P150 Glued Research Articles

Kif3a interacts with Dynactin subunit p150Glued to organize centriole subdistal appendages

Formation of cilia, microtubule-based structures that function in propulsion and sensation, requires Kif3a, a subunit of Kinesin II essential for intraflagellar transport (IFT). We have found that, Kif3a is also required to organize centrioles. In the absence of Kif3a, the subdistal appendages of centrioles are disorganized and lack p150(Glued) and Ninein. Consequently, microtubule anchoring, centriole cohesion and basal foot formation are abrogated by loss of Kif3a. Kif3a localizes to the mother centriole and interacts with the Dynactin subunit p150(Glued). Depletion of p150(Glued) phenocopies the effects of loss of Kif3a, indicating that Kif3a recruitment of p150(Glued) is critical for subdistal appendage formation. The transport functions of Kif3a are dispensable for subdistal appendage organization as mutant forms of Kif3a lacking motor activity or the motor domain can restore p150(Glued) localization. Comparison to cells lacking Ift88 reveals that the centriolar functions of Kif3a are independent of IFT. Thus, in addition to its ciliogenic roles, Kif3a recruits p150(Glued) to the subdistal appendages of mother centrioles, critical for centrosomes to function as microtubule-organizing centres.

Tubulin-binding cofactor B is a direct interaction partner of the dynactin subunit p150Glued

The dynactin p150(Glued) subunit, encoded by the gene DCTN1, is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport in motor neurons. The p150 subunit is a candidate gene for neurodegenerative diseases, in particular motor neuron and extrapyramidal diseases. Tubulin-binding cofactors are believed to be involved in tubulin biogenesis and degradation and therefore to contribute to microtubule functional diversity and regulation. A yeast-two-hybrid screen for putative interacting proteins of dynactin p150(Glued) has revealed tubulin-folding cofactor B (TBCB). We analyzed the interaction of these proteins and investigated the impact of this complex on the microtubule network in cell lines and primary hippocampal neurons in vitro. We especially concentrated on neuronal morphology and synaptogenesis. Overexpression of both proteins or depletion of TBCB alone does not alter the microtubule network and/or neuronal morphology. The demonstration of the interaction of the transport molecule dynactin and the tubulin-regulating factor TBCB is thought to have an impact on several cellular mechanisms. TBCB expression levels have been found to have only a subtle influence on the microtubule network and neuronal morphology. However, overexpression of TBCB leads to the decreased localization of p150 to the microtubule network that might result in a functional modulation of this protein complex.

The p150Glued CAP-Gly Domain Regulates Initiation of Retrograde Transport at Synaptic Termini

p150(Glued) is the major subunit of dynactin, a complex that functions with dynein in minus-end-directed microtubule transport. Mutations within the p150(Glued) CAP-Gly microtubule-binding domain cause neurodegenerative diseases through an unclear mechanism. A p150(Glued) motor neuron degenerative disease-associated mutation introduced into the Drosophila Glued locus generates a partial loss-of-function allele (Gl(G38S)) with impaired neurotransmitter release and adult-onset locomotor dysfunction. Disruption of the p150(Glued) CAP-Gly domain in neurons causes a specific disruption of vesicle trafficking atterminal boutons (TBs), the distal-most ends of synapses. Gl(G38S) larvae accumulate endosomes along with dynein and kinesin motor proteins within swollen TBs, and genetic analyses show that kinesin and p150(Glued) function cooperatively at TBs to coordinate transport. Therefore, the p150(Glued) CAP-Gly domain regulates dynein-mediated retrograde transport at synaptic termini, and this function of dynactin is disrupted by a mutation that causes motorneuron disease.

TRAPPC9 Mediates the Interaction between p150Glued and COPII Vesicles at the Target Membrane

BackgroundThe transport of endoplasmic reticulum (ER)-derived COPII vesicles toward the ER-Golgi intermediate compartment (ERGIC) requires cytoplasmic dynein and is dependent on microtubules. p150Glued, a subunit of dynactin, has been implicated in the transport of COPII vesicles via its interaction with COPII coat components Sec23 and Sec24. However, whether and how COPII vesicle tether, TRAPP (Transport protein particle), plays a role in the interaction between COPII vesicles and microtubules is currently unknown.Principle FindingsWe address the functional relationship between COPII tether TRAPP and dynactin. Overexpressed TRAPP subunits interfered with microtubule architecture by competing p150Glued away from the MTOC. TRAPP subunit TRAPPC9 bound directly to p150Glued via the same carboxyl terminal domain of p150Glued that binds Sec23 and Sec24. TRAPPC9 also inhibited the interaction between p150Glued and Sec23/Sec24 both in vitro and in vivo, suggesting that TRAPPC9 serves to uncouple p150Glued from the COPII coat, and to relay the vesicle-dynactin interaction at the target membrane.ConclusionsThese findings provide a new perspective on the function of TRAPP as an adaptor between the ERGIC membrane and dynactin. By preserving the connection between dynactin and the tethered and/or fused vesicles, TRAPP allows nascent ERGIC to continue the movement along the microtubules as they mature into the cis-Golgi.

Motor Coordination via a Tug-of-War Mechanism Drives Bidirectional Vesicle Transport

The microtubule motors kinesin and dynein function collectively to drive vesicular transport. High-resolution tracking of vesicle motility in the cell indicates that transport is often bidirectional, characterized by frequent directional changes. However, the mechanisms coordinating the collective activities of oppositely oriented motors bound to the same cargo are not well understood. To examine motor coordination, we purified neuronal transport vesicles and analyzed their motility via automated particle tracking with nanometer resolution. The motility of purified vesicles reconstituted in vitro closely models the movement of LysoTracker-positive vesicles in primary neurons, where processive bidirectional motility is interrupted with frequent directional switches, diffusional movement, and pauses. Quantitative analysis indicates that vesicles copurify with a low number of stably bound motors: one to five dynein and one to four kinesin motors. These observations compare well to predictions from a stochastic tug-of-war model, where transport is driven by the force-dependent kinetics of teams of opposing motors in the absence of external regulation. Together, these observations indicate that vesicles move robustly with a small complement of tightly bound motors and suggest an efficient regulatory scheme for bidirectional motility where small changes in the number of engaged motors manifest in large changes in the motility of cargo.

The retromer component SNX6 interacts with dynactin p150Glued and mediates endosome-to-TGN transport

The retromer is a protein complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network (TGN). It is comprised of a cargo-selection subcomplex of Vps26, Vps29 and Vps35 and a membrane-binding coat subcomplex of sorting nexins (SNXs). Previous studies identified SNX1/2 as one of the components of the SNX subcomplex, and SNX5/6 as candidates for the second SNX. How the retromer-associated cargoes are recognized and transported by molecular motors are largely unknown. In this study, we found that one of SNX1/2's dimerization partners, SNX6, interacts with the p150(Glued) subunit of the dynein/dynactin motor complex. We present evidence that SNX6 is a component of the retromer, and that recruitment of the motor complex to the membrane-associated retromer requires the SNX6-p150(Glued) interaction. Disruption of the SNX6-p150(Glued) interaction causes failure in formation and detachment of the tubulovesicular sorting structures from endosomes and results in block of CI-MPR retrieval from endosomes to the TGN. These observations indicate that in addition to SNX1/2, SNX6 in association with the dynein/dynactin complex drives the formation and movement of tubular retrograde intermediates.

Dynactin is essential for growth cone advance

Dynactin is a multi-subunit complex that serves as a critical cofactor of the microtubule motor cytoplasmic dynein. We previously identified dynactin in the nerve growth cone. However, the function of dynactin in the growth cone is still unclear. Here we show that dynactin in the growth cone is required for constant forward movement of the growth cone. Chromophore-assisted laser inactivation (CALI) of dynamitin, a dynactin subunit, within the growth cone markedly decreases the rate of growth cone advance. CALI of dynamitin in vitro dissociates another dynactin subunit, p150 Glued, from dynamitin. These results indicate that dynactin, especially the interaction between dynamitin and p150 Glued, plays an essential role in growth cone advance.

The microtubule plus end-binding protein EB1 is involved in Sertoli cell plasticity in testicular seminiferous tubules

Sertoli cells of testis belong to a unique type of polarized epithelial cells and are essential for spermatogenesis. They form the blood–testis barrier at the base of seminiferous tubule. Their numerous long, microtubule-rich processes extend inward and associate with developing germ cells to sustain germ cell growth and differentiation. How Sertoli cells develop and maintain their elaborate processes has been an intriguing question. Here we showed that, by microinjecting lentiviral preparations into mouse testes of 29 days postpartum, we were able to specifically label individual Sertoli cells with GFP, thus achieving a clear view of their natural configurations together with associated germ cells in situ. Moreover, compared to other microtubule plus end-tracking proteins such as CLIP-170 and p150 Glued, EB1 was highly expressed in Sertoli cells and located along microtubule bundles in Sertoli cell processes. Stable overexpression of a GFP-tagged dominant-negative EB1 mutant disrupted microtubule organizations in cultured Sertoli cells. Furthermore, its overexpression in testis Sertoli cells altered their shapes. Sertoli cells in situ became rod-like, with decreased basal and lateral cell processes. Seminiferous tubule circularity and germ cell number were also reduced. These data indicate a requirement of proper microtubule arrays for Sertoli cell plasticity and function in testis.

Requirement of Dynactin p150Glued Subunit for the Functional Integrity of the Keratinocyte Microparasol

The keratinocyte microparasol, composed of a perinuclear microtubular/melano-phagolysosomal complex, protects the nucleus from UV-induced DNA damage. We have previously demonstrated that cytoplasmic dynein is the motor involved in the perinuclear-directed aggregation of phagocytosed melanosomes. Dynactin, of which p150(Glued) is the major subunit, can link directly to microtubules and links organelles to dynein at different domains. To further define the mechanism of the microparasol, we transfected siRNA targeted against p150(Glued) into human keratinocytes cultured with 0.5 mm fluorescent microspheres and performed time-lapse analysis, confocal immunolocalization, and Western immunoblotting after 24 and 48 hours. Western blots revealed a significant knockdown of the p150(Glued) subunit. The knockdown decreased p150(Glued) colocalization with microtubules and decreased perinuclear positioning of the convergent microtubular framework. It also inhibited perinuclear aggregation of phagocytosed fluorescent microspheres and reduced mean centripetal microsphere displacement. The findings provide evidence that dynactin p150(Glued) plays an important role in the functional integrity of the keratinocyte microparasol.

A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport.

A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed p150Glued by live-cell imaging. Time-lapse analysis of p150Glued revealed targeting to the plus ends of growing microtubules, requiring the NH2-terminal cytoskeleton-associated protein–glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150Glued phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150Glued phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150Glued phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150Glued during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150Glued–labeled microtubules just prior to transport, implicating microtubules and dynactin in a search–capture mechanism for minus-end–directed organelles.

M Phase Phosphorylation of Cytoplasmic Dynein Intermediate Chain and p150Glued

To understand how the dramatic cell biological changes of oocyte maturation are brought about, we have begun to identify proteins whose phosphorylation state changes during Xenopus oocyte maturation. Here we have focused on one such protein, p83. We partially purified p83, obtained peptide sequence, and identified it as the intermediate chain of cytoplasmic dynein. During oocyte maturation, dynein intermediate chain became hyperphosphorylated at the time of germinal vesicle breakdown and remained hyperphosphorylated throughout the rest of meiosis and early embryogenesis. p150(Glued), a subunit of dynactin that has been shown to bind to dynein intermediate chain, underwent similar changes in its phosphorylation. Both dynein intermediate chain and p150(Glued) also became hyperphosphorylated during M phase in XTC-2 cells and HeLa cells. Thus, two components of the dynein-dynactin complex undergo coordinated phosphorylation changes at two G2/M transitions (maturation in oocytes and mitosis in cells in culture) but remain constitutively in their M phase forms during early embryogenesis. Dynein intermediate chain and p150(Glued) phosphorylation may positively regulate mitotic processes, such as spindle assembly or orientation, or negatively regulate interphase processes such as minus-end-directed organelle trafficking.

The APC-associated protein EB1 associates with components of the dynactin complex and cytoplasmic dynein intermediate chain

Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein [1], a domain of APC that is commonly deleted in colorectal neoplasia [2]. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 [3] and Saccharomyces cerevisiae Bim1p [4]. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex [5]. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle [6], in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis [7] [8–10]. Here, we demonstrate that human EB1 can be coprecipitated with p150 Glued, a member of the dynactin protein complex. EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.

Interaction of Huntingtin-Associated Protein with Dynactin P150Glued

Huntingtin is the protein product of the gene for Huntington's disease (HD) and carries a polyglutamine repeat that is expanded in HD (>36 units). Huntingtin-associated protein (HAP1) is a neuronal protein and binds to huntingtin in association with the polyglutamine repeat. Like huntingtin, HAP1 has been found to be a cytoplasmic protein associated with membranous organelles, suggesting the existence of a protein complex including HAP1, huntingtin, and other proteins. Using the yeast two-hybrid system, we found that HAP1 also binds to dynactin P150(Glued) (P150), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent retrograde transport of membranous organelles. An in vitro binding assay showed that both huntingtin and P150 selectively bound to a glutathione transferase (GST)-HAP1 fusion protein. An immunoprecipitation assay demonstrated that P150 and huntingtin coprecipitated with HAP1 from rat brain cytosol. Western blot analysis revealed that HAP1 was enriched in rat brain microtubules and comigrated with P150 and huntingtin in sucrose gradients. Immunofluorescence showed that transfected HAP1 colocalized with P150 and huntingtin in human embryonic kidney (HEK) 293 cells. We propose that HAP1, P150, and huntingtin are present in a protein complex that may participate in dynein-dynactin-associated intracellular transport.

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.