Mitotic control by mRNA splicing regulators ensures primary cilia formation

The biogenesis of the primary cilium is coordinated with cell cycle exit/re-entry in most types of cells. After serum starvation, the cilia-generating cells enter quiescence and produce the primary cilium; upon re-addition of serum, they re-enter the cell cycle and resorb the cilium. We previously identified novel mechanisms to link cell cycle progression and ciliogenesis by high-content genome-wide RNAi cell-based screening. In the present study, we pay attention to reveal the impact of mRNA splicing on cilia assembly after mitosis of cell cycle. We demonstrate that splicing regulators such as SON and XAB2 play an important role in mitosis exit, and thus affect ciliogenesis in G1/G0 phases. Knockdown of the splicing regulators in hTERT-RPE1 cells caused abnormal G2/M arrest under both serum addition and serum starvation, indicating defects in mitosis exit. Moreover, the knockdown cells failed to assemble the cilia under serum starvation and an inhibition of mRNA splicing using SSA, a spliceosome inhibitor, also revealed ciliogenesis defect. Finally, we show that the SSA-treated zebrafish display abnormal vascular development as a ciliary defect. These findings suggest the pivotal role of mRNA splicing regulators in cilia assembly and underscore the importance of mitotic regulation in ciliogenesis.     

Orientation and three-dimensional organization of actin filaments in dividing cultured cells

The current hypothesis of cytokinesis suggests that contractile forces in the cleavage furrow are generated by a circumferential band of actin filaments. However, relatively little is known about the global organization of actin filaments in dividing cells. To approach this problem we have used fluorescence-detected linear dichroism (FDLD) microscopy to measure filament orientation, and digital optical sectioning microscopy to perform three-dimensional reconstructions of dividing NRK cells stained with rhodamine-phalloidin. During metaphase, actin filaments in the equatorial region show a slight orientation along the spindle axis, while those in adjacent regions appear to be randomly distributed. Upon anaphase onset and through cytokinesis, the filaments become oriented along the equator in the furrow region, and along the spindle axis in adjacent regions. The degree of orientation appears to be dependent on cell-cell and cell-substrate adhesions. By performing digital optical sectioning microscopy on a highly spread NRK subclone, we show that actin filaments organize as a largely isotropic cortical meshwork in metaphase cells and convert into an anisotropic network shortly after anaphase onset, becoming more organized as cytokinesis proceeds. The conversion is most dramatic on the adhering ventral surface which shows little or no cleavage activity, and results in the formation of large bundles along the equator. On the dorsal surface, where cleavage occurs actively, actin filaments remain isotropic, showing only subtle alignment late in cytokinesis. In addition, stereo imaging has led to the discovery of a novel set of filaments that are associated with the cortex and traverse through the cytoplasm. Together, these studies provide important insights into the process of actin remodeling during cell division and point to possible additional mechanisms for force generation.     

The assembly and function of perinuclear actin cap in migrating cells

Stress fibers are actin bundles encompassing actin filaments, actin-crosslinking, and actin-associated proteins that represent the major contractile system in the cell. Different types of stress fibers assemble in adherent cells, and they are central to diverse cellular processes including establishment of the cell shape, morphogenesis, cell polarization, and migration. Stress fibers display specific cellular organization and localization, with ventral fibers present at the basal side, and dorsal fibers and transverse actin arcs rising at the cell front from the ventral to the dorsal side and toward the nucleus. Perinuclear actin cap fibers are a specific subtype of stress fibers that rise from the leading edge above the nucleus and terminate at the cell rear forming a dome-like structure. Perinuclear actin cap fibers are fixed at three points: both ends are anchored in focal adhesions, while the central part is physically attached to the nucleus and nuclear lamina through the linker of nucleoskeleton and cytoskeleton (LINC) complex. Here, we discuss recent work that provides new insights into the mechanism of assembly and the function of these actin stress fibers that directly link extracellular matrix and focal adhesions with the nuclear envelope.     

Cyclic expression of the voltage‐gated potassium channel KV10.1 promotes disassembly of the primary cilium

The primary cilium, critical for morphogenic and growth factor signaling, is assembled upon cell cycle exit, but the links between ciliogenesis and cell cycle progression are unclear. K_V10.1 is a voltage‐gated potassium channel frequently overexpressed in tumors. We have previously reported that expression of K_V10.1 is temporally restricted to a time period immediately prior to mitosis in healthy cells. Here, we provide microscopical and biochemical evidence that K_V10.1 localizes to the centrosome and the primary cilium and promotes ciliary disassembly. Interference with K_V10.1 ciliary localization abolishes not only the effects on ciliary disassembly, but also K_V10.1‐induced tumor progression in vivo. Conversely, upon knockdown of K_V10.1, ciliary disassembly is impaired, proliferation is delayed, and proliferating cells show prominent primary cilia. Thus, modulation of ciliogenesis by K_V10.1 can explain the influence of K_V10.1 expression on the proliferation of normal cells and is likely to be a major mechanism underlying its tumorigenic effects.     

Soluble levels of cytosolic tubulin regulate ciliary length control

The primary cilium is an evolutionarily conserved dynamic organelle important for regulating numerous signaling pathways, and, as such, mutations disrupting ciliogenesis result in a variety of developmental abnormalities and postnatal disorders. The length of the cilium is regulated by the cell through largely unknown mechanisms. Normal cilia length is important, as either shortened or elongated cilia have been associated with disease and developmental defects. Here we explore the importance of cytoskeletal dynamics in regulating cilia length. Using pharmacological approaches in different cell types, we demonstrate that actin depolymerization or stabilization and protein kinase A activation result in a rapid elongation of the primary cilium. The effects of pharmacological agents on cilia length are associated with a subsequent increase in soluble tubulin levels and can be impaired by depletion of soluble tubulin with taxol. In addition, subtle nocodazole treatment was able to induce ciliogenesis under conditions in which cilia are not normally formed and also increases cilia length on cells that have already established cilia. Together these data indicate that cilia length can be regulated through changes in either the actin or microtubule network and implicate a possible role for soluble tubulin levels in cilia length control.     

Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry

The primary cilium is an antenna-like organelle that is dynamically regulated during the cell cycle. Ciliogenesis is initiated as cells enter quiescence, whereas resorption of the cilium precedes mitosis. The mechanisms coordinating ciliogenesis with the cell cycle are unknown. Here we identify the centrosomal protein Nde1 (nuclear distribution gene E homologue 1) as a negative regulator of ciliary length. Nde1 is expressed at high levels in mitosis, low levels in quiescence and localizes at the mother centriole, which nucleates the primary cilium. Cells depleted of Nde1 have longer cilia and a delay in cell cycle re-entry that correlates with ciliary length. Knockdown of Nde1 in zebrafish embryos results in increased ciliary length, suppression of cell division, reduction of the number of cells forming the Kupffer's vesicle and left-right patterning defects. These data suggest that Nde1 is an integral component of a network coordinating ciliary length with cell cycle progression and have implications for understanding the transition from a quiescent to a proliferative state.     

HDAC2 promotes loss of primary cilia in pancreatic ductal adenocarcinoma

Loss of primary cilia is frequently observed in tumor cells, including pancreatic ductal adenocarcinoma (PDAC) cells, suggesting that the absence of this organelle may promote tumorigenesis through aberrant signal transduction and the inability to exit the cell cycle. However, the molecular mechanisms that explain how PDAC cells lose primary cilia are still ambiguous. In this study, we found that inhibition or silencing of histone deacetylase 2 (HDAC2) restores primary cilia formation in PDAC cells. Inactivation of HDAC2 results in decreased Aurora A expression, which promotes disassembly of primary cilia. We further showed that HDAC2 controls ciliogenesis independently of Kras, which facilitates Aurora A expression. These studies suggest that HDAC2 is a novel regulator of primary cilium formation in PDAC cells.     

Stages of ciliogenesis and regulation of ciliary length

Cilia and flagella are highly conserved eukaryotic microtubule-based organelles that protrude from the surface of most mammalian cells. These structures require large protein complexes and motors for distal addition of tubulin and extension of the ciliary membrane. In order for ciliogenesis to occur, coordination of many processes must take place. An intricate concert of cell cycle regulation, vesicular trafficking, and ciliary extension must all play out with accurate timing to produce a cilium. Here, we review the stages of ciliogenesis as well as regulation of the length of the assembled cilium. Regulation of ciliogenesis during cell cycle progression centers on centrioles, from which cilia extend upon maturation into basal bodies. Centriole maturation involves a shift from roles in cell division to cilium nucleation via migration to the cell surface and docking at the plasma membrane. Docking is dependent on a variety of proteinaceous structures, termed distal appendages, acquired by the mother centriole. Ciliary elongation by the process of intraflagellar transport (IFT) ensues. Direct modification of ciliary structures, as well as modulation of signal transduction pathways, play a role in maintenance of the cilium. All of these stages are tightly regulated to produce a cilium of the right size at the right time. Finally, we discuss the implications of abnormal ciliogenesis and ciliary length control in human disease as well as some open questions.     

A transmembrane relationship between fibronectin and vinculin (130 kd protein): Serum modulation in normal and transformed hamster fibroblasts

Using electron microscopy, we had previously demonstrated a very close transmembrane relationship between actin microfilaments and fibronectin fibrils, termed the fibronexus. Since vinculin, a recently discovered intracellular protein, is localized at the membrane-insertion regions of actin fibers, we studied its possible relationship to fibronectin and the fibronexus. Using double-label immunofluorescence microscopy, we have observed that the distributions of vinculin and fibronectin are strikingly coincident in normal Nil 8 hamster fibroblasts arrested in the G1 phase of the cell cycle, and in HSV-transformed Nil hamster cells treated with purified fibronectin after culturing in 0.3% serum. Extensively spread Nil 8 cells have numerous vinculin-positive focal patches, which are localized either directly over or in tandem with fibronectin fibers at the ventral surface. However, fibronectin and vinculin do not exhibit this relationship in Nil 8 cells grown in 5% serum. These vinculin patches closely resemble the vinculin plaques that Geiger found to be dark under interference-reflection microscopy, suggesting that fibronectin is associated with substrate-adhesion plaques in arrested cells. Fibronectin treatment of the HSV-transformed Nil cells cultured in a low concentration of serum results in the formation of ventral microprocesses, exhibiting an extraordinary congruence of vinculin and fibronectin staining. In addition, these cells bind matrix-like arrangements of fibronectin on their dorsal surface at sites of cell-cell interaction that are vinculin-negative. These results imply that two distinct types of fibronexuses may exist: a ventral substrate-adhesive nexus consisting of fibronectin, vinculin and actin, and a dorsal association between actin and intercellular fibronectin matrix fibers. Transmembrane vinculin-fibronectin associations are evidently sensitive to the growth state of the cell.     

The TBC1D31/praja2 complex controls primary ciliogenesis through PKA‐directed OFD1 ubiquitylation

The primary cilium is a microtubule‐based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X‐linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin‐proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G‐protein‐coupled receptor (GPCR)‐cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2‐UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non‐phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders.     

Peritubular myoid cells from rat seminiferous tubules contain actin and myosin filaments distributed in two independent layers

In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.     

The mammalian Nek1 kinase is involved in primary cilium formation

Recent studies implicate primary cilium (PC) proteins in the etiologies of various polycystic kidney diseases (PKD). NIMA-related kinases (NRKs) are conserved serine/threonine kinases, which are usually defined as 'mitotic kinases'. Murine mutants for the NRKs, nek1 (kat mice) suffer from PKD, suggesting that it may be involved in cilium control. We demonstrated herein that Nek1 is localized to basal body region and that Nek1 overexpression inhibits ciliogenesis in Madin-Darby canine kidney epithelial cells. The number of primary cilia is dramatically reduced in kat2J mouse embryonic fibroblasts culture. It is thus hypothesized that Nek1 links cell cycle progression and the PC cycle.     

Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow

Nonmuscle myosin IIA (NMM-IIA) is involved in the formation of focal adhesions and neurite retraction. However, the role of NMM-IIA in these functions remains largely unknown. Using RNA interference as a tool to decrease NMM-IIA expression, we have found that NMM-IIA is the major myosin involved in traction force generation and retrograde F-actin flow in mouse embryonic fibroblast cells. Quantitative analyses revealed that approximately 60% of traction force on fibronectin-coated surfaces is contributed by NMM-IIA and approximately 30% by NMM-IIB. The retrograde F-actin flow decreased dramatically in NMM-IIA-depleted cells, but seemed unaffected by NMM-IIB deletion. In addition, we found that depletion of NMM-IIA caused cells to spread at a higher rate and to a greater area on fibronectin substrates during the early spreading period, whereas deletion of NMM-IIB appeared to have no effect on spreading. The distribution of NMM-IIA was concentrated on the dorsal surface and approached the ventral surface in the periphery, whereas NMM-IIB was primarily concentrated around the nucleus and to a lesser extent at the ventral surface in cell periphery. Our results suggest that NMM-IIA is involved in generating a coherent cytoplasmic contractile force from one side of the cell to the other through the cross-linking and the contraction of dorsal actin filaments.     

Ciliogenesis and centriole formation in the mouse embryonic nervous system. An ultrastructural analysis

Serial ultrathin sections were used to study the formation of the primary cilium and the centriolar apparatus, basal body, and centriole in the neuroepithelial primordial cell of the embryonic nervous system in the mouse. At the end of mitosis, the centrioles seem to migrate toward the ventricular process of the neuroepithelial cell, near the ventricular surface. One of these centrioles, the nearest to the ventricular surface, begins to mature to form a basal body, since its tip is capped by a vesicle probably originating in the cytoplasm. This vesicle fuses with the plasmalemma and the cilium growth by the centrifugal extension of the 9 sets of microtubule doublets. These 9 sets invade the thick base of the cilium which is initially capped by a ball-shaped tip with the appearance of a mushroom cilium. The secondary extension of 7, then 5, and finally 2 sets of microtubule doublets contribute to form the tip of the mature cilium, which is associated with a mature centriolar apparatus formed by a basal body and a centriole. Centriologenesis occurs before mitosis and is concomitant with the progressive resorption of the cilium. The daughter centriole, or procentriole, begins to take form near the tips of fibrils that extend perpendicularly and at a short distance from the wall of the parent centriole. Osmiophilic material accumulates around these fibrils, and gives rise to the microtubules of the mature daughter centriole. These centrioles formed by a centriolar process are further engaged in mitosis, after the total resorption of the cilium. This pattern of development suggests that in the primordial cells of the embryonic nervous system, centriologenesis and ciliogenesis are 2 independent phenomena.     

Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity

The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video-enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three-dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament populations suggest that they differ in stability and function. The population of bundled long filaments, which appeared to be more ventrally located and in contact with membrane proteins, may be more stable than the population of short branched filaments. The location, organization, and polarity of the long bundled filaments suggest that they may be necessary for the expansion of lamellipodia and for the production of tension mediated by receptors to substrate adhesion molecules.     

Asymmetric distribution of Echinoid defines the epidermal leading edge during Drosophila dorsal closure

During Drosophila melanogaster dorsal closure, lateral sheets of embryonic epidermis assemble an actomyosin cable at their leading edge and migrate dorsally over the amnioserosa, converging at the dorsal midline. We show that disappearance of the homophilic cell adhesion molecule Echinoid (Ed) from the amnioserosa just before dorsal closure eliminates homophilic interactions with the adjacent dorsal-most epidermal (DME) cells, which comprise the leading edge. The resulting planar polarized distribution of Ed in the DME cells is essential for the localized accumulation of actin regulators and for actomyosin cable formation at the leading edge and for the polarized localization of the scaffolding protein Bazooka/PAR-3. DME cells with uniform Ed fail to assemble a cable and protrude dorsally, suggesting that the cable restricts dorsal migration. The planar polarized distribution of Ed in the DME cells thus provides a spatial cue that polarizes the DME cell actin cytoskeleton, defining the epidermal leading edge and establishing its contractile properties.     

Histological structure of human nail as studied by synchrotron X-ray microdiffraction.

Three layers (characterized by different orientations of the keratin molecules) from the outer to the inner side of human nail were observed by synchrotron X-ray microdiffraction. These layers are associated with the histological dorsal, intermediate and ventral plates. The hair-like type alpha-keratin filaments (81 A in diameter), are only present in the intermediate layer (accounting for approximately 2/3 of the nail width) and are perfectly oriented perpendicular to the growth axis, in the nail plane. Keratin filaments of stratum corneum (epidermis) type, found in the dorsal and ventral cells, are oriented in two privileged directions; parallel and perpendicular to the growth axis. This "sandwich" structure in the corneocytes and the strong intercellular junctions, gives the nail high mechanical rigidity and hardness, both in the curvature direction and in the growth direction. Lipid bilayers (49 A thick) parallel to the nail surface fill certain ampullar dilations of the dorsal plate and intercellular spaces in the ventral plate. Using X-ray micro-diffraction, we show that onychomycosis disrupts the keratin structure, probably during the synthesis phase.