RESUMEN
Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.
Asunto(s)
Arabidopsis/química , Arabidopsis/citología , Polaridad Celular/fisiología , Células Vegetales/fisiología , Polimerizacion , Dominios Proteicos , Animales , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteína Axina/química , Proteína Axina/metabolismo , Bryopsida/química , Bryopsida/citología , Bryopsida/genética , Bryopsida/crecimiento & desarrollo , Células COS , Chlorocebus aethiops , Proteínas Dishevelled/metabolismo , Células HEK293 , Humanos , Marchantia/química , Marchantia/citología , Marchantia/genética , Marchantia/crecimiento & desarrollo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Plantas Modificadas Genéticamente , Proteínas Represoras/metabolismo , Vía de Señalización WntRESUMEN
The fission yeast Schizosaccharomycespombe serves as a good genetic model organism for the molecular dissection of the microtubule (MT) cytoskeleton. However, analysis of the number and distribution of individual MTs throughout the cell cycle, particularly during mitosis, in living cells is still lacking, making quantitative modelling imprecise. We use quantitative fluorescent imaging and analysis to measure the changes in tubulin concentration and MT number and distribution throughout the cell cycle at a single MT resolution in living cells. In the wild-type cell, both mother and daughter spindle pole body (SPB) nucleate a maximum of 23 ± 6 MTs at the onset of mitosis, which decreases to a minimum of 4 ± 1 MTs at spindle break down. Interphase MT bundles, astral MT bundles, and the post anaphase array (PAA) microtubules are composed primarily of 1 ± 1 individual MT along their lengths. We measure the cellular concentration of αß-tubulin subunits to be ~5 µM throughout the cell cycle, of which one-third is in polymer form during interphase and one-quarter is in polymer form during mitosis. This analysis provides a definitive characterization of αß-tubulin concentration and MT number and distribution in fission yeast and establishes a foundation for future quantitative comparison of mutants defective in MTs.
Asunto(s)
Ciclo Celular , Microtúbulos/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Tubulina (Proteína)/análisis , Tubulina (Proteína)/metabolismo , Microtúbulos/químicaRESUMEN
During plant cytokinesis a radially expanding membrane-enclosed cell plate is formed from fusing vesicles that compartmentalizes the cell in two. How fusion is spatially restricted to the site of cell plate formation is unknown. Aggregation of cell-plate membrane starts near regions of microtubule overlap within the bipolar phragmoplast apparatus of the moss Physcomitrella patens Since vesicle fusion generally requires coordination of vesicle tethering and subsequent fusion activity, we analyzed the subcellular localization of several subunits of the exocyst, a tethering complex active during plant cytokinesis. We found that the exocyst complex subunit Sec6 but not the Sec3 or Sec5 subunits localized to microtubule overlap regions in advance of cell plate construction in moss. Moreover, Sec6 exhibited a conserved physical interaction with an ortholog of the Sec1/Munc18 protein KEULE, an important regulator for cell-plate membrane vesicle fusion in Arabidopsis Recruitment of the P. patens protein KEULE and vesicles to the early cell plate was delayed upon Sec6 gene silencing. Our findings, thus, suggest that vesicle-vesicle fusion is, in part, enabled by a pool of exocyst subunits at microtubule overlaps, which is recruited independently of vesicle delivery.
Asunto(s)
Bryopsida/genética , Citocinesis/genética , Regulación de la Expresión Génica de las Plantas , Microtúbulos/metabolismo , Proteínas de Plantas/genética , Proteínas de Transporte Vesicular/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Bryopsida/metabolismo , Bryopsida/ultraestructura , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Silenciador del Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Células Vegetales/metabolismo , Células Vegetales/ultraestructura , Proteínas de Plantas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteína Fluorescente RojaRESUMEN
Different from animal cells that divide by constriction of the cortex inward, cells of land plants divide by initiating a new cell-wall segment from their center. For this, a disk-shaped, membrane-enclosed precursor termed the cell plate is formed that radially expands toward the parental cell wall [1-3]. The synthesis of the plate starts with the fusion of vesicles into a tubulo-vesicular network [4-6]. Vesicles are putatively delivered to the division plane by transport along microtubules of the bipolar phragmoplast network that guides plate assembly [7-9]. How vesicle immobilization and fusion are then locally triggered is unclear. In general, a framework for how the cytoskeleton spatially defines cell-plate formation is lacking. Here we show that membranous material for cell-plate formation initially accumulates along regions of microtubule overlap in the phragmoplast of the moss Physcomitrella patens. Kinesin-4-mediated shortening of these overlaps at the onset of cytokinesis proved to be required to spatially confine membrane accumulation. Without shortening, the wider cell-plate membrane depositions evolved into cell walls that were thick and irregularly shaped. Phragmoplast assembly thus provides a regular lattice of short overlaps on which a new cell-wall segment can be scaffolded. Since similar patterns of overlaps form in central spindles of animal cells, involving the activity of orthologous proteins [10, 11], we anticipate that our results will help uncover universal features underlying membrane-cytoskeleton coordination during cytokinesis.
Asunto(s)
Bryopsida/fisiología , Citocinesis , Citoesqueleto/fisiología , Cinesinas/metabolismo , Microtúbulos/metabolismo , Proteínas de Plantas/metabolismo , Pared Celular/fisiologíaRESUMEN
The bipolar kinesin-5 motors are one of the major players that govern mitotic spindle dynamics. Their bipolar structure enables them to cross-link and slide apart antiparallel microtubules (MTs) emanating from the opposing spindle poles. The budding yeast kinesin-5 Cin8 was shown to switch from fast minus-end- to slow plus-end-directed motility upon binding between antiparallel MTs. This unexpected finding revealed a new dimension of cellular control of transport, the mechanism of which is unknown. Here we have examined the role of the C-terminal tail domain of Cin8 in regulating directionality. We first constructed a stable dimeric Cin8/kinesin-1 chimera (Cin8Kin), consisting of head and neck linker of Cin8 fused to the stalk of kinesin-1. As a single dimeric motor, Cin8Kin switched frequently between plus and minus directionality along single MTs, demonstrating that the Cin8 head domains are inherently bidirectional, but control over directionality was lost. We next examined the activity of a tetrameric Cin8 lacking only the tail domains (Cin8Δtail). In contrast to wild-type Cin8, the motility of single molecules of Cin8Δtail in high ionic strength was slow and bidirectional, with almost no directionality switches. Cin8Δtail showed only a weak ability to cross-link MTs in vitro. In vivo, Cin8Δtail exhibited bias toward the plus-end of the MTs and was unable to support viability of cells as the sole kinesin-5 motor. We conclude that the tail of Cin8 is not necessary for bidirectional processive motion, but is controlling the switch between plus- and minus-end-directed motility.
Asunto(s)
Cinesinas/química , Cinesinas/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cinesinas/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Eliminación de SecuenciaRESUMEN
Cytoskeletal remodeling is essential to eukaryotic cell division and morphogenesis. The mechanical forces driving the restructuring are attributed to the action of molecular motors and the dynamics of cytoskeletal filaments, which both consume chemical energy. By contrast, non-enzymatic filament crosslinkers are regarded as mere friction-generating entities. Here, we experimentally demonstrate that diffusible microtubule crosslinkers of the Ase1/PRC1/Map65 family generate directed microtubule sliding when confined between partially overlapping microtubules. The Ase1-generated forces, directly measured by optical tweezers to be in the piconewton-range, were sufficient to antagonize motor-protein driven microtubule sliding. Force generation is quantitatively explained by the entropic expansion of confined Ase1 molecules diffusing within the microtubule overlaps. The thermal motion of crosslinkers is thus harnessed to generate mechanical work analogous to compressed gas propelling a piston in a cylinder. As confinement of diffusible proteins is ubiquitous in cells, the associated entropic forces are likely of importance for cellular mechanics beyond cytoskeletal networks.
Asunto(s)
Microtúbulos/metabolismo , Animales , Fenómenos Biomecánicos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fricción , Proteínas Fluorescentes Verdes/metabolismo , Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Pinzas Ópticas , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
During cytokinesis the cytoplasm of a cell is divided to form two daughter cells. In animal cells, the existing plasma membrane is first constricted and then abscised to generate two individual plasma membranes. Plant cells on the other hand divide by forming an interior dividing wall, the so-called cell plate, which is constructed by localized deposition of membrane and cell wall material. Construction starts in the centre of the cell at the locus of the mitotic spindle and continues radially towards the existing plasma membrane. Finally the membrane of the cell plate and plasma membrane fuse to form two individual plasma membranes. Two microtubule-based cytoskeletal networks, the phragmoplast and the pre-prophase band (PPB), jointly control cytokinesis in plants. The bipolar microtubule array of the phragmoplast regulates cell plate deposition towards a cortical position that is templated by the ring-shaped microtubule array of the PPB. In contrast to most animal cells, plants do not use centrosomes as foci of microtubule growth initiation. Instead, plant microtubule networks are striking examples of self-organizing systems that emerge from physically constrained interactions of dispersed microtubules. Here we will discuss how microtubule-based activities including growth, shrinkage, severing, sliding, nucleation and bundling interrelate to jointly generate the required ordered structures. Evidence mounts that adapter proteins sense the local geometry of microtubules to locally modulate the activity of proteins involved in microtubule growth regulation and severing. Many of the proteins and mechanisms involved have roles in other microtubule assemblies as well, bestowing broader relevance to insights gained from plants.
RESUMEN
The phragmoplast, a plant-specific apparatus that mediates cytokinesis, mainly consists of microtubules (MTs) arranged in a bipolar fashion, such that their plus ends interdigitate at the equator. Membrane vesicles are thought to move along the MTs toward the equator and fuse to form the cell plate. Although several genes required for phragmoplast MT organization have been identified, the mechanisms that maintain the bipolarity of phragmoplasts remain poorly understood. Here, we show that engaging phragmoplast MTs in a bipolar fashion in protonemal cells of the moss Physcomitrella patens requires the conserved MT cross-linking protein MICROTUBULE-ASSOCIATED PROTEIN65 (MAP65). Simultaneous knockdown of the three MAP65s expressed in those cells severely compromised MT interdigitation at the phragmoplast equator after anaphase onset, resulting in the collapse of the phragmoplast in telophase. Cytokinetic vesicles initially localized to the anaphase midzone as normal but failed to further accumulate in the next several minutes, although the bipolarity of the MT array was preserved. Our data indicate that the presence of bipolar MT arrays is insufficient for vesicle accumulation at the equator and further suggest that MAP65-mediated MT interdigitation is a prerequisite for maintenance of bipolarity of the phragmoplast and accumulation and/or fusion of cell plate-destined vesicles at the equatorial plane.
Asunto(s)
Bryopsida/citología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Plantas/metabolismo , Bryopsida/genética , Bryopsida/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Familia de Multigenes , Filogenia , Proteínas de Plantas/genética , Plantas Modificadas GenéticamenteRESUMEN
Short regions of overlap between ends of antiparallel microtubules are central elements within bipolar microtubule arrays. Although their formation requires motors, recent in vitro studies demonstrated that stable overlaps cannot be generated by molecular motors alone. Motors either slide microtubules along each other until complete separation or, in the presence of opposing motors, generate oscillatory movements. Here, we show that Ase1, a member of the conserved MAP65/PRC1 family of microtubule-bundling proteins, enables the formation of stable antiparallel overlaps through adaptive braking of Kinesin-14-driven microtubule-microtubule sliding. As overlapping microtubules start to slide apart, Ase1 molecules become compacted in the shrinking overlap and the sliding velocity gradually decreases in a dose-dependent manner. Compaction is driven by moving microtubule ends that act as barriers to Ase1 diffusion. Quantitative modelling showed that the molecular off-rate of Ase1 is sufficiently low to enable persistent overlap stabilization over tens of minutes. The finding of adaptive braking demonstrates that sliding can be slowed down locally to stabilize overlaps at the centre of bipolar arrays, whereas sliding proceeds elsewhere to enable network self-organization.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/metabolismo , Animales , Proteínas de Drosophila/metabolismo , Cinesinas/metabolismo , Microscopía Fluorescente , Microscopía por Video , Proteínas Asociadas a Microtúbulos/genética , Modelos Biológicos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Factores de TiempoRESUMEN
Microtubule (MT) crosslinking proteins of the ase1p/PRC1/Map65 family play a major role in the construction of MT networks such as the mitotic spindle. Most homologs in this family have been shown to localize with a remarkable specificity to sets of MTs that overlap with an antiparallel relative orientation [1-4]. Regulatory proteins bind to ase1p/PRC1/Map65 and appear to use the localization to set up precise spatial signals [5-10]. Here, we present evidence for a mechanism of localized protein multimerization underlying the specific targeting of ase1p, the fision yeast homolog. In controlled in vitro experiments, dimers of ase1-GFP diffused along the surface of single MTs and, at concentrations above a certain threshold, assembled into static multimeric structures. We observed that this threshold was significantly lower on overlapping MTs. We also observed diffusion and multimerization of ase1-GFP on MTs inside living cells, suggesting that a multimerization-driven localization mechanism is relevant in vivo. The domains responsible for MT binding and multimerization were identified via a series of ase1p truncations. Our findings show that cells use a finely tuned cooperative localization mechanism that exploits differences in the geometry and concentration of ase1p binding sites along single and overlapping MTs.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Multimerización de Proteína , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/ultraestructura , Animales , Células COS , Chlorocebus aethiops , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Schizosaccharomyces/metabolismoRESUMEN
Sets of overlapping microtubules support the segregation of chromosomes by linking the poles of mitotic spindles. Recent work examines the effect of putting these linkages under pressure by the activation of dicentric chromosomes and sheds new light on the structural role of several well-known spindle midzone proteins.
Asunto(s)
Segregación Cromosómica/fisiología , Huso Acromático/fisiología , Cromosomas Fúngicos/fisiología , Microtúbulos/fisiología , Saccharomyces cerevisiaeRESUMEN
Microtubule (MT) nucleation not only occurs from centrosomes, but also in large part from dispersed nucleation sites. The subsequent sorting of short MTs into networks like the mitotic spindle requires molecular motors that laterally slide overlapping MTs and bundling proteins that statically connect MTs. How bundling proteins interfere with MT sliding is unclear. In bipolar MT bundles in fission yeast, we found that the bundler ase1p localized all along the length of antiparallel MTs, whereas the motor klp2p (kinesin-14) accumulated only at MT plus ends. Consequently, sliding forces could only overcome resistant bundling forces for short, newly nucleated MTs, which were transported to their correct position within bundles. Ase1p thus regulated sliding forces based on polarity and overlap length, and computer simulations showed these mechanisms to be sufficient to generate stable bipolar bundles. By combining motor and bundling proteins, cells can thus dynamically organize stable regions of overlap between cytoskeletal filaments.
Asunto(s)
Proteínas Portadoras/metabolismo , Microtúbulos/metabolismo , Mitosis/fisiología , Proteínas Motoras Moleculares/metabolismo , Schizosaccharomyces/metabolismo , Huso Acromático/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Sitios de Unión , Proteínas Portadoras/genética , Polaridad Celular/fisiología , Simulación por Computador , Corriente Citoplasmática/fisiología , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/genética , Microtúbulos/ultraestructura , Proteínas Motoras Moleculares/genética , Schizosaccharomyces/genética , Schizosaccharomyces/ultraestructura , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/genética , Huso Acromático/ultraestructura , Estrés MecánicoRESUMEN
Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells.
Asunto(s)
Microtúbulos/química , Microtúbulos/metabolismo , Algoritmos , Tampones (Química) , Dimerización , Guanosina Trifosfato/metabolismo , Rayos Láser , Óptica y Fotónica , Estructura Cuaternaria de Proteína , Sensibilidad y Especificidad , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMEN
The mechanism for forming linear microtubule (MT) arrays in cells such as neurons, polarized epithelial cells, and myotubes is not well understood. A simpler bipolar linear array is the fission yeast interphase MT bundle, which in its basic form contains two MTs that are bundled at their minus ends. Here, we characterize mto2p as a novel fission yeast protein required for MT nucleation from noncentrosomal gamma-tubulin complexes (gamma-TuCs). In interphase mto2Delta cells, MT nucleation was strongly inhibited, and MT bundling occurred infrequently and only when two MTs met by chance in the cytoplasm. In wild-type 2, we observed MT nucleation from gamma-TuCs bound along the length of existing MTs. We propose a model on how these nucleation events can more efficiently drive the formation of bipolar MT bundles in interphase. Key to the model is our observation of selective antiparallel binding of MTs, which can both explain the generation and spatial separation of multiple bipolar bundles.
Asunto(s)
División Celular/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/metabolismo , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , División Celular/genética , Eliminación de Gen , Interfase/fisiología , Modelos Biológicos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
The assembly and disassembly of microtubules can generate pushing and pulling forces that, together with motor proteins, contribute to the correct positioning of chromosomes, mitotic spindles and nuclei in cells. In vitro experiments combined with modeling have shed light on the intrinsic capability of dynamic microtubules to generate force, and various observations of positioning processes in cells and model systems have shown how pushing and pulling forces are used in different situations. A sophisticated set of microtubule-end-binding proteins is responsible for steering dynamic microtubules toward their cellular target and regulating the pushing and/or pulling forces that are generated once contact is established.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Microtúbulos/química , Núcleo Celular/metabolismo , Cromosomas/metabolismo , Dimerización , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Saccharomycetales , Schizosaccharomyces , Huso Acromático/metabolismoRESUMEN
Microtubules are dynamic protein polymers that continuously switch between elongation and rapid shrinkage. They have an exceptional bending stiffness that contributes significantly to the mechanical properties of eukaryotic cells. Measurements of the persistence length of microtubules have been published since 10 years but the reported values vary over an order of magnitude without an available explanation. To precisely measure the rigidity of microtubules in their native growing state, we adapted a previously developed bending mode analysis of thermally driven shape fluctuations to the case of an elongating filament that is clamped at one end. Microtubule shapes were quantified using automated image processing, allowing for the characterization of up to five bending modes. When taken together with three other less precise measurements, our rigidity data suggest that fast-growing microtubules are less stiff than slow-growing microtubules. This would imply that care should be taken in interpreting rigidity measurements on stabilized microtubules whose growth history is not known. In addition, time analysis of bending modes showed that higher order modes relax more slowly than expected from simple hydrodynamics, possibly by the effects of internal friction within the microtubule.
Asunto(s)
Cristalización/métodos , Cristalografía/métodos , Interpretación de Imagen Asistida por Computador , Microtúbulos/química , Microtúbulos/ultraestructura , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructura , Algoritmos , Elasticidad , Sustancias Macromoleculares/química , Conformación Molecular , Movimiento (Física) , Conformación Proteica , Estrés Mecánico , ViscosidadRESUMEN
We present a single curve that describes the decay in average growth velocity for microtubules in response to a mechanical force. Curves obtained at two new and one previously studied tubulin concentrations coalesce when normalized with the growth velocity at zero load. This scaling provides direct evidence for a force-independent molecular off rate, in agreement with Brownian ratchet models. In addition, microtubule length changes were measured with a precision up to 10 nm, revealing that microtubules under load abruptly switch between different growth velocities.
Asunto(s)
Microtúbulos/química , Tubulina (Proteína)/química , Dimerización , Modelos Biológicos , Proteínas Motoras Moleculares/químicaRESUMEN
In living cells, dynamic microtubule ends interact with specialized protein complexes located on microtubule targets such as chromosomes and the cell cortex. A significant role in coupling microtubule ends to these complexes has been attributed to motor proteins, which are thought to provide a physical link while at the same time allowing for microtubule growth or shrinkage. In the past, motor-coated beads have been shown to be able to follow the ends of depolymerizing microtubules, in a direction opposite to their natural walking direction. Here we show that beads coated with plus-end-directed motors can also stay attached for several seconds to the ends of growing microtubules. Upon arrival at the microtubule end, fast-moving beads reduce their velocity to the microtubule growth velocity. We show that the tendency to stay attached depends on the initial bead velocity and that the microtubule growth velocity is unaffected by the presence of the bead.
Asunto(s)
Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Animales , DrosophilaRESUMEN
Microtubules are long filamentous protein structures that randomly alternate between periods of elongation and shortening in a process termed dynamic instability. The average time a microtubule spends in an elongation phase, known as the catastrophe time, is regulated by the biochemical machinery of the cell throughout the cell cycle. In this light, observed changes in the catastrophe time near cellular boundaries (Brunner, D., and P. Nurse. 2000. Cell. 102:695-704; Komarova, Y.A., I.A. Vorobjev, and G.G. Borisy. 2002. J. Cell Sci. 115:3527-3539) may be attributed to regulatory effects of localized proteins. Here, we argue that the pushing force generated by a microtubule when growing against a cellular object may itself provide a regulatory mechanism of the catastrophe time. We observed an up to 20-fold, force-dependent decrease in the catastrophe time when microtubules grown from purified tubulin were polymerizing against microfabricated barriers. Comparison with catastrophe times for microtubules growing freely at different tubulin concentrations leads us to conclude that force reduces the catastrophe time only by limiting the rate of tubulin addition.