Dynactin integrity depends upon direct binding of dynamitin to Arp1

Dynactin is a multiprotein complex that works with cytoplasmic dynein and other motors to support a wide range of cell functions. It serves as an adaptor that binds both dynein and cargoes and enhances single-motor processivity. The dynactin subunit dynamitin (also known as p50) is believed to be integral to dynactin structure because free dynamitin displaces the dynein-binding p150^Glued subunit from the cargo-binding Arp1 filament. We show here that the intrinsically disordered dynamitin N-terminus binds to Arp1 directly. When expressed in cells, dynamitin amino acids (AA) 1–87 causes complete release of endogenous dynamitin, p150, and p24 from dynactin, leaving behind Arp1 filaments carrying the remaining dynactin subunits (CapZ, p62, Arp11, p27, and p25). Tandem-affinity purification–tagged dynamitin AA 1–87 binds the Arp filament specifically, and binding studies with purified native Arp1 reveal that this fragment binds Arp1 directly. Neither CapZ nor the p27/p25 dimer contributes to interactions between dynamitin and the Arp filament. This work demonstrates for the first time that Arp1 can directly bind any protein besides another Arp and provides important new insight into the underpinnings of dynactin structure.     

Dynein and Dynactin Leverage Their Bivalent Character to Form a High-Affinity Interaction

Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150^Glued; however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10–44, is sufficient for binding p150^Glued. Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150^Glued form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150^Glued fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD) experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150^Glued. Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.     

Dynactin Subunit p150Glued Is a Neuron-Specific Anti-Catastrophe Factor

Regulation of microtubule dynamics in neurons is critical, as defects in the microtubule-based transport of axonal organelles lead to neurodegenerative disease. The microtubule motor cytoplasmic dynein and its partner complex dynactin drive retrograde transport from the distal axon. We have recently shown that the p150^Glued subunit of dynactin promotes the initiation of dynein-driven cargo motility from the microtubule plus-end. Because plus end-localized microtubule-associated proteins like p150^Glued may also modulate the dynamics of microtubules, we hypothesized that p150^Glued might promote cargo initiation by stabilizing the microtubule track. Here, we demonstrate in vitro using assembly assays and TIRF microscopy, and in primary neurons using live-cell imaging, that p150^Glued is a potent anti-catastrophe factor for microtubules. p150^Glued alters microtubule dynamics by binding both to microtubules and to tubulin dimers; both the N-terminal CAP-Gly and basic domains of p150^Glued are required in tandem for this activity. p150^Glued is alternatively spliced in vivo, with the full-length isoform including these two domains expressed primarily in neurons. Accordingly, we find that RNAi of p150^Glued in nonpolarized cells does not alter microtubule dynamics, while depletion of p150^Glued in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a mutation in p150^Glued causal for the lethal neurodegenerative disorder Perry syndrome abrogates this anti-catastrophe activity. Thus, we find that dynactin has multiple functions in neurons, both activating dynein-mediated retrograde axonal transport and enhancing microtubule stability through a novel anti-catastrophe mechanism regulated by tissue-specific isoform expression; disruption of either or both of these functions may contribute to neurodegenerative disease.     

Alanine scanning of Arp1 delineates a putative binding site for Jnm1/dynamitin and Nip100/p150Glued

Arp1p is the only actin-related protein (ARP) known to form actin-like filaments. Unlike actin, Arp1p functions with microtubules, as part of the dynein regulator, dynactin. Arp1p's dissimilar functions imply interactions with a distinct set of proteins. To distinguish surface features relating to Arp1p's core functions and to identify the footprint of protein interactions essential for dynactin function, we performed the first complete charge-cluster-to-alanine scanning mutagenesis of an ARP and compared the results with a similar study of actin. The Arp1p mutations revealed three nonoverlapping surfaces with distinct genetic properties. One of these surfaces encompassed a region unique to Arp1p that is crucial for Jnm1p (dynamitin/p50) and Nip100p (p150^Glued) association as well as pointed-end associations. Unlike the actin mutations, none of the ARP1 alleles disrupt filament formation; however, one pointed-end allele delayed the elution of Arp1p on gel filtration, consistent with loss of additional subunits.     

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.     

Transient Tertiary Structures of Disordered Dynein Intermediate Chain Regulate its Interactions with Multiple Partners

The N-terminal domain of dynein intermediate chain (N–IC) is central to the cytoplasmic dynein ‘cargo attachment subcomplex’ and regulation of motor activity. It is a prototypical intrinsically disordered protein (IDP), serving as a primarily disordered polybivalent molecular scaffold for numerous binding partners, including three dimeric dynein light chains and coiled coil domains of dynein partners dynactin p150^Glued and NudE. At the very N-terminus, a 40 amino acid single alpha helix (SAH) forms the major binding site for both p150^Glued and NudE, while a shorter nascent helix (H2) separated from SAH by a disordered linker, is necessary for tight binding to dynactin p150^Glued but not to NudE. Here we demonstrate that transient tertiary interactions in this highly dynamic protein underlie the differences in its interactions with p150^Glued and NudE. NMR paramagnetic relaxation enhancement experiments and restrained molecular dynamics simulations identify interactions between the two non-contiguous SAH and H2 helical regions, the extent of which correlates with the length and stability of H2, showing clearly that tertiary and secondary structure formation are coupled in IDPs. These interactions are significantly attenuated when N–IC is bound to NudE, suggesting that NudE binding shifts the conformational ensemble to one that is more extended and with less structure in H2. While the intrinsic disorder and flexibility in N–IC modulate its ability to serve as a binding platform for numerous partners, deviations of this protein from random-coil behavior provide a process for regulating these binding interactions and potentially the dynein motor.     

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.     

Ste20-like protein kinase SLK (LOSK) regulates microtubule organization by targeting dynactin to the centrosome

The microtubule- and centrosome-associated Ste20-like kinase (SLK; long Ste20-like kinase [LOSK]) regulates cytoskeleton organization and cell polarization and spreading. Its inhibition causes microtubule disorganization and release of centrosomal dynactin. The major function of dynactin is minus end–directed transport along microtubules in a complex with dynein motor. In addition, dynactin is required for maintenance of the microtubule radial array in interphase cells, and depletion of its centrosomal pool entails microtubule disorganization. Here we demonstrate that SLK (LOSK) phosphorylates the p150^Glued subunit of dynactin and thus targets it to the centrosome, where it maintains microtubule radial organization. We show that phosphorylation is required only for centrosomal localization of p150^Glued and does not affect its microtubule-organizing properties: artificial targeting of nonphosphorylatable p150^Glued to the centrosome restores microtubule radial array in cells with inhibited SLK (LOSK). The phosphorylation site is located in a microtubule-binding region that is variable for two isoforms (1A and 1B) of p150^Glued expressed in cultured fibroblast-like cells (isoform 1B lacks 20 amino acids in the basic microtubule-binding domain). The fact that SLK (LOSK) phosphorylates only a minor isoform 1A of p150^Glued suggests that transport and microtubule-organizing functions of dynactin are distinctly divided between the two isoforms. We also show that dynactin phosphorylation is involved in Golgi reorientation in polarized cells.     

Dynactin polices two-way organelle traffic

How is the bidirectional motion of organelles controlled? In this issue, Deacon et al. (2003) reveal the unexpected finding that dynactin (previously known to control dynein-based motility) binds to kinesin II and regulates anterograde movement of Xenopus melanosomes. This result suggests that dynactin may be a key player in coordinating vesicle traffic in this system.The movement of intracellular cargo is essential for cell survival. In animal cells, membranous organelles are propelled through the cytoplasm by microtubule-based motor proteins. Anterograde movement toward microtubule plus ends at the cell periphery is driven by motor proteins of the kinesin superfamily, whereas retrograde movement toward minus ends at the cell center is largely accomplished by cytoplasmic dynein. In most cells, organelles do not travel smoothly in one direction but frequently switch between plus and minus end–directed travel. The net time spent traveling in the plus versus the minus end direction determines the steady-state distribution of an organelle population within a cell. A long-standing question for those studying organelle transport is how this bidirectional trafficking is coordinated. Is the binding of kinesin and dynein to vesicles mutually exclusive, or are these motors bound at the same time but with their activities coordinately regulated? What molecule(s) might be responsible for linking kinesin and dynein activities? In this issue, Vladimir Gelfand''s group (Deacon et al., 2003) addresses these questions by studying the motor proteins kinesin II and cytoplasmic dynein that move pigment granules in Xenopus melanophore cells. Their results are surprising; the dynactin complex, previously known to bind to cytoplasmic dynein and anchor it to organelles, also interacts with kinesin II and is necessary for plus end–directed motion. The ability of dynactin to physically interact with these two opposite polarity motors suggests that it may be the long sought-after molecular switch that coordinates bidirectional movement in this system.Previous studies hinted that the actions of dynein and kinesin may be controlled via dynactin. Dynactin is a large, multimeric protein complex. Its p150^Glued subunit has binding sites for both microtubules and the intermediate chain of dynein and is thought to be responsible for the association of dynein with many of its cargo organelles (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995; Waterman-Storer et al., 1995). Curiously, the treatment of extruded squid axoplasm with antibodies against p150^Glued inhibited both the anterograde and retrograde movement of organelles along microtubules (Waterman-Storer et al., 1997). These antibodies were known to inhibit the interaction of dynactin with dynein, but their effect on anterograde movement was more difficult to explain. However, genetic studies yielded similar results. Martin et al. (1999) found that mutations in either p150^Glued, the cytoplasmic dynein heavy chain, or kinesin I inhibited both retrograde and anterograde fast axonal transport in Drosophila larvae. This phenotype potentially could be explained by stalled retrograde vesicles sterically blocking the movement of anterograde cargo, but the authors also suggested the possibility of a physical linkage between kinesin, dynein, and dynactin. This theory was further tested by tracking the movement of lipid droplets in Drosophila embryos (Gross et al., 2002b). A mild defect in the dynein heavy chain impaired several aspects of minus end–directed transport of lipid droplets: run lengths, velocities, and the opposing optical trap force required to halt droplet movement were all decreased. Surprisingly, this mutation produced similar effects on droplets moving toward the microtubule plus ends. Embryos expressing a mutant p150^Glued protein that partially impaired dynactin function also exhibited impaired movement in both the plus and minus end directions. Collectively, these results suggested that dynactin might be involved in coordinating the bidirectional movement of organelles. However, these studies did not provide a molecular explanation of how this mechanism might work.To study the mechanism of coordination of bidirectional vesicle movement, Deacon et al. (2003) used Xenopus melanophores due to the unique ability to experimentally control the directional movement of their pigmented melanosomes (Daniolos et al., 1990). Upon treatment of melanophores with melatonin, the cAMP concentration in the cytoplasm drops and the melanosomes move with a net minus end–directed bias and aggregate toward the cell center. Treatment with melanocyte-stimulating hormone (MSH)^* restores cAMP levels, and the melanosomes exhibit a plus end–directed bias and disperse throughout the cell. Aggregation is accomplished by cytoplasmic dynein (Nilsson and Wallin, 1997), whereas dispersion requires the combined actions of kinesin II and the actin-based motor myosin V (Rogers and Gelfand, 1998; Tuma et al., 1998; Gross et al., 2002a). Kinesin II is a heterotrimeric protein consisting of two motor subunits and a third nonmotor subunit known as kinesin-associated protein (KAP) (Cole et al., 1992). KAP is thought to be involved in binding kinesin II to its cargo, although the mechanism for this interaction is not known.The role of dynactin in melanosome transport was investigated by disrupting dynactin function via the overexpression of dynamitin (Echeverri et al., 1996), a crucial subunit that holds the dynactin complex together. To ensure that all observed melanosome movement occurred on the microtubule cytoskeleton, actin filaments were depolymerized with latrunculin B. Here, the authors report that melanosome movement to both the plus and minus ends of microtubules was inhibited by dynamitin overexpression, suggesting a role for dynactin in coordinating bidirectional movement. They considered whether this result might be explained if both kinesin II and dynein bound to dynactin and thereby docked onto membranes. To test this idea, kinesin II was immunoprecipitated with a series of antibodies, and the authors found that dynactin was pulled down along with this kinesin motor in all cases. The reverse experiment of immunoprecipitating with p150^Glued antibodies also brought down kinesin II. Blot overlays of purified melanosomes with p150^Glued detected an interaction with a 115-kD protein, the expected size of Xenopus KAP. Subsequent overlay and affinity pull-down experiments with purified proteins confirmed the direct binding of p150^Glued to KAP. By constructing a series of GST fusion proteins, Deacon et al. (2003) were able to map the site of this interaction to residues 600–811 of p150^Glued and the COOH-terminal domain of KAP. Interestingly, this region of p150^Glued also interacts with the dynein intermediate chain, which raised the question of whether kinesin II and dynein might compete for binding to dynactin. Using a blot overlay competition assay, the authors found that the COOH-terminal KAP domain blocked the binding of p150^Glued to the dynein intermediate chain, whereas the NH₂-terminal KAP domain, used as a control, did not. This result confirms that the two motors cannot bind dynactin simultaneously.If these biochemical results are relevant to melanosome movement, then overexpression of KAP should inhibit both anterograde and retrograde traffic. Indeed, overexpession of Xenopus KAP or just its COOH-terminal fragment inhibited bidirectional melanosome movement. As a control, NH₂-terminal KAP had no effect on retrograde movement and only a small effect on anterograde movement, perhaps due to interactions with the kinesin II motor subunits. Together, the results of Deacon et al. (2003) demonstrate that kinesin II, via its KAP subunit, binds to the p150^Glued subunit of dynactin and that this interaction is important for kinesin II–mediated movement of melanosomes.Although the authors identify the p150^Glued subunit of dynactin as a key player in coordinating the bidirectional movement of melanosomes, the mechanism is still unclear. Their biochemical results showing competitive binding to dynactin suggest that binding of kinesin II and dynein to melanosomes may be mutually exclusive events; however, previous work has shown that this is not the case. In a recent paper from the same authors (Gross et al., 2002a), as well as an earlier study from Reese and Haimo (Reese and Haimo, 2000), the relative amounts of kinesin II and dynein bound to purified melanosomes did not change when cells were treated with melatonin to stimulate aggregation or with MSH to stimulate dispersion. Thus, it is possible that proteins other than dynactin might bind kinesin II and dynein to melanosomes. This question also could be addressed by determining if motor binding to melanophores is diminished in cells overexpressing KAP or dynamitin. Unfortunately, Deacon et al. (2003) were not able to answer this question by biochemical isolation of melanosomes and motor quantitation because transfected cells were only a small percentage of the total population. Another possible model is that dynactin is not needed for recruiting kinesin II and dynein to melanosomes but is somehow involved in regulating the activation or organization of motors already bound to the membrane.Future studies will no doubt explore whether dynactin is involved in bidirectional transport in systems other than melanophores. In intraflagellar transport, kinesin II and cytoplasmic dynein 2 are involved in moving nonmembranous particles between the cell body and the tip of the flagella or cilia (Rosenbaum and Witman, 2002). It will be interesting to determine whether dynactin plays a role in this type of cargo transport. In neurons, kinesin I is responsible for moving organelles from the cell body to the axon terminal. As discussed above, Martin et al. (1999) found that mutations in either kinesin I heavy chain, dynein, or p150^Glued all produced the same phenotype in Drosophila larvae neurons, suggesting that dynactin may play a role in coordinating bidirectional movement in this system as well. Immunoprecipitation of neuronal p150^Glued, however, brought down only dynein but not kinesin I. This finding may result from the fact that kinesin I, which possesses a light chain unrelated to the KAP subunit, could be linked indirectly to dynactin by another protein. Thus, this study by Deacon et al. (2003) has opened up a new area of exploration and dynactin will undoubtedly receive closer scrutiny from kinesin researchers in the future.     

Arp11 affects dynein-dynactin interaction and is essential for dynein function in Aspergillus nidulans

The dynactin complex contains proteins including p150 that interacts with cytoplasmic dynein and an actin-related protein Arp1 that forms a minifilament. Proteins including Arp11 and p62 locate at the pointed end of the Arp1 filament, but their biochemical functions are unclear (Schroer TA. Dynactin. Annu Rev Cell Dev Biol 2004;20:759–779). In Aspergillus nidulans , loss of Arp11 or p62 causes the same nu clear d istribution (nud) defect displayed by dynein mutants, indicating that these pointed-end proteins are essential for dynein function. We constructed a strain with S-tagged p150 of dynactin that allows us to pull down components of the dynactin and dynein complexes. Surprisingly, while the ratio of pulled-down Arp1 to S-p150 in Arp11-depleted cells is clearly lower than that in wild-type cells, the ratio of pulled-down dynein to S-p150 is significantly higher. We further show that the enhanced dynein–dynactin interaction in Arp11-depleted cells is also present in the soluble fraction and therefore is not dependent upon the affinity of these proteins to the membrane. We suggest that loss of the pointed-end proteins alters the Arp1 filament in a way that affects the conformation of p150 required for its proper interaction with the dynein motor.     

HookA is a novel dynein–early endosome linker critical for cargo movement in vivo

Cytoplasmic dynein transports membranous cargoes along microtubules, but the mechanism of dynein–cargo interaction is unclear. From a genetic screen, we identified a homologue of human Hook proteins, HookA, as a factor required for dynein-mediated early endosome movement in the filamentous fungus Aspergillus nidulans. HookA contains a putative N-terminal microtubule-binding domain followed by coiled-coil domains and a C-terminal cargo-binding domain, an organization reminiscent of cytoplasmic linker proteins. HookA–early endosome interaction occurs independently of dynein–early endosome interaction and requires the C-terminal domain. Importantly, HookA interacts with dynein and dynactin independently of HookA–early endosome interaction but dependent on the N-terminal part of HookA. Both dynein and the p25 subunit of dynactin are required for the interaction between HookA and dynein–dynactin, and loss of HookA significantly weakens dynein–early endosome interaction, causing a virtually complete absence of early endosome movement. Thus, HookA is a novel linker important for dynein–early endosome interaction in vivo.     

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.     

Molecular and Functional Basis for the Scaffolding Role of the p50/Dynamitin Subunit of the Microtubule-associated Dynactin Complex

p50/dynamitin (DM) is a major subunit of the microtubule-associated dynactin complex that is required for stabilization and attachment of its two distinct structural domains, namely the Arp1 rod and the shoulder/sidearm. Here, we define the determinants of p50/DM required for self-oligomerization of the protein and for interactions with other subunits of the dynactin complex. Whereas the N-terminal 1–91-amino acid region of the protein is required and sufficient for binding to the Arp1 rod, additional determinants contained within the first half of the protein are required for optimal recruitment of the p150^Glued subunit of the shoulder/sidearm. Overexpression experiments confirmed that the N-terminal 1–91-amino acid region of p50/DM is critical for dynactin functionality, because this fragment acts as a dominant negative to inhibit both dynein-dependent and -independent functions of the complex. Furthermore, the first two predicted coiled-coil motifs of p50/DM contain determinants that mediate self-association of the protein. Interestingly, p50/DM self-association does not contribute to p50/DM-induced disruption of the dynactin complex, but most likely participates in the stabilization of the complex.     

A Neurospora crassa Arp1 mutation affecting cytoplasmic dynein and dynactin localization

Of the actin-related proteins, Arp1 is the most similar to conventional actin, and functions solely as a component of the multisubunit complex dynactin. Dynactin has been identified as an activator of the microtubule-associated motor cytoplasmic dynein. The role of Arp1 within dynactin is two-fold: (1) it serves as a structural scaffold protein for other dynactin subunits; and (2) it has been proposed to link dynactin, and thereby dynein, with membranous cargo via interaction with spectrin. Using the filamentous fungus Neurospora crassa, we have identified genes encoding subunits of cytoplasmic dynein and dynactin. In this study, we describe a genetic screen for N. crassa Arp1 (ro-4) mutants that are defective for dynactin function. We report that the ro-4(E8) mutant is unusual in that it shows alterations in the localization of cytoplasmic dynein and dynactin and in microtubule organization. In the mutant, dynein/dynactin complexes co-localize with bundled microtubules at hyphal tips. Given that dynein transports membranous cargo from hyphal tips to distal regions, the cytoplasmic dynein and dynactin complexes that accumulate along microtubule tracts at hyphal tips in the ro-4(E8) mutant may have either reduced motor activity or be delayed for activation of motor activity following cargo binding.     

Dynactin's pointed-end complex is a cargo-targeting module

Dynactin is an essential part of the cytoplasmic dynein motor that enhances motor processivity and serves as an adaptor that allows dynein to bind cargoes. Much is known about dynactin''s interaction with dynein and microtubules, but how it associates with its diverse complement of subcellular binding partners remains mysterious. It has been suggested that cargo specification involves a group of subunits referred to as the “pointed-end complex.” We used chemical cross-linking, RNA interference, and protein overexpression to characterize interactions within the pointed-end complex and explore how it contributes to dynactin''s interactions with endomembranes. The Arp11 subunit, which caps one end of dynactin''s Arp1 filament, and p62, which binds Arp11 and Arp1, are necessary for dynactin stability. These subunits also allow dynactin to bind the nuclear envelope prior to mitosis. p27 and p25, by contrast, are peripheral components that can be removed without any obvious impact on dynactin integrity. Dynactin lacking these subunits shows reduced membrane binding. Depletion of p27 and p25 results in impaired early and recycling endosome movement, but late endosome movement is unaffected, and mitotic spindles appear normal. We conclude that the pointed-end complex is a bipartite structural domain that stabilizes dynactin and supports its binding to different subcellular structures.     

Colocalization studies of Arp1 and p150Glued to spindle microtubules during mitosis: the effect of cytochalasin on the organization of microtubules and motor proteins in PtK1 cells

Motor proteins play a fundamental role in the congression and segregation of chromosomes in mitosis as well as the formation of the mitotic spindle. In particular, the dynein/dynactin complex is involved in the maintenance of the spindle, formation of astral microtubules, chromosome motion, and chromosome segregation. Dynactin is a multisubunit, high molecular weight protein that is responsible for the attachment of cargo to dynein. There are a number of major subunits in dynactin that are presumed to be important during mitosis. Arp1 is thought to be the attachment site for cargo to the complex while p150(Glued), a side arm of this complex regulates binding to MTs and the binding of dynactin to dynein. We performed colocalization studies of Arp1 and p150(Glued) to spindle microtubules. Both Arp1 and p150(Glued) colocalize with spindle MTs as well as cytoplasmic components. When treated with cytochalasin J, Arp1 concentrates at the centrosomes and is less co-localized with spindle MTs. Cytochalasin J has less of an effect on the colocalization of p150(Glued) with spindle MTs, suggesting that Arp1 may have a cytochalasin J sensitive site.     

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

Background

The transport of endoplasmic reticulum (ER)-derived COPII vesicles toward the ER-Golgi intermediate compartment (ERGIC) requires cytoplasmic dynein and is dependent on microtubules. p150^Glued, 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 Findings

We address the functional relationship between COPII tether TRAPP and dynactin. Overexpressed TRAPP subunits interfered with microtubule architecture by competing p150^Glued away from the MTOC. TRAPP subunit TRAPPC9 bound directly to p150^Glued via the same carboxyl terminal domain of p150^Glued that binds Sec23 and Sec24. TRAPPC9 also inhibited the interaction between p150^Glued and Sec23/Sec24 both in vitro and in vivo, suggesting that TRAPPC9 serves to uncouple p150^Glued from the COPII coat, and to relay the vesicle-dynactin interaction at the target membrane.

Conclusions

These 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.     

Characterization of the p22 Subunit of Dynactin Reveals the Localization of Cytoplasmic Dynein and Dynactin to the Midbody of Dividing Cells

Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. We have characterized p22 as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150^Glued subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures. We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.     

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 p150^Glued 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 p150^Glued 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.     

Null Mutants of the Neurospora Actin-related Protein 1 Pointed-End Complex Show Distinct Phenotypes

Dynactin is a multisubunit complex that regulates the activities of cytoplasmic dynein, a microtubule-associated motor. Actin-related protein 1 (Arp1) is the most abundant subunit of dynactin, and it forms a short filament to which additional subunits associate. An Arp1 filament pointed-end--binding subcomplex has been identified that consists of p62, p25, p27, and Arp11 subunits. The functional roles of these subunits have not been determined. Recently, we reported the cloning of an apparent homologue of mammalian Arp11 from the filamentous fungus Neurospora crassa. Here, we report that N. crassa ro-2 and ro-12 genes encode the respective p62 and p25 subunits of the pointed-end complex. Characterization of Delta ro-2, Delta ro-7, and Delta ro-12 mutants reveals that each has a distinct phenotype. All three mutants have reduced in vivo vesicle trafficking and have defects in vacuole distribution. We showed previously that in vivo dynactin function is required for high-level dynein ATPase activity, and we find that all three mutants have low dynein ATPase activity. Surprisingly, Delta ro-12 differs from Delta ro-2 and Delta ro-7 and other previously characterized dynein/dynactin mutants in that it has normal nuclear distribution. Each of the mutants shows a distinct dynein/dynactin localization pattern. All three mutants also show stronger dynein/dynactin-membrane interaction relative to wild type, suggesting that the Arp1 pointed-end complex may regulate interaction of dynactin with membranous cargoes.