Length limitation of astral microtubules orients cell divisions in murine intestinal crypts.

Planar spindle orientation is critical for epithelial tissue organization and is generally instructed by the long cell-shape axis or cortical polarity domains. We introduced mouse intestinal organoids in order to study spindle orientation in a monolayered mammalian epithelium. Although spindles were planar, mitotic cells remained elongated along the apico-basal (A-B) axis, and polarity complexes were segregated to basal poles, so that spindles oriented in an unconventional manner, orthogonal to both polarity and geometric cues. Using high-resolution 3D imaging, simulations, and cell-shape and cytoskeleton manipulations, we show that planar divisions resulted from a length limitation in astral microtubules (MTs) which precludes them from interacting with basal polarity, and orient spindles from the local geometry of apical domains. Accordingly, lengthening MTs affected spindle planarity, cell positioning, and crypt arrangement. We conclude that MT length regulation may serve as a key mechanism for spindles to sense local cell shapes and tissue forces to preserve mammalian epithelial architecture.

Regulation and functions of cell division in the intestinal tissue.

In multicellular organisms, epithelial cells are key elements of tissue organization. In developing epithelial tissues, cellular proliferation and differentiation are under the tight regulation of morphogenetic programs to ensure correct organ formation and functioning. In these processes, proliferation rates and division orientation regulate the speed, timing and direction of tissue expansion but also its proper patterning. Moreover, tissue homeostasis relies on spatio-temporal modulations of daughter cell behavior and arrangement. These aspects are particularly crucial in the intestine, which is one of the most proliferative tissues in adults, making it a very attractive adult organ system to study the role of cell division on epithelial morphogenesis and organ function. Although epithelial cell division has been the subject of intense research for many years in multiple models, it still remains in its infancy in the context of the intestinal tissue. In this review, we focus on the current knowledge on cell division and regulatory mechanisms at play in the intestinal epithelial tissue, as well as their importance in developmental biology and physiopathology.

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.

Modulation of designer biomimetic matrices for optimized differentiated intestinal epithelial cultures.

The field of intestinal biology is thirstily searching for different culture methods that complement the limitations of organoids, particularly the lack of a differentiated intestinal compartment. While being recognized as an important milestone for basic and translational biological studies, many primary cultures of intestinal epithelium (IE) rely on empirical trials using hydrogels of various stiffness, whose mechanical impact on epithelial organization remains vague until now. Here, we report the development of hydrogel scaffolds with a range of elasticities and their influence on IE expansion, organization, and differentiation. On stiff substrates (>5 kPa), mouse IE cells adopt a flat cell shape and detach in the short-term. In contrast, on soft substrates (80-500 Pa), they sustain for a long-term, pack into high density, develop columnar shape with improved apical-basal polarity and differentiation marker expression, a phenotype reminiscent of features in vivo mouse IE. We then developed a soft gel molding process to produce 3D Matrigel scaffolds of close-to-nature stiffness, which support and maintain a culture of mouse IE into crypt-villus architecture. Thus, the present work is up-to-date informative for the design of biomaterials for ex vivo intestinal models, offering self-renewal in vitro culture that emulates the mouse IE.

EpCAM promotes endosomal modulation of the cortical RhoA zone for epithelial organization.

At the basis of cell shape and behavior, the organization of actomyosin and its ability to generate forces are widely studied. However, the precise regulation of this contractile network in space and time is unclear. Here, we study the role of the epithelial-specific protein EpCAM, a contractility modulator, in cell shape and motility. We show that EpCAM is required for stress fiber generation and front-rear polarity acquisition at the single cell level. In fact, EpCAM participates in the remodeling of a transient zone of active RhoA at the cortex of spreading epithelial cells. EpCAM and RhoA route together through the Rab35/EHD1 fast recycling pathway. This endosomal pathway spatially organizes GTP-RhoA to fine tune the activity of actomyosin resulting in polarized cell shape and development of intracellular stiffness and traction forces. Impairment of GTP-RhoA endosomal trafficking either by silencing EpCAM or by expressing Rab35/EHD1 mutants prevents proper myosin-II activity, stress fiber formation and ultimately cell polarization. Collectively, this work shows that the coupling between co-trafficking of EpCAM and RhoA, and actomyosin rearrangement is pivotal for cell spreading, and advances our understanding of how biochemical and mechanical properties promote cell plasticity.

Human Microphysiological Models of Intestinal Tissue and Gut Microbiome.

The gastrointestinal (GI) tract is a complex system responsible for nutrient absorption, digestion, secretion, and elimination of waste products that also hosts immune surveillance, the intestinal microbiome, and interfaces with the nervous system. Traditional in vitro systems cannot harness the architectural and functional complexity of the GI tract. Recent advances in organoid engineering, microfluidic organs-on-a-chip technology, and microfabrication allows us to create better in vitro models of human organs/tissues. These micro-physiological systems could integrate the numerous cell types involved in GI development and physiology, including intestinal epithelium, endothelium (vascular), nerve cells, immune cells, and their interplay/cooperativity with the microbiome. In this review, we report recent progress in developing micro-physiological models of the GI systems. We also discuss how these models could be used to study normal intestinal physiology such as nutrient absorption, digestion, and secretion as well as GI infection, inflammation, cancer, and metabolism.

Galectin-3 modulates epithelial cell adaptation to stress at the ER-mitochondria interface.

Cellular stress response contributes to epithelial defense in adaptation to environment changes. Galectins play a pivotal role in the regulation of this response in malignant cells. However, precise underlying mechanisms are largely unknown. Here we demonstrate that Galectin-3, a pro and anti-apoptotic lectin, is required for setting up a correct cellular response to stress by orchestrating several effects. First, Galectin-3 constitutes a key post-transcriptional regulator of stress-related mRNA regulons coordinating the cell metabolism, the mTORC1 complex or the unfolded protein response (UPR). Moreover, we demonstrated the presence of Galectin-3 with mitochondria-associated membranes (MAM), and its interaction with proteins located at the ER or mitochondrial membranes. There Galectin-3 prevents the activation and recruitment at the mitochondria of the regulator of mitochondria fission DRP-1. Accordingly, loss of Galectin-3 impairs mitochondrial morphology, with more fragmented and round mitochondria, and dynamics both in normal and cancer epithelial cells in basal conditions. Importantly, Galectin-3 deficient cells also display changes of the activity of the mitochondrial respiratory chain complexes, of the mTORC1/S6RP/4EBP1 translation pathway and reactive oxygen species levels. Regarding the ER, Galectin-3 did not modify the activities of the 3 branches of the UPR in basal conditions. However, Galectin-3 favours an adaptative UPR following ER stress induction by Thapsigargin treatment. Altogether, at the ER-mitochondria interface, Galectin-3 coordinates the functioning of the ER and mitochondria, preserves the integrity of mitochondrial network and modulates the ER stress response.

Galectin 3 Deficiency Alters Chondrocyte Primary Cilium Formation and Exacerbates Cartilage Destruction via Mitochondrial Apoptosis.

Mechanical overload and aging are the main risk factors of osteoarthritis (OA). Galectin 3 (GAL3) is important in the formation of primary cilia, organelles that are able to sense mechanical stress. The objectives were to evaluate the role of GAL3 in chondrocyte primary cilium formation and in OA in mice. Chondrocyte primary cilium was detected in vitro by confocal microscopy. OA was induced by aging and partial meniscectomy of wild-type (WT) and Gal3-null 129SvEV mice (Gal3-/-). Primary chondrocytes were isolated from joints of new-born mice. Chondrocyte apoptosis was assessed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), caspase 3 activity and cytochrome c release. Gene expression was assessed by qRT-PCR. GAL3 was localized at the basal body of the chondrocyte primary cilium. Primary cilia of Gal3-/- chondrocytes were frequently abnormal and misshapen. Deletion of Gal3 triggered premature OA during aging and exacerbated joint instability-induced OA. In both aging and surgery-induced OA cartilage, levels of chondrocyte catabolism and hypertrophy markers and apoptosis were more severe in Gal3-/- than WT samples. In vitro, Gal3 knockout favored chondrocyte apoptosis via the mitochondrial pathway. GAL3 is a key regulator of cartilage homeostasis and chondrocyte primary cilium formation in mice. Gal3 deletion promotes OA development.

Spindle pole cohesion requires glycosylation-mediated localization of NuMA.

Glycosylation is critical for the regulation of several cellular processes. One glycosylation pathway, the unusual O-linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) has been shown to be required for proper mitosis, likely through a subset of proteins that are O-GlcNAcylated during metaphase. As lectins bind glycosylated proteins, we asked if specific lectins interact with mitotic O-GlcNAcylated proteins during metaphase to ensure correct cell division. Galectin-3, a small soluble lectin of the Galectin family, is an excellent candidate, as it has been previously described as a transient centrosomal component in interphase and mitotic epithelial cells. In addition, it has recently been shown to associate with basal bodies in motile cilia, where it stabilizes the microtubule-organizing center (MTOC). Using an experimental mouse model of chronic kidney disease and human epithelial cell lines, we investigate the role of Galectin-3 in dividing epithelial cells. Here we find that Galectin-3 is essential for metaphase where it associates with NuMA in an O-GlcNAcylation-dependent manner. We provide evidence that the NuMA-Galectin-3 interaction is important for mitotic spindle cohesion and for stable NuMA localization to the spindle pole, thus revealing that Galectin-3 is a novel contributor to epithelial mitotic progress.

Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells.

Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3'UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3-/- mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally.

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity.

Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture.

Mechanics of epithelial tissues during gap closure.

The closure of gaps is crucial to maintaining epithelium integrity during developmental and repair processes such as dorsal closure and wound healing. Depending on biochemical as well as physical properties of the microenvironment, gap closure occurs through assembly of multicellular actin-based contractile cables and/or protrusive activity of cells lining the gap. This review discusses the relative contributions of 'purse-string' and cell crawling mechanisms regulated by cell-substrate and cell-cell interactions, cellular mechanics and physical constraints from the environment.

Plasticity of the brush border - the yin and yang of intestinal homeostasis.

The brush border on the apical surface of enterocytes is a highly specialized structure well-adapted for efficient digestion and nutrient transport, whilst at the same time providing a protective barrier for the intestinal mucosa. The brush border is constituted of a densely ordered array of microvilli, protrusions of the plasma membrane, which are supported by actin-based microfilaments and interacting proteins and anchored in an apical network of actomyosin and intermediate filaments, the so-called terminal web. The highly dynamic, specialized apical domain is both an essential partner for the gut microbiota and an efficient signalling platform that enables adaptation to physiological stimuli from the external and internal milieu. Nevertheless, genetic alterations or various pathological stresses, such as infection, inflammation, and mechanical or nutritional alterations, can jeopardize this equilibrium and compromise intestinal functions. Long-time neglected, the intestinal brush-border shall be enlightening again as the central actor of the complex but essential intestinal homeostasis. Here, we review the processes and components involved in brush border organization and discuss pathological mechanisms that can induce brush border defects and their physiological consequences.

Actin-Based Transport Adapts Polarity Domain Size to Local Cellular Curvature.

Intracellular structures and organelles such as the nucleus, the centrosome, or the mitotic spindle typically scale their size to cell size [1]. Similarly, cortical polarity domains built around the active form of conserved Rho-GTPases, such as Cdc42p, exhibit widths that may range over two orders of magnitudes in cells with different sizes and shapes [2-6]. The establishment of such domains typically involves positive feedback loops based on reaction-diffusion and/or actin-mediated vesicle transport [3, 7, 8]. How these elements may adapt polarity domain size to cellular geometry is not known. Here, by tracking the width of successive oscillating Cdc42-GTP domains in fission yeast spores [9], we find that domain width scales with local cell-surface radii of curvature over an 8-fold range, independently of absolute cell volume, surface, or Cdc42-GTP concentration. This local scaling requires formin-nucleated cortical actin cables and the fusion of secretory vesicles transported along these cables with the membrane. These data suggest that reaction-diffusion may set a minimal domain size and that secretory vesicle transport along actin cables may dilute and extend polarity domains to adapt their size to local cell-surface curvature. This work reveals that actin networks may act as micrometric curvature sensors and uncovers a generic morphogenetic principle for how polarity domains define their size according to cell morphologies.

In vivo examination of the cortical cytoskeleton in multiciliated cells using electron tomography.

Multiciliated cells are characterized by coordinated arrays of motile cilia. In the respiratory tract, the maintenance of this array is essential to ensure proper ciliary and mucus clearance. The establishment and the maintenance of the ciliary set are mediated by the correct positioning of basal bodies at the cell cortex. While microtubule and actin cytoskeletons have been reported to regulate basal body lattices, an understanding of their detailed organization was missing until recently. Here, we describe how electron tomography can highlight the arrangement of the cytoskeletal networks and their interplay with basal bodies in ciliated cells in their tissular environment. Thanks to this approach, information in fine detail on large parts of the cell, dense in organelles, is provided. In combination with other approaches, such as transgenic animal models, electron tomography constitutes a powerful technique giving an overview of tissues and cells concomitantly with acquisition of three-dimensional detail.

A Chlamydia effector recruits CEP170 to reprogram host microtubule organization.

The obligate intracellular bacterial pathogen Chlamydia trachomatis deploys virulence effectors to subvert host cell functions enabling its replication within a specialized membrane-bound compartment termed an inclusion. The control of the host cytoskeleton is crucial for Chlamydia uptake, inclusion biogenesis and cell exit. Here, we demonstrate how a Chlamydia effector rearranges the microtubule (MT) network by initiating organization of the MTs at the inclusion surface. We identified an inclusion-localized effector that is sufficient to interfere with MT assembly, which we named inclusion protein acting on MTs (IPAM). We established that IPAM recruits and stimulates the centrosomal protein 170 kDa (CEP170) to hijack the MT organizing functions of the host cell. We show that CEP170 is essential for chlamydial control of host MT assembly, and is required for inclusion morphogenesis and bacterial infectivity. Together, we demonstrate how a pathogen effector reprograms the host MT network to support its intracellular development.

Overexpression of galectin-7 in mouse epidermis leads to loss of cell junctions and defective skin repair.

BACKGROUND: The proteins of the galectin family are implicated in many cellular processes, including cell interactions, polarity, intracellular trafficking, and signal transduction. In human and mouse, galectin-7 is almost exclusively expressed in stratified epithelia, notably in the epidermis. Galectin-7 expression is also altered in several human tumors of epithelial origin. This study aimed at dissecting the consequences of galectin-7 overexpression on epidermis structure and functions in vivo. METHODS: We established transgenic mice specifically overexpressing galectin-7 in the basal epidermal keratinocytes and analyzed the consequences on untreated skin and after UVB irradiation or mechanical injury. RESULTS: The intercellular cohesion of the epidermis is impaired in transgenic animals, with gaps developing between adjacent keratinocytes, associated with loss of adherens junctions. The epidermal architecture is aberrant with perturbations in the multilayered cellular organisation of the tissue, and structural defects in the basement membrane. These transgenic animals displayed a reduced re-epithelialisation potential following superficial wound, due to a defective collective migration of keratinocytes. Finally, a single mild dose of UVB induced an abnormal apoptotic response in the transgenic epidermis. CONCLUSION: These results indicate that an excess of galectin-7 leads to a destabilisation of adherens junctions associated with defects in epidermal repair. As this phenotype shares similarities with that of galectin-7 null mutant mice, we conclude that a critical level of this protein is required for maintaining proper epidermal homeostasis. This study brings new insight into the mode of action of galectins in normal and pathological situations.