mNUDC is required for plus‐end‐directed transport of cytoplasmic dynein and dynactins by kinesin‐1

Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. The LIS1 (or PAFAH1B1) gene was identified as the gene mutated in lissencephaly patients, and was found to regulate cytoplasmic dynein function and localization. In particular, LIS1 is essential for anterograde transport of cytoplasmic dynein as a part of the cytoplasmic dynein–LIS1–microtubule complex in a kinesin‐1‐dependent manner. However, the underlying mechanism by which a cytoplasmic dynein–LIS1–microtubule complex binds kinesin‐1 is unknown. Here, we report that mNUDC (mammalian NUDC) interacts with kinesin‐1 and is required for the anterograde transport of a cytoplasmic dynein complex by kinesin‐1. mNUDC is also required for anterograde transport of a dynactin‐containing complex. Inhibition of mNUDC severely suppressed anterograde transport of distinct cytoplasmic dynein and dynactin complexes, whereas motility of kinesin‐1 remained intact. Reconstruction experiments clearly demonstrated that mNUDC mediates the interaction of the dynein or dynactin complex with kinesin‐1 and supports their transport by kinesin‐1. Our findings have uncovered an essential role of mNUDC for anterograde transport of dynein and dynactin by kinesin‐1.     

An RNA‐binding atypical tropomyosin recruits kinesin‐1 dynamically to oskar mRNPs

Localization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport‐competent mRNPs. We show that the posterior‐targeting kinesin‐1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein‐dependent transport from the nurse cells into the oocyte. We demonstrate that kinesin‐1 recruitment requires the DmTropomyosin1‐I/C isoform, an atypical RNA‐binding tropomyosin that binds directly to dimerizing oskar 3′UTRs. Finally, we show that a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin‐1 and that the motor is activated during mid‐oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo‐dependent motor activation constitutes an optimized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo‐transport processes and between the cargo‐associated dynein and kinesin‐1.     

Kinesin-like proteins are involved in postmitotic nuclear migration of the unicellular green alga Micrasterias denticulata

The unicellular green alga Micrasterias denticulata performs a two-directional postmitotic nuclear migration during development, a passive migration into the growing semicell, and a microtubule mediated backward migration towards the cell centre. The present study provides first evidence for force generation by motor proteins of the kinesin family in this process. The new kinesin specific inhibitor adociasulfate-2 causes abnormal nuclear displacement at 18 microM. AMP-PNP, a non hydrolyseable ATP analogue or the general ATPase inhibitors calyculin A and sodium orthovanadate also disturb nuclear migration. In addition kinesin-like proteins are detected by means of immunoblotting using antibodies against brain kinesin, plant derived antibodies to kinesin-like proteins and a calmodulin binding kinesin-like protein. Immunoelectron microscopy suggests a correlation of conventional kinesin-like proteins, but not of the calmodulin binding kinesin-like protein to the microtubule apparatus associated with the migrating nucleus.     

UNC-83 coordinates kinesin-1 and dynein activities at the nuclear envelope during nuclear migration

Nuclei migrate during many events, including fertilization, establishment of polarity, differentiation, and cell division. The Caenorhabditis elegans KASH protein UNC-83 localizes to the outer nuclear membrane where it recruits kinesin-1 to provide the major motor activity required for nuclear migration in embryonic hyp7 cells. Here we show that UNC-83 also recruits two dynein-regulating complexes to the cytoplasmic face of the nucleus that play a regulatory role. One consists of the NudE homolog NUD-2 and the NudF/Lis1/Pac1 homolog LIS-1, and the other includes dynein light chain DLC-1, the BicaudalD homolog BICD-1, and the Egalitarian homologue EGAL-1. Genetic disruption of any member of these two complexes caused nuclear migration defects that were enhanced in some double mutant animals, suggesting that BICD-1 and EGAL-1 function in parallel to NUD-2. Dynein heavy chain mutant animals also had a nuclear migration defect, suggesting these complexes function through dynein. Deletion analysis indicated that independent domains of UNC-83 interact with kinesin and dynein. These data suggest a model where UNC-83 acts as the cargo-specific adaptor between the outer nuclear membrane and the microtubule motors kinesin-1 and dynein. Kinesin-1 functions as the major force generator during nuclear migration, while dynein is involved in regulation of bidirectional transport of the nucleus.     

Miro proteins coordinate microtubule‐ and actin‐dependent mitochondrial transport and distribution

In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho‐GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double‐knockout mouse embryos and single‐ and double‐knockout embryonic fibroblasts, we demonstrate the essential and non‐redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double‐knockout cells. In contrast, we show that TRAK2‐mediated retrograde mitochondrial transport is Miro1‐dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin‐dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double‐knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine‐tune actin‐ and tubulin‐dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation.     

Nuclear envelope‐associated dynein drives prophase centrosome separation and enables Eg5‐independent bipolar spindle formation

The microtubule motor protein kinesin‐5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti‐cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5‐independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)‐associated dynein that drives centrosome separation in prophase. This NE‐dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5‐dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin‐5 inhibitors.     

Velocity of myosin-based actin sliding depends on attachment and detachment kinetics and reaches a maximum when myosin-binding sites on actin saturate

Molecular motors such as kinesin and myosin often work in groups to generate the directed movements and forces critical for many biological processes. Although much is known about how individual motors generate force and movement, surprisingly, little is known about the mechanisms underlying the macroscopic mechanics generated by multiple motors. For example, the observation that a saturating number, N, of myosin heads move an actin filament at a rate that is influenced by actin–myosin attachment and detachment kinetics is accounted for neither experimentally nor theoretically. To better understand the emergent mechanics of actin–myosin mechanochemistry, we use an in vitro motility assay to measure and correlate the N-dependence of actin sliding velocities, actin-activated ATPase activity, force generation against a mechanical load, and the calcium sensitivity of thin filament velocities. Our results show that both velocity and ATPase activity are strain dependent and that velocity becomes maximized with the saturation of myosin-binding sites on actin at a value that is 40% dependent on attachment kinetics and 60% dependent on detachment kinetics. These results support a chemical thermodynamic model for ensemble motor mechanochemistry and imply molecularly explicit mechanisms within this framework, challenging the assumption of independent force generation.     

Regulatory Control of Both Microtubule‐ and Actin‐dependent Fish Melanosome Movement

In fish melanophores, melanosomes can either aggregate around the cell centre or disperse uniformly throughout the cell. This organelle transport involves microtubule‐ and actin‐dependent motors and is regulated by extracellular stimuli that modulate levels of intracellular cyclic adenosine 3‐phosphate (cAMP). We analysed melanosome dynamics in Atlantic cod melanophores under different experimental conditions in order to increase the understanding of the regulation and relative contribution of the transport systems involved. By inhibiting dynein function via injection of inhibitory antidynein IgGs, and modulating cAMP levels using forskolin, we present cellular evidence that dynein is inactivated by increased cAMP during dispersion and that the kinesin‐related motor is inactivated by low cAMP levels during aggregation. Inhibition of dynein further resulted in hyperdispersed melanosomes, which subsequently reversed movement towards a more normal dispersed state, pointing towards a peripheral feedback regulation in maintaining the evenly dispersed state. This reversal was blocked by noradrenaline. Analysis of actin‐mediated melanosome movements shows that actin suppresses aggregation and dispersion, and indicates the possibility of down‐regulating actin‐dependent melanosome movement by noradrenaline. Data from immuno‐electron microscopy indicate that myosinV is associated with fish melanosomes. Taken together, our study presents evidence that points towards a model where both microtubule‐ and actin‐mediated melanosome transport are synchronously regulated during aggregation and dispersion, and this provides a cell physiological explanation behind the exceptionally fast rate of background adaptation in fish.     

Autonomous circular and radial motions of the nucleus inPleurenterium tumidum and their relation to cytoskeletal elements and the plasma membrane

Summary InPleurenterium tumidum the nucleus leaves its central position at the end of cell development and moves centrifugally towards the cortical cytoplasm of the isthmus area. It passes between the chloroplast lobes and starts to perform circular motions along the cell wall ring of the isthmus independently from other cell organelles and cytoplasmic streaming. This autonomous nuclear motion is a unique phenomenon in plant cells which is reported here for the first time. One turn of the nucleus which may occur either clockwise or counter-clockwise lasts an average of 60 minutes. The velocity of circular nuclear motion lies between 0.03 and 0.08 m per second and increases with increasing number of nuclear turns. The nucleus undergoes at least 12 but sometimes up to 70 turns and may change its direction of motion several times. When circular nuclear motion is finished the nucleus migrates centripetally towards the cell center where the next mitosis takes place.Ultrastructural studies demonstrate that a distinct arrangement of the plasma membrane forming a ring-shaped fold together with the adjacent isthmus system of microtubules (IS) serves as a hoop-like track for the nucleus during the stage of circular motion. The nucleus moves along this track by surrounding it in a deep furrow which develops parallel to its longitudinal axis at its cell wall facing side. The spatial arrangement of the plasma membrane fold and the nuclear furrow are only present during the stage of circular nuclear motion. No actin filaments seem to be involved in the nuclear circulations since the nucleus continues its circular motions after cytochalasin B (CB) treatment even at concentrations which arrest cytoplasmic streaming. Amiprophos-methyl (APM) leads to an inhibition of circular nuclear motion which resumes when the APM solution is removed. Microtubules appear to be primarily responsible also for both the radial nuclear motions as well as the anchoring of the nucleus in its central position. The meaning of circular and radial nuclear motions for thePleurenterium cell is not yet clear, a relation between the nuclear behavior and the inner cell architecture is discussed and compared to that of other desmids.     

Apical myosin XI anticipates F‐actin during polarized growth of Physcomitrella patens cells

Tip growth is essential for land colonization by bryophytes, plant sexual reproduction and water and nutrient uptake. Because this specialized form of polarized cell growth requires both a dynamic actin cytoskeleton and active secretion, it has been proposed that the F‐actin‐associated motor myosin XI is essential for this process. Nevertheless, a spatial and temporal relationship between myosin XI and F‐actin during tip growth is not known in any plant cell. Here, we use the highly polarized cells of the moss Physcomitrella patens to show that myosin XI and F‐actin localize, in vivo, at the same apical domain and that both signals fluctuate. Surprisingly, phase analysis shows that increase in myosin XI anticipates that of F‐actin; in contrast, myosin XI levels at the tip fluctuate in identical phase with a vesicle marker. Pharmacological analysis using a low concentration of the actin polymerization inhibitor latrunculin B showed that the F‐actin at the tip can be significantly diminished while myosin XI remains elevated in this region, suggesting that a mechanism exists to cluster myosin XI‐associated structures at the cell's apex. In addition, this approach uncovered a mechanism for actin polymerization‐dependent motility in the moss cytoplasm, where myosin XI‐associated structures seem to anticipate and organize the actin polymerization machinery. From our results, we inferred a model where the interaction between myosin XI‐associated vesicular structures and F‐actin polymerization‐driven motility function at the cell's apex to maintain polarized cell growth. We hypothesize this is a general mechanism for the participation of myosin XI and F‐actin in tip growing cells.     

The myosin ATPase inhibitor 2,3-butanedione-2-monoxime disorganizes microtubules as well as F-actin in Saccharomyces cerevisiae

Interactions between microtubules and filamentous actin (F-actin) are essential to many cellular processes, but their mechanisms are poorly understood. We investigated possible roles of the myosin family of proteins in the interactions between filamentous actin (F-actin) and microtubules of budding yeast Saccharomyces cerevisiae with the general myosin ATPase inhibitor 2,3-butanedione-2-monoxime (BDM). The growth of S. cerevisiae was completely inhibited by BDM at 20 mmol/L and the effect of BDM on cell growth was reversible. In more than 80% of BDM-treated budding yeast cells, the polarized distribution of F-actin was lost and fewer F-actin dots were observed. When cells were synchronized in G₁ with α-factor and released in the presence of BDM, cell number did not increase and cells were mainly arrested in G₁ DNA content without any bud, suggesting that myosin activity is required for new bud formation and the start of a new cell cycle. More than 10% of the BDM-treated cells also revealed defects in nuclear migration to the bud neck as well as in nuclear shape. Consistent with these defects, the orientation of mitotic spindles was random in the 57% of cells treated with 20 mmol/L BDM and immunostained with anti-tubulin antibody. Furthermore, microtubule structures were completely disorganized in most of the cells incubated in 50 mmol/L BDM, while similar amounts of tubulin proteins were present in both BDM-treated and untreated cells. These results show that the general myosin inhibitor BDM disorganizes microtubule structures as well as F-actin, and suggest that BDM-sensitive myosin activities are necessary for the interaction of F-actin and microtubules to coordinate polarized bud growth and the shape and migration of the nucleus in S. cerevisiae. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nuclear spectrin-like proteins are structural actin-binding proteins in plants

Background information. Although actin is a relevant component of the plant nucleus, only three nuclear ABPs (actin‐binding proteins) have been identified in plants to date: cofilin, profilin and nuclear myosin I. Although plants lack orthologues of the main structural nuclear ABPs in animals, such as lamins, lamin‐associated proteins and nesprins, their genome does contain sequences with spectrin repeats and N‐terminal calponin homology domains for actin binding that might be distant relatives of spectrin. We investigated here whether spectrin‐like proteins could act as structural nuclear ABPs in plants. Results. We have investigated the presence of spectrins in Allium cepa meristematic nuclei by Western blotting, confocal and electron microscopy, using antibodies against α‐ and β‐spectrin chains that cross‐react in plant nuclei. Their role as nuclear ABPs was analysed by co‐immunoprecipitation and IF (immunofluorescence) co‐localization and their association with the nuclear matrix was investigated by sequential extraction of nuclei with non‐ionic detergent, and in low‐ and high‐salt buffers after nuclease digestion. Our results demonstrate the existence of several spectrin‐like proteins in the nucleus of onion cells that have different intranuclear distributions in asynchronous meristematic populations and associate with the nuclear matrix. These nuclear proteins co‐immunoprecipitate and co‐localize with actin. Conclusions. These results reveal that the plant nucleus contains spectrin‐like proteins that are structural nuclear components and function as ABPs. Their intranuclear distribution suggests that plant nuclear spectrin‐like proteins could be involved in multiple nuclear functions.     

2‐methoxyestradiol inhibits atorvastatin‐induced rounding of human vascular smooth muscle cells

The cardiovascular benefits of statins, including atorvastatin (ATV), have been reported to be gender‐dependent, but the underlying mechanism is unclear. In this study we examine whether estrogen and its metabolite, 2‐methoxyestradiol (2ME), affect the rounding response of human vascular smooth muscle cells (SMCs) induced by ATV. Twenty‐four hour treatment with ATV (10–100 µM) induced rounding of cultured human SMCs. Addition of 2ME (1–20 µM), but not 17β‐estradiol, for 2 h induced re‐spreading of rounded cells. Our further studies showed that the effects of 2ME were mimicked by microtubule‐disrupting drugs and inhibited by taxol. Inhibition of RhoA and ROCK (Rho‐kinase) by C3‐toxin and H‐1152, respectively, blocked 2ME effects. 2ME effects were also blocked by treatment with either actin‐interfering drugs, such as cytochalasin D and jasplakinolide, or myosin inhibitor blebbistatin. ML‐7 and ‐9, the inhibitors for myosin light chain kinase, inhibited 2ME effect as well. ATV treatment induced a decrease of F‐actin content and Thr18/Ser19 dual phosphorylation of myosin regulatory light chain (MRLC), which was rescued by 2ME or mevalonate. The rescue effects of 2ME on F‐actin content and MRLC dual phosphorylation were abolished by taxol or H‐1152. In addition, kinesin Eg5 inhibitor monastrol and dynein inhibitor erythro‐9‐3‐(2‐hydroxynonyl) adenine (EHNA) significantly blocked 2ME effects. Finally, our results revealed that 2ME inhibited the migration of SMCs induced by ATV (0.1 µM) in wound healing assay and Boyden chamber assay. In summary, our data show that 2ME, but not estrogen, inhibits ATV‐induced rounding of human SMCs through induction of microtubule disassembly and activation of the Rho‐ROCK‐actinomyosin pathway. J. Cell. Physiol. 222: 556–564, 2010. © 2009 Wiley‐Liss, Inc.     

Symposium 4: Dynamics of the Neuronal Cytoskeleton. Actin‐based vesicle transport in nerve cell extracts: implications for neurodegenerative diseases

Neurodegenerative diseases may result in part from defects in motor‐driven vesicle transport in neuronal cells. Myosin‐V, an actin‐based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin‐V interacts with the microtubule‐based motor, kinesin, to form a ‘hetero‐motor’ complex on vesicles. The complex of these two motors, one microtubule‐based and the other actin‐based, facilitates the movement of vesicles from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons, we hypothesize that one of the functions of the tail–tail interaction is to provide feedback between the two proteins to allow seamless transition of vesicles from microtubules to actin filaments. To study the interactions of the globular tail domain of myosin‐V to kinesin and to neuronal vesicles, we used a GST‐tagged globular tail fragment in motility assays. The MyoV tail fragment inhibited vesicle transport by 81–91% and thereby exhibited a dominant negative effect. These data show that the recombinant protein blocked the activity of native myosin‐V presumably by binding to vesicles and competing away the native myosin‐V motors. The GST‐MyoV‐tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates and therefore it exhibited binding properties of native myosin‐V. These data show that the headless myosin‐V fragment is an effective inhibitor of vesicle transport in cell extracts. These studies support the hypothesis that tail–tail interactions may be a mechanism for feedback between myosin‐V and kinesin to allow transition of vesicles from microtubules to actin filaments. Acknowledgements: Supported by NSF grant MCB9974709.     

A Role for Spectrin in Dynactin‐dependent Melanosome Transport in Xenopus laevis Melanophores

The bi‐directional movement of pigment granules in frog melanophores involves the microtubule‐based motors cytoplasmic dynein, which is responsible for aggregation, and kinesin  II and myosin  V, which are required for dispersion of pigment. It was recently shown that dynactin acts as a link between dynein and kinesin  II and melanosomes, but it is not fully understood how this is regulated and if more proteins are involved. Here, we suggest that spectrin, which is known to be associated with Golgi vesicles as well as synaptic vesicles in a number of cells, is of importance for melanosome movements in Xenopus laevis melanophores. Large amounts of spectrin were found on melanosomes isolated from both aggregated and dispersed melanophores. Spectrin and two components of the oligomeric dynactin complex, p150^glued and Arp1/centractin, co‐localized with melanosomes during aggregation and dispersion, and the proteins were found to interact as determined by co‐immunoprecipitation. Spectrin has been suggested as an important link between cargoes and motor proteins in other cell types, and our new data indicate that spectrin has a role in the specialized melanosome transport processes in frog melanophores, in addition to a more general vesicle transport.     

Load‐dependent detachment kinetics plays a key role in bidirectional cargo transport by kinesin and dynein

Bidirectional cargo transport along microtubules is carried out by opposing teams of kinesin and dynein motors. Despite considerable study, the factors that determine whether these competing teams achieve net anterograde or retrograde transport in cells remain unclear. The goal of this work is to use stochastic simulations of bidirectional transport to determine the motor properties that most strongly determine overall cargo velocity and directionality. Simulations were carried out based on published optical tweezer characterization of kinesin‐1 and kinesin‐2, and for available data for cytoplasmic dynein and the dynein‐dynactin‐BicD2 (DDB) complex. By varying dynein parameters and analyzing cargo trajectories, we find that net cargo transport is predicted to depend minimally on the dynein stall force, but strongly on dynein load‐dependent detachment kinetics. In simulations, dynein is dominated by kinesin‐1, but DDB and kinesin‐1 are evenly matched, recapitulating recent experimental work. Kinesin‐2 competes less well against dynein and DDB, and overall, load‐dependent motor detachment is the property that most determines a motor's ability to compete in bidirectional transport. It follows that the most effective intracellular regulators of bidirectional transport are predicted to be those that alter motor detachment kinetics rather than motor velocity or stall force.      

Homo‐C‐nucleoside analogs IV. Synthesis of 4‐(2,5‐anhydro‐D‐altro‐pentitol‐1‐yl)‐2‐phenyl‐2H‐1,2,3‐triazole homo‐C‐nucleoside. CD assignment of the absolute configuration of the carbon bridge

Dehydrative cyclization of 4‐(D‐altro ‐pentitol‐1‐yl)2‐phenyl‐2H ‐1,2,3‐triazole in basic medium with one moler equivalent of p‐toluene sulfonyl chloride in pyridine solution gave the homo‐C‐ nucleoside 4‐(2,5‐anhydro‐D‐altro ‐1‐yl)‐2‐phenyl‐2H ‐1,2,3‐triazole. The structure and anomeric configuration was determined by acylation, nuclear magnetic resonance (NMR), and mass spectroscopy. The stereochemistry at the carbon bridge of homo‐C‐ nucleoside 2‐phenyl‐2H ‐1,2,3‐triazoles was determined by circular dichroism (CD) spectroscopy.     

Endogenous GSK‐3/Shaggy Regulates Bidirectional Axonal Transport of the Amyloid Precursor Protein

Neurons rely on microtubule (MT) motor proteins such as kinesin‐1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK‐3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin‐1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, we find that endogenous GSK‐3 is a required negative regulator of both kinesin‐1‐mediated and dynein‐mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK‐3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, we find that GSK‐3 regulates transport by altering the activity of kinesin‐1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK‐3 and APP, and demonstrate that endogenous GSK‐3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK‐3 controls the number of active motors that are moving a cargo .     

Engineered Tug‐of‐War Between Kinesin and Dynein Controls Direction of Microtubule Based Transport In Vivo

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus‐end directed kinesins and minus‐end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT‐based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug‐of‐war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug‐of‐war between opposing MT motors alone, by attaching a large number of kinesin‐1 motors to organelles transported by dynein to minus‐ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus‐end‐directed dynein‐dependent MT runs, leading to a reversal of the overall direction of dynein‐driven organelles in vivo. Therefore, in the absence of external regulators tug‐of‐war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo.