Minus Ends Of Microtubules Research Articles

The Mechanism of Cytoplasmic Dynein Processivity

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos towards the microtubule minus end. It is currently unknown how the two catalytic head domains interact and move relative to each other during processive movement. We have tracked the relative positions of both heads with nanometer precision and directly observed that the heads move independently along the microtubule. The heads remain widely separated and the stepping behavior of the heads varies as a function of interhead separation. Consistent with a lack of tight coordination, only one active head is sufficient for processive movement and the active head drags its inactive partner head forward. Only a single active ATPase ring is sufficient for processivity, and the linker swing provides required force to drive minus end directed motion. These results show that dynein is the first dimeric motor that moves processively without interhead coordination, a mechanism fundamentally distinct from hand-over-hand motion of kinesin and myosin.

Lis1 Uncouples Allosteric Communication Between Dynein's AAA+ Motor and Microtubule Binding Domains

Cytoplasmic dynein, the large microtubule-based motor protein, is highly regulated in cells, enabling it to be deployed to specific sites, collect cargos, and translocate them towards the microtubule minus-end at specific times. Defects in the regulation of dynein can cause neurological diseases; for example, mutations in the dynein co-factor Lis1 cause lissencephaly. Dynein’s “engine” is evolved from ring-shaped AAA+ ATPases, and its microtubule-binding domain lies at the tip of a coiled-coil stalk, however how these elements might be acted upon to achieve control remains unknown.

Drosophila javelin-like encodes a novel microtubule-associated protein and is required for mRNA localization during oogenesis

Asymmetrical localization of mRNA transcripts during Drosophila oogenesis determines the anteroposterior and dorsoventral axes of the Drosophila embryo. Correct localization of these mRNAs requires both microtubule (MT) and actin networks. In this study, we have identified a novel gene, CG43162, that regulates mRNA localization during oogenesis and also affects bristle development. We also showed that the Drosophila gene javelin-like, which was identified based on its bristle phenotype, is an allele of the CG43162 gene. We demonstrated that female mutants for jvl produce ventralized eggs owing to the defects in the localization and translation of gurken mRNA during mid-oogenesis. Mutations in jvl also affect oskar and bicoid mRNA localization. Analysis of cytoskeleton organization in the mutants reveal defects in both MT and actin networks. We showed that Jvl protein colocalizes with MT network in Schneider cells, in mammalian cells and in the Drosophila oocyte. Both in the oocyte and in the bristle cells, the protein localizes to a region where MT minus-ends are enriched. Jvl physically interacts with SpnF and is required for its localization. We found that overexpression of Jvl in the germline affects MT-dependent processes: oocyte growth and oocyte nucleus anchoring. Thus, our results show that we have identified a novel MT-associated protein that affects mRNA localization in the oocyte by regulating MT organization.

Cytoplasmic dynein

The organization and function of eukaryotic cells rely on the action of many different molecular motor proteins. Cytoplasmic dynein drives the movement of a wide range of cargoes towards the minus ends of microtubules, and these events are needed, not just at the single-cell level, but are vital for correct development. In the present paper, I review recent progress on understanding dynein's mechanochemistry, how it is regulated and how it binds to such a plethora of cargoes. The importance of a number of accessory factors in these processes is discussed.

Roles of dynein and dynactin in early endosome dynamics revealed using automated tracking and global analysis.

Microtubule-dependent movement is crucial for the spatial organization of endosomes in most eukaryotes, but as yet there has been no systematic analysis of how a particular microtubule motor contributes to early endosome dynamics. Here we tracked early endosomes labeled with GFP-Rab5 on the nanometer scale, and combined this with global, first passage probability (FPP) analysis to provide an unbiased description of how the minus-end microtubule motor, cytoplasmic dynein, supports endosome motility. Dynein contributes to short-range endosome movement, but in particular drives 85–98% of long, inward translocations. For these, it requires an intact dynactin complex to allow membrane-bound p150Glued to activate dynein, since p50 over-expression, which disrupts the dynactin complex, inhibits inward movement even though dynein and p150Glued remain membrane-bound. Long dynein-dependent movements occur via bursts at up to ∼8 µms−1 that are linked by changes in rate or pauses. These peak speeds during rapid inward endosome movement are still seen when cellular dynein levels are 50-fold reduced by RNAi knock-down of dynein heavy chain, while the number of movements is reduced 5-fold. Altogether, these findings identify how dynein helps define the dynamics of early endosomes.

Hysteresis-Based Mechanism for the Directed Motility of the Ncd Motor

Ncd is a Kinesin-14 family protein that walks to the microtubule's minus end. Although available structures show its α-helical neck in either pre- or post-stroke orientations, little is known about the transition between these two states. Using a combination of molecular dynamics simulations and structural analyses, we find that the neck sequentially makes intermediate contacts with the motor head along its mostly longitudinal path, and it develops a 24° twist in the post-stroke orientation. The forward (pre-stroke to post-stroke) motion has an ∼4.5 k B T (where k B is the Boltzmann constant, and T = 300 K) free-energy barrier and is a diffusion guided by the intermediate contacts. The post-stroke free-energy minimum is higher and is formed ∼10° before reaching the orientation in the post-stroke crystal structure, consistent with previous structural data. The importance of intermediate contacts correlates with the existing motility data, including those for mutant Ncds. Unlike the forward motion, the recovery stroke goes nearly downhill in free energy, powered in part by torsional relaxation of the neck. The hysteresis in the energetics of the neck motion arises from the mechanical compliance of the protein, and together with guided diffusion, it may be key to the directed motility of Ncd.

Stimulation of the CLIP-170–dependent capture of membrane organelles by microtubules through fine tuning of microtubule assembly dynamics

Cytoplasmic microtubules (MTs) continuously grow and shorten at their free plus ends, a behavior that allows them to capture membrane organelles destined for MT minus end-directed transport. In Xenopus melanophores, the capture of pigment granules (melanosomes) involves the +TIP CLIP-170, which is enriched at growing MT plus ends. Here we used Xenopus melanophores to test whether signals that stimulate minus end MT transport also enhance CLIP-170-dependent binding of melanosomes to MT tips. We found that these signals significantly (>twofold) increased the number of growing MT plus ends and their density at the cell periphery, thereby enhancing the likelihood of interaction with dispersed melanosomes. Computational simulations showed that local and global increases in the density of CLIP-170-decorated MT plus ends could reduce the half-time of melanosome aggregation by ~50%. We conclude that pigment granule aggregation signals in melanophores stimulate MT minus end-directed transport by the increasing number of growing MT plus ends decorated with CLIP-170 and redistributing these ends to more efficiently capture melanosomes throughout the cytoplasm.

Wolbachia bacteria reside in host Golgi-related vesicles whose position is regulated by polarity proteins.

Wolbachia pipientis are intracellular symbiotic bacteria extremely common in various organisms including Drosophila melanogaster, and are known for their ability to induce changes in host reproduction. These bacteria are present in astral microtubule-associated vesicular structures in host cytoplasm, but little is known about the identity of these vesicles. We report here that Wolbachia are restricted only to a group of Golgi-related vesicles concentrated near the site of membrane biogenesis and minus-ends of microtubules. The Wolbachia vesicles were significantly mislocalized in mutant embryos defective in cell/planar polarity genes suggesting that cell/tissue polarity genes are required for apical localization of these Golgi-related vesicles. Furthermore, two of the polarity proteins, Van Gogh/Strabismus and Scribble, appeared to be present in these Golgi-related vesicles. Thus, establishment of polarity may be closely linked to the precise insertion of Golgi vesicles into the new membrane addition site.

Golgi Positioning

The Golgi apparatus in mammalian cells is positioned near the centrosome-based microtubule-organizing center (Fig.1). Secretory cargo moves inward in membrane carriers for delivery to Golgi membranes in which it is processed and packaged for transport outward to the plasma membrane. Cytoplasmic dynein motor proteins (herein termed dynein) primarily mediate inward cargo carrier movement and Golgi positioning. These motors move along microtubules toward microtubule minus-ends embedded in centrosomes. Centripetal motility is controlled by a host of regulators whose precise functions remain to be determined. Significantly, a specific Golgi receptor for dynein has not been identified. This has impaired progress toward elucidation of membrane-motor-microtubule attachment in the periphery and, after inward movement, recycling of the motor for another round. Pericentrosomal positioning of the Golgi apparatus is dynamic. It is regulated during critical cellular processes such as mitosis, differentiation, cell polarization, and cell migration. Positioning is also important as it aligns the Golgi along an axis of cell polarity. In certain cell types, this promotes secretion directed to the proximal plasma membrane domain thereby maintaining specializations critical for diverse processes including wound healing, immunological synapse formation, and axon determination.

Microtubule stabilization in vivo by nucleation-incompetent γ-tubulin complex

Although the fission yeast Schizosaccharomyces pombe contains many of the γ-tubulin ring complex (γ-TuRC)-specific proteins of the γ-tubulin complex (γ-TuC), several questions about the organizational state and function of the fission yeast γ-TuC in vivo remain unresolved. Using 3×GFP-tagged γ-TuRC-specific proteins, we show here that γ-TuRC-specific proteins are present at all microtubule organizing centers in fission yeast and that association of γ-TuRC-specific proteins with the γ-tubulin small complex (γ-TuSC) does not depend on Mto1, which is a key regulator of the γ-TuC. Through sensitive imaging in mto1Δ mutants, in which cytoplasmic microtubule nucleation is abolished, we unexpectedly found that γ-TuC incapable of nucleating microtubules can nevertheless associate with microtubule minus-ends in vivo. The presence of γ-TuC at microtubule ends is independent of γ-TuRC-specific proteins and strongly correlates with the stability of microtubule ends. Strikingly, microtubule bundles lacking γ-TuC at microtubule ends undergo extensive treadmilling in vivo, apparently induced by geometrical constraints on plus-end growth. Our results indicate that microtubule stabilization by the γ-TuC, independently of its nucleation function, is important for maintaining the organization and dynamic behavior of microtubule arrays in vivo.

CK1 activates minus-end–directed transport of membrane organelles along microtubules

Microtubule (MT)-based organelle transport is driven by MT motor proteins that move cargoes toward MT minus-ends clustered in the cell center (dyneins) or plus-ends extended to the periphery (kinesins). Cells are able to rapidly switch the direction of transport in response to external cues, but the signaling events that control switching remain poorly understood. Here, we examined the signaling mechanism responsible for the rapid activation of dynein-dependent MT minus-end-directed pigment granule movement in Xenopus melanophores (pigment aggregation). We found that, along with the previously identified protein phosphatase 2A (PP2A), pigment aggregation signaling also involved casein kinase 1ε (CK1ε), that both enzymes were bound to pigment granules, and that their activities were increased during pigment aggregation. Furthermore we found that CK1ε functioned downstream of PP2A in the pigment aggregation signaling pathway. Finally, we discovered that stimulation of pigment aggregation increased phosphorylation of dynein intermediate chain (DIC) and that this increase was partially suppressed by CK1ε inhibition. We propose that signal transduction during pigment aggregation involves successive activation of PP2A and CK1ε and CK1ε-dependent phosphorylation of DIC, which stimulates dynein motor activity and increases minus-end-directed runs of pigment granules.

Unbinding Force of Cytoplasmic Dynein

Dynein is a molecular motor that moves toward the minus-end of microtubules using the chemical energy of ATP hydrolysis. Cytoplasmic dynein play roles in positioning the Golgi complex and other organelles in cells, movement of chromosomes, and positioning the mitotic spindles during mitosis. Force generation by a dynein molecule, thus, is one of the important factors for understanding molecular properties of dynein. To examine the binding mode between dynein and a microtubule, we measured the unbinding force of dynein in various nucleotide conditions. We used truncated C-terminal motor domain, thus we can eliminate possible effects of tail region and/or accessory proteins on the motor activity of dynein. Dynein with the biotin-tag was attached to avidin-coated polystyrene bead. The bead was trapped by optical tweezers, and the external load was imposed by moving the stage. When the stage was moved at 160nm/sec (loading rate is 5.9 pN/sec), the mean value of the unbinding force of strongly bound state of dynein was 5.7 - 6.6pN upon backward loading. The unbinding force was 20-35% smaller when force was applied to the minus end of microtubules. These data indicate that dynein unbinds from microtubules easily toward the minus end of microtubules to which dynein moves.

Detailed Analysis of the Dynein Stepping Mechanism using Multicolor Tracking

Cytoplasmic dynein is a motor protein that moves processively towards the microtubule minus end. To characterize dynein's step size at both limiting (5 µM) and saturating (1 mM) ATP concentrations, we have improved the temporal resolution of FIONA to 2 milliseconds and tracked the movement of single yeast cytoplasmic dynein motors, each labeled with one quantum dot. In contrast to kinesin, dynein's step size is highly variable, and backwards and sideways steps are frequently observed. As the tail domain takes ∼8 nm steps, the heads advance by taking both short (∼8 nm) and long (∼16 nm) steps. These data indicate that the heads do not move in a strictly alternating manner, as is the case with kinesin and myosin. To further characterize how the dynein heads move relative to each other, we have tracked the movement of both heads simultaneously. To precisely measure the head-head separation vector, we have developed novel small quantum dots (10 nm diameter) that specifically attach to each dynein head. The fluorescent signals of the differently-colored quantum dots attached to dynein are then registered with 2 nm precision. Using this technique, we find the heads to be widely separated, mostly in the off-axis, indicating that the two heads walk along separate filaments. Our two-color stepping data also show that the dynein heads advance mostly by taking alternating steps, as is the case with kinesin. However, the heads may also move independently of each other, and one head may take multiple steps before its partner moves forward. Both the leading head and the trailing head may initiate a step. Taken together, these unprecedented behaviors indicate that cytoplasmic dynein achieves processive movement via a mechanism unique among the molecular motors.

Superresolution Studies to Reveal the Interactions between Motor Proteins and Individual Cargos in Chlamydomonas Flagella

Our research aims to understand the molecular mechanism of cytoplasmic dynein, which is involved in the transport of cargo towards the microtubule minus end of eukaryotic cells. Specifically, we use intraflagellar transport (IFT) in Chlamydomonas cells as a model system to study interaction of IFT dynein with kinesin II, opposite polarity motor that moves cargos toward the plus end. To detect the distribution and cargo interaction of single motor proteins along the flagellum, we employed Stochastic Optical Reconstruction Microscopy (STROM) microscopy. This technique has proven to construct superresolution images of precisely positioned fluorophores from single-molecule images. In order to facilitate STORM imaging, photoswitchable cyanine reporter and an activator molecule were coupled to antibodies against kinesin II and IFT dynein. The quantitative analysis of the STORM images will allow us to determine how many kinesin and dynein motors actively work on a cellular cargo, and how the cargo direction is determined by kinesin-dynein interactions. IFT is a universal process in all eukaryotic cilia and flagella. Defects in this process are the primary causes of polycystic kidney disease and retinal degeneration.

Enclosing, Deformable Membrane Aids in Focusing and Stabilizing the Mitotic Spindle

Mitosis involves the interaction of microtubules, motor and associated proteins to assemble a mitotic spindle. Recent evidence suggests that the assembly of the mitotic spindle maybe be aided through the contributions of an external membranous matrix. A multi-agent computational model was constructed to study the possible mechanisms by which an external membrane could aid and influence the assembly of the mitotic spindle. A systematic study of the model parameters examined the effect motor and crosslinker proteins at varying concentrations have on the elongation rate and assembly of a mitotic spindle. The effect on spindle assembly and overall spindle length by varying membrane elasticity, microtubule length and microtubule orientation was also investigated. We show that overall; an enclosing, elastic membrane around the mitotic assembly machinery helps to focus microtubule minus ends, and provides a resistive force to regulate the size of the assembled spindle.

Mitotic Membrane Helps to Focus and Stabilize the Mitotic Spindle

During mitosis, microtubules (MTs), aided by motors and associated proteins, assemble into a mitotic spindle. Recent evidence supports the notion that a membranous spindle matrix aids spindle formation; however, the mechanisms by which the matrix may contribute to spindle assembly are unknown. To search for a mechanism by which the presence of a mitotic membrane might help spindle morphology, we built a computational model that explores the interactions between these components. We show that an elastic membrane around the mitotic apparatus helps to focus MT minus ends and provides a resistive force that acts antagonistically to plus-end-directed MT motors such as Eg5.

Kinesins at a glance.

Kinesin was discovered in 1985 – 25 years ago – based on its motility in cytoplasm extruded from the giant axon of the squid ([Allen et al., 1982][1]; [Brady et al., 1982][2]; [Vale et al., 1985][3]). The purified protein, now known as kinesin-1, consisted of two heavy chains with motor activity

Patronin Regulates the Microtubule Network by Protecting Microtubule Minus Ends

Tubulin assembles into microtubule polymers that have distinct plus and minus ends. Most microtubule plus ends in living cells are dynamic; the transitions between growth and shrinkage are regulated by assembly-promoting and destabilizing proteins. In contrast, minus ends are generally not dynamic, suggesting their stabilization by some unknown protein. Here, we have identified Patronin (also known as ssp4) as a protein that stabilizes microtubule minus ends in Drosophila S2 cells. In the absence of Patronin, minus ends lose subunits through the actions of the Kinesin-13 microtubule depolymerase, leading to a sparse interphase microtubule array and short, disorganized mitotic spindles. In vitro, the selective binding of purified Patronin to microtubule minus ends is sufficient to protect them against Kinesin-13-induced depolymerization. We propose that Patronin caps and stabilizes microtubule minus ends, an activity that serves a critical role in the organization of the microtubule cytoskeleton.

How cellular membranes can regulate microtubule network

Microtubule array in eukaryotic cells supports directed transport of various cargoes driven by motor proteins. The arrangement of microtubules in cytoplasm is not stochastic; they are organized in a certain way setting a system of coordinates for intracellular transport. Most cultured fibroblast-like cells possess a radial microtubule array with the minus ends of microtubules gathered on the centrosome and plus ends directed towards the periphery of the cell. Mechanisms that regulate the formation of radial microtubule system remain unclear. Usually centrosome works as a microtubule-organizing center; however, the radial system of microtubules can be formed without centrosome participation. At least in some cases microtubule network can be organized by dynein-dynactin complexes associated with membrane vesicles. Membrane vesicles can nucleate microtubules, anchor them and move along them. However, the role of membrane organelles in microtubule organization began to attract attention of researches only recently. It this review we summarize the data indicating that membrane organelles can organize microtubules, providing “tracks” for their subsequent transport.

Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded theCENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which α,β-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.