Microtubules in Transcellular Movement of Procollagen

Alteration of microtubular funtion modifies the rate at which procollagen is converted to collagen.     

Diffusive Movement of Processive Kinesin-1 on Microtubules

The processive motor kinesin-1 moves unidirectionally toward the plus end of microtubules. This process can be visualized by total internal reflection fluorescence microscopy of kinesin bound to a carboxylated quantum dot (Qdot), which acts both as cargo and label. Surprisingly, when kinesin is bound to an anti-HIS Qdot, it shows diffusive movement on microtubules, which decreased in favor of processive runs with increasing salt concentration. This observation implies that kinesin movement on microtubules is governed by its conformation, as it is well established that kinesin undergoes a salt-dependent transition from a folded (inactive) to an extended (active) molecule. A truncated kinesin lacking the last 75 amino acids (kinesin-ΔC) showed both processive and diffusive movement on microtubules. The extent of each behavior depends on the relative amounts of ADP and ATP, with purely diffusive movement occurring in ADP alone. Taken together, these data imply that folded kinesin.ADP can exist in a state that diffuses along the microtubule lattice without expending energy. This mechanism may facilitate the ability of kinesin to pick up cargo, and/or allow the kinesin/cargo complex to stay bound after encountering obstacles.     

Structural and Molecular Basis for Katanin-Mediated Severing of Glutamylated Microtubules

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Intracellular Transformation of Phagosomes

The transformation of nascent phagosomes into forms capable of interacting with antimicrobial organelles of phagocytes, peroxisomes, depends on certain interactions between phagosomes and other vacuolar organelles. Phagosomes repeatedly interact with early and late endosomes through temporary contacts, which allows them to gain and lose complex sets of proteins. In addition, certain polypeptides are eliminated from phagosomes through recycling. New proteins enter phagosomes from the organelles of the biosynthetic pathway or are recruited from the cytoplasm. In addition, phagosomes receive proteins in the process of interaction with endosomes. The overall result of such transformation is acquiring new properties that make possible their interaction with peroxisomes.     

Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of Lysosomes

An earlier report suggested that actin and myosin I alpha (MMIalpha), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIalpha were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIalpha. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIalpha impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIalpha contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors.     

Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation

It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements.     

Modelling the Role of Intrinsic Electric Fields in Microtubules as an Additional Control Mechanism of Bi-directional Intracellular Transport

Active transport is essential for cellular function, while impaired transport has been linked to diseases such as neuronal degeneration. Much long distance transport in cells uses opposite polarity molecular motors of the kinesin and dynein families to move cargos along microtubules. It is clear that many types of cargo are moved by both sets of motors, and frequently in a reverse direction. The general question of how the direction of transport is regulated is still open. The mechanism of the cell's differential control of diverse cargos within the same cytoplasmic background is still unclear as is the answer to the question how endosomes and mitochondria move to different locations within the same cell. To answer these questions we postulate the existence of a local signaling mechanism used by the cell to specifically control different cargos. In particular, we propose an additional physical mechanism that works through the use of constant and alternating intrinsic (endogenous) electric fields as a means of controlling the speed and direction of microtubule-based transport. A specific model is proposed and analyzed in this paper. The model involves the rotational degrees of freedom of the C-termini of tubulin, their interactions and the coupling between elastic and dielectric degrees of freedom. Viscosity of the solution is also included and the resultant equation of motion is found as a nonlinear elliptic equation with dissipation. A particular analytical solution of this equation is obtained in the form of a kink whose properties are analyzed. It is concluded that this solution can be modulated by the presence of electric fields and hence may correspond to the observed behavior of motor protein transport along microtubules.     

Moose Movement Rates Along Highways and Crossing Probability Models

ABSTRACT We developed and validated a density-adjusted spatial model to predict moose (Alces alces) highway-crossing probability to see if the model could be used as an index of moose-vehicle collision risk. We installed Global Positioning System telemetry collars on 47 moose in the north of the Laurentides Wildlife Reserve, Québec, for 2–36 months. We recorded only 84 highway crossings in spring (0.29% of 28,967 2-hr steps) and 122 crossings in summer (0.18% of 68,337 2-hr steps), despite a high sampling effort and having captured moose close to highways. Moose movement rates during movement steps crossing a highway were on average 3 times higher than during the steps preceding or following highway crossing. Paths used by moose when crossing a highway were characterized by a high proportion of food stands, low proportion of lakes and rivers, and topography typical of a valley. Highway-crossing sites were located in valleys with brackish pools and forest stands providing coniferous cover but a low proportion of lakes and rivers. We adjusted moose crossing probability for local variation in moose density using aerial survey data and assessed crossing probability along the highways in the entire Laurentides Wildlife Reserve. We tested the model using moose-vehicle accident data from 1990 to 2002. The relationship between the density-adjusted crossing probability and number of accidents was relatively loose at the 1-km scale but improved markedly when using longer highway sections (5–15 km; r > 0.80). Our results demonstrate that roads and their surroundings are perceived as low-quality habitat by moose. We also conclude that road segments installed along secondary valleys could be a highly strategic site to deploy mitigation measures such as fences and that it could be desirable to increase the width of road shoulders to reduce forest cover and to eliminate brackish pools to reduce cervid-vehicle collisions. We suggest using empirical data such as location of vehicle-wildlife collisions to plan mitigation measures at a fine scale.     

Vaccinia Virus Intracellular Movement Is Associated with Microtubules and Independent of Actin Tails

Two mechanisms have been proposed for the intracellular movement of enveloped vaccinia virus virions: rapid actin polymerization and microtubule association. The first mechanism is used by the intracellular pathogens Listeria and Shigella, and the second is used by cellular vesicles transiting from the Golgi network to the plasma membrane. To distinguish between these models, two recombinant vaccinia viruses that express the B5R membrane protein fused to enhanced green fluorescent protein (GFP) were constructed. One had Tyr(112) and Tyr(132) of the A36R membrane protein, which are required for phosphorylation and the nucleation of actin tails, conservatively changed to Phe residues; the other had the A36R open reading frame deleted. Although the Tyr mutant was impaired in Tyr phosphorylation and actin tail formation, digital video and time-lapse confocal microscopy demonstrated that virion movement from the juxtanuclear region to the periphery was saltatory with maximal speeds of >2 microm/s and was inhibited by the microtubule-depolymerizing drug nocodazole. Moreover, this actin tail-independent movement was indistinguishable from that of a control virus with an unmutated A36R gene and closely resembled the movement of vesicles on microtubules. However, in the absence of actin tails, the Tyr mutant did not induce the formation of motile, virus-tipped microvilli and had a reduced ability to spread from cell to cell. The deletion mutant was more severely impaired, suggesting that the A36R protein has additional roles. Optical sections of unpermeabilized, B5R antibody-stained cells that expressed GFP-actin and were infected with wild-type vaccinia virus revealed that all actin tails were associated with virions on the cell surface. We concluded that the intracellular movement of intracellular enveloped virions occurs on microtubules and that the motile actin tails enhance extracellular virus spread to neighboring cells.     

The Mechanism of Bactericidal Activity in Phagosomes of Neutrophils

Myeloperoxidase plays the key role in antimicrobial of phagocytes. This enzyme uses hydrogen peroxide and chloride to catalyze hypochlorous acid formation. HOCl is the most probable agent in the oxygen-dependent bactericidal activity in the phagocyte phagosome. Chlorination markers indicate HOCl generation in the quantities lethal for bacteria. Enzymatic assay for myeloperoxidase indicates proceeding of other reactions involved in bactericidal activity. Superoxide integrates many activities of this kind and is important for physiological function of myeloperoxidase. Elucidation of phagosomes biochemistry can help us to understand why certain pathogens survive in such unfavorable environment.     

Modulating Microtubules: A Molecular Perspective on the Effects of Tail Modifications

Microtubules (MTs), an essential component of the eukaryotic cytoskeleton, are a lattice of polymerized tubulin dimers and are crucial for various cellular processes. The genetic and chemical diversity of tubulin and their disordered tails gives rise to a “tubulin code”. The functional role of tubulin post-translational modifications (PTMs), which contribute to the chemical diversity of the tubulin code, is gradually being unraveled. However, variation in the length and spatial organization of tubulin poly-modifications leads to an enormous combinatorial PTM space, which is difficult to study experimentally. Hence, the impact of the combinatorial tubulin PTM space on the biophysical properties of tubulin tails and their interactions with other proteins remains elusive.Here, we combine all-atom and coarse-grained molecular dynamics simulations to elucidate the biophysical implications of the large combinatorial tubulin PTM space in the context of an MT lattice. We find that tail–body interactions are more dominant in the tubulin dimer than in an MT lattice, and are more significant for the tails of α compared with β tubulin. In addition, polyglutamylation, but not polyglycylation, expands the dimensions of the tubulin tails. Polyglutamylation also leads to a decrease in the diffusion rate of MT-associated protein EB1 on MTs, while polyglycylation often increases diffusion rate. These observations are generally not sensitive to the organization of the polymodifications. The effect of PTMs on MT charge density and tail dynamics are also discussed. Overall, this study presents a molecular quantification of the biophysical properties of tubulin tails and their polymodifications, and provides predictions on the functional importance of tubulin PTMs.     

植物微管骨架参与下胚轴伸长调节机制研究进展

微管作为细胞骨架的重要成员,在植物生长发育过程中起重要作用。下胚轴作为研究细胞伸长的模式系统之一,其伸长受到多种信号的调节。该文综述了微管骨架在响应环境和生长发育信号调节下胚轴伸长过程中的作用及机制,旨在帮助读者深入理解微管骨架响应上游信号在植物下胚轴伸长中的作用机理。     

Age-Dependent Changes in the Ultrastructure and in the Molecular Composition of Rat Brain Microtubules

An age-dependent increase of a cathepsin D-like protease activity that preferentially degrades high molecular weight microtubule-associated proteins (MAPs) has been previously described. Microtubules (MT) purified from rat brain of different ages in the presence of several protease inhibitors retained undegraded MAPs through cycles of polymerization, and revealed several age-dependent changes in the relative amounts of MAPs and MT-associated kinases. MAP2 immunoreactivity was found significantly lower in MT preparations from aged animals in contrast with a relative increase of tau molecules. In addition, the phosphorylation of MAP2 by its associated cyclic AMP-dependent protein kinase was also altered, consecutively to the partial loss of the enzyme during polymerization cycles and an age-dependent decrease in the ability of the cyclic nucleotide to stimulate MAP2-bound kinase activity. The evidence of an unusually high packing density of sedimented MT from old rat brains further suggested the modification with aging of the physical structure of the arm-like projections of MAPs, in addition to a lower amount in high molecular weight MAPs. These results support the hypothesis of a selective alteration with aging of the mechanical and regulatory properties of brain MT, consecutive to a change in the composition and/or the structure of MAPs.     

Magnetic Separation of Phagosomes of Defined Age from Tetrahymena thermophila

ABSTRACT. We describe a new mass isolation procedure for both pure and stage-specific phagosomes from Tetrahymena thermophila . We prepared magnetic iron dextran particles about 1 μm in diameter to label the phagosomes. The oral apparatus of the cells concentrated these particles so readily that after 1 min the majority of the cells had formed a single phagosome. A short wash removed non-ingested particles, enabling us to follow the age-dependent changes of a single labeled phagosome through the cell. Phagosomes of different ages, including very young and nascent phagosomes, were removed easily from the non-magnetic cell debris of mechanically homogenized cells by means of a permanent magnet. The isolated phagosomes are pure as tested by enzymatic assays and light and electron microscopy. Since the yield of pure phagosomes of all ages is high (∼ 90%), this method could be generally applied for phagosome isolation from ciliates.     

Microtubules: a ring for the depolymerization motor

Newly discovered rings around microtubules, assembled from the Dam1 protein complex, may provide the dynamic linkage at microtubule ends for force generation coupled to microtubule depolymerization and polymerization.     

Evidence for Opposing Effects of Calmodulin on Cortical Microtubules

Microtubule integrity within the cortical array was visualized in detergent-lysed carrot (Daucus carota L.) protoplasts that were exposed to various exogenous levels of Ca2+ and calmodulin (CaM). CaM appears to help stabilize cortical microtubules against the destabilizing action of Ca2+/CaM complexes at low Ca2+ concentrations, but not at higher Ca2+ concentrations. The hypothesis that CaM interacts with microtubules at two different sites, determined by the concentration of Ca2+, is supported by the effects of the CaM antagonists N-(6-aminohexyl)-1-naphthalene-sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfanamide (20 [mu]M) and by affinity chromatography. Two classes of proteins were identified that interact with tubulin and bind to CaM. One class required Ca2+ for CaM binding, whereas the second class bound only when Ca2+ concentrations were low (<320 nM). Thus, CaM's ability to have two opposing effects upon microtubules may be regulated by the concentration of intracellular Ca2+ and its differential interactions with microtubule-associated proteins. Experimental manipulation of intracellular Ca2+ concentrations, as monitored by Indo-1, revealed that the effect of Ca2+ is specific to the cortical microtubules and does not affect actin microfilaments in these cells.     

Microtubules are necessary for lamellae retraction of Vero cells

Behavior of Vero cells under the 2,3-butaneodione monoxime (BDM) treatment was examined using video-microscopy with contrast enhancement. After addition of BDM to the culture medium the area of cell contact with substratum gradually reduced--within 5 min of treatment cell lamellae became thicker, after 60 min the cell area decreased approximately 70 %, and the cells became nearly rounded. At the same time actin bundles (stress fibers) depolymerized, and microtubule network became denser. Partial depolymerization of microfilaments by treatment with latrunculin B at a concentration of 5 nM resulted in complete loss of stress fibers, yet cells slightly change their form, and microtubule system remained the same as in the control cells. However, after addition of BDM in the presence of latrunculin B cells retracted their lamellae more quickly then under BDM sole treatment. To evaluate the role of microtubules in the process of cell retraction we depolymerized them with nocodazole taken at the concentration of 5 ng/ml. Under nocodazole treatment the cell area decreased approximately 20 %, and stress fibers became more thick and abandon. The cells did not change their form, and stress fibers depolymerized very slowly under BDM treatment in the absence of microtubules. After 1 h of BDM treatment in the presence ofnocodazole stress fibers were still more numerous than in the control cells. Complete depolymerization of stress fibers happened in 90 % of cells only in 24 h after addition of BDM. When nocodazole had been washed out of the culture medium in the presence of BDM, lamellae started shrinking in 6 min. This time corresponds to the time required for the partial restoration of microtubule system. On the bases of the results obtained we conclude that retraction of the lamellae in Vero cells is guided rather mainly by microtubules, than stress-fibers.     

Selective Fusion of Azurophilic Granules with Leishmania-containing Phagosomes in Human Neutrophils

Leishmania parasites use polymorphonuclear neutrophils as intermediate hosts before their ultimate delivery to macrophages following engulfment of parasite-infected neutrophils. This leads to a silent and unrecognized entry of Leishmania into the macrophage host cell. Neutrophil function depends on its cytoplasmic granules, but their mobilization and role in how Leishmania parasites evade intracellular killing in neutrophils remain undetermined. Here, we have found by ultrastructural approaches that neutrophils ingested Leishmania major promastigotes, and azurophilic granules fused in a preferential way with parasite-containing phagosomes, without promoting parasite killing. Azurophilic granules, identified by the granule marker myeloperoxidase, also fused with Leishmania donovani-engulfed vacuoles in human neutrophils. In addition, the azurophilic membrane marker CD63 was also detected in the vacuole surrounding the parasite, and in the fusion of azurophilic granules with the parasite-engulfed phagosome. Tertiary and specific granules, involved in vacuole acidification and superoxide anion generation, hardly fused with Leishmania-containing phagosomes. L. major interaction with neutrophils did not elicit production of reactive oxygen species or mobilization of tertiary and specific granules. By using immunogold electron microscopy approaches in the engulfment of L. major and L. donovani by human neutrophils, we did not find a significant contribution of endoplasmic reticulum to the formation of Leishmania-containing vacuoles. Live Leishmania parasites were required to be optimally internalized by neutrophils. Our data suggest that Leishmania promastigotes modulate their uptake by neutrophils, and regulate granule fusion processes in a rather selective way to favor parasite survival in human neutrophils.