Understanding a molecular motor walking along a microtubule: an asymmetric Brownian motor driven by bubble formation with a focus on binding affinity

In recent years, many studies on a molecular motor have been conducted in the fields of biorheology and nanoengineering. The molecular motor is a molecule that converts the chemical energy obtained by ATP hydrolysis into mechanical energy. Explaining this mechanism is important for nanoengineering. A kinesin, which is a type of molecular motor, has the characteristics to move on a microtubule with hand-over-hand steps. The kinesin walking behaviour is explained by the ‘asymmetric Brownian ratchet model’. Previously, we had suggested that the walking mechanism was achieved by the bubble formation in a nanosized channel surrounded by hydrophobic atoms with the transition between the two states – bubble state and liquid state. However, the walking behaviour of the model motor was different from that of a single molecule measurement of a kinesin. In this study, we constructed a new motor system focused on the asymmetric binding affinity of a motor protein and performed a model simulation using the dissipative particle dynamics method. As a result, it was observed that hand-over-hand walking depends on the transition position ratio and the transition frequency coefficient. Moreover, the efficiency of the new motor system is higher than that of the previous motor systems. The new motor model can provide a simulation guide for the design of biomimetic nanomachines.     

The reconstituted isolated uncoupling protein is a membrane potential driven H+ translocator.

The isolated uncoupling protein (UCP) from brown fat adipose tissue mitochondria has been reconstituted into artificial phospholipid vesicles. Because of the high lability of H+ transport, several new steps have been introduced in the reconstitution; the detergent octyl-POE, the addition of phospholipids to mitochondria prior to solubilization and purification, the vesicle formation by rapid removal of detergent with polystyrene beads and of external salts by a mixed ion exchange. In the K+-loaded proteoliposomes, H+ influx can be induced by a diffusion potential on addition of valinomycin. H+ influx is inhibited to more than 90% by GTP addition, in the assay for UCP activity. By reversing delta psi with external K+, H+ efflux is measured, however, at a four times lower rate. In vesicles loaded with internal GTP, H+ influx is fully inhibited but can be activated by Dowex-OH treatment to an even higher rate than that found in the GTP-free vesicles. Binding studies with GTP show that most of the active UCP are oriented with the binding site outside as in mitochondria, and that in GTP-loaded vesicles GTP is also bound at the outside. The rate of H+ transport is linearly dependent on the membrane potential. Despite the ordered orientation, there is no 'valve' mechanism, since there is H+ efflux with a reversed potential. pH dependency is only small between pH 6.5 and 7.5, indicating that the H+-translocating site differs from the highly pH-dependent nucleotide-binding site. The turnover number of reconstituted UCP is commensurate with mitochondrial function and indicates a carrier instead of a channel-type H+ transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Minus-end-directed motor Ncd exhibits processive movement that is enhanced by microtubule bundling in vitro

Drosophila Ncd, a kinesin-14A family member, is essential for meiosis and mitosis. Ncd is a minus-end-directed motor protein that has an ATP-independent microtubule binding site in the tail region, which enables it to act as a dynamic crosslinker of microtubules to assemble and maintain the spindle. Although a tailless Ncd has been shown to be nonprocessive, the role of the Ncd tail in single-molecule motility is unknown. Here, we show that individual Ncd dimers containing the tail region can move processively along microtubules at very low ionic strength, which provides the first evidence of processivity for minus-end-directed kinesins. The movement of GFP-Ncd consists of both a unidirectional and a diffusive element, and it was sensitive to ionic strength. Motility of a truncation series of Ncd and removal of the tubulin tail suggested that the Ncd tail serves as an electrostatic tether to microtubules. Under higher ionic conditions, Ncd showed only a small bias in diffusion along "single" microtubules, whereas it exhibited processive movement along "bundled" microtubules. This property may allow Ncd to accumulate preferentially in the vicinity of focused microtubules and then to crosslink and slide microtubules, possibly contributing to dynamic spindle self-organization.     

Cell cycle control of microtubule-based membrane transport and tubule formation in vitro

When higher eukaryotic cells enter mitosis, membrane organization changes dramatically and traffic between membrane compartments is inhibited. Since membrane transport along microtubules is involved in secretion, endocytosis, and the positioning of organelles during interphase, we have explored whether the mitotic reorganization of membrane could involve a change in microtubule-based membrane transport. This question was examined by reconstituting organelle transport along microtubules in Xenopus egg extracts, which can be converted between interphase and metaphase states in vitro in the absence of protein synthesis. Interphase extracts support the microtubule-dependent formation of abundant polygonal networks of membrane tubules and the transport of small vesicles. In metaphase extracts, however, the plus end- and minus end-directed movements of vesicles along microtubules as well as the formation of tubular membrane networks are all reduced substantially. By fractionating the extracts into soluble and membrane components, we have shown that the cell cycle state of the supernatant determines the extent of microtubule-based membrane movement. Interphase but not metaphase Xenopus soluble factors also stimulate movement of membranes from a rat liver Golgi fraction. In contrast to above findings with organelle transport, the minus end-directed movements of microtubules on glass surfaces and of latex beads along microtubules are similar in interphase and metaphase extracts, suggesting that cytoplasmic dynein, the predominant soluble motor in frog extracts, retains its force-generating activity throughout the cell cycle. A change in the association of motors with membranes may therefore explain the differing levels of organelle transport activity in interphase and mitotic extracts. We propose that the regulation of organelle transport may contribute significantly to the changes in membrane structure and function observed during mitosis in living cells.     

Fluctuation in the microtubule sliding movement driven by kinesin in vitro.

We studied the fluctuation in the translational sliding movement of microtubules driven by kinesin in a motility assay in vitro. By calculating the mean-square displacement deviation from the average as a function of time, we obtained motional diffusion coefficients for microtubules and analyzed the dependence of the coefficients on microtubule length. Our analyses suggest that 1) the motional diffusion coefficient consists of the sum of two terms, one that is proportional to the inverse of the microtubule length (as the longitudinal diffusion coefficient of a filament in Brownian movement is) and another that is independent of the length, and 2) the length-dependent term decreases with increasing kinesin concentration. This latter term almost vanishes within the length range we studied at high kinesin concentrations. From the length-dependence relationship, we evaluated the friction coefficient for sliding microtubules. This value is much larger than the solvent friction and thus consistent with protein friction. The length independence of the motional diffusion coefficient observed at sufficiently high kinesin concentrations indicates the presence of correlation in the sliding movement fluctuation. This places significant constraint on the possible mechanisms of the sliding movement generation by kinesin motors in vitro.     

Rab11-FIP3 is a cell cycle-regulated phosphoprotein

Background

Rab11 and its effector molecule, Rab11-FIP3 (FIP3), associate with recycling endosomes and traffic into the furrow and midbody of cells during cytokinesis. FIP3 also controls recycling endosome distribution during interphase. Here, we examine whether phosphorylation of FIP3 is involved in these activities.

Results

We identify four sites of phosphorylation of FIP3 in vivo, S-102, S-280, S-347 and S-450 and identify S-102 as a target for Cdk1-cyclin B in vitro. Of these, we show that S-102 is phosphorylated in metaphase and is dephosphorylated as cells enter telophase. Over-expression of FIP3-S102D increased the frequency of binucleate cells consistent with a role for this phospho-acceptor site in cytokinesis. Mutation of S-280, S-347 or S-450 or other previously identified phospho-acceptor sites (S-488, S-538, S-647 and S-648) was without effect on binucleate cell formation and did not modulate the distribution of FIP3 during the cell cycle. In an attempt to identify a functional role for FIP3 phosphorylation, we report that the change in FIP3 distribution from cytosolic to membrane-associated observed during progression from anaphase to telophase is accompanied by a concomitant dephosphorylation of FIP3. However, the phospho-acceptor sites identified here did not control this change in distribution.

Conclusions

Our data thus identify FIP3 as a cell cycle regulated phosphoprotein and suggest dephosphorylation of FIP3 accompanies its translocation from the cytosol to membranes during telophase. S102 is dephosphorylated during telophase; mutation of S102 exerts a modest effect on cytokinesis. Finally, we show that de/phosphorylation of the phospho-acceptor sites identified here (S-102, S-280, S-347 and S-450) is not required for the spatial control of recycling endosome distribution or function.     

The human nuclear SRcyp is a cell cycle-regulated cyclophilin

Cyclophilins of the Moca family (Cavarec, L., Kamphausen, T., Dubourg, B., Callebaut, I., Lemeunier, F., Metivier, D., Feunteun, J., Fischer, G., and Modjtahedi, N. (2002) J. Biol. Chem. 277, 41171-41182) are found only in organisms of the animal kingdom and share several structural and enzymatic features. The presence of serine/arginine (S/R) dipeptide repeats in their C-terminal tail suggests that these enzymes belong to the SR protein family involved in the regulation of gene expression. The function of this group of cyclophilins is currently unknown. However, their C-terminal tails contain a highly conserved polypeptide signature segment (the moca domain), which may well be involved in the functional regulation of these proteins. We report here the identification of five Cdc2-type phosphorylation sites gathered in and around the moca domain of SRcyp, a human cyclophilin belonging to the Moca family. The segment of SRcyp containing the identified sites is specifically phosphorylated in mitotic cells. This mitosis-specific phosphorylation was inhibited by treatment of the cells with roscovitine, a specific inhibitor of cyclin-dependent kinases, suggesting that the unknown activity of the moca domain of SRcyp requires mitotic regulation by the Cdc2-cyclin B kinase complex. The Cdc2-cyclin B complex was found to phosphorylate four of the five identified phosphorylation sites in vitro, providing further support for this possibility. Like many factors stored in nuclear speckles and involved in the regulation of gene expression, this nuclear cyclophilin displays a predominantly diffuse cytoplasmic distribution at the onset of mitosis. Only in late telophase is SRcyp recruited to the newly formed nuclei. The transit of SRcyp through mitotic interchromatin granule clusters, before re-entering the nucleus, suggests that the timing of the appearance of this cyclophilin in the telophasic nuclei is tightly coordinated with post-mitotic events. Human SRcyp is the first cell cycle-regulated cyclophilin to be described.     

Formation of membrane networks in vitro by kinesin-driven microtubule movement

Certain intracellular organelles such as the endoplasmic reticulum (Terasaki, M., L. B. Chen, and K. Fujiwara. 1986. J. Cell Biol. 103:1557-1568) and lysosomes (Swanson, J., A. Bushnell, and S. C. Silverstein. Proc. Natl. Acad. Sci. USA. 84:1921-1925) form tubular networks that are closely aligned with microtubules. Here we describe the formation of polygonal networks composed of interconnected membrane tubules that occurs when a preparation of microtubule affinity-purified squid kinesin is combined with microtubules and ATP on a glass surface. The membrane, which is a minor contaminant in the microtubule affinity- purified kinesin preparation, binds to microtubules translocating along kinesin-coated glass surfaces. Force exerted by kinesin upon the microtubule is transmitted to the membrane and a tubular extension of the membrane is produced. As the membrane tubule elongates, membrane tension exerts an opposing force upon the translocating microtubule that can alter its direction of movement by dissociating or partially dissociating the microtubule from the kinesin-coated surface. Membrane tubules that come in contact appear to fuse with one another, and thus give rise to two-dimensional polygonal networks of tubules that have similar features to endoplasmic reticulum networks in cells. Artificial liposomes composed of dimyristoylphosphatidylcholine and yolk phosphatidylglycerol also form stable tubular structures when subjected to shear forces, but do not interact with microtubules or form polygonal networks, suggesting that such phenomena may require membrane- associated proteins. These findings indicate that kinesin generates sufficient force to form tubular membrane extensions in vitro and suggest that this microtubule-based motility protein may also be responsible for creating tubular membrane networks within cells.     

Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage

T cells rapidly reposition their centrosome to the center of the immunological synapse (IS) to drive polarized secretion in the direction of the bound target cell. Using an optical trap for spatial and temporal control over target presentation, we show that centrosome repositioning in Jurkat T cells exhibited kinetically distinct polarization and docking phases and required calcium flux and signaling through both the T cell receptor and integrin to be robust. In “frustrated” conjugates where the centrosome is stuck behind the nucleus, the center of the IS invaginated dramatically to approach the centrosome. Consistently, imaging of microtubules during normal repositioning revealed a microtubule end-on capture-shrinkage mechanism operating at the center of the IS. In agreement with this mechanism, centrosome repositioning was impaired by inhibiting microtubule depolymerization or dynein. We conclude that dynein drives centrosome repositioning in T cells via microtubule end-on capture-shrinkage operating at the center of the IS and not cortical sliding at the IS periphery, as previously thought.     

Aggregation during fruiting body formation in Myxococcus xanthus is driven by reducing cell movement

When starved, Myxococcus xanthus cells assemble themselves into aggregates of about 10(5) cells that grow into complex structures called fruiting bodies, where they later sporulate. Here we present new observations on the velocities of the cells, their orientations, and reversal rates during the early stages of fruiting body formation. Most strikingly, we find that during aggregation, cell velocities slow dramatically and cells orient themselves in parallel inside the aggregates, while later cell orientations are circumferential to the periphery. The slowing of cell velocity, rather than changes in reversal frequency, can account for the accumulation of cells into aggregates. These observations are mimicked by a continuous agent-based computational model that reproduces the early stages of fruiting body formation. We also show, both experimentally and computationally, how changes in reversal frequency controlled by the Frz system mutants affect the shape of these early fruiting bodies.     

Basement membrane formation and lung cell differentiation in vitro

In high density cultures of mouse fetal lung cells, so-called "mass cultures", development of organoid structures, formation of a basement membrane (BM), and differentiation of pneumocytes type II occur accompanied by synthesis and secretion of lamellar bodies. The relationship between the formation of a BM, on the one hand, and morphogenesis as well as differentiation of pneumocytes type II, on the other hand, has been investigated by use of antibodies against BM components in the lung mass culture. It is shown here that anti-laminin antibodies prevented BM formation, but morphogenesis and pneumocyte differentiation occurred as in untreated cultures. Short-term treatment with the antibody revealed that the BM is formed only during the first 2 to 3 days in vitro. Already formed BM could not be removed by anti-laminin. Anti-collagen type IV antibodies showed no effect in the lung mass culture except for a stronger staining of the BM. Anti-BM-1 antibodies caused no changes in morphogenesis, cell differentiation and BM formation either, but the mesenchymal intercellular space exhibited a dark staining, which is probably due to antigen-antibody complexes. The results obtained with anti-laminin antibodies indicate that a BM is not necessary for lung cell differentiation in vitro.     

Bleb formation during anoxia is not a prerequisite for eventual cell death in renal tubule cells

Membrane deformations, generally termed blebs, are a hallmark of cells subjected to noxious stimuli or injurious conditions. Although not lethal itself, bleb formation has been used as an indicator of the stage of cell injury. Whether or not bleb formation is an integral part of the sequence of events that lead to irreversible injury, or occurs in all cells within a group subjected to the same conditions is not known. We demonstrate here that renal epithelial cells in culture produce blebs when subjected to sustained periods of anoxia and substrate deprivation. However, the occurrence of blebs does not correlate with eventual cell death. The data suggest that bleb formation may be associated with prolonged survival.     

Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion

Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G₂/M phases, whereas the G₁ phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G₂/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G₂/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G₂/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.     

Nuclear congression is driven by cytoplasmic microtubule plus end interactions in S. cerevisiae

Nuclear movement before karyogamy in eukaryotes is known as pronuclear migration or as nuclear congression in Saccharomyces cerevisiae. In this study, S. cerevisiae is used as a model system to study microtubule (MT)-dependent nuclear movements during mating. We find that nuclear congression occurs through the interaction of MT plus ends rather than sliding and extensive MT overlap. Furthermore, the orientation and attachment of MTs to the shmoo tip before cell wall breakdown is not required for nuclear congression. The MT plus end-binding proteins Kar3p, a class 14 COOH-terminal kinesin, and Bik1p, the CLIP-170 orthologue, localize to plus ends in the shmoo tip and initiate MT interactions and depolymerization after cell wall breakdown. These data support a model in which nuclear congression in budding yeast occurs by plus end MT capture and depolymerization, generating forces sufficient to move nuclei through the cytoplasm. This is the first evidence that MT plus end interactions from oppositely oriented organizing centers can provide the force for organelle transport in vivo.     

A new identity for MLK3 as an NIMA-related,cell cycle-regulated kinase that is localized near centrosomes and influences microtubule organization

Although conserved counterparts for most proteins involved in the G(2)/M transition of the cell cycle have been found in all eukaryotes, a notable exception is the essential but functionally enigmatic fungal kinase NIMA. While a number of vertebrate kinases have been identified with catalytic domain homology to NIMA, none of these resemble NIMA within its extensive noncatalytic region, a region critical for NIMA function in Aspergillus nidulans. We used a bioinformatics approach to search for proteins with homology to the noncatalytic region of NIMA and identified mixed lineage kinase 3 (MLK3). MLK3 has been proposed to serve as a component in MAP kinase cascades, particularly those resulting in the activation of the c-Jun N-terminal kinase (JNK). Here we describe the first in-depth study of endogenous MLK3 and report that, like NIMA, MLK3 phosphorylation and activity are enhanced during G(2)/M, whereas JNK remains inactive. Coincident with the G(2)/M transition, a period marked by dramatic reorganization of the cytoplasmic microtubule network, endogenous MLK3 transiently disperses away from the centrosome and centrosomal-proximal sites where it is localized during interphase. Furthermore, when overexpressed, MLK3, like NIMA, localizes to the centrosomal region, induces profound disruption of cytoplasmic microtubules and a nuclear distortion phenotype that differs from mitotic chromosome condensation. Cellular depletion of MLK3 protein using siRNA technology results in an increased sensitivity to the microtubule-stabilizing agent taxol. Our studies suggest a new role for MLK3, separable from its function in the JNK pathway, that may contribute to promoting microtubule instability, a hallmark of M phase entry.     

ch-TOGp is required for microtubule aster formation in a mammalian mitotic extract

Microtubules induced to polymerize with taxol in a mammalian mitotic extract organize into aster-like arrays in a centrosome-independent process that is driven by microtubule motors and structural proteins. These microtubule asters accurately reflect the noncentrosomal aspects of mitotic spindle pole formation. We show here that colonic-hepatic tumor-overexpressed gene (ch-TOGp) is an abundant component of these asters. We have prepared ch-TOGp-specific antibodies and show by immunodepletion that ch-TOGp is required for microtubule aster assembly. Microtubule polymerization is severely inhibited in the absence of ch-TOGp, and silver stain analysis of the ch-TOGp immunoprecipitate indicates that it is not present in a preformed complex and is the only protein removed from the extract during immunodepletion. Furthermore, the reduction in microtubule polymerization efficiency in the absence of ch-TOGp is dependent on ATP. These results demonstrate that ch-TOGp is a major constituent of microtubule asters assembled in a mammalian mitotic extract and that it is required for robust microtubule polymerization in an ATP-dependent manner in this system even though taxol is present. These data, coupled with biochemical and genetic data derived from analysis of ch-TOGp-related proteins in other organisms, indicate that ch-TOGp is a key factor regulating microtubule dynamics during mitosis.     

The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor

The Drosophila ncd gene is required for chromosome segregation during female meiosis. Previous analyses suggested that the ncd gene encoded a protein with sequence similarity to the kinesin motor domain, which suggested that, like kinesin, the ncd protein might be a plus end-directed microtubule motor. Here we describe the expression of ncd protein in E. coli and the initial characterization of the ncd protein's motor properties. The ncd protein is indeed a microtubule motor, but the polarity of movement is minus end directed. The ncd protein also has microtubule bundling activity. These findings limit possible models for the in vivo functions of the ncd protein and suggest that motor proteins with similar sequence can generate movement in opposite directions along a microtubule.