The emergence of spontaneous coordinated epithelial rotation on cylindrical curved surfaces.

Three-dimensional collective epithelial rotation around a given axis represents a coordinated cellular movement driving tissue morphogenesis and transformation. Questions regarding these behaviors and their relationship with substrate curvatures are intimately linked to spontaneous active matter processes and to vital morphogenetic and embryonic processes. Here, using interdisciplinary approaches, we study the dynamics of epithelial layers lining different cylindrical surfaces. We observe large-scale, persistent, and circumferential rotation in both concavely and convexly curved cylindrical tissues. While epithelia of inverse curvature show an orthogonal switch in actomyosin network orientation and opposite apicobasal polarities, their rotational movements emerge and vary similarly within a common curvature window. We further reveal that this persisting rotation requires stable cell-cell adhesion and Rac-1-dependent cell polarity. Using an active polar gel model, we unveil the different relationships of collective cell polarity and actin alignment with curvatures, which lead to coordinated rotational behavior despite the inverted curvature and cytoskeleton order.

Non-ciliary Roles of IFT Proteins in Cell Division and Polycystic Kidney Diseases.

Cilia are small organelles present at the surface of most differentiated cells where they act as sensors for mechanical or biochemical stimuli. Cilia assembly and function require the Intraflagellar Transport (IFT) machinery, an intracellular transport system that functions in association with microtubules and motors. If IFT proteins have long been studied for their ciliary roles, recent evidences indicate that their functions are not restricted to the cilium. Indeed, IFT proteins are found outside the ciliary compartment where they are involved in a variety of cellular processes in association with non-ciliary motors. Recent works also provide evidence that non-ciliary roles of IFT proteins could be responsible for the development of ciliopathies related phenotypes including polycystic kidney diseases. In this review, we will discuss the interactions of IFT proteins with microtubules and motors as well as newly identified non-ciliary functions of IFT proteins, focusing on their roles in cell division. We will also discuss the potential contribution of non-ciliary IFT proteins functions to the etiology of kidney diseases.

Intraflagellar Transport Complex B Proteins Regulate the Hippo Effector Yap1 during Cardiogenesis.

Cilia and the intraflagellar transport (IFT) proteins involved in ciliogenesis are associated with congenital heart diseases (CHDs). However, the molecular links between cilia, IFT proteins, and cardiogenesis are yet to be established. Using a combination of biochemistry, genetics, and live-imaging methods, we show that IFT complex B proteins (Ift88, Ift54, and Ift20) modulate the Hippo pathway effector YAP1 in zebrafish and mouse. We demonstrate that this interaction is key to restrict the formation of the proepicardium and the myocardium. In cellulo experiments suggest that IFT88 and IFT20 interact with YAP1 in the cytoplasm and functionally modulate its activity, identifying a molecular link between cilia-related proteins and the Hippo pathway. Taken together, our results highlight a noncanonical role for IFT complex B proteins during cardiogenesis and shed light on a mechanism of action for ciliary proteins in YAP1 regulation.

mTOR and S6K1 drive polycystic kidney by the control of Afadin-dependent oriented cell division.

mTOR activation is essential and sufficient to cause polycystic kidneys in Tuberous Sclerosis Complex (TSC) and other genetic disorders. In disease models, a sharp increase of proliferation and cyst formation correlates with a dramatic loss of oriented cell division (OCD). We find that OCD distortion is intrinsically due to S6 kinase 1 (S6K1) activation. The concomitant loss of S6K1 in Tsc1-mutant mice restores OCD but does not decrease hyperproliferation, leading to non-cystic harmonious hyper growth of kidneys. Mass spectrometry-based phosphoproteomics for S6K1 substrates revealed Afadin, a known component of cell-cell junctions required to couple intercellular adhesions and cortical cues to spindle orientation. Afadin is directly phosphorylated by S6K1 and abnormally decorates the apical surface of Tsc1-mutant cells with E-cadherin and α-catenin. Our data reveal that S6K1 hyperactivity alters centrosome positioning in mitotic cells, affecting oriented cell division and promoting kidney cysts in conditions of mTOR hyperactivity.

IFT proteins interact with HSET to promote supernumerary centrosome clustering in mitosis.

Centrosome amplification is a hallmark of cancer, and centrosome clustering is essential for cancer cell survival. The mitotic kinesin HSET is an essential contributor to this process. Recent studies have highlighted novel functions for intraflagellar transport (IFT) proteins in regulating motors and mitotic processes. Here, using siRNA knock-down of various IFT proteins or AID-inducible degradation of endogenous IFT88 in combination with small-molecule inhibition of HSET, we show that IFT proteins together with HSET are required for efficient centrosome clustering. We identify a direct interaction between the kinesin HSET and IFT proteins, and we define how IFT proteins contribute to clustering dynamics during mitosis using high-resolution live imaging of centrosomes. Finally, we demonstrate the requirement of IFT88 for efficient centrosome clustering in a variety of cancer cell lines naturally harboring supernumerary centrosomes and its importance for cancer cell proliferation. Overall, our data unravel a novel role for the IFT machinery in centrosome clustering during mitosis in cells harboring supernumerary centrosomes.

IFT88 controls NuMA enrichment at k-fibers minus-ends to facilitate their re-anchoring into mitotic spindles.

To build and maintain mitotic spindle architecture, molecular motors exert spatially regulated forces on microtubules (MT) minus-ends. This spatial regulation is required to allow proper chromosomes alignment through the organization of kinetochore fibers (k-fibers). NuMA was recently shown to target dynactin to MT minus-ends and thus to spatially regulate dynein activity. However, given that k-fibers are embedded in the spindle, our understanding of the machinery involved in the targeting of proteins to their minus-ends remains limited. Intraflagellar transport (IFT) proteins were primarily studied for their ciliary roles but they also emerged as key regulators of cell division. Taking advantage of MT laser ablation, we show here that IFT88 concentrates at k-fibers minus-ends and is required for their re-anchoring into spindles by controlling NuMA accumulation. Indeed, IFT88 interacts with NuMA and is required for its enrichment at newly generated k-fibers minus-ends. Combining nocodazole washout experiments and IFT88 depletion, we further show that IFT88 is required for the reorganization of k-fibers into spindles and thus for efficient chromosomes alignment in mitosis. Overall, we propose that IFT88 could serve as a mitotic MT minus-end adaptor to concentrate NuMA at minus-ends thus facilitating k-fibers incorporation into the main spindle.

IFT proteins spatially control the geometry of cleavage furrow ingression and lumen positioning.

Cytokinesis mediates the physical separation of dividing cells and, in 3D epithelia, provides a spatial landmark for lumen formation. Here, we unravel an unexpected role in cytokinesis for proteins of the intraflagellar transport (IFT) machinery, initially characterized for their ciliary role and their link to polycystic kidney disease. Using 2D and 3D cultures of renal cells, we show that IFT proteins are required to correctly shape the central spindle, to control symmetric cleavage furrow ingression and to ensure central lumen positioning. Mechanistically, IFT88 directly interacts with the kinesin MKLP2 and is essential for the correct relocalization of the Aurora B/MKLP2 complex to the central spindle. IFT88 is thus required for proper centralspindlin distribution and central spindle microtubule organization. Overall, this work unravels a novel non-ciliary mechanism for IFT proteins at the central spindle, which could contribute to kidney cyst formation by affecting lumen positioning.

Flotillins control zebrafish epiboly through their role in cadherin-mediated cell-cell adhesion.

Zebrafish gastrulation and particularly epiboly that involves coordinated movements of several cell layers is a dynamic process for which regulators remain to be identified. We show here that Flotillin 1 and 2, ubiquitous and highly conserved proteins, are required for epiboly. Flotillins knockdown compromised embryo survival, strongly delayed epiboly and impaired deep cell radial intercalation and directed collective migration without affecting enveloping layer cell movement. At the molecular level, we identified that Flotillins are required for the formation of E-cadherin-mediated cell-cell junctions. These results provide the first in vivo evidence that Flotillins regulate E-cadherin-mediated cell-cell junctions to allow epiboly progression.

The use of the NEDD8 inhibitor MLN4924 (Pevonedistat) in a cyclotherapy approach to protect wild-type p53 cells from MLN4924 induced toxicity.

Targetting the ubiquitin pathway is an attractive strategy for cancer therapy. The inhibitor of the ubiquitin-like molecule NEDD8 pathway, MLN4924 (Pevonedistat) is in Phase II clinical trials. Protection of healthy cells from the induced toxicity of the treatment while preserving anticancer efficacy is a highly anticipated outcome in chemotherapy. Cyclotherapy was proposed as a promising approach to achieve this goal. We found that cytostatic activation of p53 protects cells against MLN4924-induced toxicity and importantly the effects are reversible. In contrast, cells with mutant or no p53 remain sensitive to NEDD8 inhibition. Using zebrafish embryos, we show that MLN4924-induced apoptosis is reduced upon pre-treatment with actinomycin D in vivo. Our studies show that the cellular effects of NEDD8 inhibition can be manipulated based on the p53 status and that NEDD8 inhibitors can be used in a p53-based cyclotherapy protocol to specifically target cancer cells devoid of wild type p53 function, while healthy cells will be protected from the induced toxicity.

New frontiers: discovering cilia-independent functions of cilia proteins.

In most vertebrates, mitotic spindles and primary cilia arise from a common origin, the centrosome. In non-cycling cells, the centrosome is the template for primary cilia assembly and, thus, is crucial for their associated sensory and signaling functions. During mitosis, the duplicated centrosomes mature into spindle poles, which orchestrate mitotic spindle assembly, chromosome segregation, and orientation of the cell division axis. Intriguingly, both cilia and spindle poles are centrosome-based, functionally distinct structures that require the action of microtubule-mediated, motor-driven transport for their assembly. Cilia proteins have been found at non-cilia sites, where they have distinct functions, illustrating a diverse and growing list of cellular processes and structures that utilize cilia proteins for crucial functions. In this review, we discuss cilia-independent functions of cilia proteins and re-evaluate their potential contributions to "cilia" disorders.

[Non-ciliary functions of cilia proteins]. / De nouvelles fonctions extraciliaires pour les protéines ciliaires - quelles conséquences sur l'apparition de ciliopathies ?

Cilia proteins have long been characterized for their role in cilia formation and function, and their implications in ciliopathies. However, several cellular defects induced by cilia proteins deregulation suggest that they could have non-ciliary roles. Indeed, several non-ciliary functions have been recently characterized for cilia proteins including roles in intra-cellular and in vesicular transport, in spindle orientation or in the maintenance of genomic stability. These observations thus raise the crucial question of the contribution of non-ciliary functions of cilia proteins to the pathological manifestations associated with ciliopathies such as polycystic kidney disease.

Identification of a mitotic Rac-GEF, Trio, that counteracts MgcRacGAP function during cytokinesis.

The Rho GTPases RhoA and Rac1 function as master regulators of cytokinesis by controlling the actomyosin cytoskeleton. RhoA and Rac1 have to be respectively activated and inactivated at the division plane for cytokinesis to occur properly. The inactivation of Rac1 at the cleavage furrow is controlled by MgcRacGAP. However, the guanine-nucleotide exchange factor (GEF) that activates Rac1 during cell division remains unknown. Here, using a siRNA screening approach in HeLa cells, we identify Trio as a mitotic GEF of Rac1. We demonstrate that Trio controls Rac1 activation and subsequent F-actin remodeling in dividing cells. Moreover, Trio depletion specifically rescues the cytokinesis failure induced by MgcRacGAP depletion. Of importance, we demonstrate that this rescue is mediated by the Trio-Rac1 pathway, using GEF-dead mutants of Trio and a specific inhibitor of Rac1 activation by Trio. Overall this work identifies for the first time a GEF controlling Rac1 activation in dividing cells that counteracts MgcRacGAP function in cytokinesis.

Centrin depletion causes cyst formation and other ciliopathy-related phenotypes in zebrafish.

Most bona fide centrosome proteins including centrins, small calcium-binding proteins, participate in spindle function during mitosis and play a role in cilia assembly in non-cycling cells. Although the basic cellular functions of centrins have been studied in lower eukaryotes and vertebrate cells in culture, phenotypes associated with centrin depletion in vertebrates in vivo has not been directly addressed. To test this, we depleted centrin2 in zebrafish and found that it leads to ciliopathy phenotypes including enlarged pronephric tubules and pronephric cysts. Consistent with the ciliopathy phenotypes, cilia defects were observed in differentiated epithelial cells of ciliated organs such as the olfactory bulb and pronephric duct. The organ phenotypes were also accompanied by cell cycle deregulation namely mitotic delay resulting from mitotic defects. Overall, this work demonstrates that centrin2 depletion causes cilia-related disorders in zebrafish. Moreover, given the presence of both cilia and mitotic defects in the affected organs, it suggests that cilia disorders may arise from a combination of these defects.

The cilia protein IFT88 is required for spindle orientation in mitosis.

Cilia dysfunction has long been associated with cyst formation and ciliopathies. More recently, misoriented cell division has been observed in cystic kidneys, but the molecular mechanism leading to this abnormality remains unclear. Proteins of the intraflagellar transport (IFT) machinery are linked to cystogenesis and are required for cilia formation in non-cycling cells. Several IFT proteins also localize to spindle poles in mitosis, indicating uncharacterized functions for these proteins in dividing cells. Here, we show that IFT88 depletion induces mitotic defects in human cultured cells, in kidney cells from the IFT88 mouse mutant Tg737(orpk) and in zebrafish embryos. In mitosis, IFT88 is part of a dynein1-driven complex that transports peripheral microtubule clusters containing microtubule-nucleating proteins to spindle poles to ensure proper formation of astral microtubule arrays and thus proper spindle orientation. This work identifies a mitotic mechanism for a cilia protein in the orientation of cell division and has important implications for the etiology of ciliopathies.

Pericentrin in cellular function and disease.

Pericentrin is an integral component of the centrosome that serves as a multifunctional scaffold for anchoring numerous proteins and protein complexes. Through these interactions, pericentrin contributes to a diversity of fundamental cellular processes. Recent studies link pericentrin to a growing list of human disorders. Studies on pericentrin at the cellular, molecular, and, more recently, organismal level, provide a platform for generating models to elucidate the etiology of these disorders. Although the complexity of phenotypes associated with pericentrin-mediated disorders is somewhat daunting, insights into the cellular basis of disease are beginning to come into focus. In this review, we focus on human conditions associated with loss or elevation of pericentrin and propose cellular and molecular models that might explain them.

Distinct roles of BARD1 isoforms in mitosis: full-length BARD1 mediates Aurora B degradation, cancer-associated BARD1beta scaffolds Aurora B and BRCA2.

The BRCA1-associated ring domain protein 1 (BARD1) interacts with BRCA1 via its RING finger domain. The BARD1-BRCA1 complex participates in DNA repair, cell cycle control, genomic stability, and mitotic spindle formation through its E3 ubiquitin ligase activity. Cancer cells express several BARD1 protein isoforms, including the RING finger-deficient variant BARD1beta. Here, we show that BARD1 has BRCA1-dependent and BRCA1-independent functions in mitosis. BARD1, but not BRCA1, localizes to the midbody at telophase and cytokinesis, where it colocalizes with Aurora B. The 97-kDa full-length (FL) BARD1 coimmunoprecipates with BRCA1, but the 82-kDa BARD1beta coimmunoprecipitates with Aurora B and BRCA2. We used selective small interfering RNAs to distinguish the functions of FL BARD1 and BARD1beta. Depletion of FL BARD1 had only minor effects on cell growth and did not abolish midbody localization of BARD1 staining, but resulted in massive up-regulation of Aurora B. In contrast, suppression of FL BARD1 and BARD1beta led to growth arrest and correlated with various mitotic defects and disappearance of midbody localization of BARD1 staining. Our data suggest a novel function of FL BARD1 in Aurora B ubiquitination and degradation, opposing a proproliferative function of BARD1beta in scaffolding Aurora B and BRCA2. Thus, loss of FL BARD1 and up-regulation of Aurora B, as observed in cancer cells, can be explained by an imbalance of FL BARD1 and BARD1beta.

Chromatin remodeling proteins interact with pericentrin to regulate centrosome integrity.

Pericentrin is an integral centrosomal component that anchors regulatory and structural molecules to centrosomes. In a yeast two-hybrid screen with pericentrin we identified chromodomain helicase DNA-binding protein 4 (CHD4/Mi2beta). CHD4 is part of the multiprotein nucleosome remodeling deacetylase (NuRD) complex. We show that many NuRD components interacted with pericentrin by coimmunoprecipitation and that they localized to centrosomes and midbodies. Overexpression of the pericentrin-binding domain of CHD4 or another family member (CHD3) dissociated pericentrin from centrosomes. Depletion of CHD3, but not CHD4, by RNA interference dissociated pericentrin and gamma-tubulin from centrosomes. Microtubule nucleation/organization, cell morphology, and nuclear centration were disrupted in CHD3-depleted cells. Spindles were disorganized, the majority showing a prometaphase-like configuration. Time-lapse imaging revealed mitotic failure before chromosome segregation and cytokinesis failure. We conclude that pericentrin forms complexes with CHD3 and CHD4, but a distinct CHD3-pericentrin complex is required for centrosomal anchoring of pericentrin/gamma-tubulin and for centrosome integrity.

Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest.

Centrosomes organize the microtubule cytoskeleton for both interphase and mitotic functions. They are implicated in cell-cycle progression but the mechanism is unknown. Here, we show that depletion of 14 out of 15 centrosome proteins arrests human diploid cells in G1 with reduced Cdk2-cyclin A activity and that expression of a centrosome-disrupting dominant-negative construct gives similar results. Cell-cycle arrest is always accompanied by defects in centrosome structure and function (for example, duplication and primary cilia assembly). The arrest occurs from within G1, excluding contributions from mitosis and cytokinesis. The arrest requires p38, p53 and p21, and is preceded by p38-dependent activation and centrosomal recruitment of p53. p53-deficient cells fail to arrest, leading to centrosome and spindle dysfunction and aneuploidy. We propose that loss of centrosome integrity activates a checkpoint that inhibits G1-S progression. This model satisfies the definition of a checkpoint in having three elements: a perturbation that is sensed, a transducer (p53) and a receiver (p21).