p150Glued Subunit Of Dynactin Research Articles

Phosphorylation Controls Autoinhibition of Cytoplasmic Linker Protein-170

Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150(Glued) (J. Cell Biol. 2004: 166, 1003-1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an "open" conformation and a higher binding affinity for growing MT ends and p150(Glued) as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the "folded back" conformation shows decreased MT association and does not interact with p150(Glued). We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.

Dynactin Subunit p150Glued Isoforms Notable for Differential Interaction with Microtubules

Dynactin is a multiprotein complex that enhances dynein activity. The largest dynactin subunit, p150Glued, interacts with microtubules through its N-terminal region that contains a globular cytoskeleton-associated protein (CAP)-Gly domain and basic microtubule-binding domain of unknown structure. The p150Glued gene has a complicated intron-exon structure, and many splice isoforms of p150Glued protein have been predicted. Here we describe novel natural 150 kDa isoforms: the p150Glued-1A isoform, whose basic domain is composed of 41 amino acids, and p150Glued-1B with a basic domain of 21 aa because of the lack of exons 5-7 in the corresponding messenger RNA (mRNA). According to reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot data, p150Glued-1A is expressed in nerve tissues, in cultured cells and in embryonic tissues, while 1B is expressed ubiquitously. Overexpression of GFP-p150Glued-1A and -1B fusion proteins and immunostaining of cultured cells with 1A-specific antibodies show that the p150Glued-1A isoform is distributed along microtubules, whereas 1B is associated with microtubule plus-ends. The higher affinity of the p150Glued-1A isoform for microtubules is confirmed by a co-pelleting assay. In fibroblast-like cells, the interaction of p150Glued-1A with microtubules is less dependent on EB1/EB3 and CLIP170 proteins, compared with p150Glued-1B. In polarized cells, p150Glued-1A decorates microtubules that face the leading edge of the cell. The pattern of p150Glued-1A and p150Glued-1B interaction with microtubules and their tissue-specific expression patterns suggest that these isoforms might be involved in cell differentiation and proliferation.

Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7–RILP–p150Glued and late endosome positioning

Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150Glued bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)–LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7–RILP complex to remove p150Glued and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.

Lysosomal proliferation and distal degeneration in motor neurons expressing the G59S mutation in the p150Glued subunit of dynactin

An increasing number of neurodegenerative diseases are being linked to mutations in genes encoding proteins required for axonal transport and intracellular trafficking. A mutation in p150(Glued), a component of the cytoplasmic dynein/dynactin microtubule motor complex, results in the human neurodegenerative disease distal spinal and bulbar muscular atrophy (dSBMA). We have developed a transgenic mouse model of dSBMA; these mice exhibit late-onset, slowly progressive muscle weakness but do not have a shortened lifespan, consistent with the human phenotype. Examination of motor neurons from the transgenic model reveals the proliferation of enlarged tertiary lysosomes and lipofuscin granules, indicating significant alterations in the cellular degradative pathway. In addition, we observe deficits in axonal caliber and neuromuscular junction (NMJ) integrity, indicating distal degeneration of motor neurons. However, sciatic nerve ligation studies reveal that inhibition of axonal transport is not evident in this model. Together, these data suggest that mutant p150(Glued) causes neurodegeneration in the absence of significant changes in axonal transport, and therefore other functions of dynein/dynactin, such as trafficking in the degradative pathway and stabilization of the NMJ are likely to be critical in maintaining the health of motor neurons.

Dynein-dependent nuclear dynamics affect morphogenesis inCandida albicansby means of the Bub2p spindle checkpoint

Candida albicans, the most prevalent fungal pathogen of humans, grows with multiple morphologies. The dynamics of nuclear movement are similar in wild-type yeast and pseudohyphae: nuclei divide across the bud neck. By contrast, in hyphae, nuclei migrate 10-20 microm into the growing germ tube before dividing. We analyzed the role of the dynein-dynactin complex in hyphal and yeast cells using time-lapse fluorescence microscopy. Cells lacking the heavy chain of cytoplasmic dynein or the p150(Glued) subunit of dynactin were defective in the position and orientation of the spindle. Hyphal cells often failed to deliver a nucleus to the daughter cell, resulting in defects in morphogenesis. Under yeast growth conditions, cultures included a mixture of yeast and pseudohyphal-like cells that exhibited distinctive defects in nuclear dynamics: in yeast, nuclei divided within the mother cell, and the spindle position checkpoint protein Bub2p ensured the delivery of the daughter nucleus to the daughter cell before cytokinesis; in pseudohyphal-like cells, pre-mitotic nuclei migrated into the daughter and no checkpoint ensured return of a nucleus to the mother cell before cytokinesis. Analysis of double mutants indicated that Bub2p also mediated the pre-anaphase arrest and polarization of pseudohyphal-like cells. Thus, Bub2p has two distinct roles in C. albicans cells lacking dynein: it mediates pre-anaphase arrest and it coordinates spindle disassembly with mitotic exit.

The centrosome keeps nucleating microtubules but looses the ability to anchor them after the inhibition of dynein-dynactin complex

We inhibited dynein in cells either by the expression of coiled coil-1 (CC1) fragment of dynactin p150Glued subunit or by the microinjection of CC1 protein synthesized in Escherichia coli. CC1 impeded the aggregation of pigment granules in fish melanophores and caused the dispersion of Golgi in Vero and HeLa cells. These data demonstrated the inhibiting effect of CC1 on dynein. In cultured cells, CC1 expression caused the disruption of microtubule array, while the nucleation of new microtubules remained unaltered. This was proved both with in vivo microtubule recovery after nocodazole treatment and with in vitro microtubule polymerization on centrosomes, when the number of nucleated microtubules marginally reduced after the incubation with CC1. Moreover, the inhibiting anti-dynein 74.1 antibodies caused the same effect. Thus we have shown that though dynein is not important for microtubule nucleation, it is essential for the radial organization of microtubules presumably being involved in microtubule anchoring on the centrosome.

Dynactin targets Pavarotti-KLP to the central spindle during anaphase and facilitates cytokinesis inDrosophilaS2 cells

The dynactin complex cooperates with the dynein complex in various systems for mitotic completion. Here we analysed the mitotic phenotype of Drosophila S2 cells following the knockdown of the dynactin subunit p150(Glued). We found that p150(Glued)-depleted cells were delayed in metaphase and that the centrosomes were poorly connected to mitotic spindle poles. In addition, anaphase occurred with asynchronous chromosome segregation. Although cyclin B was degraded in these anaphase cells, Aurora B, MEI-S322 and BubR1 were not released from the non-segregating chromosomes. We also found that the density and organisation of the central spindle were compromised, with Aurora B and polo kinases absent from the diminished number of microtubules. Pavarotti-KLP, a component of the centralspindlin complex required for the formation of stable microtubule bundles, was not immediately targeted to the plus ends of the microtubules following anaphase onset as happened in controls. Instead, it accumulated transiently at the cell cortex during early anaphase and its targeting to the central spindle was delayed. These data suggest that the dynactin complex contributes to cytokinesis by promoting stable targeting of the centralspindlin complex to microtubule plus ends at anaphase onset. The contribution of the dynein-dynactin complex to synchronous chromosome segregation and cytokinesis is discussed.

Structural Basis for the Activation of Microtubule Assembly by the EB1 and p150 Glued Complex

Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. Here we report the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued. Each molecule of the EB1 dimer contains two helices forming a conserved four-helix bundle, while also providing p150Glued binding sites in its flexible tail region. Combining crystallography, NMR, and mutational analyses, our studies reveal the critical interacting elements of both EB1 and p150Glued, whose mutation alters microtubule polymerization activity. Moreover, removal of the key flexible tail from EB1 activates microtubule assembly by EB1 alone, suggesting that the flexible tail negatively regulates EB1 activity. We, therefore, propose that EB1 possesses an auto-inhibited conformation, which is relieved by p150Glued as an allosteric activator.

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.

Trapping of normal EB1 ligands in aggresomes formed by an EB1 deletion mutant

BackgroundEB1 is a microtubule tip-associated protein that interacts with the APC tumour suppressor protein and the p150glued subunit of dynactin. We previously reported that an EB1 deletion mutant that retains both of these interactions but does not directly associate with microtubules (EB1-ΔN2-GFP) spontaneously formed perinuclear aggregates when expressed in COS-7 cells.ResultsIn the present study live imaging indicated that EB1-ΔN2-GFP aggregates underwent dynamic microtubule-dependent changes in morphology and appeared to be internally cohesive. EB1-ΔN2-GFP aggregates were phase-dense structures that displayed microtubule-dependent accumulation around the centrosome, were immunoreactive for both the 20s subunit of the proteasome and ubiquitin, and induced the collapse of the vimentin cytoskeleton. Fractionation studies revealed that a proportion of EB1-ΔN2-GFP was detergent-insoluble and ubiquitylated, indicating that EB1-ΔN2-GFP aggregates are aggresomes. Immunostaining also revealed that APC and p150glued were present in EB1-ΔN2-GFP aggregates, whereas EB3 was not. Furthermore, evidence for p150glued degradation was found in the insoluble fraction of EB1-ΔN2-GFP transfected cultures.ConclusionOur data indicate that aggresomes can be internally cohesive and may not represent a simple "aggregate of aggregates" assembled around the centrosome. Our observations also indicate that a partially misfolded protein may retain the ability to interact with its normal physiological ligands, leading to their co-assembly into aggresomes. This supports the idea that the trapping and degradation of co-aggregated proteins might contribute to human pathologies characterised by aggresome formation.

Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization.

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150Glued are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH2 terminus of p150Glued binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150Glued and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH2-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH2 and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.

Mutant Huntingtin Blocks BDNF Transport

The mechanisms by which polyglutamine (polyQ) expansion in the huntingtin protein (htt) cause neuronal pathogenesis are unclear (see Ross). Gauthier et al. report that wild-type huntingtin is required for proper vesicular transport of brain-derived neurotrophic factor (BDNF) through its interactions with huntingtin-associated protein-1 (HAP1), which in turn interacts with the p150 Glued subunit of dynactin, a required regulator of the microtubule motor dynein. Vesicle movement was monitored by recording the movement of BDNF-eGFP (enhanced green fluorescent protein)-tagged vesicles by ultrafast 3D videomicroscopy in NG108-15 neuroblastoma cells expressing either wild-type htt or polyQ-htt. Only wild-type htt promoted the movement of vesicles, measured as either the proportion of moving vesicles or the velocity of vesicle movement. To determine the relevance to Huntington's disease, the authors analyzed BDNF vesicular transport rates in wild-type cells, cells with one mutant htt allele (polyQ-htt), and cells with two mutant htt alleles. Cells with polyQ-htt showed decreased vesicle velocity, and overexpression of wild-type htt increased the velocity of vesicle movement. HAP1 was required for the htt stimulation of vesicle movement, because reduction of HAP1 by small interfering RNA blocked htt's stimulation of transport, and htt truncation mutants that did not interact with HAP1 did not stimulate transport. Sucrose gradient sedimentation analysis of wild-type cells or polyQ-htt cells indicated that htt was present in the dynein and dynactin protein complex, whereas polyQ-htt was present in a lower density fraction. In cells from Huntington patients, a disruption in the dynein and dynactin complex was observed. PolyQ-htt inhibited the interaction of HAP1 and various motor proteins (components of both kinesin and dynein) with microtubules. Release of BDNF after an initial stimulation was compromised in cells with polyQ-htt. Thus, the disruption of vesicular transport by polyQ-htt appears to block the refilling of the releasable pool of BDNF, which results in loss of trophic support and cell death. C. A. Ross, Huntington's disease: New paths to pathogenesis. Cell 118 , 4-7 (2004). [Online Journal] L. R. Gauthier, B. C. Charrin, M. Borrell-Pagès, J. P. Dompierre, H. Rangone, F. P. Cordelières, J. de Mey, M. E. MacDonald, V. Leßmann, S. Humbert, F. Saudou, Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules. Cell 118 , 127-138 (2004). [Online Journal]

Analysis of the dynein-dynactin interaction in vitro and in vivo.

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.

A glue for microtubule cargos

Vaughan et al., reporting on page 305, have used live-cell imaging to characterize the dynamic interactions of dynactin with growing microtubule plus-ends. The results provide substantial new insights into the regulation of these interactions, and suggest a new model for organelle transport and anchoring. Figure Golgi membranes (yellow) move after encountering microtubule-bound GFP-p150Glued. Previous work identified the p150Glued subunit of dynactin as a specific binding partner for cytoplasmic dynein. To characterize this protein further, the authors tracked GFP-tagged p150Glued using time-lapse imaging. P150Glued associated with growing microtubule plus-ends to form “comet tails,” which reflect binding followed by delayed release. The delay suggests that p150Glued binding to the growing plus-ends may be followed by multiple molecular events at the microtubule tip, including the phosphorylation of p150Glued that mediates its release. Contact of p150Glued comet tails with Golgi-derived membranes was correlated with the initiation of transport of these membranes. Vaughan et al. propose that growing microtubules probe the cytoplasm for organelles in need of transport. Dynactin on the organelles captures the microtubules, and also recruits motor proteins through its specific interaction with cytoplasmic dynein. A similar search-and-capture model has been proposed for kinetochore–microtubule interactions, potentially revealing a universal mechanism for microtubule-based transport. ▪

A molecular genetic analysis of the interaction between the cytoplasmic dynein intermediate chain and the glued (dynactin) complex.

The microtubule motor cytoplasmic dynein performs multiple cellular functions; however, the regulation and targeting of the motor to different cargoes is not well understood. A biochemical interaction between the dynein intermediate chain subunit and the p150-Glued component of the dynein regulatory complex, dynactin, has supported the hypothesis that the intermediate chain is a key modulator of dynein attachment to cellular cargoes. In this report, we identify multiple intermediate chain polypeptides that cosediment with the 19S dynein complex and two differentially expressed transcripts derived from the single cytoplasmic dynein intermediate chain (Cdic) gene that differ in the 3' untranslated region sequence. These results support previous observations of multiple Cdic gene products that may contribute to the specialization of dynein function. Most significantly, we provide genetic evidence that the interaction between the dynein intermediate chain and p150-Glued is functionally relevant. We use a genomic Cdic transgene to show that extra copies of the dynein intermediate chain gene act to suppress the rough eye phenotype of the mutant Glued(1), a mutation in the p150-Glued subunit of dynactin. Furthermore, we show that the interaction between the dynein intermediate chain and p150-Glued is dependent on the dosage of the Cdic gene. This result suggests that the dynein intermediate chain may be a limiting component in the assembly of the dynein complex and that the regulation of the interaction between the dynein intermediate chain and dynactin is critical for dynein function.

Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine repeat in the HD protein huntingtin. Huntingtin's localization within the cell includes an association with cytoskeletal elements and vesicles. We previously identified a protein (HAP1) which binds to huntingtin in a glutamine repeat length-dependent manner. We now report that HAP1 interacts with cytoskeletal proteins, namely the p150 Glued subunit of dynactin and the pericentriolar protein PCM-1. Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils. We examined the interaction of HAP1 with p150 Glued . Binding of HAP1 to p150 Glued (amino acids 879-1150) was confirmed in vitro by binding of p150 Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads. Also, HAP1 co-immunoprecipitated with p150 Glued from brain extracts, indicating that the interaction occurs in vivo . Like HAP1, p150 Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction. Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150 Glued partially co-localize. These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins. Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD.

Phosphorylation by p34cdc2 Protein Kinase Regulates Binding of the Kinesin-related Motor HsEg5 to the Dynactin Subunit p150Glued

The kinesin-related motor HsEg5 is essential for centrosome separation, and its association with centrosomes appears to be regulated by phosphorylation of tail residue threonine 927 by the p34(cdc2) protein kinase. To identify proteins able to interact with the tail of HsEg5, we performed a yeast two-hybrid screen with a HsEg5 stalk-tail construct as bait. We isolated a cDNA coding for the central, alpha-helical region of human p150(Glued), a prominent component of the dynactin complex. The interaction between HsEg5 and p150(Glued) was enhanced upon activation of p34(CDC28), the budding yeast homolog of p34(cdc2), provided that HsEg5 had a phosphorylatable residue at position 927. Phosphorylation also enhanced the specific binding of p150(Glued) to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly. Immunofluorescence microscopy revealed co-localization of HsEg5 and p150(Glued) during mitosis but not during interphase, consistent with a cell cycle-dependent association between the two proteins. Taken together, these results suggest that HsEg5 and p150(Glued) may interact in mammalian cells in vivo and that p34(cdc2) may regulate this interaction. Furthermore, they imply that the dynactin complex may functionally interact not only with dynein but also with kinesin-related motors.

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 p150Gluedsubunit 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 of32P into p150Glued; the increased phosphorylation correlated with activated vesicular transport. Phosphoamino-acid analysis of p150Gluedisolated 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.

Actin-related protein 1 and cytoplasmic dynein-based motility - what's the connection?

The actin-related protein Arp1 works in conjunction with the microtubule-based motor cytoplasmic dynein to drive many types of intracellular motility. In vertebrate cells, Arp1 is present exclusively in the form of a 37-nm filament that constitutes the backbone of dynactin, a 1.2-MDa macromolecular complex containing nine other polypeptides. Dynactin has been proposed to function as the link between dynein and its cargo. Recent work indicates that the dynactin subunit p150 Glued mediates the interaction of the dynactin molecule with dynein and microtubules, leaving the Arp1 filament as a possible cargo-binding domain. Mechanisms for binding of F-actin to membranes are discussed as models of the Arp1-membrane interaction.