevelopmental features. Here, we describe an Italian pediatric patient with typical features of Joubert Syndrome (JBTS): "Molar Tooth Sign", global developmental delay, nystagmus, mild hypotonia, and oculomotor apraxia. Whole exome sequencing and segregation analysis identified in our infant patient a novel heterozygous germline missense variant c.3626C > T; p.(Pro1209Leu) inherited from the father and a novel 7.16 kb deletion inherited from the mother. To the best of our knowledge, this is the first report showing a novel missense and deletion variant involving exon 30 of the CC2D2A gene.

Chondrocyte primary cilia lengthening and shortening in response to mediators of osteoarthritis; a role for integrin α1β1 and focal adhesions

ObjectiveIntegrin α1β1 protects against osteoarthritis when it is upregulated in the early stages of disease, however, the mechanism behind this is currently unknown. Hypo-osmotic stress, interleukin-1 (IL-1) and transforming growth factor β (TGFβ) influence chondrocyte signaling and are important mediators of osteoarthritis. Evidence for primary cilia as a signaling hub for these factors and the involvement of the F-actin cytoskeleton in this response is growing. The purpose of this study was to investigate the role of integrin α1β1 in the response of primary cilia and the F-actin cytoskeleton to these osteoarthritic mediators. DesignPrimary cilia length and the number of F-actin peaks were measured in ex vivo wild type and itga1-null chondrocytes in response to hypo-osmotic stress, IL-1, and TGFβ alone or in combination, and with or without focal adhesion kinase inhibitor. ResultsWe show that integrin α1β1 and focal adhesions are necessary for cilial lengthening and increases in F-actin peaks with hypo-osmotic stress and IL-1, but are not required for cilial shortening with TGFβ. Furthermore, we established that the chondrocyte primary cilium has a resting length of 2.4 ​μm, a minimum length of 2.1 ​μm corresponding to the thickness of the pericellular matrix, and a maximum length of 3.0 ​μm. ConclusionsWhile integrin α1β1 is not necessary for the formation of chondrocyte primary cilia and cilial shortening in response to TGFβ, it is necessary for the mediation of cilial lengthening and the formation of F-actin peaks in response to hypo-osmotic stress and IL-1.

Septin-mediated RhoA activation engages the exocyst complex to recruit the cilium transition zone.

Septins are filamentous GTPases that play important but poorly characterized roles in ciliogenesis. Here, we show that SEPTIN9 regulates RhoA signaling at the base of cilia by binding and activating the RhoA guanine nucleotide exchange factor, ARHGEF18. GTP-RhoA is known to activate the membrane targeting exocyst complex, and suppression of SEPTIN9 causes disruption of ciliogenesis and mislocalization of an exocyst subunit, SEC8. Using basal body-targeted proteins, we show that upregulating RhoA signaling at the cilium can rescue ciliary defects and mislocalization of SEC8 caused by global SEPTIN9 depletion. Moreover, we demonstrate that the transition zone components, RPGRIP1L and TCTN2, fail to accumulate at the transition zone in cells lacking SEPTIN9 or depleted of the exocyst complex. Thus, SEPTIN9 regulates the recruitment of transition zone proteins on Golgi-derived vesicles by activating the exocyst via RhoA to allow the formation of primary cilia.

A direct interaction between CENTLEIN and RABIN8 is required for primary cilium formation.

Primary cilia are formed in nearly all growth-arrested cells and are essential for mammalian development and tissue homeostasis. Defects in primary cilia result in a range of disorders in humans, named ciliopathies. The spatiotemporal localization of RABIN8 on the pericentrosome is an early step in ciliogenesis. Here, we show that CENTLEIN depletion causes the persistent accumulation of RABIN8 on the pericentrosome and primary cilium loss in hTERT-immortalized retinal pigment epithelial cells and murine embryonic fibroblasts. CENTLEIN interacts with RABIN8 directly. A stretch of a 31-amino acid sequence located in the 200‒230 region of the RABIN8 GEF domain is responsible for its physical interaction with CENTLEIN, while expression of the full-length but not the internal deletion lacking the RABIN8-binding site of CENTLEIN largely rescues the ciliogenesis defect provoked by CENTLEIN depletion. Expression of activated RAB8A partially reverses cilium loss in CENTLEIN-null RPE1 cells, so the functional importance of the CENTLEIN-RABIN8 interaction is defined.

Three-dimensional single-molecule super-resolution imaging of proteins involved in primary cilia formation.

Primary cilia are organelles that protrude from the cell membrane of most human cells where they perform critical sensory and signaling functions. Defects and mutations in proteins in the primary cilium cause a range of diseases and syndromes collectively termed ciliopathies. Intracellular primary cilium formation begins with the recruitment of Golgi-derived pre-ciliary vesicles to the distal appendages of the mother centriole, but the molecular mechanisms behind the recruitment of the distal appendage proteins are not fully understood. The proteins Rab34 and MyosinVa were found through structured illumination microscopy (SIM) to be closely associated with these pre-ciliary vesicles during this step. However, because of the sub-diffraction size and three-dimensional (3D) structures of the centriole and of the involved proteins, higher resolution multi-target 3D microscopy is required to resolve their organization. To address this limitation, 3D two-color direct stochastic optical reconstruction microscopy (dSTORM) was implemented, providing a single-molecule localization precision of roughly 20 nm in all dimensions. A two-channel 4f emissions system was constructed using a 2-μm axial range double helix phase mask for single-molecule localization in 3D through point spread function engineering. This system allowed for 3D single-molecule super-resolution imaging of Rab34 and MyosinVa in two different color-channels together with imaging of the centriolar protein FOP in both color-channels for context and precise channel registration. The resulting 3D super-resolved reconstructions revealed the nanoscale structures formed by these proteins at the pre-ciliary vesicles, the structural relation between Rab34 and MyosinVa, and their position in relation to the mother centriole.

Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development.

Trisomy 21, the genetic cause of Down syndrome, disrupts primary cilia formation and function, in part through elevated Pericentrin, a centrosome protein encoded on chromosome 21. Yet how trisomy 21 and elevated Pericentrin disrupt cilia-related molecules and pathways, and the in vivo phenotypic relevance remain unclear. Utilizing ciliogenesis time course experiments combined with light microscopy and electron tomography, we reveal that chromosome 21 polyploidy elevates Pericentrin and microtubules away from the centrosome that corral MyosinVA and EHD1, delaying ciliary membrane delivery and mother centriole uncapping essential for ciliogenesis. If given enough time, trisomy 21 cells eventually ciliate, but these ciliated cells demonstrate persistent trafficking defects that reduce transition zone protein localization and decrease sonic hedgehog signaling in direct anticorrelation with Pericentrin levels. Consistent with cultured trisomy 21 cells, a mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers at P4. Our work reveals that elevated Pericentrin from trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding mechanism results in signaling deficiencies consistent with the neurological phenotypes found in individuals with Down syndrome.

A novel 1.38-kb deletion combined with a single nucleotide variant in KIAA0586 as a cause of Joubert syndrome

BackgroundKIAA0586, also known as Talpid3, plays critical roles in primary cilia formation and hedgehog signaling in humans. Variants in KIAA0586 could cause some different ciliopathies, including Joubert syndrome (JBTS), which is a clinically and genetically heterogeneous group of autosomal recessive neurological disorders.Methods and ResultsA 9-month-old girl was diagnosed as JBTS by the “molar tooth sign” of the mid-brain and global developmental delay. By whole-exome sequencing, we identified a single nucleotide variant c.3303G > A and a 1.38-kb deletion in KIAA0586 in the proband. These two variants of KIAA0586 were consistent with the mode of autosomal recessive inheritance in the family, which was verified using Sanger sequencing.ConclusionsThis finding of a compound heterozygote with a 1.38-kb deletion and c.3303G > A gave a precise genetic diagnosis for the patient, and the novel 1.38-kb deletion also expanded the pathogenic variation spectrum of JBTS caused by KIAA0586.

Proximity Mapping of CCP6 Reveals Its Association with Centrosome Organization and Cilium Assembly.

The cytosolic carboxypeptidase 6 (CCP6) catalyzes the deglutamylation of polyglutamate side chains, a post-translational modification that affects proteins such as tubulins or nucleosome assembly proteins. CCP6 is involved in several cell processes, such as spermatogenesis, antiviral activity, embryonic development, and pathologies like renal adenocarcinoma. In the present work, the cellular role of CCP6 has been assessed by BioID, a proximity labeling approach for mapping physiologically relevant protein-protein interactions (PPIs) and bait proximal proteins by mass spectrometry. We used HEK 293 cells stably expressing CCP6-BirA* to identify 37 putative interactors of this enzyme. This list of CCP6 proximal proteins displayed enrichment of proteins associated with the centrosome and centriolar satellites, indicating that CCP6 could be present in the pericentriolar material. In addition, we identified cilium assembly-related proteins as putative interactors of CCP6. In addition, the CCP6 proximal partner list included five proteins associated with the Joubert syndrome, a ciliopathy linked to defects in polyglutamylation. Using the proximity ligation assay (PLA), we show that PCM1, PIBF1, and NudC are true CCP6 physical interactors. Therefore, the BioID methodology confirms the location and possible functional role of CCP6 in centrosomes and centrioles, as well as in the formation and maintenance of primary cilia.

Hedgehog signaling regulates Wolffian duct development through the primary cilium†.

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). Although mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; IFT88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix, mesenchymal-epithelial transition, canonical Hh and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

FOXA1 is a transcriptional activator of Odf2/Cenexin and regulates primary ciliation

Primary cilia are sensory organelles essential for embryonic and postnatal development, and tissue homeostasis in adulthood. They are generated in a cell cycle-dependent manner and found on most cells of the body. Although cilia formation is intensively investigated virtually nothing is known about the transcriptional regulation of primary ciliation. We used here Odf2/Cenexin, encoding a protein of the mother centriole and the basal body that is mandatory for primary cilia formation, as the target gene for the identification of transcriptional activators. We identified a consensus binding site for Fox transcription factors (TFs) in its promoter region and focused here on the Fox family. We found transcriptional activation of Odf2 neither by FOXO TFs nor by the core TF for multiciliation, FOXJ1. However, we identified FOXA1 as a transcriptional activator of Odf2 by reporter gene assays and qRT-PCR, and showed by qWB that Foxa1 knockdown caused a decrease in ODF2 and CP110 proteins. We verified the binding sequence of FOXA1 in the Odf2 promoter by ChIP. Finally, we demonstrated that knockdown of FOXA1 affected primary cilia formation. We, thus, showed for the first time, that FOXA1 regulates primary ciliation by transcriptional activation of ciliary genes.

Nuclear factor (erythroid-derived 2)-like 2 counter-regulates thymosin beta-4 expression and primary cilium formation for HeLa cervical cancer cell survival

We investigated the function of thymosin beta-4 (TB4) expression and primary cilium (PC) formation via the underlying Nrf2-dependent mechanism for cervical cancer cell (CC) survival under conditions of serum deprivation (SD). TB4 silencing was achieved using RNA interference. The percentage of PC formation was analyzed by immunofluorescence staining. Nrf2 expression was modified by the preparation of stable Nrf2-knockdown cells with shNrf2 and the overexpression of Nrf2 with pcDNA-Nrf2 plasmids. Gene expression was measured using reverse-transcription PCR, Gaussia luciferase assay, and western blotting. Cell viability was assessed using the MTT assay or CellTiter Glo assay. Reactive oxygen species (ROS) were detected with flow cytometry. CCs incubated in SD without fetal bovine serum remained viable, and SD increased PC formation and TB4 transcription. CC viability was further decreased by treatment with ciliobrevin A to inhibit PC formation or TB4-siRNA. SD increased ROS, including H2O2. N-acetylcysteine inhibited ROS production following H2O2 treatment or SD, which also decreased PC formation and TB4 transcription. Meanwhile, H2O2 increased PC formation, which was attenuated in response to TB4 siRNA. Treatment with H2O2 increased Nrf2 expression, antioxidant responsive element (ARE) activity, and PC formation, which were inhibited by the Nrf2 inhibitor clobestasol propionate. Nrf2 knockdown via expression of Tet-On shNrf2 enhanced ROS production, leading to increased PC formation and decreased TB4 expression; these effects were counteracted by Nrf2 overexpression. Our data demonstrate that Nrf2 counter-regulates TB4 expression and PC formation for CC survival under conditions of SD, suggesting cervical CC survival could be upregulated by PC formation via Nrf2 activation and TB4 expression.

HeLa Cervical Cancer Cells Are Maintained by Nephronophthisis 3-Associated Primary Cilium Formation via ROS-Induced ERK and HIF-1α Activation under Serum-Deprived Normoxic Condition.

The primary cilium (PC) is a microtubule-based antenna-like organelle projecting from the surface of the cell membrane. We previously reported that PC formation could be regulated by nephronophthisis 3 (NPHP3) expression followed by its interaction with thymosin β4. Here, we investigated whether cancer cell viability is regulated by NPHP3-mediated PC formation. The total and viable cell number were reduced by incubating cells under serum deprivation (SD) without fetal bovine serum (-FBS). PC frequency was increased by SD which enhanced NPHP3 expression and hypoxia inducible factor (HIF)-1α. The role of HIF-1α on NPHP3 expression and PC formation was confirmed by the binding of HIF-1α to the NPHP3 promoter and siRNA-based inhibition of HIF-1α (siHIF-1α), respectively. HIF-1α-stabilizing dimethyloxallyl glycine (DMOG) and hypoxic conditions increased NPHP3 expression and PC formation. In addition, as SD elevated the reactive oxygen species (ROS), PC frequency and NPHP3 expression were inhibited by a treatment with N-acetylcysteine (NAC), a ROS scavenger. PC formation was increased by H2O2 treatment, which was inhibited by siHIF-1α. The inhibition of ERK with P98059 decreased the frequency of PC formation and NPHP3 expression. Cell viability was reduced by a treatment with ciliobrevin A (CilioA) to inhibit PC formation, which was re-affirmed by using PC-deficient IFT88-/- cells. Taken together, the results imply that PC formation in cancer cells could be controlled by NPHP3 expression through ROS-induced HIF-1α and ERK activation under SD conditions. It suggests that cancer cell viability under SD conditions could be maintained by NPHP3 expression to regulate PC formation.

Knockdown of Ift88 in fibroblasts causes extracellular matrix remodeling and decreases conduction velocity in cardiomyocyte monolayers.

Background: Atrial fibrosis plays an important role in the development and persistence of atrial fibrillation by promoting reentry. Primary cilia have been identified as a regulator of fibroblasts (FB) activation and extracellular matrix (ECM) deposition. We hypothesized that selective reduction of primary cilia causes increased fibrosis and facilitates reentry. Aim: The aim of this study was to disrupt the formation of primary cilia in FB and examine its consequences on ECM and conduction in a co-culture system of cardiomyocytes (CM) and FB. Materials: Using short interfering RNA (siRNA), we removed primary cilia in neonatal rat ventricular FB by reducing the expression of Ift88 gene required for ciliary assembly. We co-cultured neonatal rat ventricular cardiomyocytes (CM) with FB previously transfected with Ift88 siRNA (siIft88) or negative control siRNA (siNC) for 48h. We examined the consequences of ciliated fibroblasts reduction on conduction and tissue remodeling by performing electrical mapping, microelectrode, and gene expression measurements. Results: Transfection of FB with siIft88 resulted in a significant 60% and 30% reduction of relative Ift88 expression in FB and CM-FB co-cultures, respectively, compared to siNC. Knockdown of Ift88 significantly increased the expression of ECM genes Fn1, Col1a1 and Ctgf by 38%, 30% and 18%, respectively, in comparison to transfection with siNC. Conduction velocity (CV) was significantly decreased in the siIft88 group in comparison to siNC [11.12 ± 4.27cm/s (n = 10) vs. 17.00 ± 6.20 (n = 10) respectively, p < 0.05]. The fraction of sites with interelectrode activation block was larger in the siIft88 group than in the siNC group (6.59 × 10-2 ± 8.01 × 10-2 vs. 1.18 × 10-2 ± 3.72 × 10-2 respectively, p < 0.05). We documented spontaneous reentrant arrhythmias in two cultures in the siIft88 group and in none of the siNC group. Action potentials were not significantly different between siNC and siIft88 groups. Conclusion: Disruption of cilia formation by siIft88 causes ECM remodeling and conduction abnormalities. Prevention of cilia loss could be a target for prevention of arrhythmias.

ODP651 Abnormal Primary Ciliogenesis in the Developing Mouse Kidney of Glucocorticoid Receptor-Deficient Mice

Abstract Glucocorticoid (GC) hormones have well characterised regulatory roles for the development and maturation of several fetal organs, however, specific developmental roles of GCs in the fetal kidney have not been described (1). We have explored both the expression and localisation of the glucocorticoid receptor (GR) within the developing fetal mouse kidney during organogenesis and the effect on the transcriptome of eliminating GR expression in gene-targeted GR-deficient mice. Staining for the GR by immunohistochemistry at E14.5, E16.5 and E18.5 demonstrated widespread expression of the GR in the renal tubule and the interstitial renal mesenchyme, with strong staining in collecting ducts and increasing expression in mesenchyme up to E18.5. Loss of GR expression in GR-null mice had a profound effect on the renal transcriptome with altered expression (at an FDR of 0.5 and fold change &amp;gt;2. 0) for 454 genes in the GR-null mouse kidney at E18.5, with a greater number of genes showing significantly reduced mRNA levels (305) than increased mRNA levels (99) relative to wild-type controls. Interestingly these deregulated genes included key primary cilia-associated genes, such as centrosomal protein 290 (Cep290), with the largest fold change of -3.6, Cep97, Ccp110, and the primary cilia-associated kinesin Kif3A. In addition, two renal tubule markers, Kidney androgen regulated protein (Kap) (proximal tubule marker) and S100 calcium binding protein G (S100g) (distal tubule marker) were also strongly downregulated with fold changes of -8.61 and -2.5 respectively. Analysis of primary cilia in sections of E18.5 kidney using multicolour confocal microscopy demonstrated that primary cilium length was significantly decreased and abnormally projected from kidney proximal tubule cells in E18.5 GR-null mice (5.23 + 0.12, µm + SEM) compared with wild type controls (6.27+ 0.15, µm + SEM). Finally, in cultured IMCD3 mouse collecting duct cells dexamethasone treatment was shown to increase the length of primary cilia compared to vehicle-treated control cells (vehicle 2.49 + 0. 03, µm + SEM vs dexamethasone 3.17 + 0. 05 µm + SEM), an effect that could be blocked by pre-treatment with the GR antagonist RU486. Taken together these results indicate that GC signalling mediated via the GR contributes either directly or indirectly to the normal formation of primary cilia in the proximal tubule and is therefore required for normal kidney development. This also suggests that abnormalities in GR-regulation of ciliogenesis may underpin or contribute to causes of human ciliopathy conditions. 1. Cole TJ, Short KL, Hooper SB (2019) The Science of Steroids. Sem. Fetal and Neonatal Med. 24: 170-175. Presentation: No date and time listed

The centrosomal protein 83 (CEP83) regulates human pluripotent stem cell differentiation toward the kidney lineage.

During embryonic development, the mesoderm undergoes patterning into diverse lineages including axial, paraxial, and lateral plate mesoderm (LPM). Within the LPM, the so-called intermediate mesoderm (IM) forms kidney and urogenital tract progenitor cells, while the remaining LPM forms cardiovascular, hematopoietic, mesothelial, and additional progenitor cells. The signals that regulate these early lineage decisions are incompletely understood. Here, we found that the centrosomal protein 83 (CEP83), a centriolar component necessary for primary cilia formation and mutated in pediatric kidney disease, influences the differentiation of human-induced pluripotent stem cells (hiPSCs) toward IM. We induced inactivating deletions of <i>CEP83</i> in hiPSCs and applied a 7-day in vitro protocol of IM kidney progenitor differentiation, based on timed application of WNT and FGF agonists. We characterized induced mesodermal cell populations using single-cell and bulk transcriptomics and tested their ability to form kidney structures in subsequent organoid culture. While hiPSCs with homozygous <i>CEP83</i> inactivation were normal regarding morphology and transcriptome, their induced differentiation into IM progenitor cells was perturbed. Mesodermal cells induced after 7 days of monolayer culture of <i>CEP83</i>-deficient hiPCS exhibited absent or elongated primary cilia, displayed decreased expression of critical IM genes (<i>PAX8</i>, <i>EYA1</i>, <i>HOXB7</i>), and an aberrant induction of LPM markers (e.g. <i>FOXF1</i>, <i>FOXF2</i>, <i>FENDRR</i>, <i>HAND1</i>, <i>HAND2</i>). Upon subsequent organoid culture, wildtype cells differentiated to form kidney tubules and glomerular-like structures, whereas <i>CEP83</i>-deficient cells failed to generate kidney cell types, instead upregulating cardiomyocyte, vascular, and more general LPM progenitor markers. Our data suggest that <i>CEP83</i> regulates the balance of IM and LPM formation from human pluripotent stem cells, identifying a potential link between centriolar or ciliary function and mesodermal lineage induction.

Human Cholangiocytes Form a Polarized and Functional Bile Duct on Hollow Fiber Membranes.

Liver diseases affect hundreds of millions of people worldwide; most often the hepatocytes or cholangiocytes are damaged. Diseases of the biliary tract cause severe patient burden, and cholangiocytes, the cells lining the biliary tract, are sensitive to numerous drugs. Therefore, investigations into proper cholangiocyte functions are of utmost importance, which is restricted, in vitro, by the lack of primary human cholangiocytes allowing such screening. To investigate biliary function, including transepithelial transport, cholangiocytes must be cultured as three-dimensional (3D) ductular structures. We previously established murine intrahepatic cholangiocyte organoid-derived cholangiocyte-like cells (CLCs) and cultured them onto polyethersulfone hollow fiber membranes (HFMs) to generate 3D duct structures that resemble native bile ducts at the structural and functional level. Here, we established an efficient, stepwise method for directed differentiation of human intrahepatic cholangiocyte organoids (ICOs) into CLCs. Human ICO-derived CLCs showed key characteristics of cholangiocytes, such as the expression of structural and functional markers, formation of primary cilia, and P-glycoprotein-mediated transport in a polarized fashion. The organoid cultures exhibit farnesoid X receptor (FXR)-dependent functions that are vital to liver bile acid homeostasis in vivo. Furthermore, human ICO-derived CLCs cultured on HFMs in a differentiation medium form tubular architecture with some tight, confluent, and polarized monolayers that better mimic native bile duct characteristics than differentiated cultures in standard 2D or Matrigel-based 3D culture plates. Together, our optimized differentiation protocol to obtain CLC organoids, when applied on HFMs to form bioengineered bile ducts, will facilitate studying cholangiopathies and allow developing therapeutic strategies.

Proteolytic control in ciliogenesis: Temporal restriction or early initiation?

Cellular processes are highly dependent on a dynamic proteome that undergoes structural and functional rearrangements to allow swift conversion between different cellular states. By inducing proteasomal degradation of inhibitory or stimulating factors, ubiquitylation is particularly well suited to trigger such transitions. One prominent example is the remodelling of the centrosome upon cell cycle exit, which is required for the formation of primary cilia - antenna-like structures on the surface of most cells that act as integrative hubs for various extracellular signals. Over the last decade, many reports on ubiquitin-related events involved in the regulation of ciliogenesis have emerged. Very often, these processes are considered to be initiated ad hoc, that is, directly before its effect on cilia biogenesis becomes evident. While such a temporal restriction may hold true for the majority of events, there is evidence that some of them are initiated earlier during the cell cycle. Here, we provide an overview of ubiquitin-dependent processes in ciliogenesis and discuss available data that indicate such an early onset of proteolytic regulation within preceding cell cycle stages.

Abstract 5029: A computational analysis of NEK10 and its novel protein-protein interaction with HspB1

Abstract The NEK kinase family of proteins consists of 11 serine/threonine kinases that participate in the disjunction of the centrosome, mitotic spindle assembly, and primary cilium formation. NEK10 is the most divergent member of the NEK family. It is unique in having a catalytic domain that is centrally positioned and flanked by two coiled-coil domains, while all the other NEKs have their catalytic domain near the N-terminus. Instead, NEK10 has four armadillo repeats of unexplored function in its N-terminus. NEK10 seems to play a key role in carcinogenesis and has been linked in melanoma, breast cancer, and a variety of ciliopathies. As part of our long-term goal to build interactomes of all the NEK members, we have previously reported data for known and predicted NEK10 interacting proteins, including novel protein-protein interactions such as HspB1 and MAP3K1, which have not been previously reported in the literature. In this study, we focused on understanding the molecular mechanism underlying NEK10’s interaction with HspB1 and its functional consequences. HspB1 is involved in various cellular functions such as maintaining cytoskeletal integrity and cell death. It is also a well-studied cancer protein and contains a highly conserved α-crystallin domain flanked by disordered N- and C-terminal domains. Many proteins are targeted by HspB1 to promote resistance to cell death, and malignant phenotypes; high levels of HspB1 have been identified in many cancer stem cells, such as those from lung and breast cancers. We used state-of-the-art computational approaches to model and characterize the full-length NEK10 as well as HspB1 protein. Our results offer robust full-length three-dimensional models of NEK10 and HspB1 and their biophysical characterization. In addition, we delineate the role of the armadillo repeats that are unique to NEK10 in the NEK family, in its interaction with HspB1. Our docking analysis shows that H301 of NEK10 located within its Armadillo Repeats forms a salt bridge with I179 of HspB1. Our results, therefore, suggest a scenario of Nek10 interaction with HspB1 using its ARM motif, which frees the catalytic domain for auto-phosphorylation and/or phosphorylation of other targets. Since HspB1 regulates the architecture of the cellular microtubular network (MT) by facilitating the formation of non-centrosomal MT, we speculate that the interaction of NEK10 with HspB1 plays a role in centrosome dynamics. Overall, this study shows novel and intriguing information about NEK10’s structure-function relationships, especially in the context of its interaction with HspB1. Furthermore, it establishes the framework for elucidating the detailed molecular mechanisms of NEK10 interactions with other proteins to further explore its potential as a therapeutic target. Citation Format: Andriele Silva, Shaneen Singh. A computational analysis of NEK10 and its novel protein-protein interaction with HspB1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5029.