Oriented Cell Division Research Articles

Cell shape and intercellular adhesion regulate mitotic spindle orientation.

Cell division orientation plays an essential role in tissue morphogenesis and cell fate decision. Recent studies showed that either cell shape or adhesion geometry can regulate the orientation of mitotic spindles and thereby the cell division orientation. However, how they together regulate the spindle orientation remains largely unclear. In this work, we use a general computational model to investigate the competitive mechanism of determining the spindle orientation between cell shape and intercellular adhesion in epithelial cells. We find the spindle orientation is dominated by the intercellular adhesion when the cell shape anisotropy is small, but dominated by the cell shape when the shape anisotropy is large. A strong adhesion and moderate adhesive size can ensure the planar division of epithelial cells with large apico-basal elongation. We also find the spindle orientation could be perpendicular to the adhesive region when only one side of the cell is adhered to an E-cadherin–coated matrix. But after the cell is compressed, the spindle orientation is governed by the cell shape and the spindle will be parallel to the adhesive region when the cell shape anisotropy is large. Finally, we demonstrate the competition between cell shape and tricellular junctions can also effectively regulate the spindle orientation.

Extracellular matrix stiffness cues junctional remodeling for 3D tissue elongation

Organs are sculpted by extracellular as well as cell-intrinsic forces, but how collective cell dynamics are orchestrated in response to environmental cues is poorly understood. Here we apply advanced image analysis to reveal extracellular matrix-responsive cell behaviors that drive elongation of the Drosophila follicle, a model system in which basement membrane stiffness instructs three-dimensional tissue morphogenesis. Through in toto morphometric analyses of wild type and round egg mutants, we find that neither changes in average cell shape nor oriented cell division are required for appropriate organ shape. Instead, a major element is the reorientation of elongated cells at the follicle anterior. Polarized reorientation is regulated by mechanical cues from the basement membrane, which are transduced by the Src tyrosine kinase to alter junctional E-cadherin trafficking. This mechanosensitive cellular behavior represents a conserved mechanism that can elongate edgeless tubular epithelia in a process distinct from those that elongate bounded, planar epithelia.

FAM83D directs protein kinase CK1α to the mitotic spindle for proper spindle positioning.

The concerted action of many protein kinases helps orchestrate the error‐free progression through mitosis of mammalian cells. The roles and regulation of some prominent mitotic kinases, such as cyclin‐dependent kinases, are well established. However, these and other known mitotic kinases alone cannot account for the extent of protein phosphorylation that has been reported during mammalian mitosis. Here we demonstrate that CK1α, of the casein kinase 1 family of protein kinases, localises to the spindle and is required for proper spindle positioning and timely cell division. CK1α is recruited to the spindle by FAM83D, and cells devoid of FAM83D, or those harbouring CK1α‐binding‐deficient FAM83D F283A/F283A knockin mutations, display pronounced spindle positioning defects, and a prolonged mitosis. Restoring FAM83D at the endogenous locus in FAM83D −/− cells, or artificially delivering CK1α to the spindle in FAM83D F283A/F283A cells, rescues these defects. These findings implicate CK1α as new mitotic kinase that orchestrates the kinetics and orientation of cell division.

The interaction between CASK and the tumour suppressor Dlg1 regulates mitotic spindle orientation in mammalian epithelia.

ABSTRACTOriented cell divisions are important for the formation of normal epithelial structures. Dlg1, a tumour suppressor, is required for mitotic spindle orientation in Drosophila epithelia and chick neuroepithelia, but how Dlg1 is localised to the membrane and its importance in mammalian epithelia are unknown. We show that Dlg1 is required in non-transformed mammalian epithelial cells for oriented cell divisions and normal lumen formation. We demonstrate that the MAGUK protein CASK, a membrane-associated scaffold, is the factor responsible for Dlg1 membrane localisation during spindle orientation, thereby identifying a new cellular function for CASK. Depletion of CASK leads to misoriented divisions in 3D, and to the formation of multilumen structures in cultured kidney and breast epithelial cells. Blocking the CASK–Dlg1 interaction with an interfering peptide, or by deletion of the CASK-interaction domain of Dlg1, disrupts spindle orientation and causes multilumen formation. We show that the CASK–Dlg1 interaction is important for localisation of the canonical LGN–NuMA complex known to be required for spindle orientation. These results establish the importance of the CASK–Dlg1 interaction in oriented cell division and epithelial integrity.This article has an associated First Person interview with the first author of the paper.

NDP52 tunes cortical actin interaction with astral microtubules for accurate spindle orientation.

Oriented cell divisions are controlled by a conserved molecular cascade involving Gαi, LGN, and NuMA. Here, we show that NDP52 regulates spindle orientation via remodeling the polar cortical actin cytoskeleton. siRNA-mediated NDP52 suppression surprisingly revealed a ring-like compact subcortical F-actin architecture surrounding the spindle in prophase/prometaphase cells, which resulted in severe defects of astral microtubule growth and an aberrant spindle orientation. Remarkably, NDP52 recruited the actin assembly factor N-WASP and regulated the dynamics of the subcortical F-actin ring in mitotic cells. Mechanistically, NDP52 was found to bind to phosphatidic acid-containing vesicles, which absorbed cytoplasmic N-WASP to regulate local filamentous actin growth at the polar cortex. Our TIRFM analyses revealed that NDP52-containing vesicles anchored N-WASP and shortened the length of actin filaments in vitro. Based on these results we propose that NDP52-containing vesicles regulate cortical actin dynamics through N-WASP to accomplish a spatiotemporal regulation between astral microtubules and the actin network for proper spindle orientation and precise chromosome segregation. In this way, intracellular vesicles cooperate with microtubules and actin filaments to regulate proper mitotic progression. Since NDP52 is absent from yeast, we reason that metazoans have evolved an elaborate spindle positioning machinery to ensure accurate chromosome segregation in mitosis.

Epithelial geometry regulates spindle orientation and progenitor fate during formation of the mammalian epidermis.

The control of cell fate through oriented cell division is imperative for proper organ development. Basal epidermal progenitor cells divide parallel or perpendicular to the basement membrane to self-renew or produce differentiated stratified layers, but the mechanisms regulating the choice between division orientations are unknown. Using time-lapse imaging to follow divisions and fates of basal progenitors, we find that mouse embryos defective for the planar cell polarity (PCP) gene, Vangl2, exhibit increased perpendicular divisions and hyperthickened epidermis. Surprisingly, this is not due to defective Vangl2 function in the epidermis, but to changes in cell geometry and packing that arise from the open neural tube characteristic of PCP mutants. Through regional variations in epidermal deformation and physical manipulations, we show that local tissue architecture, rather than cortical PCP cues, regulates the decision between symmetric and stratifying divisions, allowing flexibility for basal cells to adapt to the needs of the developing tissue.

Driving Cells with Light-Controlled Topographies.

Cell–substrate interactions can modulate cellular behaviors in a variety of biological contexts, including development and disease. Light‐responsive materials have been recently proposed to engineer active substrates with programmable topographies directing cell adhesion, migration, and differentiation. However, current approaches are affected by either fabrication complexity, limitations in the extent of mechanical stimuli, lack of full spatio‐temporal control, or ease of use. Here, a platform exploiting light to plastically deform micropatterned polymeric substrates is presented. Topographic changes with remarkable relief depths in the micron range are induced in parallel, by illuminating the sample at once, without using raster scanners. In few tens of seconds, complex topographies are instructed on demand, with arbitrary spatial distributions over a wide range of spatial and temporal scales. Proof‐of‐concept data on breast cancer cells and normal kidney epithelial cells are presented. Both cell types adhere and proliferate on substrates without appreciable cell damage upon light‐induced substrate deformations. User‐provided mechanical stimulation aligns and guides cancer cells along the local deformation direction and constrains epithelial colony growth by biasing cell division orientation. This approach is easy to implement on general‐purpose optical microscopy systems and suitable for use in cell biology in a wide variety of applications.

Re-activation of Stem Cell Pathways for Pattern Restoration in Plant Wound Healing

SummaryPatterning in plants relies on oriented cell divisions and acquisition of specific cell identities. Plants regularly endure wounds caused by abiotic or biotic environmental stimuli and have developed extraordinary abilities to restore their tissues after injuries. Here, we provide insight into a mechanism of restorative patterning that repairs tissues after wounding. Laser-assisted elimination of different cells in Arabidopsis root combined with live-imaging tracking during vertical growth allowed analysis of the regeneration processes in vivo. Specifically, the cells adjacent to the inner side of the injury re-activated their stem cell transcriptional programs. They accelerated their progression through cell cycle, coordinately changed the cell division orientation, and ultimately acquired de novo the correct cell fates to replace missing cells. These observations highlight existence of unknown intercellular positional signaling and demonstrate the capability of specified cells to re-acquire stem cell programs as a crucial part of the plant-specific mechanism of wound healing.

Electrical Stimulation Directs Migration, Enhances and Orients Cell Division and Upregulates the Chemokine Receptors CXCR4 and CXCR2 in Endothelial Cells

Natural direct current electric fields (DC EFs) within tissues undergoing angiogenesis have the potential to influence vessel formation, but how they affect endothelial cells is not clear. We therefore quantified behaviours of human umbilical vein endothelial cells (HUVEC) and human microvasculature endothelial cells (HMEC) stimulated by EFsin vitro. Both cell types migrated faster and toward the cathode; HUVECs responded to fields as low as 50mV/mm, but the HMEC threshold was 100 mV/mm. Mitosis was stimulated at 50 mV/mm for HMEC and at 150 mV/mm for HUVECs, but the cleavage plane was oriented orthogonal to the field vector at 200 mV/mm for both cell types. That different field strengths induced different cell responses suggests distinct underlying cellular mechanisms. A physiological electric field also upregulated expression of CXCR4 and CXCR2 chemokine receptors and upregulated phosphorylation of both chemokines in HUVEC and HMEC cells. Evidence that DC EFs direct endothelial cell migration, proliferation and upregulate chemokines involved in wound healing suggests a key role for electrical control of capillary production during healing. Our data contribute to the molecular mechanisms by which DC EFs direct endothelial cell behaviour and present a novel signalling paradigm in wound healing, tissue regeneration and angiogenesis-related diseases.

Anionic Lipids: A Pipeline Connecting Key Players of Plant Cell Division.

How cells position their division plane is a critical component of cell division. Indeed, it defines whether the two daughter cells divide symmetrically (with equal volumes) or not, and as such is critical for cell differentiation and lineage specification across eukaryotes. However, oriented cell divisions are of special significance for organisms with cell walls, such as plants, because their cells are embedded and cannot relocate. Correctly positioning the division plane is therefore of prevailing importance in plants, as it controls not only the occurrence of asymmetric cell division, but also tissue morphogenesis and organ integrity. While cytokinesis is executed in radically different manners in animals and plants, they both rely on the dynamic interplay between the cytoskeleton and membrane trafficking to precisely deliver molecular components to the future site of cell division. Recent research has shown that strict regulation of the levels and distribution of anionic lipids, which are minor components of the cell membrane’s lipids, is required for successful cytokinesis in non-plant organisms. This review focused on the recent evidence pointing to whether such signaling lipids have roles in plant cell division.

Oriented Cell Divisions Are Not Required for Drosophila Wing Shape

Formation of correctly shaped organs is vital for normal function. The Drosophila wing has an elongated shape, which has been attributed in part to a preferential orientation of mitotic spindles along the proximal-distal axis [1, 2]. Orientation of mitotic spindles is believed to be a fundamental morphogenetic mechanism in multicellular organisms [3-6]. Acontribution of spindle orientation to wing shape was inferred from observations that mutation of Dachsous-Fat pathway genes results in both rounder wings and loss of the normal proximal-distal bias in spindle orientation [1, 2, 7]. To directly evaluate the potential contribution of spindle orientation to wing morphogenesis, we assessed the consequences of loss of the Drosophila NuMA homolog Mud, which interacts with the dynein complex and has a conserved role in spindle orientation [8, 9]. Loss of Mud randomizes spindle orientation but does not alter wing shape. Analysis of growth and cell dynamics in developing discs and in exvivo culture suggests that theabsence of oriented cell divisions is compensated for by an increased contribution of cell rearrangements to wing shape. Our results indicate that oriented cell divisions are not required for wingmorphogenesis, nor are they required for the morphogenesis of other Drosophila appendages. Moreover, our results suggest that normal organ shape is not achieved through locally specifying and then summing up individual cell behaviors, like oriented cell division. Instead, wing shape might be specified through tissue-wide stresses that dictate an overall arrangement of cells without specifying the individual cell behaviors needed to achieve it.

Cell geometry determines symmetric and asymmetric division plane selection in Arabidopsis early embryos.

Plant tissue architecture and organ morphogenesis rely on the proper orientation of cell divisions. Previous attempts to predict division planes from cell geometry in plants mostly focused on 2D symmetric divisions. Using the stereotyped division patterns of Arabidopsis thaliana early embryogenesis, we investigated geometrical principles underlying plane selection in symmetric and in asymmetric divisions within complex 3D cell shapes. Introducing a 3D computational model of cell division, we show that area minimization constrained on passing through the cell centroid predicts observed divisions. Our results suggest that the positioning of division planes ensues from cell geometry and gives rise to spatially organized cell types with stereotyped shapes, thus underlining the role of self-organization in the developing architecture of the embryo. Our data further suggested the rule could be interpreted as surface minimization constrained by the nucleus position, which was validated using live imaging of cell divisions in the stomatal cell lineage.

A SOSEKI-based coordinate system interprets global polarity cues in Arabidopsis

Multicellular development requires coordinated cell polarization relative to body axes, and translation to oriented cell division1–3. In plants, it is unknown how cell polarities are connected to organismal axes and translated to division. Here, we identify Arabidopsis SOSEKI (SOK) proteins that integrate apical-basal and radial organismal axes to localize to polar cell edges. Localization does not depend on tissue context, requires cell wall integrity and is defined by a transferrable, protein-specific motif. A Domain of Unknown Function in SOK proteins resembles the DIX oligomerization domain in the animal Dishevelled polarity regulator. The DIX-like domain self-interacts and is required for edge localization and for influencing division orientation, together with a second domain that defines the polar membrane domain. Our work shows that SOK proteins locally interpret global polarity cues and can influence cell division orientation. Furthermore, this work reveals that despite fundamental differences, cell polarity mechanisms in plants and animals converge upon a similar protein domain.

Selective inhibition of the FcεRI-induced de novo synthesis of mediators by an inhibitory receptor.

Selective inhibition of the FcεRI-induced de novo synthesis of mediators by an inhibitory receptor.

Minimal in vitro systems shed light on cell polarity.

Cell polarity - the morphological and functional differentiation of cellular compartments in a directional manner - is required for processes such as orientation of cell division, directed cellular growth and motility. How the interplay of components within the complexity of a cell leads to cell polarity is still heavily debated. In this Review, we focus on one specific aspect of cell polarity: the non-uniform accumulation of proteins on the cell membrane. In cells, this is achieved through reaction-diffusion and/or cytoskeleton-based mechanisms. In reaction-diffusion systems, components are transformed into each other by chemical reactions and are moving through space by diffusion. In cytoskeleton-based processes, cellular components (i.e. proteins) are actively transported by microtubules (MTs) and actin filaments to specific locations in the cell. We examine how minimal systems - in vitro reconstitutions of a particular cellular function with a minimal number of components - are designed, how they contribute to our understanding of cell polarity (i.e. protein accumulation), and how they complement in vivo investigations. We start by discussing the Min protein system from Escherichia coli, which represents a reaction-diffusion system with a well-established minimal system. This is followed by a discussion of MT-based directed transport for cell polarity markers as an example of a cytoskeleton-based mechanism. To conclude, we discuss, as an example, the interplay of reaction-diffusion and cytoskeleton-based mechanisms during polarity establishment in budding yeast.

DOF2.1 Controls Cytokinin-Dependent Vascular Cell Proliferation Downstream of TMO5/LHW

SummaryTo create a three-dimensional structure, plants rely on oriented cell divisions and cell elongation. Oriented cell divisions are specifically important in procambium cells of the root to establish the different vascular cell types [1, 2]. These divisions are in part controlled by the auxin-controlled TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) transcription factor complex [3, 4, 5, 6, 7]. Loss-of-function of tmo5 or lhw clade members results in strongly reduced vascular cell file numbers, whereas ectopic expression of both TMO5 and LHW can ubiquitously induce periclinal and radial cell divisions in all cell types of the root meristem. TMO5 and LHW interact only in young xylem cells, where they promote expression of two direct target genes involved in the final step of cytokinin (CK) biosynthesis, LONELY GUY3 (LOG3) and LOG4 [8, 9] Therefore, CK was hypothesized to act as a mobile signal from the xylem to trigger divisions in the neighboring procambium cells [3, 6]. To unravel how TMO5/LHW-dependent cytokinin regulates cell proliferation, we analyzed the transcriptional responses upon simultaneous induction of both transcription factors. Using inferred network analysis, we identified AT2G28510/DOF2.1 as a cytokinin-dependent downstream target gene. We further showed that DOF2.1 controls specific procambium cell divisions without inducing other cytokinin-dependent effects such as the inhibition of vascular differentiation. In summary, our results suggest that DOF2.1 and its closest homologs control vascular cell proliferation, thus leading to radial expansion of the root.

Getting into shape: tissue tension drives oriented cell divisions duringorganogenesis.

The shapes and architecture of the organs in the animal body develop through complex coordination of cell shape change, cell migration, and cell proliferation. Finegan et al (2019) use the simple model of the Drosophila ovary and elongation of each egg chamber to explore this process. They find that a gradient of myosin‐driven tension exists along the axis of organ elongation and that this orients the division of the constituent cells. This and other recent work highlight how tissue‐level tension plays a key role in regulating tissue architecture.

Epithelial DNA methyltransferase-1 regulates cell survival, growth and maturation in developing prostatic buds

DNA methyltransferase 1 (DNMT1) is required for embryogenesis but roles in late forming organ systems including the prostate, which emerges from the urethral epithelium, have not been fully examined. We used a targeted genetic approach involving a Shhcre recombinase to demonstrate requirement of epithelial DNA methyltransferase-1 (Dnmt1) in mouse prostate morphogenesis. Dnmt1 mutant urethral cells exhibit DNA hypomethylation, DNA damage, p53 accumulation and undergo cell cycle arrest and apoptosis. Urethral epithelial cells are disorganized in Dnmt1 mutants, leading to impaired prostate growth and maturation and failed glandular development. We evaluated oriented cell division as a mechanism of bud elongation and widening by demonstrating that mitotic spindle axes typically form parallel or perpendicular to prostatic bud elongation axes. We then deployed a ShhcreERT allele to delete Dnmt1 from a subset of urethral epithelial cells, creating mosaic mutants with which to interrogate the requirement for cell division in specific prostatic bud epithelial populations. DNMT1- cell distribution within prostatic buds is not random as would be expected in a process where DNMT1 was not required. Instead, replication competent DNMT1 + cells primarily accumulate in prostatic bud margins and tips while replication impeded DNMT1- cells accumulate in prostatic bud cores. Together, these results highlight the role of DNMT1 in regulating epithelial bud formation by maintaining cell cycle progression and survival of rapidly dividing urethral epithelial cells, which can be extended to the study of other developing epithelial organs. In addition, our results show that prostatic buds consist of two epithelial cell populations with distinct molecular and functional characteristics that could potentially contribute to specialized lineages in the adult prostate.

Stathmin Is Required for Normal Mouse Mammary Gland Development and Δ16HER2-Driven Tumorigenesis.

Postnatal development of the mammary gland relies on the maintenance of oriented cell division and apicobasal polarity, both of which are often deregulated in cancer. The microtubule (MT) network contributes to control these processes; however, very little is known about the impact of altered MT dynamics in the development of a complex organ and on the role played by MT-interacting proteins such as stathmin. In this study, we report that female stathmin knock-out (STM KO) mice are unable to nurse their litters due to frank impairment of mammary gland development. In mouse mammary epithelial cells, loss of stathmin compromised the trafficking of polarized proteins and the achievement of proper apicobasal polarity. In particular, prolactin receptor internalization and localization was altered in STM KO mammary epithelial cells, leading to decreased protein stability and downmodulation of the Prl/PrlR/STAT5 signaling pathway. Absence of stathmin induced alterations in mitotic spindle orientation, accumulation of mitotic defects, and apoptosis, overall contributing to tissue disorganization and further decreasing the expansion of the mammary epithelial compartment. Loss of stathmin in MMTV-Δ16HER2 transgenic mice decreased the incidence and increased the latency of these very aggressive mammary carcinomas. Collectively, these data identify the essential mammary protein stathmin as protumorigenic and suggest it may serve as a potential therapeutic target in breast cancer. SIGNIFICANCE: Stathmin expression is critical to maintain oriented cell division and apicobasal polarity in normal mammary glands and to establish a protumorigenic program that eventually sustains HER2-positive breast cancer formation in mice.

GSK3β controls the timing and pattern of the fifth spiral cleavage at the 2-4 cell stage in Lymnaea stagnalis.

Establishment of the body plan of multicellular organisms by the primary body axis determination and cell-fate specification is a key issue in biology. We have examined the mRNA localization of three Wnt pathway components gsk3β, β-catenin, and disheveled and investigated the effects of four selective inhibitors of these proteins on the early developmental stages of the spiral cleavage embryo of the fresh water snail Lymnaea (L.) stagnalis. mRNAs for gsk3β and β-catenin were distributed uniformly throughout the embryo during development whereas disheveled mRNA showed specific localization with intra- and inter-blastomere differences in concentration along the A-V axis during spiral cleavages. Remarkably, through inhibitor studies, we identified a short sensitive period from the 2- to 4-cell stage in which GSK3β inhibition by the highly specific 1-azakenpaullone (AZ) and by LiCl induced a subsequent dramatic developmental delay and alteration of the cleavage patterns of blastomeres at the fifth cleavage (16- to 24-cell stage) resulting in exogastrulation and other abnormalities in later stages. Inhibition of β-Catenin or Disheveled had no effect. Our inhibitor experiments establish a novel role for GSK3β in the developmental timing and orientated cell division of the snail embryo. Further work will be needed to identify the downstream targets of the kinase.