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PubMed2002

Motor-cargo interactions: the key to transport specificity.

Eukaryotic cells organize their cytoplasm by moving different organelles and macromolecular complexes along microtubules and actin filaments. These movements are powered by numerous motor proteins that must recognize their respective cargoes in order to function. Recently, several proteins that interact with motors have been identified by yeast two-hybrid and biochemical analyses, and their roles in transport are now being elucidated. In several cases, analysis of the binding partners helped to identify new transport pathways, new types of cargo, and transport regulated at the level of motor-cargo binding. We discuss here how different motors of the kinesin, dynein and myosin families recognize their cargo and how motor-cargo interactions are regulated.

Animals↗

PubMed2003

Microtubule-dependent regulation of Rho GTPases during internalisation of Yersinia pseudotuberculosis.

Internalisation of the human pathogen Yersinia pseudotuberculosis via interaction of bacterial invasin with host beta1 integrins depends on the actin cytoskeleton and involves Src family kinases, focal adhesion kinase, p130Crk-associated substrate, proline-rich tyrosine kinase 2, Rac, Arp 2/3 complex and WASP family members. We show here that Rho GTPases are regulated by the microtubule system during bacterial uptake. Interfering with microtubule organisation using nocodazole or paclitaxel suppressed uptake by HeLa cells. The nocodazole effect on microtubule depolymerisation was partially inhibited through overexpression of Rac, Cdc42, RhoG or RhoA and completely prevented by expression of Vav2. This suggests that microtubules influence Rho GTPases during invasin-mediated phagocytosis and in the absence of functional microtubules Vav2 can mimic their effect on one, or more, of the Rho family GTPases. Lastly, overexpression of p50 dynamitin partially inhibited bacterial uptake and this effect was also blocked by co-expression of Vav2, thus further implicating this guanine nucleotide exchange factor in activating Rho GTPases for internalisation during loss of microtubule function.

Biological Transport, Active↗

PubMed1991

Homology of a 150K cytoplasmic dynein-associated polypeptide with the Drosophila gene Glued.

Cytoplasmic dynein is a microtubule-activated ATPase which produces force towards the minus ends of microtubules. It is thought to be responsible for retrograde axonal transport and other aspects of organelle motility and may have a role in the poleward movement of mitotic chromosomes. Cytoplasmic dynein is an oligomeric complex of two catalytic heavy chains and a number of accessory subunits. We now report the cloning and sequencing of a complementary DNA for one of these species, a cytoplasmic dynein-associated polypeptide of relative molecular mass 150,000 (Mr 150K). A full-length cDNA was found to contain an open reading frame of 4.0 kilobases, which is predicted to encode a polypeptide of Mr 145K. It has extensive homology with the product of the Drosophila gene Glued, which encodes a polypeptide of Mr 148K. The Glued mutation is dominant, with pleiotropic developmental defects in heterozygotes and an embryonic lethal phenotype in homozygotes. As dominant mutations may involve disruption of normal protein-protein interactions, the Glued mutation should provide insight into the mode of action of cytoplasmic dynein in vivo.

Amino Acid Sequence↗

PubMed2002

Microtubule release from the centrosome in migrating cells.

In migrating cells, force production relies essentially on a polarized actomyosin system, whereas the spatial regulation of actomyosin contraction and substrate contact turnover involves a complex cooperation between the microtubule (MT) and the actin filament networks (Goode, B.L., D.G. Drubin, and G. Barnes. 2000. Curr. Opin. Cell Biol., 12:63-71). Targeting and capture of MT plus ends at the cell periphery has been described, but whether or not the minus ends of these MTs are anchored at the centrosome is not known. Here, we show that release of short MTs from the centrosome is frequent in migrating cells and that their transport toward the cell periphery is blocked when dynein activity is impaired. We further show that MT release, but not MT nucleation or polymerization dynamics, is abolished by overexpression of the centrosomal MT-anchoring protein ninein. In addition, a dramatic inhibition of cell migration was observed; but, contrary to cells treated by drugs inhibiting MT dynamics, polarized membrane ruffling activity was not affected in ninein overexpressing cells. We thus propose that the balance between MT minus-end capture and release from the centrosome is critical for efficient cell migration.

Animals↗

PubMed2004

Human Zwint-1 specifies localization of Zeste White 10 to kinetochores and is essential for mitotic checkpoint signaling.

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that Zwint-1 is required and is sufficient for kinetochore localization of Zeste White 10 (ZW10) in HeLa cells. Zwint-1 specifies the kinetochore association of ZW10 by interacting with its N-terminal domain. Suppression of synthesis of Zwint-1 by small interfering RNA abolishes the localization of ZW10 to the kinetochore, demonstrating the requirement of Zwint-1 for ZW10 kinetochore localization. In addition, depletion of Zwint-1 affects no mitotic arrest but causes aberrant premature chromosome segregation. These Zwint-1-suppressed cells display chromosome bridge phenotype with sister chromatids inter-connected. Moreover, Zwint-1 is required for stable association of CENP-F and dynamitin but not BUB1 with the kinetochore. Finally, our studies show that Zwint-1 is a new component of the mitotic check-point, as cells lacking Zwint-1 fail to arrest in mitosis when exposed to microtubule inhibitors, yielding interphase cells with multinuclei. As ZW10 and Zwint-1 are absent from yeast, we reasoned that metazoans evolved an elaborate spindle checkpoint machinery to ensure faithful chromosome segregation in mitosis.

Animals↗

PubMed2002

Dynamitin controls Beta 2 integrin avidity by modulating cytoskeletal constraint on integrin molecules.

Dynamitin, a subunit of the microtubule-dependent motor complex, was implicated in cell adhesion by binding to MacMARCKS (Macrophage-enriched myristoylated alanine-rice C kinase substrate). However, how dynamitin is involved in cell adhesion is unclear despite the fact that both MacMARCKS and microtubules regulate beta(2) integrin activation. We report that dynamitin regulates beta(2) integrin avidity toward iC3b by modulating the lateral mobility of beta(2) integrin molecules. Using the single particle tracking method, we found that integrin molecular mobility in cells expressing the fusion protein CFP (cyan fluorescent protein)-dynamitin or CFP-MB (the MacMARCKS binding domain peptide of dynamitin) increased 6-fold over the control cells, suggesting that disturbing dynamitin function dramatically altered the cytoskeletal constraint on beta(2) integrin molecules. Further mechanistic studies revealed that overexpression of dynamitin stimulated the phosphorylation of endogenous MacMARCKS protein, which lead to the enhanced tyrosine phosphorylation of paxillin. This effect of dynamitin correlates with the observation that higher concentration of PKC inhibitor is required to block beta(2) integrin mobility in dynamitin-expressing cells. Although dynamitin acts at the point of MacMARCKS phosphorylation, it is upstream of RhoA, because its effect was blocked by RhoA inhibitor. Thus, we conclude that dynamitin is a part of the cytoskeletal constraint that locks beta(2) integrin in the inactive form.

Animals↗

PubMed1997

The involvement of the intermediate chain of cytoplasmic dynein in binding the motor complex to membranous organelles of Xenopus oocytes.

Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74-1, m74-2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 micrograms/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74-1 and m74-2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column binding assay, both antibodies inhibited the IC-p150Glued interaction. Thus these findings demonstrate that direct IC-p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.

Animals↗

PubMed2006

Role of NuMA in vertebrate cells: review of an intriguing multifunctional protein.

The 236 kDa large coiled-coil protein NuMA plays diverse important roles in vertebrate cells. It is an important component of the nuclear matrix in interphase cells, and is possibly involved in nuclear re-assembly after mitosis. In dividing cells, upon phosphorylation, NuMA disperses into the cytoplasm, associates with cytoplasmic dynein/dynactin to form a complex, and translocates along microtubules to the spindle poles where it organizes and tethers microtubules to spindle poles. It is thought that the stable complex of NuMA/dynein/dynactin is needed to focus microtubule minus ends to the spindle poles. But, it has also been reported that NuMA can organize microtubules in the absence of centrosomes and dynein. Another hypothesis suggests that once localized to the spindle poles, spindle-associated NuMA's exchange with cytoplasmic soluble pools and its stable crosslinking with the microtubule fibers are independent of dynein/dyactin. NuMA's function in spindle microtubule organization is regulated by RanGTP and Pins-related protein LGN. NuMA becomes dephosphorylated, loses its association with dynein/dynactin, and releases from spindle poles after anaphase onset to allow spindle disassembly and reformation of interphase daughter nuclei. The cell-cycle-dependent phosphorylation of NuMA is regulated by the balanced activities of protein kinases and phosphatases. It has been shown that phosphorylation of NuMA by cyclin B/cdc2 kinase allows NuMA to release from the nucleus and to associate with centrosomes and/or microtubules at the spindle poles, while NuMA's dephosphorylation due to the cyclin B degradation allows NuMA to dissociate from the spindle poles after anaphase onset. Overexpression of NuMA interferes with spindle-associated dynein localization and promotes multipolar spindle formation and cancer. On the other hand, NuMA is absent in many kinds of non-proliferating cells and highly differentiated cells. NuMA also functions during meiotic spindle organization in male and female germ cells. Degradation of NuMA results in the breakdown of normal nuclear structure, and has been used as a marker of cell apoptosis. The implications of NuMA protein in somatic cell animal cloning by nuclear transfer are discussed.

Active Transport, Cell Nucleus↗

PubMed2001

In vitro reconstitution of fish melanophore pigment aggregation.

Movement and positioning of melanophore pigment organelles depend on microtubule- and actin-dependent motors, but the regulation of these forces are poorly understood. Here, we describe a cell free and fixed time motility assay for the study of the regulation of microtubule-dependent pigment organelle positioning in vitro. The assay involves introduction of microtubule-asters assembled in clam oocyte lysates into lysates prepared from Fundulus heteroclitus melanophores with either aggregated or dispersed pigment. When asters were introduced in lysates prepared from melanophores with dispersed pigment, pigment organelles bound astral microtubules and were evenly distributed throughout the aster. In contrast, when asters were introduced into lysates prepared from melanophores with aggregated pigment, pigment organelles accumulated around the centrosome, mimicking a pigment aggregate. The addition of anti-dynein intermediate chain antibody (m74-1), previously shown to interfere with binding of dynactin to dynein and thereby causing detachment of dynein from organelles, blocked the ATP-dependent aggregation of pigment in vitro and induced a depletion of pigment from the centrosomal area. The results show that dynein is essential for pigment aggregation and involved in maintenance of evenly dispersed pigment in vitro, analogous to cellular evidence, and suggest a possible role for dynactin in these processes as well.

Actins↗

PubMed2005

Distal spinal and bulbar muscular atrophy caused by dynactin mutation.

Impaired axonal transport has been postulated to play a role in the pathophysiology of multiple neurodegenerative disorders. In this report, we describe the results of clinical and neuropathological studies in a family with an inherited form of motor neuron disease caused by mutation in the p150Glued subunit of dynactin, a microtubule motor protein essential for retrograde axonal transport. Affected family members had a distinct clinical phenotype characterized by early bilateral vocal fold paralysis affecting the adductor and abductor laryngeal muscles. They later experienced weakness and atrophy in the face, hands, and distal legs. The extremity involvement was greater in the hands than in the legs, and it had a particular predilection for the thenar muscles. No clinical or electrophysiological sensory abnormality existed; however, skin biopsy results showed morphological abnormalities of epidermal nerve fibers. An autopsy study of one patient showed motor neuron degeneration and axonal loss in the ventral horn of the spinal cord and hypoglossal nucleus of the medulla. Immunohistochemistry showed abnormal inclusions of dynactin and dynein in motor neurons. This mutation of dynactin, a ubiquitously expressed protein, causes a unique pattern of motor neuron degeneration that is associated with the accumulation of dynein and dynactin in neuronal inclusions.

Action Potentials↗

PubMed2005

Heterozygous R1101K mutation of the DCTN1 gene in a family with ALS and FTD.

A heterozygous R1101K mutation of the p150 subunit of dynactin (DCTN1) is reported in a family with amyotrophic lateral sclerosis (ALS) and co-occurrence of frontotemporal dementia (FTD). Two members of our kindred were affected with motor neuron disease and two with dementia in an autosomal dominant pattern of inheritance. We excluded the involvement of the ALS and FTD-linked genes for copper/zinc superoxide dismutase (SOD1) and tau. The R1101K sequence alteration of the DCTN1 gene may predispose subjects to ALS and FTD.

Amyotrophic Lateral Sclerosis↗

PubMed2004

LIMK1 and CLIP-115: linking cytoskeletal defects to Williams syndrome.

Williams Syndrome is a developmental disorder that is characterized by cardiovascular problems, particular facial features and several typical behavioral and neurological abnormalities. In Williams Syndrome patients, a heterozygous deletion is present of a region on chromosome 7q11.23 (the Williams Syndrome critical region), which spans approximately 20 genes. Two of these genes encode proteins that regulate dynamic aspects of the cytoskeleton of the cell, either via the actin filament system (LIM kinase 1, or LIMK1), or through the microtubule network (cytoplasmic linker protein of 115 kDa, or CLIP-115). The recent findings that knockout mice lacking LIMK1 or CLIP-115 have distinct neurological and behavioural phenotypes, indicates that cytoskeletal defects might play a role in the development of neurological symptoms in Williams Syndrome patients. In this review, we discuss the properties of LIMK and CLIP family proteins, their function in the regulation of the actin and microtubule cytoskeletal systems, respectively, and the relationship with neurodevelopmental aspects of Williams Syndrome.

Actins↗

PubMed2004

Interaction of Cep135 with a p50 dynactin subunit in mammalian centrosomes.

Cep135 is a 135-kDa, coiled-coil centrosome protein important for microtubule organization in mammalian cells [Ohta et al., 2002: J. Cell Biol. 156:87-99]. To identify Cep135-interacting molecules, we screened yeast two-hybrid libraries. One clone encoded dynamitin, a p50 dynactin subunit, which localized at the centrosome and has been shown to be involved in anchoring microtubules to centrosomes. The central domain of p50 binds to the C-terminal sequence of Cep135; this was further confirmed by immunoprecipitation and immunostaining of CHO cells co-expressing the binding domains for Cep135 and p50. Exogenous p50 lacking the Cep 135-binding domain failed to locate at the centrosome, suggesting that Cep135 is required for initial targeting of the centrosome. Altered levels of Cep135 and p50 by RNAi and protein overexpression caused the release of endogenous partner molecules from centrosomes. This also resulted in dislocation of other centrosomal molecules, such as gamma-tubulin and pericentrin, ultimately leading to disorganization of microtubule patterns. These results suggest that Cep135 and p50 play an important role in assembly and maintenance of functional microtubule-organizing centers.

Animals↗

PubMed2001

Spindle positioning in fibroblasts supports an astral microtubule length dependent force generation at the basal membrane.

V79 Chinese hamster fibroblasts that maintain an elongated shape in metaphase occur at a low frequency and often show the spindle asymmetrically positioned. We show here that this aberrant position is corrected in anaphase by an external force, pulling the spindle into place. The force was applied on astral microtubules because spindle motility was hampered when astral microtubules were poorly developed spontaneously, or destroyed by colcemid. Colcemid also abolished the observed downward positioning of centrosomes in anaphase. One pole of the spindle was usually dominant during correction, but occasionally both poles could become subject to pulling making the spindle move perpendicular to the long axis of the cell, which induced reshaping of the cell. The pulling force acted unevenly with short intervals of resting between the pulling. Spindle elongation also varied in rate but showed a different periodicity than translocation of the spindle, and therefore appeared independently regulated. The length of the spindle increased with the length of the cell, and the rate of spindle elongation and pole movement increased with distance moved, indicating that the forces mediated by astral microtubules increase with their length. Arp1/dynactin, not colocalising with tubulin, was more often continuous with microtubules in anaphase B than in metaphase, and was primarily located at the bottom of the cell. Further, shifts in the geometric gravity centre of the cell occurred in the same direction as migration of the spindle. To explain these results, we suggest that astral microtubles transiently anchored at the bottom of the cell are of particular importance for spindle translocation in fibroblasts.

Anaphase↗

PubMed2006

Batten disease (JNCL) is linked to disturbances in mitochondrial, cytoskeletal, and synaptic compartments.

Intracellular pathways leading to neuronal degeneration are poorly understood in the juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease), caused by mutations in the CLN3 gene. To elucidate the early pathology, we carried out comparative global transcript profiling of the embryonic, primary cultures of the Cln3-/- mouse neurons. Statistical and functional analyses delineated three major cellular pathways or compartments affected: mitochondrial glucose metabolism, cytoskeleton, and synaptosome. Further functional studies showed a slight mitochondrial dysfunction and abnormalities in the microtubule cytoskeleton plus-end components. Synaptic dysfunction was also indicated by the pathway analysis, and by the gross upregulation of the G protein beta 1 subunit, known to regulate synaptic transmission via the voltage-gated calcium channels. Intracellular calcium imaging showed a delay in the recovery from depolarization in the Cln3-/- neurons, when the N-type Ca2+ channels had been blocked. The data suggests a link between the mitochondrial dysfunction and cytoskeleton-mediated presynaptic inhibition, thus providing a foundation for further investigation of the disease mechanism underlying JNCL disease.

Animals↗

PubMed2006

Phenotypic changes associated with DYNACTIN-2 (DCTN2) over expression characterise SJSA-1 osteosarcoma cells.

DYNACTIN-2 (DCTN2) localises to chromosome 12q13-q15, a region prone to stable amplification in several cancers. Transient DCTN2 overexpression has a significant impact on cellular phenotype primarily due to disruption of the DYNEIN-dynactin motor. Changes reported include alterations of microtubule-directed movement of molecular (e.g. TP53) and organelle (e.g. Golgi) cargoes towards the nucleus, centrosome biology, cellular movement and mitosis with a potential predisposition to mitotic block and polyploidy. These changes would be expected to be of relevance to carcinogenesis. To investigate this, we report the first study of DCTN2 genomic amplification and sustained DCTN2 overexpression in cancer cells. QFMPCR was employed to characterise the extent of chromosome 12q13-q15 amplicons in SJSA-1, SJRH30, U373MG and CCF-STTG1 cancer cells. DCTN2 amplification was present in SJSA-1, U373MG and SJRH30 cells, yet was incomplete at the 5'-end in SJRH30 cells. Only SJSA-1 cells were characterised by DCTN2 overexpression on Western blot analyses. Microscopy studies distinguished SJSA-1 cells by greater DCTN2 immunofluorescence and diminished centrosome and 58K protein Golgi-marker focus compared to SJRH30 cells. Indirect evidence derived from the published work of others indicated that TP53 transport into the nucleus was unimpaired. Furthermore, we observed that SJSA-1 cells were easy to propagate. In conclusion, persistent DCTN2 overexpression can be tolerated in SJSA-1 cancer cells despite phenotypic abnormalities predicted from transient overexpression studies. This preliminary study does not support a major role for DCTN2 overexpression in carcinogenesis, although further studies would be necessary to confirm this.

Adenocarcinoma↗

PubMed1997

Dynactin phosphorylation is modulated in response to cellular effectors.

Reversible protein phosphorylation has been implicated in the regulation of organelle transport by cytoplasmic dynein. Motor function may be modulated directly by the phosphorylation of dynein or through the phosphorylation of an accessory factor. Dynactin binds to cytoplasmic dynein and is a required activator for dynein-driven vesicular motility. In metabolic labeling studies we have determined that the p150Glued subunit of dynactin is a phosphoprotein. Treatment of Rat2 cells with okadaic acid or with activators of protein kinase A or protein kinase C caused a marked increase in the incorporation of 32P into p150Glued; the increased phosphorylation correlated with activated vesicular transport. Phosphoamino-acid analysis of p150Glued isolated from cells treated with okadaic acid or with activators of either protein kinase A or protein kinase C indicated exclusive labeling of phosphoserine. These results suggest that the phosphorylation of dynactin may serve to regulate intracellular transport catalyzed by cytoplasmic dynein.

Animals↗

PubMed2001

Identification of novel molecular components of the photoreceptor connecting cilium by immunoscreens.

The connecting cilium of photoreceptor cells is the only intracellular link between the morphologically, functionally and biochemically different compartments of the inner and outer segments. The non-motile modified cilium plays an important role in the organization and the function of photoreceptor cells, namely in delivery and turnover of enzymes and substrates of the visual transduction cascade, and the photosensitive membranes of the outer segment. The protein components of the cilium participate in the intracellular transport through the cilium, in the outer segment disk morphogenesis and in the maintenance of discrete membrane domains. In order to identify yet unknown cytoskeletal components of the connecting cilium, a combined biochemical and molecular biological strategy was applied. For this purpose, antibodies were raised against proteins of photoreceptor cell axonemes. Using this AX-4-antiserum, a rat retina cDNA expression library was immunoscreened and clones encoding partial sequences of (i) already known photoreceptor specific proteins; (ii) ubiquitously expressed proteins; (iii) clones with homologies to retinal ESTs; and (iv) clones coding for cytoskeletal proteins were isolated. Further analysis revealed that these candidate clones have homologies to Drosophila flightless I, mouse APC-binding protein EB2, human microtubule associated protein 4 (MAP4), human centrin 3, human cytoplasmic dynein intermediate chain 2C, and human dynamitin.The immunoscreening approach used here was successfully applied to isolate genes encoding yet unknown cytoskeletal proteins of photoreceptor cell axonemes. The obtained information will provide further insight into the role of the connecting cilium in photoreceptor cell function.

Adenomatous Polyposis Coli Protein↗