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Results for “Dynactin Complex”

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PubMed2006

Examining how the spatial organization of chromatin signals influences metaphase spindle assembly.

During cell division, the proper assembly of a microtubule-based bipolar spindle depends on signals from chromatin. However, it is unknown how the spatial organization of chromatin signals affects spindle morphology. Here, we use paramagnetic chromatin beads, and magnetic fields for their alignment in cell-free extracts, to examine the spatial components of signals that regulate spindle assembly. We find that for linear chromatin-bead arrays that vary by eightfold in length, metaphase spindle size and shape are constant. Our findings indicate that, although chromatin provides cues for microtubule formation, metaphase spindle organization, which is controlled by microtubule-based motors, is robust to changes in the shape of chromatin signals.

Animals↗

PubMed2003

Mutant dynactin in motor neuron disease.

Impaired axonal transport in motor neurons has been proposed as a mechanism for neuronal degeneration in motor neuron disease. Here we show linkage of a lower motor neuron disease to a region of 4 Mb at chromosome 2p13. Mutation analysis of a gene in this interval that encodes the largest subunit of the axonal transport protein dynactin showed a single base-pair change resulting in an amino-acid substitution that is predicted to distort the folding of dynactin's microtubule-binding domain. Binding assays show decreased binding of the mutant protein to microtubules. Our results show that dysfunction of dynactin-mediated transport can lead to human motor neuron disease.

Amino Acid Motifs↗

PubMed2002

Targeted mutation of Cyln2 in the Williams syndrome critical region links CLIP-115 haploinsufficiency to neurodevelopmental abnormalities in mice.

Williams syndrome is a neurodevelopmental disorder caused by the hemizygous deletion of 1.6 Mb on human chromosome 7q11.23. This region comprises the gene CYLN2, encoding CLIP-115, a microtubule-binding protein of 115 kD. Using a gene-targeting approach, we provide evidence that mice with haploinsufficiency for Cyln2 have features reminiscent of Williams syndrome, including mild growth deficiency, brain abnormalities, hippocampal dysfunction and particular deficits in motor coordination. Absence of CLIP-115 also leads to increased levels of CLIP-170 (a closely related cytoplasmic linker protein) and dynactin at the tips of growing microtubules. This protein redistribution may affect dynein motor regulation and, together with the loss of CLIP-115-specific functions, underlie neurological alterations in Williams syndrome.

Animals↗

PubMed2002

Degenerate mice.

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Animals↗

PubMed2004

Par6alpha signaling controls glial-guided neuronal migration.

Neuronal migrations along glial fibers provide a primary pathway for the formation of cortical laminae. To examine the mechanisms underlying glial-guided migration, we analyzed the dynamics of cytoskeletal and signaling components in living neurons. Migration involves the coordinated two-stroke movement of a perinuclear tubulin 'cage' and the centrosome, with the centrosome moving forward before nuclear translocation. Overexpression of mPar6alpha disrupts the perinuclear tubulin cage, retargets PKCzeta and gamma-tubulin away from the centrosome, and inhibits centrosomal motion and neuronal migration. Thus, we propose that during neuronal migration the centrosome acts to coordinate cytoskeletal dynamics in response to mPar6alpha-mediated signaling.

Animals↗

PubMed2001

Refinement of the PARK3 locus on chromosome 2p13 and the analysis of 14 candidate genes.

Parkinson's disease (PD) is a common neurodegenerative disorder with clinical features of bradykinesia, rigidity, resting tremor and postural instability resulting from the deficiency of dopamine in the nigrostriatal system. Previously we mapped a susceptibility gene for an autosomal dominant form of PD to a 10.6 cM region of chromosome 2p (PARK3; OMIM 602404). A common haplotype shared by two North American kindreds (Families B and C) genealogically traced to Southern Denmark and Northern Germany suggested a founder effect. Here we report progress in the refinement of the PARK3 locus and sequence analysis of candidate genes within the region. Members of families B and C were genotyped using polymorphic markers, reducing the minimum common haplotype to eight markers spanning a physical distance of 2.5 Mb. Analysis of 14 genes within the region did not reveal any potentially pathogenic mutations segregating with the disease, implying that none of these genes are likely candidates for PARK3.

Adaptor Proteins, Signal Transducing↗

PubMed2006

A dynein loading zone for retrograde endosome motility at microtubule plus-ends.

In the fungus Ustilago maydis, early endosomes move bidirectionally along microtubules (MTs) and facilitate growth by local membrane recycling at the tip of the infectious hypha. Here, we set out to elucidate the molecular mechanism of this process. We show that endosomes travel by Kinesin-3 activity into the hyphal apex, where they reverse direction and move backwards in a dynein-dependent manner. Our data demonstrate that dynein, dynactin and Lis1 accumulate at MT plus-ends within the hyphal tip, where they provide a reservoir of inactive motors for retrograde endosome transport. Consistently, endosome traffic is abolished after depletion of the dynein activator Lis1 and in Kinesin-1 null mutants, which was due to a defect in targeting of dynein and dynactin to the apical MT plus-ends. Furthermore, biologically active GFP-dynein travels on endosomes in retrograde and not in anterograde direction. Surprisingly, a CLIP170 homologue was neither needed for dynein localization nor for endosome transport. These results suggest an apical dynein loading zone in the hyphal tip, which ensure that endosomes reach the expanding growth region before they reverse direction.

Biological Transport↗

PubMed2004

Cyclin B degradation leads to NuMA release from dynein/dynactin and from spindle poles.

The protein NuMA localizes to mitotic spindle poles where it contributes to the organization of microtubules. In this study, we demonstrate that NuMA loses its stable association with the spindle poles after anaphase onset. Using extracts from Xenopus laevis eggs, we show that NuMA is dephosphorylated in anaphase and released from dynein and dynactin. In the presence of a nondegradable form of cyclin B (Delta90), NuMA remains phosphorylated and associated with dynein and dynactin, and remains localized to stable spindle poles that fail to disassemble at the end of mitosis. Inhibition of NuMA or dynein allows completion of mitosis, despite inducing spindle pole abnormalities. We propose that NuMA functions early in mitosis during the formation of spindle poles, but is released from the spindle after anaphase, to allow spindle disassembly and remodelling of the microtubule network.

Anaphase↗

PubMed2003

Biochemical changes contributing to functional quiescence in lacrimal gland acinar cells after chronic ex vivo exposure to a muscarinic agonist.

Profound secretory dysfunction can be associated with relatively modest lymphocytic infiltration of the lacrimal and salivary glands of Sjögren's syndrome (SjS) patients. SjS patients' sera contain autoantibodies to M3 muscarinic acetylcholine receptors (MAChR) that have variously been reported to have agonistic and antagonistic effects. We sought to identify consequences of chronic agonist stimulation by maintaining acinar cells from rabbit lacrimal glands for 20 h in the presence or absence of 10 microM carbachol (CCh). Exposure to CCh diminished the cells' ability to elevate cytosolic Ca2+ and secrete beta-hexosaminidase in response to acute stimulation with 100 microM CCh, but it enhanced their secretory responses to phenylephrine and ionomycin. Secretory vesicles appeared normal by electron microscopy, but confocal fluorescence microscopy revealed depletion of the secretory vesicle membrane marker, rab3D, and decreased ability to recruit secretory transport vesicles in response to acute 100 microM CCh. Additionally, the apical cortical actin cytoskeleton was disrupted and diminished compared to the basal-lateral cortical network. Subcellular fractionation analyses revealed that total membrane phase protein content was increased. The contents of beta-hexosaminidase and MAChR relative to total protein were not significantly altered, and MAChR abundance in the plasma membrane fraction was increased as the result of redistribution from endomembrane pools. However, relative cellular contents of the heterotrimeric guanosine triphosphate (GTP)-binding proteins, Gq and G11, were decreased. Additional biochemical changes included decreased contents of 47 kDa Gs and Gi3, protein kinase Calpha and rab3D and polymeric immunoglobulin (Ig) receptors; internalization of Na,K-ATPase from the plasma membranes to endomembrane compartments and decreased content of beta-hexosaminidase in the lysosomes. The observations demonstrate that chronic exposure to a MAChR agonist induces refractoriness to optimal stimulation, without causing receptor downregulation, by downregulating postreceptor-signalling mediators and effectors. The cells' secretory mechanisms for IgA and electrolytes also appear to be impaired, as does their ability to properly sort proteins to the lysosomes.

Animals↗

PubMed2000

Interaction of peroxisomes with microtubules. In vitro studies using a novel peroxisome-microtubule binding assay.

The association of membrane-bounded cell organelles to microtubules is crucial for determination of their shape, intracellular localization and translocation. We have shown previously the high affinity binding of peroxisomes to microtubules which appears to be of static nature as in vivo studies indicate that only a few peroxisomes move along the microtubular tracks. In order to characterize the interactions of peroxisomes with microtubules, we have developed a semiquantitative in vitro binding assay, which is based on the association of highly purified rat liver peroxisomes to microtubules coated onto microtiterplates. The binding was visualized by differential interference contrast and immunofluorescence using a confocal laser scanning microscope. The binding was concentration dependent and saturable, being affected by time, temperature, and pH. Addition of ATP or the motor proteins kinesin and dynein increased the binding capacity, while ATP-depletion or microtubule associated proteins (MAPs) decreased it. KCl treatment of peroxisomes reduced the binding, which was restored by dialyzed KCl-stripping eluate as well as by rat liver cytosol. The reconstituting effect of cytosol was abolished by its pretreatment with proteases or N-ethylmaleimide. Moreover, the treatment of peroxisomes with proteases or N-ethylmaleimide reduced their binding, which was not reversed by cytosol. These results suggest the involvement of a peroxisomal membrane protein and cytosolic factor(s) in the binding of peroxisomes to microtubules. This notion is supported by the observation that distinct subfractions of dialyzed KCl-stripping eluate obtained by gel chromatography augmented the binding. Those subfractions, as well as purified peroxisome fractions, exhibited strong immunoreactivity with an antibody to cytoplasmic linker protein (CLIP)-115, revealing a 70-kDa polypeptide. Moreover, immunodepletion of KCl-stripping eluate and its subfractions with an antibody to the conserved microtubule binding domain of CLIPs, abolished their promoting effect on the binding, thus suggesting the involvement of a CLIP-related protein in the binding of peroxisomes to microtubules.

Adenosine Triphosphate↗

PubMed1997

Casein kinase II binds to and phosphorylates cytoplasmic dynein.

We have isolated a 27-kDa protein that binds to cytoplasmic dynein. Microsequencing of a 17-amino acid peptide of this polypeptide yielded a sequence which completely matched the predicted sequence of the beta subunit of casein kinase II, a highly conserved serine/threonine kinase. Affinity chromatography using a dynein column indicates that both the alpha and beta subunits of casein kinase II are retained by the column from rat brain cytosol. Although dynactin is also bound to the column, casein kinase II is not a dynactin subunit. Casein kinase II does not co-immunoprecipitate with dynactin, and it binds to a dynein intermediate chain column which has been preblocked with excess p150(Glued), a treatment that inhibits the binding of dynactin from cytosol. Bacterially expressed and purified rat dynein intermediate chain can be phosphorylated by casein kinase II in vitro. Further, native cytoplasmic dynein purified from rat brain can also be phosphorylated by casein kinase II in vitro. We propose that CKII may be involved in the regulation of dynein function possibly by altering its cargo specificity or its ability to interact with dynactin.

Animals↗

PubMed2001

Cytoplasmic dynein intermediate chain phosphorylation regulates binding to dynactin.

Previously, we identified dynactin as a cargo receptor or adaptor for cytoplasmic dynein, mediated by an interaction between the dynein intermediate chain and p150(Glued). To test phosphorylation as a potential regulatory mechanism for this interaction, we analyzed cytoplasmic dynein by two-dimensional gel analysis and detected two intermediate chain variants, one of which was eliminated by phosphatase treatment. Overlay assays demonstrated that p150(Glued) bound dephosphorylated but not phosphorylated intermediate chains. We then subjected the purified cytoplasmic dynein intermediate chain to mass spectrometry and identified a single phosphorylated tryptic fragment corresponding to the p150(Glued)-binding domain. Fragmentation and retention time analysis mapped the phosphorylation site to serine 84. Site-directed mutants designed to mimic the dephosphorylated or phosphorylated intermediate chain disrupted both in vitro phosphorylation and in vivo phosphorylation of transfected proteins. Mutants mimicking the dephosphorylated form bound p150(Glued) in vitro and overexpression perturbed transport of dynein-dependent membranes. Mutants mimicking the phosphorylated form displayed diminished p150(Glued) binding in vitro and did not disrupt dynein-mediated transport when expressed in vivo. These findings represent the first mapping of an intermediate chain phosphorylation site and suggest that this phosphorylation plays an important role in regulating the binding of cytoplasmic dynein to dynactin.

Amino Acid Sequence↗

PubMed2004

STAT5B-mediated growth hormone signaling is organized by highly dynamic microtubules in hepatic cells.

In the last decade, the notion that microtubules are critical to the spatial organization of signal transduction and contribute to the transmission of signals to downstream targets has been proposed. Because the STAT5B transduction and transcription factor is the major STAT protein activated by growth hormone stimulation in hepatocytes and is a crossroads between many signaling pathways, we studied the involvement of microtubules in STAT5B-mediated growth hormone signaling pathway in the highly differentiated and polarized WIF-B hepatic cell line. We showed that depolymerization of the microtubule network impaired STAT5B translocation to the nucleus upon growth hormone treatment. A significant amount of STAT5B binds to microtubules, while STAT5A and STAT3 are exclusively compartmentalized in the cytosol. Moreover, taxol-induced stabilization of microtubules released STAT5B from its binding, and we show that STAT5B binds specifically to the highly dynamic microtubules and is absent of the stable microtubule subpopulation. The specific involvement of dynamic microtubule subpopulation in growth hormone signaling pathway was confirmed by the inhibition of growth hormone-induced STAT5B nuclear translocation after stabilization of microtubules or specific disruption of highly dynamic microtubules. Upon growth hormone treatment, MT-bound STAT5B was rapidly released from microtubules by a dynein-dependent transport to the nucleus. Altogether, our findings indicate that the labile microtubule subpopulation specifically and dynamically organizes STAT5B-mediated growth hormone signaling in hepatic cells.

Active Transport, Cell Nucleus↗

PubMed2005

Targeting and trafficking of the human thiamine transporter-2 in epithelial cells.

Humans lack biochemical pathways for thiamine synthesis, so cellular requirements are met via specific carrier-mediated uptake pathways. Two proteins from the solute carrier SLC19A gene family have been identified as human thiamine transporters (hTHTRs), SLC19A1 (hTHTR1) and SLC19A2 (hTHTR2). Both of these transporters are co-expressed but are differentially targeted in polarized cell types that mediate vectorial thiamine transport (e.g. renal and intestinal epithelia). It is important to understand the domain structure of these proteins, namely which regions within the polypeptide sequence are important for physiological delivery to the cell surface, in order to understand the impact of clinically relevant mutations on thiamine transport. Here we have characterized the mechanisms regulating hTHTR2 distribution by using live cell imaging methods that resolve the targeting and trafficking dynamics of full-length hTHTR2, a series of hTHTR2 truncation mutants, as well as chimeras comprising the hTHTR1 and hTHTR2 sequence. We showed the following: (i) that the cytoplasmic COOH-tail of hTHTR2 is not essential for apical targeting in polarized cells; (ii) that delivery of hTHTR2 to the cell surface is critically dependent on the integrity of the transmembrane backbone of the polypeptide so that minimal truncations abrogate cell surface expression of hTHTR2; and (iii) video rate images of hTHTR2-containing intracellular vesicles displayed rapid bi-directional trafficking events to and from the cell surface impaired by microtubule-disrupting but not microfilament-disrupting agents as well as by overexpression of the dynactin subunit dynamitin (p50). Finally, we compared the behavior of hTHTR2 with that of hTHTR1 and the human reduced folate carrier (SLC19A1) to underscore commonalities in the cell surface targeting mechanisms of the entire SLC19A gene family.

Animals↗

PubMed2006

Phosphoinositide 3-kinase C2alpha links clathrin to microtubule-dependent movement.

Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) is a type II PI-3-kinase that has been implicated in several important membrane transport and signaling processes. We previously found that overexpression of PI3K-C2alpha inhibits clathrin-mediated membrane trafficking and induces proliferation of novel clathrin-coated structures within the cytoplasm. Using fluorescently tagged fusions of PI3K-C2alpha and clathrin, we explored the behavior of these structures in intact cells. Both proteins are present in the structures, and using rapid image acquisition and fluorescence photoactivation probes, we find that they exhibit localized, rapid mobility (5-20 microm/s). The movement is micro-tubule-based as revealed by use of inhibitors, and PI3K-C2alpha accumulates on microtubules rapidly and reversibly following cytoplasmic acidification, which also blocks movement. Dynactin mediates the movement of these clathrin-PI3K-C2alpha structures, since disruption of dynactin function by overexpression of its p50 subunit also inhibits movement. Finally, immunoprecipitation experiments reveal an interaction between endogenous PI3K-C2alpha and dynactin subunits. Together, these results reveal a molecular linkage between PI3K-C2alpha and the microtubule motor machinery, with implications for membrane trafficking in intact cells.

Animals↗

PubMed2003

Paradigms for the identification of new genes in motor neuron degeneration.

It is estimated that between 10-20% of amyotrophic lateral sclerosis (ALS) is familial and these cases encompass recessive and dominant modes of inheritance. So far, mutations in three genes, superoxide dismutase 1 (SOD1), the p150 subunit of dynactin (DCTN1), and alsin have been shown to be directly causal for motor neuron degeneration in humans. However, clearly the disorder is genetically heterogeneous and other causal genes remain to be found that explain the vast majority of familial ALS cases. Human genetics can be problematical in that it is difficult to detect linkage in disorders in which multiple loci give similar phenotypes and where families are often small. In addition, the vertical collection of generations is often not possible with late onset disorders. An excellent genetic model of humans is provided by the mouse. We can use mouse models of neurodegeneration to find new genes in the human population. These models are not exact replicas of the human condition, but are the mouse equivalent and are incredibly valuable resources for highlighting genes and biochemical pathways disrupted in ALS and other diseases. In addition mouse models give us access to both control and affected tissues, at all stages of development and disease, thus greatly facilitating our understanding of pathogenesis. They also provide us with model systems for testing new therapies. Here we describe the approach taken to the characterization of new models of motor neuron disease and illustrate this with examples, including a recently characterized mouse model, Legs at odd angles (Loa).

Amyotrophic Lateral Sclerosis↗

PubMed1994

Molecular motors are differentially distributed on Golgi membranes from polarized epithelial cells.

Microtubules (MT) are required for the efficient transport of membranes from the trans-Golgi and for transcytosis of vesicles from the basolateral membrane to the apical cytoplasm in polarized epithelia. MTs in these cells are primarily oriented with their plus ends basally near the Golgi and their minus-ends in the apical cytoplasm. Here we report that isolated Golgi and Golgi-enriched membranes from intestinal epithelial cells possess the actin based motor myosin-I, the MT minus-end-directed motor cytoplasmic dynein and its in vitro motility activator dynactin (p150/Glued). The Golgi can be separated into stacks, possessing features of the Golgi cisternae, and small membranes enriched in the trans-Golgi network marker TGN 38/41. Whereas myosin-I is present on all membranes in the Golgi fraction, dynein is present only on the small membrane fraction. Dynein, like myosin-I, is associated with membranes as a cytoplasmic peripheral membrane protein. Dynein and myosin-I coassociate with membranes that bind to MTs and cross-link actin filaments and MTs in a nucleotide-dependent manner. We propose that cytoplasmic dynein moves Golgi membranes along MTs to the cell cortex where myosin-I provides local delivery through the actin-rich cytoskeleton to the apical membrane.

Actins↗

PubMed1996

Opposing motor activities are required for the organization of the mammalian mitotic spindle pole.

We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nu-clear protein that associates with the Mitotic Apparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletition from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end-directed activity of Eg5 antagonizes the minus end-directed activity of cytoplasmic dynein and a minus end-directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors.

Animals↗