Biomedical subjects

Erika L F Holzbaur

Publications and source records attributed to Erika L F Holzbaur.

At least 19 recordsLinked to original sources

PubMed2006

Recruitment of dynein to the Jurkat immunological synapse.

Binding of T cells to antigen-presenting cells leads to the formation of the immunological synapse, translocation of the microtubule-organizing center (MTOC) to the synapse, and focused secretion of effector molecules. Here, we show that upon activation of Jurkat cells microtubules project from the MTOC to a ring of the scaffolding protein ADAP, localized at the synapse. Loss of ADAP, but not lymphocyte function-associated antigen 1, leads to a severe defect in MTOC polarization at the immunological synapse. The microtubule motor protein cytoplasmic dynein clusters into a ring at the synapse, colocalizing with the ADAP ring. ADAP coprecipitates with dynein from activated Jurkat cells, and loss of ADAP prevents MTOC translocation and the specific recruitment of dynein to the synapse. These results suggest a mechanism that links signaling through the T cell receptor to translocation of the MTOC, in which the minus end-directed motor cytoplasmic dynein, localized at the synapse through an interaction with ADAP, reels in the MTOC, allowing for directed secretion along the polarized microtubule cytoskeleton.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Microtubule motors at the intersection of trafficking and transport.

Molecular motors drive the transport of vesicles and organelles within the cell. Traditionally, these transport processes have been considered separately from membrane trafficking events, such as regulated budding and fusion. However, recent progress has revealed mechanistic links that integrate these processes within the cell. Rab proteins, which function as key regulators of intracellular trafficking, have now been shown to recruit specific motors to organelle membranes. Rab-independent recruitment of motors by adaptor or scaffolding proteins is also a key mechanism. Once recruited to vesicles and organelles, these motors can then drive directed transport; this directed transport could in turn affect the efficiency of trafficking events. Here, we discuss this coordinated regulation of trafficking and transport, which provides a powerful mechanism for temporal and spatial control of cellular dynamics.

Animals↗

PubMed2006

Myostatin inhibition slows muscle atrophy in rodent models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease leading to motor neuron cell death, but recent studies suggest that non-neuronal cells may contribute to the pathological mechanisms involved. Myostatin is a negative regulator of muscle growth whose function can be inhibited using neutralizing antibodies. In this study, we used transgenic mouse and rat models of ALS to test whether treatment with anti-myostatin antibody slows muscle atrophy, motor neuron loss, or disease onset and progression. Significant increases in muscle mass and strength were observed in myostatin-antibody-treated SOD1(G93A) mice and rats prior to disease onset and during early-stage disease. By late stage disease, only diaphragm muscle remained significantly different in treated animals in comparison to untreated controls. Myostatin inhibition did not delay disease onset nor extend survival in either the SOD1(G93A) mouse or rat. Together, these results indicate that inhibition of myostatin does not protect against the onset and progression of motor neuron degenerative disease. However, the preservation of skeletal muscle during early-stage disease and improved diaphragm morphology and function maintained through late stage disease suggest that anti-myostatin therapy may promote some improved muscle function in ALS.

Age of Onset↗

PubMed2006

Processive bidirectional motion of dynein-dynactin complexes in vitro.

Cytoplasmic dynein is the primary molecular motor responsible for transport of vesicles, organelles, proteins and RNA cargoes from the periphery of the cell towards the nucleus along the microtubule cytoskeleton of eukaryotic cells. Dynactin, a large multi-subunit activator of dynein, docks cargo to the motor and may enhance dynein processivity. Here, we show that individual fluorescently labelled dynein-dynactin complexes exhibit bidirectional and processive motility towards both the plus and minus ends of microtubules. The dependence of this activity on substrate ATP concentration, nucleotide analogues and inhibitors suggests that bidirectional motility is an active energy-transduction property of dynein-dynactin motor mechano-chemistry. The unique motility characteristics observed may reflect the flexibility of the dynein structure that leads to an enhanced ability to navigate around obstacles in the cell.

Adenosine Triphosphate↗

PubMed2006

Axonal transport and neurodegenerative disease.

Neurons have extensive processes and communication between those processes and the cell body is crucial to neuronal function and survival. Thus, neurons are uniquely dependent on microtubule based transport. Growing evidence supports the idea that deficits in axonal transport contribute to pathogenesis in multiple neurodegenerative diseases. We describe the motor, cytoskeletal, and adaptor proteins involved in axonal transport and their interactions. Data linking disruption of axonal transport to diseases such as ALS are discussed. Finally, we explore the pathways that may cause neuronal dysfunction and death.

Amyotrophic Lateral Sclerosis↗

PubMed2006

A motor neuron disease-associated mutation in p150Glued perturbs dynactin function and induces protein aggregation.

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

Animals↗

PubMed2006

Microtubule binding proteins CLIP-170, EB1, and p150Glued form distinct plus-end complexes.

Microtubule plus-end proteins CLIP-170 and EB1 dynamically track the tips of growing microtubules in vivo. Here we examine the association of these proteins with microtubules in vitro. CLIP-170 binds tubulin dimers and co-assembles into growing microtubules. EB1 binds tubulin dimers more weakly, so no co-assembly is observed. However, EB1 binds to CLIP-170, and forms a co-complex with CLIP-170 and tubulin that is recruited to growing microtubule plus ends. The interaction between CLIP-170 and EB1 is competitively inhibited by the related CAP-Gly protein p150Glued, which also localizes to microtubule plus ends in vivo. Based on these observations, we propose a model in which the formation of distinct plus-end complexes may differentially affect microtubule dynamics in vivo.

Animals↗

PubMed2006

Cytoplasmic dynein/dynactin function and dysfunction in motor neurons.

The microtubule motor protein cytoplasmic dynein and its activator dynactin are essential in higher eukaryotes, due to critical roles in vesicular transport and cell division. Neurons are uniquely sensitive to defects in dynein/dynactin function, which affect retrograde axonal transport, neurotrophic factor signaling, neurofilament transport, mRNA localization, neuronal migration, and protein recycling and degradation. Mutations in either dynein or dynactin lead to motor neuron degeneration and loss. Recent progress in understanding the cellular mechanisms of dynein/dynactin function, and the effects of dynein/dynactin dysfunction has provided new insight into the roles of microtubule-based motility in the neuron.

Animals↗

PubMed2005

Cytoplasmic dynein nomenclature.

A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.

Animals↗

PubMed2005

Mutant superoxide dismutase disrupts cytoplasmic dynein in motor neurons.

Cytoplasmic dynein and dynactin drive retrograde axonal transport in neurons, and mutations in dynein/dynactin cause motor neuron degeneration. To test whether defects in dynein/dynactin function are involved in the neurodegenerative disease amyotrophic lateral sclerosis, we examined neurotracer transport from muscle to motor neuron in a transgenic mouse model of amyotrophic lateral sclerosis. Significant inhibition was observed, which was temporally correlated with declines in muscle strength. No decrease in dynein/dynactin expression was observed, but immunohistochemistry suggests that dynein associates with aggregates of mutant Cu/Zn superoxide dismutase 1. Expression of mutant Cu/Zn superoxide dismutase 1 in primary motor neurons altered the cellular localization of dynein, suggesting an inhibition of dynein/dynactin function. Thus, inhibition of dynein/dynactin function may have a role in motor neuron degeneration in amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis↗

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↗

PubMed2004

The interaction of neurofilaments with the microtubule motor cytoplasmic dynein.

Neurofilaments are synthesized in the cell body of neurons and transported outward along the axon via slow axonal transport. Direct observation of neurofilaments trafficking in live cells suggests that the slow outward rate of transport is due to the net effects of anterograde and retrograde microtubule motors pulling in opposition. Previous studies have suggested that cytoplasmic dynein is required for efficient neurofilament transport. In this study, we examine the interaction of neurofilaments with cytoplasmic dynein. We used fluid tapping mode atomic force microscopy to visualize single neurofilaments, microtubules, dynein/dynactin, and physical interactions between these neuronal components. AFM images suggest that neurofilaments act as cargo for dynein, associating with the base of the motor complex. Yeast two-hybrid and affinity chromatography assays confirm this hypothesis, indicating that neurofilament subunit M binds directly to dynein IC. This interaction is blocked by monoclonal antibodies directed either to NF-M or to dynein. Together these data suggest that a specific interaction between neurofilament subunit M and cytoplasmic dynein is involved in the saltatory bidirectional motility of neurofilaments undergoing axonal transport in the neuron.

Animals↗

PubMed2004

A direct interaction between cytoplasmic dynein and kinesin I may coordinate motor activity.

Cytoplasmic dynein and kinesin I are both unidirectional intracellular motors. Dynein moves cargo toward the cell center, and kinesin moves cargo toward the cell periphery. There is growing evidence that bi-directional motility is regulated in the cell, potentially through direct interactions between oppositely oriented motors. We have identified a direct interaction between cytoplasmic dynein and kinesin I. Using the yeast two-hybrid assay and affinity chromatography, we demonstrate that the intermediate chain of dynein binds to kinesin light chains 1 and 2. The interaction is both direct and specific. Co-immunoprecipitation experiments demonstrate an interaction between endogenous proteins in rat brain cytosol. Double-label immunocytochemistry reveals a partial co-localization of vesicle-associated motor proteins. Together these observations suggest that soluble motors can interact, potentially allowing kinesin I to actively localize dynein to cellular sites of function. There is also a vesicle population with both dynein and kinesin I bound that may be capable of bi-directional motility along cellular microtubules.

Animals↗

PubMed2004

Motor neurons rely on motor proteins.

The importance of active axonal transport to the neuron has been highlighted by the recent discoveries that mutations in microtubule motor proteins result in neurodegenerative diseases. Mutations affecting microtubule motor function have been shown to cause hereditary forms of Charcot-Marie-Tooth disease (type 2A), hereditary spastic paraplegia and motor neuron disease. Although motor neurons appear to be uniquely susceptible to defects in axonal transport, recent work has identified links between perturbations in axonal transport and the pathogenesis of other neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. More broadly, cytoskeletal abnormalities might also be at the root of related disorders such as spinal muscular atrophy, supporting a key role for axonal transport in the pathogenesis of many neurodegenerative diseases.

Animals↗

PubMed2004

Tangled NUDELs?

Explore the source record for details and available documents.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

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↗

PubMed2003

The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization.

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150(Glued) subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150(Glued) in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150(Glued) on microtubule polymerization, and they show that p150(Glued) has a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics.

Animals↗

PubMed2002

Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration.

To test the hypothesis that inhibition of axonal transport is sufficient to cause motor neuron degeneration such as that observed in amyotrophic lateral sclerosis (ALS), we engineered a targeted disruption of the dynein-dynactin complex in postnatal motor neurons of transgenic mice. Dynamitin overexpression was found to disassemble dynactin, a required activator of cytoplasmic dynein, resulting in an inhibition of retrograde axonal transport. Mice overexpressing dynamitin demonstrate a late-onset progressive motor neuron degenerative disease characterized by decreased strength and endurance, motor neuron degeneration and loss, and denervation of muscle. Previous transgenic mouse models of ALS have shown abnormalities in microtubule-based axonal transport. In this report, we describe a mouse model that confirms the critical role of disrupted axonal transport in the pathogenesis of motor neuron degenerative disease.

Amyotrophic Lateral Sclerosis↗