Asymmetric inheritance of centrosomally localized mRNAs during embryonic cleavages

During development, different cell fates are generated by cell-cell interactions or by the asymmetric distribution of patterning molecules. Asymmetric inheritance is known to occur either through directed transport along actin microfilaments into one daughter cell or through capture of determinants by a region of the cortex inherited by one daughter. Here we report a third mechanism of asymmetric inheritance in a mollusc embryo. Different messenger RNAs associate with centrosomes in different cells and are subsequently distributed asymmetrically during division. The segregated mRNAs are diffusely distributed in the cytoplasm and then localize, in a microtubule-dependent manner, to the pericentriolar matrix. During division, they dissociate from the core mitotic centrosome and move by means of actin filaments to the presumptive animal daughter cell cortex. In experimental cells with two interphase centrosomes, mRNAs accumulate on the correct centrosome, indicating that differences between centrosomes control mRNA targeting. Blocking the accumulation of mRNAs on the centrosome shows that this event is required for subsequent cortical localization. These events produce a complex pattern of mRNA localization, in which different messages distinguish groups of cells with the same birth order rank and similar developmental potentials.     

神经细胞树突中mRNA的运输

将mRNA靶定到神经细胞树突中, 对区域化蛋白质合成和神经功能的发挥起重要作用。神经细胞mRNA存在于包含不同种类调控mRNA 定位、稳定和翻译组分的颗粒中。该颗粒由驱动蛋白1(kinesin 1) 沿微管运输。定向运输产生的mRNA不对称分布是突触可塑性、学习和记忆所必需的。     

Coalignment of vimentin intermediate filaments with microtubules depends on kinesin.

Intermediate filaments in most types of cultured cells coalign with microtubules. Depolymerization of microtubules results in collapse of vimentin and desmin intermediate filaments to the nucleus where they form a perinuclear cap. Collapse can also be induced by microinjection of antibodies against intermediate filament or microtubule proteins. Thus, two filament systems interact with each other. But the molecules mediating this interaction are unknown. One of the candidates for this role is a microtubule motor kinesin. Recent data showed that kinesin is involved in the plus end-directed movement of the membranous organelles along microtubules such as radial extension of lysosomes in macrophages and centrifugal movement of pigment in melanophores. Here we report that injection of the anti-kinesin antibody into human fibroblasts results in the redistribution of intermediate filaments to a tight perinuclear aggregate but had no effect on the distribution of microtubules. Thus, kinesin is involved not only in organelle movement but also in interaction of the two major cytoskeletal systems, intermediate filaments and microtubules.     

The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization.

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen capable of rapid movement through the host cell cytoplasm. The biophysical basis of the motility of L. monocytogenes is an interesting question in its own right, the answer to which may shed light on the general processes of actin-based motility in cells. Moving intracellular bacteria display phase-dense 'comet tails' made of actin filaments, the formation of which is required for bacterial motility. We have investigated the dynamics of the actin filaments in the comet tails using the technique of photoactivation of fluorescence, which allows monitoring of the movement and turnover of labelled actin filaments after activation by illumination with ultraviolet light. We find that the actin filaments remain stationary in the cytoplasm as the bacterium moves forward, and that length of the comet tails is linearly proportional to the rate of movement. Our results imply that the motile mechanism involves continuous polymerization and release of actin filaments at the bacterial surface and that the rate of filament generation is related to the rate of movement. We suggest that actin polymerization provides the driving force for bacterial propulsion.     

Roles for microtubule and microfilament cytoskeletons in animal cell cytokinesis

Microtubule and microfilament cytoskeletons play key roles in the whole process of cytokinesis. Although a number of hypotheses have been proposed to elucidate the mechanism of cytokinesis by microtubule and actin flament cytoskeletons, many reports are conflicting. In our study,combining the cytoskeletons drug treatments with the time-lapse video technology, we retested the key roles of microtubule and actin filament in cytokinesis. The results showed that depolymerization of microtubules by Nocodazole after the initiation of furrowing would not inhibit the furrow ingression, but obviously decrease the stiffness of daughter cells. Depolymerizing actin filaments by Cytochalasin B before metaphase would inhibit the initiation of furrowing but not chromosome segregation, resulting in the formation of binucleate cells; however, depolymerizing actin fillaments during anaphase would prevent furrowing and lead to the regress of established furrow, also resulting in the formation of binucleate cells. Further, depolymerizing microtubules and actin filaments simultaneously after metaphase would cause the quick regress of the furrow and the formation of binudeate cells. From these results we propose that a successful cytokinesis requires functions and coordination of both the microtubule and actin filament cytoskeletons.Microtubule cytoskeleton may function in the positioning and initiation of cleavage furrow, and the actin filament cytoskeleton may play key roles in the initiation and ingression of the furrow.     

Propulsion of organelles isolated from Acanthamoeba along actin filaments by myosin-I

Eukaryotic cells are dependent on their ability to translocate membraneous elements about the cytoplasm. In many cells long translocations of organelles are associated with microtubules. In other cases, such as the rapid cytoplasmic streaming in some algae, organelles appear to be propelled along actin filaments. It has been assumed, but not proven, that myosin produces these movements. We have tested vesicles from another eukaryotic cell for their ability to move on the exposed actin bundles of Nitella as an indiction that actin-based organelle movements may be a general property of cells. We found that organelles from Acanthamoeba castellanii can move along Nitella actin filaments. Here, we report two different experiments indicating that the single-headed non-polymerizable myosin isozyme myosin-I is responsible for this organelle motility. First, monoclonal antibodies to myosin-I inhibit movement, but antibodies that inhibit double-headed myosin-II do not. Second, approximately 20% of the myosin-I in homogenates co-migrates with motile vesicles during Percoll density-gradient ultracentrifugation. This is the first indication of a role for myosin-I within the cell and supports the suggestion of Albanesi et al. that myosin-I moves vesicles in this way.     

Myosin VI is an actin-based motor that moves backwards.

Myosins and kinesins are molecular motors that hydrolyse ATP to track along actin filaments and microtubules, respectively. Although the kinesin family includes motors that move towards either the plus or minus ends of microtubules, all characterized myosin motors move towards the barbed (+) end of actin filaments. Crystal structures of myosin II (refs 3-6) have shown that small movements within the myosin motor core are transmitted through the 'converter domain' to a 'lever arm' consisting of a light-chain-binding helix and associated light chains. The lever arm further amplifies the motions of the converter domain into large directed movements. Here we report that myosin VI, an unconventional myosin, moves towards the pointed (-) end of actin. We visualized the myosin VI construct bound to actin using cryo-electron microscopy and image analysis, and found that an ADP-mediated conformational change in the domain distal to the motor, a structure likely to be the effective lever arm, is in the opposite direction to that observed for other myosins. Thus, it appears that myosin VI achieves reverse-direction movement by rotating its lever arm in the opposite direction to conventional myosin lever arm movement.     

Redistribution of intermediate filaments during capping of lymphocyte surface molecules

Intermediate filaments (IF) constitute a major cytoplasmic filamentous network of higher eukaryotic cells that is distinct from actin and myosin microfilaments or microtubules. Although structurally similar, these filaments are formed by chemically and antigenically different proteins. Vimentin is the major IF polypeptide of mesenchymal cells and cultured non-mesenchymal cell lines. Recently, we have characterized a monoclonal IgM antibody from a patient with Waldenstr?m's macroglobulinaemia which is directed against vimentin. Using this monoclonal antibody, we have shown by direct immunofluorescence that intermediate filaments of human B and T lymphocytes consist of vimentin. In cells exposed to colcemid, the intermediate filaments retracted into a juxtanuclear aggregate ('coli') characteristic of vimentin filaments. As most components of the cytoskeleton, especially actin and myosin, have been implicated in the capping phenomenon, we investigated the effect of capping of either beta 2-microglobulin or membrane immunoglobulins on the organization of the intermediate filament network. We report that capping of these surface molecules induced the redistribution of vimentin just beneath the cap. When colcemid-treated cells were allowed to cap, the location of the cap always coincided with the coil, suggesting that the anchorage point of intermediate filaments is situated within the uropod.     

Tying a molecular knot with optical tweezers.

Filamentous structures are abundant in cells. Relatively rigid filaments, such as microtubules and actin, serve as intracellular scaffolds that support movement and force, and their mechanical properties are crucial to their function in the cell. Some aspects of the behaviour of DNA, meanwhile, depend critically on its flexibility-for example, DNA-binding proteins can induce sharp bends in the helix. The mechanical characterization of such filaments has generally been conducted without controlling the filament shape, by the observation of thermal motions or of the response to external forces or flows. Controlled buckling of a microtubule has been reported, but the analysis of the buckled shape was complicated. Here we report the continuous control of the radius of curvature of a molecular strand by tying a knot in it, using optical tweezers to manipulate the strand's ends. We find that actin filaments break at the knot when the knot diameter falls below 0.4 microm. The pulling force at breakage is around 1 pN, two orders of magnitude smaller than the tensile stress of a straight filament. The flexural rigidity of the filament remained unchanged down to this diameter. We have also knotted a single DNA molecule, opening up the possibility of studying curvature-dependent interactions with associated proteins. We find that the knotted DNA is stronger than actin.     

Interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage

Many observations suggest the presence of transmembrane linkages between the cytoskeleton and the extracellular matrix. In fibroblasts both light and electron microscopic observations reveal a co-alignment between actin filaments at the cell surface and extracellular fibronectin. These associations are seen at sites of cell matrix interaction, frequently along stress fibres and sometimes where these bundles of microfilaments terminate at adhesion plaques (focal contacts). Non-morphological evidence also indicates a functional linkage between the cytoskeleton and extracellular matrix. Addition of fibronectin to transformed cells induces flattening of the cells and a reorganization of the actin cytoskeleton, with the concomitant appearance of arrays of stress fibres. Conversely, disruption of the actin cytoskeleton by treatment with cytochalasin B leads to release of fibronectin from the cell surface. As yet, there is no detailed knowledge of the molecules involved in this transmembrane linkage, although several proteins have been suggested as candidates in the chain of attachment between bundles of actin filaments and the cytoplasmic face of the plasma membrane: these include vinculin, alpha-actinin and talin, each one having been identified at regions where bundles of actin filaments interact with the plasma membrane and underlying cell-surface fibronectin. Recently, the cell-substrate attachment (CSAT) antigen has been identified as a plasma membrane receptor for fibronectin, raising the possibility that this glycoprotein complex may serve as a bridge between fibronectin and one or more of the underlying cytoskeletal components mentioned. Here we have investigated the interaction of the purified CSAT antigen with these cytoskeletal components, and we demonstrate an interaction specifically between the CSAT antigen and talin.     

Reconstitution of actin-based motility of Listeria and Shigella using pure proteins.

Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, alpha-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.     

Centrosome polarization delivers secretory granules to the immunological synapse

Cytotoxic T lymphocytes (CTLs) destroy virally infected and tumorigenic cells by releasing the contents of specialized secretory lysosomes--termed 'lytic granules'--at the immunological synapse formed between the CTL and the target. On contact with the target cell, the microtubule organizing centre of the CTL polarizes towards the target and granules move along microtubules in a minus-end direction towards the polarized microtubule organizing centre. However, the final steps of secretion have remained unclear. Here we show that CTLs do not require actin or plus-end microtubule motors for secretion, but instead the centrosome moves to and contacts the plasma membrane at the central supramolecular activation cluster of the immunological synapse. Actin and IQGAP1 are cleared away from the synapse, and granules are delivered directly to the plasma membrane. These data show that CTLs use a previously unreported mechanism for delivering secretory granules to the immunological synapse, with granule secretion controlled by centrosome delivery to the plasma membrane.     

Effect of ATP on actin filament stiffness

Actin is an adenine nucleotide-binding protein and an ATPase. The bound adenine nucleotide stabilizes the protein against denaturation and the ATPase activity, although not required for actin polymerization, affects the kinetics of this assembly Here we provide evidence for another effect of adenine nucleotides. We find that actin filaments made from ATP-containing monomers, the ATPase activity of which hydrolyses ATP to ADP following polymerization, are stiff rods, whereas filaments prepared from ADP-monomers are flexible. ATP exchanges with ADP in such filaments and stiffens them. Because both kinds of actin filaments contain mainly ADP, we suggest the alignment of actin monomers in filaments that have bound and hydrolysed ATP traps them conformationally and stores elastic energy. This energy would be available for release by actin-binding proteins that transduce force or sever actin filaments. These data support earlier proposals that actin is not merely a passive cable, but has an active mechanochemical role in cell function.