Analysis of the Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein-1 Using a Chemical "Toolkit".

Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure-activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues' effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the "glutamate switch" mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.

Dynamic Dissection of Dynein and Kinesin-1 Cooperatively Mediated Intercellular Transport of Porcine Epidemic Diarrhea Coronavirus along Microtubule Using Single Virus Tracking.

It is now clear that the intercellular transport on microtubules by dynein and kinesin-1 motors has an important role in the replication and spread of many viruses. Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded RNA virus of the Coronavirus family, which can infect swine of all ages and cause severe economic losses in the swine industry. Elucidating the molecular mechanisms of the intercellular transport of PEDV through microtubule, dynein and kinesin-1 will be crucial for understanding its pathogenesis. Here, we demonstrate that microtubule, dynein, and kinesin-1 are involved in PEDV infection and can influence PEDV fusion and accumulation in the perinuclear region but cannot affect PEDV attachment or internalization. Furthermore, we adopted a single-virus tracking technique to dynamically observe PEDV intracellular transport with five different types: unidirectional movement toward microtubule plus ends; unidirectional movement toward microtubule minus ends; bidirectional movement along the same microtubule; bidirectional movement along different microtubules and motionless state. Among these types, the functions of dynein and kinesin-1 in PEDV intercellular transport were further analyzed by single-virus tracking and found that dynein and kinesin-1 mainly transport PEDV to the minus and plus ends of the microtubules, respectively; meanwhile, they also can transport PEDV to the opposite ends of the microtubules different from their conventional transport directions and also coordinate the bidirectional movement of PEDV along the same or different microtubules through their cooperation. These results provided deep insights and references to understand the pathogenesis of PEDV as well as to develop vaccines and treatments.

Structure of LRRK2 in Parkinson's disease and model for microtubule interaction.

Leucine-rich repeat kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson's disease1 and is also linked to its idiopathic form2. LRRK2 has been proposed to function in membrane trafficking3 and colocalizes with microtubules4. Despite the fundamental importance of LRRK2 for understanding and treating Parkinson's disease, structural information on the enzyme is limited. Here we report the structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported cryo-electron tomography in situ structure5. We propose that the conformation of the LRRK2 kinase domain regulates its interactions with microtubules, with a closed conformation favouring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin 1 and cytoplasmic dynein 1 in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce the formation of LRRK2 filaments in cells, whereas inhibitors that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

Active forces shape the metaphase spindle through a mechanical instability.

The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.

Thrombin-induced cytoskeleton dynamics in spread human platelets observed with fast scanning ion conductance microscopy.

Platelets are small anucleate blood cells involved in haemostasis. Platelet activation, caused by agonists such as thrombin or by contact with the extracellular matrix, leads to platelet adhesion, aggregation, and coagulation. Activated platelets undergo shape changes, adhere, and spread at the site of injury to form a blood clot. We investigated the morphology and morphological dynamics of human platelets after complete spreading using fast scanning ion conductance microscopy (SICM). In contrast to unstimulated platelets, thrombin-stimulated platelets showed increased morphological activity after spreading and exhibited dynamic morphological changes in the form of wave-like movements of the lamellipodium and dynamic protrusions on the platelet body. The increase in morphological activity was dependent on thrombin concentration. No increase in activity was observed following exposure to other activation agonists or during contact-induced activation. Inhibition of actin polymerization and inhibition of dynein significantly decreased the activity of thrombin-stimulated platelets. Our data suggest that these morphological dynamics after spreading are thrombin-specific and might play a role in coagulation and blood clot formation.

Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action.

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.

Characterization of ciliobrevin A mediated dynein ATPase inhibition on flagellar motility of Leishmania donovani.

Axonemal dyneins are members of AAA+ proteins involved in force generation and are responsible for flagellar motility in eukaryotes. In this study, we characterized the effects of ciliobrevin A (CbA), a dynein ATPase inhibitor, on flagella driven motility of the protozoan parasite Leishmania donovani. Using fast-capture video microscopy, we observed that CbA decreased flagellar beat frequency of swimming parasites in a concentration-dependent manner. Beat frequency of live and reactivated L. donovani decreased by approximately 89% and 41% respectively in the presence of 250µM CbA. This inhibition was lost when CbA was removed, suggesting its effects were reversible. CbA also altered wavelength and amplitude of the flagellum of live parasites. Waveform analysis of live and reactivated L. donovani revealed that CbA significantly affected flagellar waveform by inducing non-uniform bends with the flagellum beating away from the cell axis. These results suggest that CbA sensitive dynein ATPases possibly are responsible for power generation and waveform maintenance of the flagellum of L. donovani. This ability to inhibit axonemal dyneins also emphasizes the use of dynein inhibitors as valuable tools in studying functional roles of axonemal dyneins of flagellated eukaryotes.

Dynein disruption perturbs post-synaptic components and contributes to impaired MuSK clustering at the NMJ: implication in ALS.

The neuromuscular junction (NMJ) allows the transformation of a neuronal message into a mechanical force by muscle contraction and is the target of several neuromuscular disorders. While the neuronal side is under extensive research, the muscle appeared recently to have a growing role in the formation and integrity of the neuromuscular junction. We used an in vitro model of mature myofibers to study the role of dynein on major postsynaptic proteins. We found that dynein affects the expression and the clustering of acetylcholine receptors (AChRs), muscle specific tyrosine kinase (MuSK) and Rapsyn. We also show that myofibers with dynein impairment or from an amyotrophic lateral sclerosis (ALS) model (SOD1(G93A)) show similar defects in myofiber formation and agrin-induced AChR clustering suggesting a role for dynein impairment in ALS progression. Finally, we found that dynein can affect MuSK traffic through the endosomal pathway. Collectively, our studies show that defects in dynein can lead to impairment of muscle NMJ components' expression and clustering. We propose that NMJ defects could happen via defective MuSK traffic and that this could be one of the pathological features involved in neurodegeneration such as ALS.

Erythroblast enucleation is a dynein-dependent process.

Mammalian erythroblasts undergo enucleation through a process thought to be similar to cytokinesis. Microtubule-organizing centers (MTOCs) mediate organization of the mitotic spindle apparatus that separates the chromosomes during mitosis and are known to be crucial for proper cytokinesis. However, the role of MTOCs in erythroblast enucleation remains unknown. We therefore investigated the effect of various MTOC inhibitors on cytokinesis and enucleation using human colony-forming units-erythroid (CFU-Es) and mature erythroblasts generated from purified CD34(+) cells. We found that erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA), a dynein inhibitor, and monastrol, a kinesin Eg5 inhibitor, as well as various inhibitors of MTOC regulators, including ON-01910 (Plk-1), MLN8237 (aurora A), hesperadin (aurora B), and LY294002 (PI3K), all inhibited CFU-E cytokinesis. Among these inhibitors, however, only EHNA blocked enucleation. Moreover, terminally differentiated erythroblasts expressed only dynein; little or none of the other tested proteins was detected. Over the course of the terminal differentiation of human erythroblasts, the fraction of cells with nuclei at the cell center declined, whereas the fraction of polarized cells, with nuclei shifted to a position near the plasma membrane, increased. Dynein inhibition impaired nuclear polarization, thereby blocking enucleation. These data indicate that dynein plays an essential role not only in cytokinesis but also in enucleation. We therefore conclude that human erythroblast enucleation is a process largely independent of MTOCs, but dependent on dynein.

A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells.

Long-distance intracellular transport of organelles, mRNA, and proteins ("cargo") occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

Co-silencing of human Bub3 and dynein highlights an antagonistic relationship in regulating kinetochore-microtubule attachments.

We previously reported that the spindle assembly checkpoint protein Bub3 is involved in regulating kinetochore-microtubule (KT-MT) attachments. Also, Bub3 was reported to interact with the microtubule motor protein dynein. Here we examined how this interaction contributes to KT-MT attachments. Depletion of Bub3 or dynein induced misaligned chromosomes, consistent with their role in KT-MT attachments. Unexpectedly, co-silencing of both proteins partially suppressed the misalignment phenotype and restored chromosome congression. Consistent with these observations, KT-MT attachments in co-depleted cells were stable, able to drive chromosome congression, and produce inter- and intra-kinetochore stretch, indicating they are functional. We suggest that a mutual antagonism exists between Bub3 and dynein to ensure optimal KT-MT attachments.

Loss-of-Function GAS8 Mutations Cause Primary Ciliary Dyskinesia and Disrupt the Nexin-Dynein Regulatory Complex.

Multiciliated epithelial cells protect the upper and lower airways from chronic bacterial infections by moving mucus and debris outward. Congenital disorders of ciliary beating, referred to as primary ciliary dyskinesia (PCD), are characterized by deficient mucociliary clearance and severe, recurrent respiratory infections. Numerous genetic defects, most of which can be detected by transmission electron microscopy (TEM), are so far known to cause different abnormalities of the ciliary axoneme. However, some defects are not regularly discernable by TEM because the ciliary architecture of the axoneme remains preserved. This applies in particular to isolated defects of the nexin links, also known as the nexin-dynein regulatory complex (N-DRC), connecting the peripheral outer microtubular doublets. Immunofluorescence analyses of respiratory cells from PCD-affected individuals detected a N-DRC defect. Genome-wide exome sequence analyses identified recessive loss-of-function mutations in GAS8 encoding DRC4 in three independent PCD-affected families.

Effects of the dynein inhibitor ciliobrevin on the flagellar motility of sea urchin spermatozoa.

Ciliobrevin has recently been found to be a membrane-permeable inhibitor that is specific to AAA+ molecular motors such as cytoplasmic dyneins. In this study, we investigated how ciliobrevin inhibited the motility of sperm from sea urchins: Hemicentrotus pulcherrimus, Pseudocentrotus depressus, and Anthocidaris crassispina. After application of 100 µM of ciliobrevin A to live spermatozoa, swimming speed decreased gradually and flagellar motion stopped almost completely within 5 to 10 min. This inhibition was reversible and the frequency of flagellar beating was reduced in a concentration-dependent manner. Ciliobrevin had similar inhibitory effects on the flagellar beating of demembranated and reactivated sperm and the sliding disintegration of trypsin-treated axonemes. We also analyzed the curvature and shear angle of the beating flagella and found that the proximal region of the sperm flagellum was less sensitive to ciliobrevin compared with more distal regions, where bending motions were blocked completely. Interestingly, the shear angle analysis of flagellar motility showed that ciliobrevin induced highly asymmetric bends in the proximal region of the flagellum. These results suggest that there is heterogeneity in the inhibitory thresholds of dynein motors, which depend on the regions along the flagellar shaft (proximal or distal) and on the sites of doublets in the flagellar cross-section (doublet numbers). We expect that it will be possible to map the functional differences in dynein subtypes along and/or around the cross-sections of flagellar axonemes by analyzing the inhibitory effects of ciliobrevin.

Habituation-Like Decrease of Acetylcholine-Induced Inward Current in Helix Command Neurons: Role of Microtubule Motor Proteins.

The role of kinesin and dynein microtubule-associated molecular motors in the cellular mechanism of depression of acetylcholine-induced inward chloride current (ACh-current) was examined in command neurons of land snails (Helix lucorum) in response to repeated applications of ACh to neuronal soma. This pharmacological stimulation imitated the protocol of tactile stimulation evoking behavioural habituation of the defensive reaction. In this system, a dynein inhibitor (erythro-9-(2-hydroxy-3-nonyl)adenine, 50 µM) decreased the ACh-current depression rate. Kinesin Eg5 inhibitors (Eg5 inhibitor III, 10 µM and Eg5 inhibitor V, trans-24, 15 µM) reduced the degree of current depression, and Eg5 inhibitor V also reduced the initial rate of depression. The results of electrophysiological experiments in combination with mathematical modelling provided evidence of the participation of dyneins and kinesin Eg5 proteins in the radial transport of acetylcholine receptors in command neurons of H. lucorum in the cellular analogue of habituation. Furthermore, these results suggest that the reciprocal interaction between dynein and kinesin proteins located on the same vesicle can lead to reverse their usual direction of transport (dyneins-in exocytosis and kinesin Eg5-in endocytosis).

The dynein inhibitor Ciliobrevin D inhibits the bidirectional transport of organelles along sensory axons and impairs NGF-mediated regulation of growth cones and axon branches.

The axonal transport of organelles is critical for the development, maintenance, and survival of neurons, and its dysfunction has been implicated in several neurodegenerative diseases. Retrograde axon transport is mediated by the motor protein dynein. In this study, using embryonic chicken dorsal root ganglion neurons, we investigate the effects of Ciliobrevin D, a pharmacological dynein inhibitor, on the transport of axonal organelles, axon extension, nerve growth factor (NGF)-induced branching and growth cone expansion, and axon thinning in response to actin filament depolymerization. Live imaging of mitochondria, lysosomes, and Golgi-derived vesicles in axons revealed that both the retrograde and anterograde transport of these organelles was inhibited by treatment with Ciliobrevin D. Treatment with Ciliobrevin D reversibly inhibits axon extension and transport, with effects detectable within the first 20 min of treatment. NGF induces growth cone expansion, axonal filopodia formation and branching. Ciliobrevin D prevented NGF-induced formation of axonal filopodia and branching but not growth cone expansion. Finally, we report that the retrograde reorganization of the axonal cytoplasm which occurs on actin filament depolymerization is inhibited by treatment with Ciliobrevin D, indicating a role for microtubule based transport in this process, as well as Ciliobrevin D accelerating Wallerian degeneration. This study identifies Ciliobrevin D as an inhibitor of the bidirectional transport of multiple axonal organelles, indicating this drug may be a valuable tool for both the study of dynein function and a first pass analysis of the role of axonal transport.

Inhibition of microtubules and dynein rescues human immunodeficiency virus type 1 from owl monkey TRIMCyp-mediated restriction in a cellular context-specific fashion.

IFN-induced restriction factors can significantly affect the replicative capacity of retroviruses in mammals. TRIM5α (tripartite motif protein 5, isoform α) is a restriction factor that acts at early stages of the virus life cycle by intercepting and destabilizing incoming retroviral cores. Sensitivity to TRIM5α maps to the N-terminal domain of the retroviral capsid proteins. In several New World and Old World monkey species, independent events of retrotransposon-mediated insertion of the cyclophilin A (CypA)-coding sequence in the trim5 gene have given rise to TRIMCyp (also called TRIM5-CypA), a hybrid protein that is active against some lentiviruses in a species-specific fashion. In particular, TRIMCyp from the owl monkey (omkTRIMCyp) very efficiently inhibits human immunodeficiency virus type 1 (HIV-1). Previously, we showed that disrupting the integrity of microtubules (MTs) and of cytoplasmic dynein complexes partially rescued replication of retroviruses, including HIV-1, from restriction mediated by TRIM5α. Here, we showed that efficient restriction of HIV-1 by omkTRIMCyp was similarly dependent on the MT network and on dynein complexes, but in a context-dependent fashion. When omkTRIMCyp was expressed in human HeLa cells, restriction was partially counteracted by pharmacological agents targeting MTs or by small interfering RNA-mediated inhibition of dynein. The same drugs (nocodazole and paclitaxel) also rescued HIV-1 from restriction in cat CRFK cells, although to a lesser extent. Strikingly, neither nocodazole, paclitaxel nor depletion of the dynein heavy chain had a significant effect on the restriction of HIV-1 in an owl monkey cell line. These results suggested the existence of cell-specific functional interactions between MTs/dynein and TRIMCyp.

Dynactin functions as both a dynamic tether and brake during dynein-driven motility.

Dynactin is an essential cofactor for most cellular functions of the microtubule motor cytoplasmic dynein, but the mechanism by which dynactin activates dynein remains unclear. Here we use single molecule approaches to investigate dynein regulation by the dynactin subunit p150(Glued). We investigate the formation and motility of a dynein-p150(Glued) co-complex using dual-colour total internal reflection fluorescence microscopy. p150(Glued) recruits and tethers dynein to the microtubule in a concentration-dependent manner. Single molecule imaging of motility in cell extracts demonstrates that the CAP-Gly domain of p150(Glued) decreases the detachment rate of the dynein-dynactin complex from the microtubule and also acts as a brake to slow the dynein motor. Consistent with this important role, two neurodegenerative disease-causing mutations in the CAP-Gly domain abrogate these functions in our assays. Together, these observations support a model in which dynactin enhances the initial recruitment of dynein onto microtubules and promotes the sustained engagement of dynein with its cytoskeletal track.

Pigment granule translocation in red ovarian chromatophores from the palaemonid shrimp Macrobrachium olfersi (Weigmann, 1836): functional roles for the cytoskeleton and its molecular motors.

The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca²âº/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean chromatophores share various features with those of vertebrate pigment cells.

Microtubules are required for efficient epithelial tight junction homeostasis and restoration.

Epithelial tight junctions are critical for creating a barrier yet allowing paracellular transport. Although it is well established that the actin cytoskeleton is critical for preserving the dynamic organization of the tight junction and maintaining normal tight junction protein recycling, contributions of microtubules to tight junction organization and function remain undefined. The aim of this study is to determine the role of microtubules in tight junction homeostasis and restoration. Our data demonstrate that occludin traffics on microtubules and that microtubule disruption perturbs tight junction structure and function. Microtubules are also shown to be required for restoring barrier function following Ca(2+) chelation and repletion. These processes are mediated by proteins participating in microtubule minus-end-directed trafficking but not plus-end-directed trafficking. These studies show that microtubules participate in the preservation of epithelial tight junction structure and function and play a vital role in tight junction restoration, thus expanding our understanding of the regulation of tight junction physiology.

Nuclear motility in glioma cells reveals a cell-line dependent role of various cytoskeletal components.

Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns--thereby forced into a bipolar morphology--displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved.