Classical cadherins control nucleus and centrosome position and cell polarity

Control of cell polarity is crucial during tissue morphogenesis and renewal, and depends on spatial cues provided by the extracellular environment. Using micropatterned substrates to impose reproducible cell–cell interactions, we show that in the absence of other polarizing cues, cell–cell contacts are the main regulator of nucleus and centrosome positioning, and intracellular polarized organization. In a variety of cell types, including astrocytes, epithelial cells, and endothelial cells, calcium-dependent cadherin-mediated cell–cell interactions induce nucleus and centrosome off-centering toward cell–cell contacts, and promote orientation of the nucleus–centrosome axis toward free cell edges. Nucleus and centrosome off-centering is controlled by N-cadherin through the regulation of cell interactions with the extracellular matrix, whereas the orientation of the nucleus–centrosome axis is determined by the geometry of N-cadherin–mediated contacts. Our results demonstrate that in addition to the specific function of E-cadherin in regulating baso-apical epithelial polarity, classical cadherins control cell polarization in otherwise nonpolarized cells.     

Excess centrosomes disrupt endothelial cell migration via centrosome scattering

Supernumerary centrosomes contribute to spindle defects and aneuploidy at mitosis, but the effects of excess centrosomes during interphase are poorly understood. In this paper, we show that interphase endothelial cells with even one extra centrosome exhibit a cascade of defects, resulting in disrupted cell migration and abnormal blood vessel sprouting. Endothelial cells with supernumerary centrosomes had increased centrosome scattering and reduced microtubule (MT) nucleation capacity that correlated with decreased Golgi integrity and randomized vesicle trafficking, and ablation of excess centrosomes partially rescued these parameters. Mechanistically, tumor endothelial cells with supernumerary centrosomes had less centrosome-localized γ-tubulin, and Plk1 blockade prevented MT growth, whereas overexpression rescued centrosome γ-tubulin levels and centrosome dynamics. These data support a model whereby centrosome–MT interactions during interphase are important for centrosome clustering and cell polarity and further suggest that disruption of interphase cell behavior by supernumerary centrosomes contributes to pathology independent of mitotic effects.     

The polarity protein Pard3 is required for centrosome positioning during neurulation

Microtubules are essential regulators of cell polarity, architecture and motility. The organization of the microtubule network is context-specific. In non-polarized cells, microtubules are anchored to the centrosome and form radial arrays. In most epithelial cells, microtubules are noncentrosomal, align along the apico-basal axis and the centrosome templates a cilium. It follows that cells undergoing mesenchyme-to-epithelium transitions must reorganize their microtubule network extensively, yet little is understood about how this process is orchestrated. In particular, the pathways regulating the apical positioning of the centrosome are unknown, a central question given the role of cilia in fluid propulsion, sensation and signaling. In zebrafish, neural progenitors undergo progressive epithelialization during neurulation, and thus provide a convenient in vivo cellular context in which to address this question. We demonstrate here that the microtubule cytoskeleton gradually transitions from a radial to linear organization during neurulation and that microtubules function in conjunction with the polarity protein Pard3 to mediate centrosome positioning. Pard3 depletion results in hydrocephalus, a defect often associated with abnormal cerebrospinal fluid flow that has been linked to cilia defects. These findings thus bring to focus cellular events occurring during neurulation and reveal novel molecular mechanisms implicated in centrosome positioning.     

Centrosome positioning in polarized cells: Common themes and variations

The centrosome position is tightly regulated during the cell cycle and during differentiated cellular functions. Because centrosome organizes the microtubule network to coordinate both intracellular organization and cell signaling, centrosome positioning is crucial to determine either the axis of cell division, the direction of cell migration or the polarized immune response of lymphocytes. Since alteration of centrosome positioning seems to promote cell transformation and tumor spreading, the molecular mechanisms controlling centrosome movement in response to extracellular and intracellular cues are under intense investigation. Evolutionary conserved pathways involving polarity proteins and cytoskeletal rearrangements are emerging as common regulators of centrosome positioning in a wide variety of cellular contexts.     

The centrosome and asymmetric cell division

Asymmetric stem cell division is a mechanism widely employed by the cell to maintain tissue homeostasis, resulting in the production of one stem cell and one differentiating cell. However, asymmetric cell division is not limited to stem cells and is widely observed even in unicellular organisms as well as in cells that make up highly complex tissues. In asymmetric cell division, cells must organize their intracellular components along the axis of asymmetry (sometimes in the context of extracellular architecture). Recent studies have described cell asymmetry in many cell types and in many cases such asymmetry involves the centrosome (or spindle pole body in yeast) as the center of cytoskeleton organization. In this review, I summarize recent discoveries in cellular polarity that lead to an asymmetric outcome, with a focus on centrosome function.Key words: stem cell, asymmetric division, niche, centrosome, spindle orientation     

The centrosome and asymmetric cell division

Asymmetric stem cell division is a mechanism widely employed by the cell to maintain tissue homeostasis, resulting in the production of one stem cell and one differentiating cell. However, asymmetric cell division is not limited to stem cells and is widely observed even in unicellular organisms as well as in cells that make up highly complex tissues. In asymmetric cell division, cells must organize their intracellular components along the axis of asymmetry(sometimes in the context of extracellular architecture). Recent studies have described cell asymmetry in many cell types, and in many cases such asymmetry involves the centrosome (or spindle pole body in yeast) as the center of cytoskeleton organization. In this review, I summarize recent discoveries in cellular polarity that lead to an asymmetric outcome, with a focus on centrosome function.     

Deubiquitylation Machinery Is Required for Embryonic Polarity in Caenorhabditis elegans

The Caenorhabditis elegans one-cell embryo polarizes in response to a cue from the paternally donated centrosome and asymmetrically segregates cell fate determinants that direct the developmental program of the worm. We have found that genes encoding putative deubiquitylating enzymes (DUBs) are required for polarization of one-cell embryos. Maternal loss of the proteins MATH-33 and USP-47 leads to variable inability to correctly establish and maintain asymmetry as defined by posterior and anterior polarity proteins PAR-2 and PAR-3. The first observable defect is variable positioning of the centrosome with respect to the cell cortex and the male pronucleus. The severity of the polarity defects correlates with distance of the centrosome from the cortex. Furthermore, polarity defects can be bypassed by mutations that bring the centrosome in close proximity to the cortex. In addition we find that polarity and centrosome positioning defects can be suppressed by compromising protein turnover. We propose that the DUB activity of MATH-33 and USP-47 stabilizes one or more proteins required for association of the centrosome with the cortex. Because these DUBs are homologous to two members of a group of DUBs that act in fission yeast polarity, we tested additional members of that family and found that another C. elegans DUB gene, usp-46, also contributes to polarity. Our finding that deubiquitylating enzymes required for polarity in Schizosaccharomyces pombe are also required in C. elegans raises the possibility that these DUBs act through an evolutionarily conserved mechanism to control cell polarity.     

The importance of a single primary cilium

The centrosome is the main microtubule-organizing center in animal cells, and helps to influence the morphology of the microtubule cytoskeleton in interphase and mitosis. The centrosome also templates the assembly of the primary cilium, and together they serve as a nexus of cell signaling that provide cells with diverse organization, motility, and sensory functions. The majority of cells in the human body contain a solitary centrosome and cilium, and cells have evolved regulatory mechanisms to precisely control the numbers of these essential organelles. Defects in the structure and function of cilia lead to a variety of complex disease phenotypes termed ciliopathies, while dysregulation of centrosome number has long been proposed to induce genome instability and tumor formation. Here, we review recent findings that link centrosome amplification to changes in cilium number and signaling capacity, and discuss how supernumerary centrosomes may be an important aspect of a set of cilia-related disease phenotypes.     

Centrosomes: The good and the bad for brain development

Centrosomes nucleate and organise the microtubule cytoskeleton in animal cells. These membraneless organelles are key structures for tissue organisation, polarity and growth. Centrosome dysfunction, defined as deviation in centrosome numbers and/or structural integrity, has major impact on brain size and functionality, as compared with other tissues of the organism. In this review, we discuss the contribution of centrosomes to brain growth during development. We discuss in particular the impact of centrosome dysfunction in Drosophila and mammalian neural stem cell division and fitness, which ultimately underlie brain growth defects.     

Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts

Cell polarity must be integrated with tissue polarity for proper development. The Drosophila embryonic central nervous system (CNS) is a highly polarized tissue; neuroblasts occupy the most apical layer of cells within the CNS, and lie just basal to the neural epithelium. Neuroblasts are the CNS progenitor cells and undergo multiple rounds of asymmetric cell division, ;budding off' smaller daughter cells (GMCs) from the side opposite the epithelium, thereby positioning neuronal/glial progeny towards the embryo interior. It is unknown whether this highly stereotypical orientation of neuroblast divisions is controlled by an intrinsic cue (e.g. cortical mark) or an extrinsic cue (e.g. cell-cell signal). Using live imaging and in vitro culture, we find that neuroblasts in contact with epithelial cells always ;bud off' GMCs in the same direction, opposite from the epithelia-neuroblast contact site, identical to what is observed in vivo. By contrast, isolated neuroblasts 'bud off' GMCs at random positions. Imaging of centrosome/spindle dynamics and cortical polarity shows that in neuroblasts contacting epithelial cells, centrosomes remained anchored and cortical polarity proteins localize at the same epithelia-neuroblast contact site over subsequent cell cycles. In isolated neuroblasts, centrosomes drifted between cell cycles and cortical polarity proteins showed a delay in polarization and random positioning. We conclude that embryonic neuroblasts require an extrinsic signal from the overlying epithelium to anchor the centrosome/centrosome pair at the site of epithelial-neuroblast contact and for proper temporal and spatial localization of cortical Par proteins. This ensures the proper coordination between neuroblast cell polarity and CNS tissue polarity.     

Cilium, centrosome and cell cycle regulation in polycystic kidney disease

Polycystic kidney disease is the defining condition of a group of common life-threatening genetic disorders characterized by the bilateral formation and progressive expansion of renal cysts that lead to end stage kidney disease. Although a large body of information has been acquired in the past years about the cellular functions that characterize the cystic cells, the mechanisms triggering the cystogenic conversion are just starting to emerge. Recent findings link defects in ciliary functions, planar cell polarity pathway, and centrosome integrity in early cystic development. Many of the signals dysregulated during cystogenesis may converge on the centrosome for its central function as a structural support for cilia formation and a coordinator of protein trafficking, polarity, and cell division. Here, we will discuss the contribution of proliferation, cilium and planar cell polarity to the cystic signal and will analyze in particular the possible role that the basal bodies/centrosome may play in the cystogenetic mechanisms. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Mapping Cellular Polarity Networks Using Mass Spectrometry-based Strategies

Cell polarity is a vital biological process involved in the building, maintenance and normal functioning of tissues in invertebrates and vertebrates. Unsurprisingly, molecular defects affecting polarity organization and functions have a strong impact on tissue homeostasis, embryonic development and adult life, and may directly or indirectly lead to diseases. Genetic studies have demonstrated the causative effect of several polarity genes in diseases; however, much remains to be clarified before a comprehensive view of the molecular organization and regulation of the protein networks associated with polarity proteins is obtained. This challenge can be approached head-on using proteomics to identify protein complexes involved in cell polarity and their modifications in a spatio-temporal manner. We review the fundamental basics of mass spectrometry techniques and provide an in-depth analysis of how mass spectrometry has been instrumental in understanding the complex and dynamic nature of some cell polarity networks at the tissue (apico-basal and planar cell polarities) and cellular (cell migration, ciliogenesis) levels, with the fine dissection of the interconnections between prototypic cell polarity proteins and signal transduction cascades in normal and pathological situations. This review primarily focuses on epithelial structures which are the fundamental building blocks for most metazoan tissues, used as the archetypal model to study cellular polarity. This field offers broad perspectives thanks to the ever-increasing sensitivity of mass spectrometry and its use in combination with recently developed molecular strategies able to probe in situ proteomic networks.     

Centrosome Function: Sometimes Less Is More

Tight regulation of centrosome duplication is critical to ensure that centrosome number doubles once and only once per cell cycle. Superimposed onto this centrosome duplication cycle is a functional centrosome cycle in which they alternate between phases of quiescence and robust microtubule (MT) nucleation and MT-anchoring activities. In vertebrate cycling cells, interphase centrioles accumulate less pericentriolar material (PCM), reducing their MT nucleation capacity. In mitosis, centrosomes mature, accumulating more PCM to increase their nucleation and anchoring capacities to form robust MT asters. Interestingly, functional cycles of centrosomes can be altered to suit the cell's needs. Some interphase centrosomes function as a microtubule-organizing center by increasing their ability to anchor MTs to form centrosomal radial arrays. Other interphase centrosomes maintain their MT nucleation capacity but reduce/eliminate their MT-anchoring capacity. Recent work demonstrates that Drosophila cells take this to the extreme, whereby centrioles lose all detectable PCM during interphase, offering an explanation as to how centrosome-deficient flies develop to adulthood. Drosophila stem cells further modify the functional cycle by differentially regulating their two centrioles – a situation that seems important for stem cell asymmetric divisions, as misregulation of centrosome duplication in stem/progenitor cells can promote tumor formation. Here, we review recent findings that describe variations in the functional cycle of centrosomes.     

Breaking the ties that bind: new advances in centrosome biology

The centrosome, which consists of two centrioles and the surrounding pericentriolar material, is the primary microtubule-organizing center (MTOC) in animal cells. Like chromosomes, centrosomes duplicate once per cell cycle and defects that lead to abnormalities in the number of centrosomes result in genomic instability, a hallmark of most cancer cells. Increasing evidence suggests that the separation of the two centrioles (disengagement) is required for centrosome duplication. After centriole disengagement, a proteinaceous linker is established that still connects the two centrioles. In G2, this linker is resolved (centrosome separation), thereby allowing the centrosomes to separate and form the poles of the bipolar spindle. Recent work has identified new players that regulate these two processes and revealed unexpected mechanisms controlling the centrosome cycle.     

A subset of centrosomal proteins are arranged in a tubular conformation that is reproduced during centrosome duplication

The centrosome plays a fundamental role in organizing the interphase cytoskeleton and the mitotic spindle, and its protein complexity is modulated to support these functions. The centrosome must also duplicate itself once during each cell cycle, thus ensuring the formation of a bipolar spindle and its continuity through successive cell divisions. In this study, we have used a battery of antibodies directed against centrosomal components to study the general organization of the centrosome during the cell cycle and during the centrosome duplication process. We demonstrate that a subset of centrosomal proteins are arranged together to form a tubular pattern within the centrosome. The tubular conformation defined by these proteins has a polarity and is closed at one end. The centriole complement of the centrosome is normally placed near this end. We show that the "wall" of the tube is enriched in proteins such as CDC2, ninein, and pericentrin as well as gamma-tubulin. In addition, a subset of gamma-tubulin is localized to the "lumen" of the tube. We also demonstrate, for the first time, that antibody staining can be used to detect centrosome duplication allowing the identification of duplication intermediates. We show that one product of centrosome duplication is the replication of the tubular structure found within the centrosome. The position of the centriole duplexes prior to and during centrosome duplication is documented and a model of the morphogenesis of the centrosome during the duplication process is proposed.     

Nucleoporin Nup62 maintains centrosome homeostasis

Centrosomes are comprised of 2 orthogonally arranged centrioles surrounded by the pericentriolar material (PCM), which serves as the main microtubule organizing center of the animal cell. More importantly, centrosomes also control spindle polarity and orientation during mitosis. Recently, we and other investigators discovered that several nucleoporins play critical roles during cell division. Here, we show that nucleoporin Nup62 plays a novel role in centrosome integrity. Knockdown of Nup62 induced mitotic arrest in G₂/M phases and mitotic cell death. Depletion of Nup62 using RNA interference results in defective centrosome segregation and centriole maturation during the G₂ phase. Moreover, Nup62 depletion in human cells leads to the appearance of multinucleated cells and induces the formation of multipolar centrosomes, centriole synthesis defects, dramatic spindle orientation defects, and centrosome component rearrangements that impair cell bi-polarity. Our results also point to a potential role of Nup62 in targeting gamma-tubulin and SAS-6 to the centrioles.     

POPX2 phosphatase regulates cell polarity and centrosome placement

Proper centrosome positioning is critical for many cellular functions, such as cell migration and maintenance of polarity. During wound healing, fibroblasts orient their centrosomes such that they face the wound edge. The centrosome orientation determines the direction of cells’ migration so that they can close the wound effectively. In this study, we investigated the regulation of centrosome polarization and have identified the phosphatase POPX2 as an important regulator of centrosome orientation. We found that POPX2 inhibits centrosome centration, but not rearward nuclear movement, by regulating multiple proteins that function in centrosome positioning. High POPX2 levels result in reduced motility of the kinesin-2 motor, which, in turn, inhibits the transport of N-cadherin to the cell periphery and cell junctions. Loss of N-cadherin localization to the cell membrane affects the localization of focal adhesions and perturbs CDC42-Par6/PKCζ signaling. In addition, overexpression of POPX2 also results in a loss of Par3 localization to the cell periphery and reduced levels of LIC2 (dynein light intermediate chain 2), leading to defects in microtubule tethering and dynamics at cell-cell contacts. Therefore, POPX2 functions as a regulator of signaling pathways to modulate the positioning of centrosome in fibroblast during wound healing.     

Nedd1 expression as a marker of dynamic centrosomal localization during mouse embryonic development

As the primary microtubule-organizing centre of the mammalian cell, the centrosome plays many important roles during cell growth and organization. This is evident across a broad range of cell types and processes, such as the proliferation, differentiation and polarity of neural cells. Additionally, given its localization and function, there are likely to be many more processes that rely on the centrosome that have not yet been characterized. Currently, little is known about centrosomal dynamics during mammalian development. In this study, we have analyzed Nedd1 protein expression to characterize the localization of the centrosome during some aspects of mouse embryogenesis. Using a Nedd1 antibody we have demonstrated the colocalization of Nedd1 with centrosomal markers. We found strong expression of Nedd1, and therefore the centrosome, in highly proliferating cells during neural development. Additionally, Nedd1 was found to have high expression in the cytoplasm of a subset of cells in the dorsal root ganglia. We have also shown a distinct, polarized centrosomal localization of Nedd1 in the developing lens, retina and other polarized tissues. This study reveals the localization of Nedd1 and the centrosome during important processes in mouse embryogenesis and provides a basis for further study into its role in development.