Cofilin is an essential component of the yeast cortical cytoskeleton

We have biochemically identified the Saccharomyces cerevisiae homologue of the mammalian actin binding protein cofilin. Cofilin and related proteins isolated from diverse organisms are low molecular weight proteins (15-20 kD) that possess several activities in vitro. All bind to monomeric actin and sever filaments, and some can stably associate with filaments. In this study, we demonstrate using viscosity, sedimentation, and actin assembly rate assays that yeast cofilin (16 kD) possesses all of these properties. Cloning and sequencing of the S. cerevisiae cofilin gene (COF1) revealed that yeast cofilin is 41% identical in amino acid sequence to mammalian cofilin and, surprisingly, has homology to a protein outside the family of cofilin- like proteins. The NH2-terminal 16kD of Abp1p, a 65-kD yeast protein identified by its ability to bind to actin filaments, is 23% identical to yeast cofilin. Immunofluorescence experiments showed that, like Abp1p, cofilin is associated with the membrane actin cytoskeleton. A complete disruption of the COF1 gene was created in diploid cells. Sporulation and tetrad analysis revealed that yeast cofilin has an essential function in vivo. Although Abp1p shares sequence similarity with cofilin and has the same distribution as cofilin in the cell, multiple copies of the ABP1 gene cannot compensate for the loss of cofilin. Thus, cofilin and Abp1p are structurally related but functionally distinct components of the yeast membrane cytoskeleton.     

Human CLASP1 is an outer kinetochore component that regulates spindle microtubule dynamics

One of the most intriguing aspects of mitosis is the ability of kinetochores to hold onto plus ends of microtubules that are actively gaining or losing tubulin subunits. Here, we show that CLASP1, a microtubule-associated protein, localizes preferentially near the plus ends of growing spindle microtubules and is also a component of a kinetochore region that we term the outer corona. A truncated form of CLASP1 lacking the kinetochore binding domain behaves as a dominant negative, leading to the formation of radial arrays of microtubule bundles that are highly resistant to depolymerization. Microinjection of CLASP1-specific antibodies suppresses microtubule dynamics at kinetochores and throughout the spindle, resulting in the formation of monopolar asters with chromosomes buried in the interior. Incubation with microtubule-stabilizing drugs rescues the kinetochore association with microtubule plus ends at the periphery of the asters. Our data suggest that CLASP1 is required at kinetochores for attached microtubules to exhibit normal dynamic behavior.     

Xenopus Cep57 is a novel kinetochore component involved in microtubule attachment

For chromosomes to congress and segregate during cell division, kinetochores must form stable attachments with spindle microtubules. We find that the centrosome protein, xCep57, localizes to kinetochores and interacts with the kinetochore proteins Zwint, Mis12, and CLIP-170. Immunodepletion of xCep57 from egg extracts yields weakened and elongated bipolar spindles which fail to align chromosomes. In the absence of xCep57, tension is lost between sister kinetochores, and spindle microtubules are no longer resistant to low doses of nocodazole. xCep57 inhibition on isolated mitotic chromosomes inhibits kinetochore-microtubule binding in vitro. xCep57 also interacts with gamma-tubulin. In xCep57 immunodepleted extracts, sperm centrosomes nucleate with normal kinetics, but are unable maintain microtubule anchorage. This characterization places xCep57 in a novel class of proteins required for stable microtubule attachments at the kinetochore and at the centrosome and suggests that the mechanism of microtubule binding at these two places is mechanistically similar.     

Gamma-tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans

We have recently discovered that the mipA gene of A. nidulans encodes gamma-tubulin, a new member of the tubulin superfamily. To determine the function of gamma-tubulin in vivo, we have created a mutation in the mipA gene by integrative transformation, maintained the mutation in a heterokaryon, and determined the phenotype of the mutation in spores produced by the heterokaryon. The mutation is lethal and recessive. It strongly inhibits nuclear division, less strongly inhibits nuclear migration, and, as judged by immunofluorescence microscopy, causes a reduction in the number and length of cytoplasmic microtubules and virtually a complete absence of mitotic apparatus. We conclude that gamma-tubulin is essential for microtubule function in general and nuclear division in particular. Immunofluorescence microscopy of wild-type hyphae with affinity-purified, gamma-tubulin-specific antibodies reveals that gamma-tubulin is a component of interphase and mitotic spindle pole bodies. We propose that gamma-tubulin attaches microtubules to the spindle pole body, nucleates microtubule assembly, and establishes microtubule polarity in vivo.     

Evidence for a ribonucleoprotein complex as a template for microtubule initiation in vivo

At the onset of mitosis in cultured mammalian cells, the centriolar region rapidly initiates the assembly of microtubules (MT) to form two asters which ultimately forms the basis of the mitotic spindle. The rapid change in the centrosphere's ability to nucleate MTs at prometaphase maybe due to the presence of an RNase sensitive component. Lysis of colcemid-blocked mitotic cells with RNase A or T₂ prior to addition of exogenous microtubule protein greatly diminishes the number of MT that can be nucleated in a lysed cell system. MT initiation can also be reduced or abolished by extended lysis in neutral buffers or brief lysis in acidic buffers. This suggests that the RNA component maybe complexed with a protein to serve as a template for MT initiation at the onset of mitosis.     

Immunostructural evidence for the template mechanism of microtubule nucleation

Two opposing models have been proposed to explain how the gamma-tubulin ring complex (gammaTuRC) induces microtubule nucleation. In the 'protofilament' model, the gammaTuRC induces nucleation as a partially or completely straightened protofilament that is incorporated longitudinally into the wall of the nascent microtubule, whereas the 'template' model proposes that the gammaTuRC acts as a helical template that constitutes the base of the newly-formed polymer. Here we appraise these two models, using high-resolution structural and immunolocalization methods. We show that components of the gammaTuRC localize to a narrow zone at the extreme minus end of the microtubule and that these ends terminate in a pointed cap. Together, these results strongly favour the template model of microtubule nucleation.     

Structure of the gamma-tubulin ring complex: a template for microtubule nucleation

The gamma-tubulin ring complex (gammaTuRC) is a protein complex of relative molecular mass approximately 2.2 x 10(6) that nucleates microtubules at the centrosome. Here we use electron-microscopic tomography and metal shadowing to examine the structure of isolated Drosophila gammaTuRCs and the ends of microtubules nucleated by gammaTuRCs and by centrosomes. We show that the gammaTuRC is a lockwasher-like structure made up of repeating subunits, topped asymmetrically with a cap. A similar capped ring is also visible at one end of microtubules grown from isolated gammaTuRCs and from centrosomes. Antibodies against gamma-tubulin label microtubule ends, but not walls, in centrosomes. These data are consistent with a template-mediated mechanism for microtubule nucleation by the gammaTuRC.     

Association of adenovirus with the microtubule organizing center

Adenoviruses (Ad) must deliver their genomes to the nucleus of the target cell to initiate an infection. Following entry into the cell and escape from the endosome, Ad traffics along the microtubule cytoskeleton toward the nucleus. In the final step in Ad trafficking, Ad must leave the microtubule and establish an association with the nuclear envelope. We hypothesized that in cells lacking a nucleus, the capsid moves to and associates with the microtubule organizing center (MTOC). To test this hypothesis, we established an experimental system to examine Ad trafficking in enucleated cells compared to Ad trafficking in intact, mock-enucleated cells. Enucleation of a monolayer of A549 human lung epithelial cells was accomplished by depolymerization of the actin cytoskeleton followed by centrifugation. Upon infection of enucleated cells with Cy3-labeled Ad, the majority of Ad capsid trafficked to a discrete, centrally located site which colocalized with pericentrin, a component of the MTOC. MTOC-associated Ad had escaped from endosomes and thus had direct access to MTOC components. Ad localization at this site was sensitive to the microtubule-depolymerizing agent nocodazole, but not to the microfilament-depolymerizing agent cytochalasin B, indicating that intact microtubules were required to maintain the localization with the MTOC. Ad localization to the MTOC in the enucleated cells was stable, as demonstrated by continuing Ad localization with pericentrin for more than 5 h after infection, a strong preference for Ad arrival at rather than Ad departure from the MTOC, and minimal redistribution of Ad between MTOCs within a single cell. In summary, the data demonstrate that the Ad capsid establishes a stable interaction with the MTOC when a nucleus is not present, suggesting that dissociation of Ad from microtubules likely requires nuclear factors.     

Dynein is a transient kinetochore component whose binding is regulated by microtubule attachment, not tension

Cytoplasmic dynein is the only known kinetochore protein capable of driving chromosome movement toward spindle poles. In grasshopper spermatocytes, dynein immunofluorescence staining is bright at prometaphase kinetochores and dimmer at metaphase kinetochores. We have determined that these differences in staining intensity reflect differences in amounts of dynein associated with the kinetochore. Metaphase kinetochores regain bright dynein staining if they are detached from spindle microtubules by micromanipulation and kept detached for 10 min. We show that this increase in dynein staining is not caused by the retraction or unmasking of dynein upon detachment. Thus, dynein genuinely is a transient component of spermatocyte kinetochores.We further show that microtubule attachment, not tension, regulates dynein localization at kinetochores. Dynein binding is extremely sensitive to the presence of microtubules: fewer than half the normal number of kinetochore microtubules leads to the loss of most kinetochoric dynein. As a result, the bulk of the dynein leaves the kinetochore very early in mitosis, soon after the kinetochores begin to attach to microtubules. The possible functions of this dynein fraction are therefore limited to the initial attachment and movement of chromosomes and/or to a role in the mitotic checkpoint.     

Apoptosis is not an invariable component of in vitro models of cortical cerebral ischaemia

Characterising the mechanisms of cell death following focal cerebral ischaemia has been hampered by a lack of an in vitro assay emulating both the apoptotic and necrotic features observed in vivo. The present study systematically characterised oxygen-glucose-deprivation (OGD) in primary rat cortical neurones to establish a reproducible model with components of both cell-death endpoints. OGD induced a time-dependent reduction in cell viability, with 80% cell death occurring 24 h after 3 h exposure to 0% O2 and 0.5 mM glucose. Indicative of a necrotic component to OGD-induced cell death, N-methyl-D-aspartate (NMDA) receptor inhibition with MK-801 attenuated neuronal loss by 60%.The lack of protection by the caspase inhibitors DEVD-CHO and z-VAD-fmk suggested that under these conditions neurones did not die by an apoptotic mechanism. Moderating the severity of the insult by decreasing OGD exposure to 60 min did not reduce the amount of necrosis, but did induce a small degree of apoptosis (a slight reduction in cell death was observed in the presence of 10 μM DEVD-CHO). In separate experiments purported to enhance the apoptotic component, cells were gradually deprived of 02, exposed to 4% 02 (as opposed to 0%) during the OGD period, or maintained in serum-containing media throughout. While NMDA receptor antagonism significantly reduced cortical cell death under all conditions, a caspase-inhibitor sensitive component of cell death was not uncovered. These studies suggest that OGD of cultured cortical cells models the excitotoxic, but not the apoptotic component of cell death observed in vivo.     

Lis1 is essential for cortical microtubule organization and desmosome stability in the epidermis

Desmosomes are cell-cell adhesion structures that integrate cytoskeletal networks. In addition to binding intermediate filaments, the desmosomal protein desmoplakin (DP) regulates microtubule reorganization in the epidermis. In this paper, we identify a specific subset of centrosomal proteins that are recruited to the cell cortex by DP upon epidermal differentiation. These include Lis1 and Ndel1, which are centrosomal proteins that regulate microtubule organization and anchoring in other cell types. This recruitment was mediated by a region of DP specific to a single isoform, DPI. Furthermore, we demonstrate that the epidermal-specific loss of Lis1 results in dramatic defects in microtubule reorganization. Lis1 ablation also causes desmosomal defects, characterized by decreased levels of desmosomal components, decreased attachment of keratin filaments, and increased turnover of desmosomal proteins at the cell cortex. This contributes to loss of epidermal barrier activity, resulting in completely penetrant perinatal lethality. This work reveals essential desmosome-associated components that control cortical microtubule organization and unexpected roles for centrosomal proteins in epidermal function.     

Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function

The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP) is caused by mutations in the SPG4 (spastin) gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ) phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, we observed the opposite phenotype: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that we observe in spastin mutants may be relevant to an understanding of human Spastin's role in maintenance of axon terminals in the spinal cord.     

Re-orientation of cortical F-actin is not necessary for wound-induced microtubule re-orientation and cell polarity establishment

Summary To assess the relative roles of cortical actin and microtubule re-orientation in the establishment of new cell polarity, we have examined the kinetics of cortical actin re-orientation around a wedge-shaped wound in pea roots. Cortical actin re-orients from a transverse alignment to an approximately longitudinal orientation between 5 and 24h after wounding, that is, after the re-alignment of microtubules, which is known to occur before 5h post-wounding. F-actin in root cortical cells does not appear to be necessary for the establishment of new cell polarity around wounds, since normal MT re-alignment, and new planes of cell division are still established around a wound in cytochalasin treated roots. The cytochalasin treatment appeared to totally disrupt cortical and cytoplasmic F-actin in cells of the root cortex. However, in the apparent absence of F-actin in these cells, the rate of wound-induced cell division, but not cell expansion, is slower, and we suggest that an effect on the phragmosomal actin is involved. Finally, we demonstrate that new cell polarity around a wound is not established if microtubules are disrupted by the herbicide oryzalin, but after re-establishment of these arrays following a wash-out of the drug, the typical new planes of cell expansion are observed. We conclude that microtubules play a critical role in establishing and maintaining cell polarity in this system, and that cortical F-actin has a minor and presently unclear function in these processes.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - DMSO dimethylsulphoxide - EGTA ethyleneglycol-bis-(-aminoethyleter)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MBS m-maleido-benzoyl N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - MT microtubule - PIPES 1,4-piperazine-dietha-nesulphonic acid - PPB pre-prophase band - Rh-ph rhodamine phalloidin     

Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos

During cell division, eukaryotic cells assemble dynamic microtubule-based spindles to segregate replicated chromosomes. Rapid spindle microtubule turnover, likely derived from dynamic instability, has been documented in yeasts, plants and vertebrates. Less studied is concerted spindle microtubule poleward translocation (flux) coupled to depolymerization at spindle poles. Microtubule flux has been observed only in vertebrates, although there is indirect evidence for it in insect spermatocytes and higher plants. Here we use fluorescent speckle microscopy (FSM) to demonstrate that mitotic spindles of syncytial Drosophila embryos exhibit poleward microtubule flux, indicating that flux is a widely conserved property of spindles. By simultaneously imaging chromosomes (or kinetochores) and flux, we provide evidence that flux is the dominant mechanism driving chromosome-to-pole movement (anaphase A) in these spindles. At 18 degrees C and 24 degrees C, separated sister chromatids moved poleward at average rates (3.6 and 6.6 microm/min, respectively) slightly greater than the mean rates of poleward flux (3.2 and 5.2 microm/min, respectively). However, at 24 degrees C the rate of kinetochore-to-pole movement varied from slower than to twice the mean rate of flux, suggesting that although flux is the dominant mechanism, kinetochore-associated microtubule depolymerization contributes to anaphase A.     

The kinesin-related protein MCAK is a microtubule depolymerase that forms an ATP-hydrolyzing complex at microtubule ends

MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins that are not motile but instead destabilize microtubules. We show that MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, 100-fold, the rate of dissociation of tubulin from microtubule ends. MCAK has one high-affinity binding site per protofilament end, which, when occupied, has both the depolymerase and ATPase activities. MCAK targets protofilament ends very rapidly (on-rate 54 micro M(-1).s(-1)), perhaps by diffusion along the microtubule lattice, and, once there, removes approximately 20 tubulin dimers at a rate of 1 s(-1). We propose that up to 14 MCAK dimers assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule.     

Population networks: a large-scale framework for modelling cortical neural networks

Artificial neural networks are usually built on rather few elements such as activation functions, learning rules, and the network topology. When modelling the more complex properties of realistic networks, however, a number of higher-level structural principles become important. In this paper we present a theoretical framework for modelling cortical networks at a high level of abstraction. Based on the notion of a population of neurons, this framework can accommodate the common features of cortical architecture, such as lamination, multiple areas and topographic maps, input segregation, and local variations of the frequency of different cell types (e.g., cytochrome oxidase blobs). The framework is meant primarily for the simulation of activation dynamics; it can also be used to model the neural environment of single cells in a multiscale approach. Received: 9 January 1996 / Accepted in revised form: 24 July 1996     

End-on microtubule-dynein interactions and pulling-based positioning of microtubule organizing centers

During important cellular processes such as centrosome and spindle positioning, dynein at the cortex interacts with dynamic microtubules in an apparent “end-on” fashion. It is well-established that dynein can generate forces by moving laterally along the microtubule lattice, but much less is known about dynein’s interaction with dynamic microtubule ends. In this paper, we review recent in vitro experiments that show that dynein, attached to an artificial cortex, is able to capture microtubule ends, regulate microtubule dynamics and mediate the generation of pulling forces on shrinking microtubules. We further review existing ideas on the involvement of dynein-mediated cortical pulling forces in the positioning of microtubule organizing centers such as centrosomes. Recent in vitro experiments have demonstrated that cortical pulling forces in combination with pushing forces can lead to reliable centering of microtubule asters in quasi two-dimensional microfabricated chambers. In these experiments, pushing leads to slipping of microtubule ends along the chamber boundaries, resulting in an anisotropic distribution of cortical microtubule contacts that favors centering, once pulling force generators become engaged. This effect is predicted to be strongly geometry-dependent, and we therefore finally discuss ongoing efforts to repeat these experiments in three-dimensional, spherical and deformable geometries.