RESUMEN
The mechanical properties and the forces involved during tissue morphogenesis have been the focus of much research in the last years. Absolute values of forces during tissue closure events have not yet been measured. This is also true for a common force-producing mechanism involving Myosin II waves that results in pulsed cell surface contractions. Our patented magnetic tweezer, CAARMA, integrated into a spinning disk confocal microscope, provides a powerful explorative tool for quantitatively measuring forces during tissue morphogenesis. Here, we used this tool to quantify the in vivo force production of Myosin II waves that we observed at the dorsal surface of the yolk cell in stage 13 Drosophila melanogaster embryos. In addition to providing for the first time to our knowledge quantitative values on an active Myosin-driven force, we elucidated the dynamics of the Myosin II waves by measuring their periodicity in both absence and presence of external perturbations, and we characterized the mechanical properties of the dorsal yolk cell surface.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Embrión no Mamífero , Morfogénesis , Miosina Tipo IIRESUMEN
Cells depend on a highly ordered organisation of their content and must develop strategies to maintain the anisotropic distribution of organelles during periods of nutrient shortage. One of these strategies is to solidify the cytoplasm, which was observed in bacteria and yeast cells with acutely interrupted energy production. Here, we describe a different type of cytoplasm solidification fission yeast cells switch to, after having run out of nutrients during multiple days in culture. It provides the most profound reversible cytoplasmic solidification of yeast cells described to date. Our data exclude the previously proposed mechanisms for cytoplasm solidification in yeasts and suggest a mechanism that immobilises cellular components in a size-dependent manner. We provide experimental evidence that, in addition to time, cells use intrinsic nutrients and energy sources to reach this state. Such cytoplasmic solidification may provide a robust means to protect cellular architecture in dormant cells.
Asunto(s)
Citoplasma/patología , Nutrientes/metabolismo , Inanición/metabolismo , Vacuolas/patología , Autofagia/fisiología , Citoplasma/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces , Inanición/patología , Vacuolas/metabolismoRESUMEN
Using correlative light and electron microscopy (CLEM), we studied the intracellular organization by of glucose-starved fission yeast cells (Schizosaccharomyces pombe) with regards to the localization of septin proteins throughout the cytoplasm. Thereby, we found that for cells carrying a deletion of the gene encoding septin-2 (spn2Δ), starvation causes a GFP-tagged version of septin-3 (spn3-GFP) and family members, to assemble into a single, prominent filamentous structure. It was previously shown that during exponential growth, spn2Δ cells form septin-3 polymers. However, the polymers we observed during exponential growth are different from the spn3p-GFP structure we observed in starved cells. Using CLEM, in combination with anti-GFP immunolabeling on plastic-sections, we could assign spn3p-GFP to the filaments we have found in EM pictures. Besides septin-3, these filamentous assemblies most likely also contain septin-1 as an RFP-tagged version of this protein forms a very similar structure in starved spn2Δ cells. Our data correlate phase-contrast and fluorescence microscopy with electron micrographs of plastic-embedded cells, and further on with detailed views of tomographic 3D reconstructions. Cryo-electron microscopy of spn2Δ cells in vitrified sections revealed a very distinct overall morphology of the spn3p-GFP assembly. The fine-structured, regular density pattern suggests the presence of assembled septin-3 filaments that are clearly different from F-actin bundles. Furthermore, we found that starvation causes substantial mitochondria fission, together with massive decoration of their outer membrane by ribosomes.
RESUMEN
Tissue morphogenesis requires coordination of multiple force-producing components. During dorsal closure in fly embryogenesis, an epidermis opening closes. A tensioned epidermal actin/MyosinII cable, which surrounds the opening, produces a force that is thought to combine with another MyosinII force mediating apical constriction of the amnioserosa cells that fill the opening. A model proposing that each force could autonomously drive dorsal closure was recently challenged by a model in which the two forces combine in a ratchet mechanism. Acute force elimination via selective MyosinII depletion in one or the other tissue shows that the amnioserosa tissue autonomously drives dorsal closure while the actin/MyosinII cable cannot. These findings exclude both previous models, although a contribution of the ratchet mechanism at dorsal closure onset remains likely. This shifts the current view of dorsal closure being a combinatorial force-component system to a single tissue-driven closure event.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Tipificación del Cuerpo/fisiología , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Células Epidérmicas , Morfogénesis/fisiología , Actomiosina/metabolismo , Animales , Movimiento Celular/fisiología , Constricción , Proteínas de Drosophila/metabolismoRESUMEN
Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-actin but not the F-actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization.
Asunto(s)
Membrana Celular/química , Schizosaccharomyces/química , Schizosaccharomyces/citología , Actinas/metabolismo , Polaridad Celular , Citoesqueleto/metabolismo , Microdominios de Membrana , Proteínas Asociadas a Microtúbulos/metabolismo , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
The forces shaping an organism are not exclusively produced by actin/myosin II networks. In part II of this SnapShot, we present various alternative mechanisms. In addition to driving morphogenesis, cells use mechanical forces to sense and react to the specific mechanical properties of their environment. Also, we present a selection of experimental tools commonly used in force analysis.
Asunto(s)
Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Animales , Fenómenos Biomecánicos , Adhesión Celular , Humanos , Microtúbulos/metabolismoRESUMEN
Cell-type-specific F-actin structures and myosin motors are key generators of the forces that drive tissue morphogenesis in developing organisms. These cytoskeletal elements mediate defined cell deformation and control the arrangement of cell-cell contacts. This SnapShot presents a selection of morphogenetic processes, the analysis of which has pioneered specific types of F-actin/myosin-mediated force generation in development.
Asunto(s)
Actinas/metabolismo , Morfogénesis , Miosinas/metabolismo , Animales , Biofisica , Adhesión Celular , Microtúbulos/metabolismoRESUMEN
We developed a method for visualizing tissues from multicellular organisms using cryo-electron tomography. Our protocol involves vitrifying samples with high-pressure freezing, thinning them with cryo-FIB-SEM (focused-ion-beam scanning electron microscopy) and applying fiducial gold markers under cryogenic conditions to the lamellae post-milling. We applied this protocol to acquire tomograms of vitrified Caenorhabditis elegans embryos and worms, which showed the intracellular organization of selected tissues at particular developmental stages in otherwise intact specimens.
Asunto(s)
Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/ultraestructura , Microscopía Electrónica de RastreoRESUMEN
The closure of epidermal openings is an essential biological process that causes major developmental problems such as spina bifida in humans if it goes awry. At present, the mechanism of closure remains elusive. Therefore, we reconstructed a model closure event, dorsal closure in fly embryos, by large-volume correlative electron tomography. We present a comprehensive, quantitative analysis of the cytoskeletal reorganization, enabling separated epidermal cells to seal the epithelium. After establishing contact through actin-driven exploratory filopodia, cells use a single lamella to generate 'roof tile'-like overlaps. These shorten to produce the force, 'zipping' the tissue closed. The shortening overlaps lack detectable actin filament ensembles but are crowded with microtubules. Cortical accumulation of shrinking microtubule ends suggests a force generation mechanism in which cortical motors pull on microtubule ends as for mitotic spindle positioning. In addition, microtubules orient filopodia and lamellae before zipping. Our 4D electron microscopy picture describes an entire developmental process and provides fundamental insight into epidermal closure.
Asunto(s)
Citoesqueleto/ultraestructura , Drosophila melanogaster/ultraestructura , Tomografía con Microscopio Electrónico , Epitelio/ultraestructura , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Citoesqueleto/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epitelio/embriología , Epitelio/metabolismo , Genes Reporteros , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Microscopía Confocal , Microscopía Fluorescente , Microscopía por Video , Microtúbulos/ultraestructura , Seudópodos/ultraestructuraRESUMEN
Mal3p and Tip1p are the fission yeast (Schizosaccharomyces pombe) homologues of EB1 and CLIP-170, two conserved microtubule plus end tracking proteins (+TIPs). These proteins are crucial regulators of microtubule dynamics. Using electron tomography, we carried out a high-resolution analysis of the phenotypes caused by mal3 and tip1 deletions. We describe the 3-dimensional microtubule organization, quantify microtubule end structures and uncover novel defects of the microtubule lattices. We also reveal unexpected structural modifications of the spindle pole bodies (SPBs), the yeast microtubule organizing centers. In both mutants we observe an increased SPB volume and a reduced number of MT/SPB attachments. The discovered defects alter previous interpretations of the mutant phenotypes and provide new insights into the molecular functions of the two protein families.
Asunto(s)
Microtúbulos/metabolismo , Schizosaccharomyces/metabolismo , Tomografía con Microscopio Electrónico , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
The two key processes in growth polarisation are the generation of a confined region and the correct positioning of that region. Fission yeast has greatly contributed to the study of cell polarisation, particularly in the aspect of growth site positioning, which involves the interphase microtubule cytoskeleton. Here we review the mechanisms of growth polarity in vegetatively growing fission yeast cells. These seemingly simple cells show astonishingly complex growth polarity behaviour, including polarity switching and integrating multiple levels of control by the cell cycle machinery. We aim to extract and highlight the underlying concepts and discuss these in context of current understanding; showing how relevant proteins are networked to integrate the various machineries.
Asunto(s)
Polaridad Celular , Schizosaccharomyces/citología , Schizosaccharomyces/crecimiento & desarrollo , Microtúbulos/metabolismoRESUMEN
The genetic integrity of every organism depends on the faithful partitioning of its genome between two daughter cells in mitosis. In all eukaryotes, chromosome segregation requires the assembly of the mitotic spindle, a bipolar array of dynamic microtubules. Perturbations in microtubule dynamics affect spindle assembly and maintenance and ultimately result in aberrant cell divisions. To identify new regulators of microtubule dynamics within the hundreds of mitotic hits, reported in RNAi screens performed in C. elegans, Drosophila and mammalian tissue culture cells [Sonnichsen et al., 2005; Goshima et al., 2007; Neumann et al., 2010], we established a fast and quantitative assay to measure microtubule dynamics in living cells. Here we present a fully automated workflow from RNAi transfection, via image acquisition and data processing, to the quantitative characterization of microtubule behaviour. Candidate genes are knocked down by solid-phase reverse transfection with siRNA oligos in HeLa cells stably expressing EB3-EGFP, a microtubule plus end marker. Mitotic cells are selected using an automatic classifier [Conrad et al., 2011] and imaged on a spinning disk confocal microscope at high temporal and spatial resolution. The time-lapse movies are analysed using a multiple particle tracking software, developed in-house, that automatically detects microtubule plus ends, tracks microtubule growth events over consecutive frames and calculates growth speeds, lengths and lifetimes of the tracked microtubules. The entire assay provides a powerful tool to analyse the effect of essential mitotic genes on microtubule dynamics in living cells and to dissect their contribution in spindle assembly and maintenance.
Asunto(s)
Microtúbulos/metabolismo , Huso Acromático/metabolismo , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/genética , Mitosis/fisiología , Interferencia de ARN/fisiologíaRESUMEN
Microtubules (MTs) exhibit dynamic instability, alternating between phases of growth and shortening, mostly at their uncapped plus ends. Based on results from cryo-electron microscopy it was proposed that growing MTs display mainly curved sheets and blunt ends; during depolymerisation curled 'ramshorns' predominate. Observations of MTs in mitotic cells have suggested that the situation in vivo differs from that in vitro, but so far, a clear comparison between in vivo and in vitro results has not been possible because MT end structures could not be correlated directly with the dynamic state of that particular MT. Here we combine light microscopy and electron tomography (ET) to show that growing MT plus ends in the fission yeast Schizosaccharomyces pombe display predominantly a flared morphology. This indicates that MT polymerisation in vivo and in vitro can follow different paths.
Asunto(s)
Tomografía con Microscopio Electrónico/métodos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Microscopía por Crioelectrón , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/química , Schizosaccharomyces/citología , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Microtubules are essential for a variety of fundamental cellular processes such as organelle positioning and control of cell shape. Schizosaccharomyces pombe is an ideal organism for studying the function and organization of microtubules into bundles in interphase cells. Using light microscopy and electron tomography we analyzed the bundle organization of interphase microtubules in S. pombe. We show that cells lacking ase1p and klp2p still contain microtubule bundles. In addition, we show that ase1p is the major determinant of inter-microtubule spacing in interphase bundles since ase1 deleted cells have an inter-microtubule spacing that differs from that observed in wild-type cells. We then identified dis1p, a XMAP215 homologue, as factor that promotes the stabilization of microtubule bundles. In wild-type cells dis1p partially co-localized with ase1p at regions of microtubule overlap. In cells deleted for ase1 and klp2, dis1p accumulated at the overlap regions of interphase microtubule bundles. In cells lacking all three proteins, both microtubule bundling and inter-microtubule spacing were further reduced, suggesting that Dis1p contributes to interphase microtubule bundling.
Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas de Xenopus/metabolismo , Citoesqueleto/metabolismo , Interfase , Microscopía Confocal/métodos , Microscopía Electrónica/métodos , Microscopía Fluorescente/métodos , Modelos Genéticos , Proteínas de Schizosaccharomyces pombe/metabolismo , Programas InformáticosRESUMEN
Dorsal closure is a tissue-modeling process in the developing Drosophila embryo during which an epidermal opening is closed. It begins with the appearance of a supracellular actin cable that surrounds the opening and provides a contractile force. Amnioserosa cells that fill the opening produce an additional critical force pulling on the surrounding epidermal tissue. We show that this force is not gradual but pulsed and occurs long before dorsal closure starts. Quantitative analysis, combined with laser cutting experiments and simulations, reveals that tension-based dynamics and cell coupling control the force pulses. These constitutively pull the surrounding epidermal tissue dorsally, but the displacement is initially transient. It is translated into dorsal-ward movement only with the help of the actin cable, which acts like a ratchet, counteracting ventral-ward epidermis relaxation after force pulses. Our work uncovers a sophisticated mechanism of cooperative force generation between two major forces driving morphogenesis.
Asunto(s)
Drosophila melanogaster/embriología , Actinas/metabolismo , Animales , Fenómenos Biomecánicos , Drosophila melanogaster/citología , Ectodermo/citología , Ectodermo/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , MorfogénesisRESUMEN
The cytoskeleton is essential for the maintenance of cell morphology in eukaryotes. In fission yeast, for example, polarized growth sites are organized by actin, whereas microtubules (MTs) acting upstream control where growth occurs. Growth is limited to the cell poles when MTs undergo catastrophes there and not elsewhere on the cortex. Here, we report that the modulation of MT dynamics by forces as observed in vitro can quantitatively explain the localization of MT catastrophes in Schizosaccharomyces pombe. However, we found that it is necessary to add length-dependent catastrophe rates to make the model fully consistent with other previously measured traits of MTs. We explain the measured statistical distribution of MT-cortex contact times and re-examine the curling behavior of MTs in unbranched straight tea1Delta cells. Importantly, the model demonstrates that MTs together with associated proteins such as depolymerizing kinesins are, in principle, sufficient to mark the cell poles.
Asunto(s)
Interfase , Microtúbulos/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Fenómenos Biomecánicos , Simulación por Computador , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Microtubules (MTs) are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology. For these purposes, MT polymerisation dynamics are tightly regulated. Using automated image analysis software, we investigate the spatial dependence of MT dynamics in interphase fission yeast cells with unprecedented statistical accuracy. We find that MT catastrophe frequencies (switches from polymerisation to depolymerisation) strongly depend on intracellular position. We provide evidence that compressive forces generated by MTs growing against the cell pole locally reduce MT growth velocities and enhance catastrophe frequencies. Furthermore, we find evidence for an MT length-dependent increase in the catastrophe frequency that is mediated by kinesin-8 proteins (Klp5/6). Given the intrinsic susceptibility of MT dynamics to compressive forces and the widespread importance of kinesin-8 proteins, we propose that similar spatial regulation of MT dynamics plays a role in other cell types as well. In addition, our systematic and quantitative data should provide valuable input for (mathematical) models of MT organisation in living cells.
Asunto(s)
Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Fenómenos Biomecánicos , Polaridad Celular , Eliminación de Gen , Cinesinas/deficiencia , Proteínas Asociadas a Microtúbulos/deficiencia , Transporte de ProteínasRESUMEN
In the presence of GTP, purified dimers of alpha- and beta-tubulin will interact longitudinally and laterally to self-assemble into microtubules (MTs). This property provides a powerful in vitro experimental system to describe MT dynamic behavior at the micrometer scale and to study effects and functioning of a large variety of microtubule associated proteins (MAPs). Despite the plethora of such data produced, the molecular mechanisms of MT assembly remain disputed. Electron microscopy (EM) studies suggested that tubulin dimers interact longitudinally to form short oligomers which form a tube by lateral interaction and which contribute to MT elongation. This idea is however challenged: Based on estimated association constants it was proposed that single dimers represent the major fraction of free tubulin. This view was recently supported by measurements suggesting that MTs elongate by addition of single tubulin dimers. To solve this discrepancy, we performed a direct measurement of the longitudinal interaction energy for tubulin dimers. We quantified the size distribution of tubulin oligomers using EM and fluorescence correlation spectroscopy (FCS). From the distribution we derived the longitudinal interaction energy in the presence of GDP and the non-hydrolysable GTP analog GMPCPP. Our data suggest that MT elongation and nucleation involves interactions of short tubulin oligomers rather than dimers. Our approach provides a solid experimental framework to better understand the role of MAPs in MT nucleation and growth.
Asunto(s)
Microtúbulos/química , Tubulina (Proteína)/metabolismo , Animales , Dimerización , Guanosina Difosfato , Guanosina Trifosfato , Microscopía Electrónica , Microtúbulos/ultraestructura , Espectrometría de Fluorescencia , Porcinos , Tubulina (Proteína)/ultraestructuraRESUMEN
Microtubules (MTs) are cytoskeletal polymers whose spatial organization is dynamically regulated, depending on their biological function during different cell cycle stages. Growing MT ends are, for example, specifically targeted towards the cortex of motile or growing cells during interphase or towards chromosomal attachment sites during mitosis. An important parameter that cells use to control the average length of MTs, and thus the distance over which these targeting processes may operate, is the so-called catastrophe frequency f(cat): the rate at which MTs switch from a growing to a shrinking state. To understand how spatial targeting and the local control of f(cat) are related, quantitative in vivo measurements are needed that allow for the measurement of f(cat) in a spatially resolved way. Since catastrophes are intrinsically stochastic events, it is essential to acquire enough statistics to obtain the underlying rate constant f(cat). Here, we present automated image processing methodology, developed using GFP-tubulin expressing fission yeast cells, that makes it possible to measure f(cat) both spatially resolved and with high statistical accuracy. Although certain aspects of the analysis are specific to the system under investigation the basic concepts of the methodology are applicable to any kind of movies of fluorescently labeled MTs.
Asunto(s)
Citoesqueleto/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Microtúbulos/fisiología , Schizosaccharomyces/fisiología , Algoritmos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/citología , Tubulina (Proteína)/metabolismoRESUMEN
The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.