RESUMEN
Cells actively position their nuclei based on their activity. In fission yeast, microtubule-dependent nuclear centering is critical for symmetrical cell division. After spindle disassembly at the end of anaphase, the nucleus recenters over an â¼90-min period, approximately half of the duration of the cell cycle. Live-cell and simulation experiments support the cooperation of two distinct microtubule competition mechanisms in the slow recentering of the nucleus. First, a push-push mechanism acts from spindle disassembly to septation and involves the opposing actions of the mitotic spindle pole body microtubules that push the nucleus away from the ends of the cell, while a postanaphase array of microtubules baskets the nucleus and limits its migration toward the division plane. Second, a slow-and-grow mechanism slowly centers the nucleus in the newborn cell by a combination of microtubule competition and asymmetric cell growth. Our work underlines how intrinsic properties of microtubules differently impact nuclear positioning according to microtubule network organization and cell size.
Asunto(s)
Anafase , Schizosaccharomyces , Humanos , Recién Nacido , Microtúbulos/metabolismo , Ciclo Celular , Citoesqueleto , Núcleo Celular , Huso AcromáticoRESUMEN
Actin-microtubule interactions are critical for cell division yet how these networks of polymers mutually influence their mechanical properties and functions in live cells remains unknown. In fission yeast, the post-anaphase array (PAA) of microtubules assembles in the plane of the contractile ring and its assembly relies on the Myp2p-dependent recruitment of Mto1p, a component of equatorial microtubule organizing centers (eMTOCs). The general organization of this array of microtubule and the impact on their physical attachment to the contractile ring remain unclear. We found that Myp2p facilitates the recruitment of Mto1p to the inner face of the contractile ring where the eMTOCs polymerize microtubules without their direct interaction. The PAA microtubules form a dynamic polygon of Ase1p crosslinked microtubules inside the contractile ring. The specific loss of PAA microtubules affects the mechanical properties of the contractile ring of actin by lowering its stiffness. This change in the mechanical properties of the ring has no measurable impact on cytokinesis or on the anchoring of the ring. Our work proposes that the PAA microtubules exploit the contractile ring for their assembly and function during cell division while the contractile ring may receive no benefit from these interactions.
RESUMEN
Actin-microtubule interactions are critical for cell division, yet how these networks of polymers mutually influence their mechanical properties and functions in live cells remains unknown. In fission yeast, the post-anaphase array (PAA) of microtubules assembles in the plane of the contractile ring, and its assembly relies on the Myp2p-dependent recruitment of Mto1p, a component of equatorial microtubule organizing centers (eMTOCs). The general organization of this array of microtubules and the impact on their physical attachment to the contractile ring remain unclear. We found that Myp2p facilitates the recruitment of Mto1p to the inner face of the contractile ring, where the eMTOCs polymerize microtubules without their direct interaction. The PAA microtubules form a dynamic polygon of Ase1p crosslinked microtubules inside the contractile ring. The specific loss of PAA microtubules affects the mechanical properties of the contractile ring of actin by lowering its stiffness. This change in the mechanical properties of the ring has no measurable impact on cytokinesis or on the anchoring of the ring. Our work proposes that the PAA microtubules exploit the contractile ring for their assembly and function during cell division, while the contractile ring may receive no benefit from these interactions.
Asunto(s)
Actinas , Schizosaccharomyces , Microtúbulos , Citoesqueleto , División CelularRESUMEN
Cells actively position their nucleus based on their activity. In fission yeast, microtubule-dependent nuclear centering is critical for symmetrical cell division. After spindle disassembly at the end of anaphase, the nucleus recenters over a ~90 min period, approximately half of the duration of the cell cycle. Live cell and simulation experiments support the cooperation of two distinct mechanisms in the slow recentering of the nucleus. First, a push-push mechanism acts from spindle disassembly to septation and involves the opposing actions of the mitotic Spindle Pole Body microtubules that push the nucleus away from the ends of the cell while post-anaphase array of microtubules basket the nucleus and limit its migration toward the division plane. Second, a slow-and-grow mechanism finalizes nuclear centering in the newborn cell. In this mechanism, microtubule competition stalls the nucleus while asymmetric cell growth slowly centers it. Our work underlines how intrinsic properties of microtubules differently impact nuclear positioning according to microtubule network organization and cell size.
RESUMEN
The contractile ring must anchor to the plasma membrane and cell wall to transmit its tension. F-BAR domain containing proteins including Imp2p and Cdc15p in fission yeast are likely candidate anchoring proteins based on their mutant phenotypes. Cdc15p is a node component, links the actin bundle to the plasma membrane, recruits Bgs1p to the division plane, prevents contractile ring sliding, and contributes to the stiffness of the contractile ring. Less is known about Imp2p. We found that similarly to Cdc15p, Imp2p contributes to the stiffness of the contractile ring and assembles into protein clusters. Imp2p clusters contain approximately eight Imp2p dimers and depend on the actin network for their stability at the division plane. Importantly, Imp2p and Cdc15p reciprocally affect the amount of each other in the contractile ring, indicating that the two proteins influence each other during cytokinesis, which may partially explain their similar phenotypes.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citocinesis , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
The mechanics that govern the constriction of the contractile ring remain poorly understood yet are critical to understanding the forces that drive cytokinesis. We used laser ablation in fission yeast cells to unravel these mechanics focusing on the role of Cdc15p as a putative anchoring protein. Our work shows that the severed constricting contractile ring recoils to a finite point leaving a gap that can heal if less than â¼1 µm. Severed contractile rings in Cdc15p-depleted cells exhibit an exaggerated recoil, which suggests that the recoil is limited by the anchoring of the ring to the plasma membrane. Based on a physical model of the severed contractile ring, we propose that Cdc15p impacts the stiffness of the contractile ring more than the viscous drag.
Asunto(s)
Terapia por Láser , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Citoesqueleto de Actina/metabolismo , Citocinesis , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Chlamydia trachomatis (Ct) causes the most prevalent bacterial sexually transmitted disease leading to ectopic pregnancy and infertility. Swine not only have many similarities to humans, but they are also susceptible to Ct. Despite these benefits and the ease of access to primary tissue from this food animal, in vitro research in swine has been underutilized. This study will provide basic understanding of the Ct host-pathogen interactions in porcine oviduct epithelial cells (pOECs)-the counterparts of human Fallopian tube epithelial cells. Using NanoString technology, flow cytometry, and confocal and transmission-electron microscopy, we studied the Ct developmental cycle in pOECs, the cellular immune response, and the expression and location of the tight junction protein claudin-4. We show that Ct productively completes its developmental cycle in pOECs and induces an immune response to Ct similar to human cells: Ct mainly induced the upregulation of interferon regulated genes and T-cell attracting chemokines. Furthermore, Ct infection induced an accumulation of claudin-4 in the Ct inclusion with a coinciding reduction of membrane-bound claudin-4. Downstream effects of the reduced membrane-bound claudin-4 expression could potentially include a reduction in tight-junction expression, impaired epithelial barrier function as well as increased susceptibility to co-infections. Thereby, this study justifies the investigation of the effect of Ct on tight junctions and the mucosal epithelial barrier function. Taken together, this study demonstrates that primary pOECs represent an excellent in vitro model for research into Ct pathogenesis, cell biology and immunity.
RESUMEN
The molecular organization of cytokinesis proteins governs contractile ring function. We used single molecule localization microscopy in live cells to elucidate the molecular organization of cytokinesis proteins and relate it to the constriction rate of the contractile ring. Wild-type fission yeast cells assemble contractile rings by the coalescence of cortical proteins complexes called nodes whereas cells without Anillin/Mid1p (Δmid1) lack visible nodes yet assemble contractile rings competent for constriction from the looping of strands. We leveraged the Δmid1 contractile ring assembly mechanism to determine how two distinct molecular organizations, nodes versus strands, can yield functional contractile rings. Contrary to previous interpretations, nodes assemble in Δmid1 cells. Our results suggest that Myo2p heads condense upon interaction with actin filaments and an excess number of Myo2p heads bound to actin filaments hinders constriction thus reducing the constriction rate. Our work establishes a predictive correlation between the molecular organization of nodes and the behavior of the contractile ring.
Asunto(s)
Citocinesis , Schizosaccharomyces/citología , Acetiltransferasas/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Modelos Biológicos , Mutación/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Factores de TiempoRESUMEN
Chytrids are early-diverging fungi that share features with animals that have been lost in most other fungi. They hold promise as a system to study fungal and animal evolution, but we lack genetic tools for hypothesis testing. Here, we generated transgenic lines of the chytrid Spizellomyces punctatus, and used fluorescence microscopy to explore chytrid cell biology and development during its life cycle. We show that the chytrid undergoes multiple rounds of synchronous nuclear division, followed by cellularization, to create and release many daughter 'zoospores'. The zoospores, akin to animal cells, crawl using actin-mediated cell migration. After forming a cell wall, polymerized actin reorganizes into fungal-like cortical patches and cables that extend into hyphal-like structures. Actin perinuclear shells form each cell cycle and polygonal territories emerge during cellularization. This work makes Spizellomyces a genetically tractable model for comparative cell biology and understanding the evolution of fungi and early eukaryotes.
Asunto(s)
Quitridiomicetos/citología , Quitridiomicetos/crecimiento & desarrollo , Quitridiomicetos/genética , Actinas/metabolismo , Evolución Biológica , Ciclo Celular , Movimiento Celular , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Microorganismos Modificados Genéticamente , Mitosis , Morfogénesis , Esporas Fúngicas/fisiología , Transformación GenéticaRESUMEN
This contribution to the Festschrift for Professor Thomas (Tom) D. Pollard focuses on his work on the elucidation of the protein organization within the cytokinetic nodes, protein assemblies, precursors to the contractile ring. In particular, this work highlights recent discoveries in the molecular organization of the proteins that make the contractile machine in fission yeast using advanced microscopy techniques. One of the main aspects of Tom's research philosophy that marked my career as one of his trainees is his embrace of interdisciplinary approaches to research. The cost of interdisciplinary research is to be willing to step out of our technical comfort zone to learn a new set of tools. The payoff of interdisciplinary research is the expansion our realm of possibilities by bringing new creative tools and ideas to push our research program forward. The rewarding outcomes of this work under Tom's mentorship were the molecular model of the cytokinetic node and the development of new techniques to unravel the structure of multi-protein complexes in live cells. Together, these findings open a new set of questions about the mechanism of cytokinesis and provide creative tools to address them.
RESUMEN
Stimulated by our 2015 Current Biology paper [1], Zambon et al. reinvestigated how three myosin isoforms participate in the formation and constriction of the contractile ring in fission yeast. Our paper presented evidence that these myosin isoforms have distinct roles: "Conventional myosin-II Myo2 is crucial to ring assembly, unconventional myosin-II Myp2 is most important for ring constriction, and type V myosin Myo51 aids the other two myosins." Zambon et al. used different markers to reexamine the contributions of the three myosins to cytokinesis and concluded "that Myo2p is the major motor involved in ring contraction in S. pombe." Here, we show that most of the differences observed by Zambon et al. can be attributed to their use of the Rlc1p-3GFP marker, which genetically interacts with myo2-E1.
Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Constricción , Cadenas Pesadas de Miosina , Miosina Tipo II , MiosinasRESUMEN
Cytokinesis in animals, fungi, and amoebas depends on the constriction of a contractile ring built from a common set of conserved proteins. Many fundamental questions remain about how these proteins organize to generate the necessary tension for cytokinesis. Using quantitative high-speed fluorescence photoactivation localization microscopy (FPALM), we probed this question in live fission yeast cells at unprecedented resolution. We show that nodes, protein assembly precursors to the contractile ring, are discrete structural units with stoichiometric ratios and distinct distributions of constituent proteins. Anillin Mid1p, Fes/CIP4 homology-Bin/amphiphysin/Rvs (F-BAR) Cdc15p, IQ motif containing GTPase-activating protein (IQGAP) Rng2p, and formin Cdc12p form the base of the node that anchors the ends of myosin II tails to the plasma membrane, with myosin II heads extending into the cytoplasm. This general node organization persists in the contractile ring where nodes move bidirectionally during constriction. We observed the dynamics of the actin network during cytokinesis, starting with the extension of short actin strands from nodes, which sometimes connected neighboring nodes. Later in cytokinesis, a broad network of thick bundles coalesced into a tight ring around the equator of the cell. The actin ring was â¼125 nm wide and â¼125 nm thick. These observations establish the organization of the proteins in the functional units of a cytokinetic contractile ring.
Asunto(s)
Citocinesis , Microscopía Fluorescente/métodos , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Interfase , Modelos Moleculares , Fenotipo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
We describe a step-by-step method for high-speed fluorescence photoactivation localization microscopy (FPALM) of live fission yeast cells. The resolution with this method is tenfold better than spinning disk confocal microscopy.
Asunto(s)
Microscopía Fluorescente/métodos , Schizosaccharomyces/citología , Marcación de Gen , Schizosaccharomyces/genética , Transformación GenéticaRESUMEN
Cytokinesis in fission yeast cells depends on conventional myosin-II (Myo2) to assemble and constrict a contractile ring of actin filaments. Less is known about the functions of an unconventional myosin-II (Myp2) and a myosin-V (Myo51) that are also present in the contractile ring. Myo2 appears in cytokinetic nodes around the equator 10 min before spindle pole body separation (cell-cycle time, -10 min) independent of actin filaments, followed by Myo51 at time zero and Myp2 at time +20 min, both located between nodes and dependent on actin filaments. We investigated the contributions of these three myosins to cytokinesis using a severely disabled mutation of the essential myosin-II heavy-chain gene (myo2-E1) and deletion mutations of the other myosin heavy-chain genes. Cells with only Myo2 assemble contractile rings normally. Cells with either Myp2 or Myo51 alone can assemble nodes and actin filaments into contractile rings but complete assembly later than normal. Both Myp2 and Myo2 contribute to constriction of fully assembled rings at rates 55% that of normal in cells relying on Myp2 alone and 25% that of normal in cells with Myo2 alone. Myo51 alone cannot constrict rings but increases the constriction rate by Myo2 in Δmyp2 cells or Myp2 in myo2-E1 cells. Three myosins function in a hierarchal, complementary manner to accomplish cytokinesis, with Myo2 and Myo51 taking the lead during contractile ring assembly and Myp2 making the greatest contribution to constriction.
Asunto(s)
Ciclo Celular , Cadenas Pesadas de Miosina/genética , Miosina Tipo II/genética , Miosinas/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/fisiología , División Celular , Citocinesis , Mutación , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo II/metabolismo , Miosinas/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Cuerpos Polares del Huso/metabolismoRESUMEN
Cytokinesis involves constriction of a contractile actomyosin ring. The mechanisms generating ring tension and setting the constriction rate remain unknown because the organization of the ring is poorly characterized, its tension was rarely measured, and constriction is coupled to other processes. To isolate ring mechanisms, we studied fission yeast protoplasts, in which constriction occurs without the cell wall. Exploiting the absence of cell wall and actin cortex, we measured ring tension and imaged ring organization, which was dynamic and disordered. Computer simulations based on the amounts and biochemical properties of the key proteins showed that they spontaneously self-organize into a tension-generating bundle. Together with rapid component turnover, the self-organization mechanism continuously reassembles and remodels the constricting ring. Ring constriction depended on cell shape, revealing that the ring operates close to conditions of isometric tension. Thus, the fission yeast ring sets its own tension, but other processes set the constriction rate.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Citocinesis/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Simulación por Computador , Miosina Tipo II/metabolismo , Polimerizacion , Schizosaccharomyces/citologíaRESUMEN
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.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Epidermis/metabolismo , Proteínas Represoras/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Secuencias de Aminoácidos , Animales , Adhesión Celular , Moléculas de Adhesión Celular/química , Movimiento Celular , Polaridad Celular , Proteínas de Drosophila/química , Células Epidérmicas , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Estructura Terciaria de Proteína , Proteínas Represoras/químicaRESUMEN
Morphogenesis of the Drosophila embryonic trachea involves a stereotyped pattern of epithelial tube branching and fusion. Here, we report unexpected phenotypes resulting from maternal and zygotic (M/Z) loss of the homophilic cell adhesion molecule Echinoid (Ed), as well as the subcellular localization of Ed in the trachea. ed(M/Z) embryos have convoluted trachea reminiscent of septate junction (SJ) and luminal matrix mutants. However, Ed does not localize to SJs, and ed(M/Z) embryos have intact SJs and show normal luminal accumulation of the matrix-modifying protein Vermiform. Surprisingly, tracheal length is not increased in ed(M/Z) mutants, but a previously undescribed combination of reduced intersegmental spacing and deep epidermal grooves produces a convoluted tracheal phenotype. In addition, ed(M/Z) mutants have unique fusion defects involving supernumerary fusion cells, ectopic fusion events and atypical branch breaks. Tracheal-specific expression of Ed rescues these fusion defects, indicating that Ed acts in trachea to control fusion cell fate.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Fusión Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Morfogénesis/fisiología , Proteínas Represoras/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/embriología , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/fisiología , Fenotipo , Proteínas Represoras/genética , Tráquea/anatomía & histología , Tráquea/embriología , Proteína Wnt1/metabolismoRESUMEN
Epithelial morphogenesis requires cell movements and cell shape changes coordinated by modulation of the actin cytoskeleton. We identify a role for Echinoid (Ed), an immunoglobulin domain-containing cell-adhesion molecule, in the generation of a contractile actomyosin cable required for epithelial morphogenesis in both the Drosophila ovarian follicular epithelium and embryo. Analysis of ed mutant follicle cell clones indicates that the juxtaposition of wild-type and ed mutant cells is sufficient to trigger actomyosin cable formation. Moreover, in wild-type ovaries and embryos, specific epithelial domains lack detectable Ed, thus creating endogenous interfaces between cells with and without Ed; these interfaces display the same contractile characteristics as the ectopic Ed expression borders generated by ed mutant clones. In the ovary, such an interface lies between the two cell types of the dorsal appendage primordia. In the embryo, Ed is absent from the amnioserosa during dorsal closure, generating an Ed expression border with the lateral epidermis that coincides with the actomyosin cable present at this interface. In both cases, ed mutant epithelia exhibit loss of this contractile structure and subsequent defects in morphogenesis. We propose that local modulation of the cytoskeleton at Ed expression borders may represent a general mechanism for promoting epithelial morphogenesis.