On and off controls within dynein-dynactin on native cargoes.
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ABSTRACT:
SUBMITTER: Sanghavi P
PROVIDER: S-EPMC8201952 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In the present paper, I report the molecular overlap of the linkage of three essential protein complexes that co-ordinate the formation of the mitotic spindle. These proteins are dynein, a large motor complex that moves machinery inside cells, and two of its regulators: a protein complex called dynactin, a dynein activator, and a protein called NudE whose depletion in mice produces a small brain and mental retardation. What is intriguing about the dynein-dynactin-NudE interplay is that dynactin and NudE bind to a common segment of dynein that is intrinsically disordered. Elucidating differences in their binding modes may explain how one regulator can be selected over the other even when both are present in the same cellular compartment. These results not only have a far-reaching impact on our understanding of processes essential for the formation and orientation of the spindle, but also offer a novel role for protein disorder in controlling cellular processes, and highlight the advantages of NMR spectroscopy in elucidating atomic-level characterization of extremely complex dynamic cellular assemblies.
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Project description:Subcellular localization of mRNAs by cytoskeletal motors plays critical roles in the spatial control of protein function. However, optical limitations of studying mRNA transport in vivo mean that there is little mechanistic insight into how transcripts are packaged and linked to motors, and how the movement of mRNA-motor complexes on the cytoskeleton is orchestrated. Here, we have reconstituted transport of mRNPs containing specific RNAs in vitro. We show directly that mRNAs that are either apically localized or non-localized in Drosophila melanogaster embryos associate with the dynein motor and move bidirectionally on individual microtubules, with localizing mRNPs exhibiting a strong minus-end-directed bias. Single-molecule fluorescence measurements reveal that RNA localization signals increase the average number of dynein and dynactin components recruited to individual mRNPs. We find that, surprisingly, individual RNA molecules are present in motile mRNPs in vitro and provide evidence that this is also the case in vivo. Thus, RNA oligomerization is not obligatory for transport. Our findings lead to a model in which RNA localization signals produce highly polarized distributions of transcript populations through modest changes in motor copy number on single mRNA molecules.
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Project description:Post-translational modifications (PTMs) of ?/?-tubulin are believed to regulate interactions with microtubule-binding proteins. A well-characterized PTM involves in the removal and re-ligation of the C-terminal tyrosine on ?-tubulin, but the purpose of this tyrosination-detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule-binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the ?-tubulin tyrosine facilitates initial motor-tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C-terminal ?-tubulin tyrosine on dynein-dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein-driven motility in cells.
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Project description:Cytoplasmic dynein is a multisubunit microtubule motor complex that, together with its activator, dynactin, drives vesicular cargo toward the minus ends of microtubules. Huntingtin (Htt) is a vesicle-associated protein found in both neuronal and nonneuronal cells that is thought to be involved in vesicular transport. In this study, we demonstrate through yeast two-hybrid and affinity chromatography assays that Htt and dynein intermediate chain interact directly; endogenous Htt and dynein co-immunoprecipitate from mouse brain cytosol. Htt RNAi in HeLa cells results in Golgi disruption, similar to the effects of compromising dynein/dynactin function. In vitro studies reveal that Htt and dynein are both present on vesicles purified from mouse brain. Antibodies to Htt inhibited vesicular transport along microtubules, suggesting that Htt facilitates dynein-mediated vesicle motility. In vivo inhibition of dynein function results in a significant redistribution of Htt to the cell periphery, suggesting that dynein transports Htt-associated vesicles toward the cell center. Together these findings indicate that Htt binds to dynein and acts in a complex along with dynactin and Htt-associated protein-1 to facilitate vesicular transport.
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Project description:A single amino acid change, F580Y (Legs at odd angles (Loa), Dync1h1(Loa)), in the highly conserved and overlapping homodimerization, intermediate chain, and light intermediate chain binding domain of the cytoplasmic dynein heavy chain can cause severe motor and sensory neuron loss in mice. The mechanism by which the Loa mutation impairs the neuron-specific functions of dynein is not understood. To elucidate the underlying molecular mechanisms of neurodegeneration arising from this mutation, we applied a cohort of biochemical methods combined with in vivo assays to systemically study the effects of the mutation on the assembly of dynein and its interaction with dynactin. We found that the Loa mutation in the heavy chain leads to increased affinity of this subunit of cytoplasmic dynein to light intermediate and a population of intermediate chains and a suppressed association of dynactin to dynein. These data suggest that the Loa mutation drives the assembly of cytoplasmic dynein toward a complex with lower affinity to dynactin and thus impairing transport of cargos that tether to the complex via dynactin. In addition, we detected up-regulation of kinesin light chain 1 (KLC1) and its increased association with dynein but reduced microtubule-associated KLC1 in the Loa samples. We provide a model describing how up-regulation of KLC1 and its interaction with cytoplasmic dynein in Loa could play a regulatory role in restoring the retrograde and anterograde transport in the Loa neurons.
| S-EPMC3000974 | biostudies-literature
Project description:Cytoplasmic dynein 1 is an important microtubule-based motor in many eukaryotic cells. Dynein has critical roles both in interphase and during cell division. Here, we focus on interphase cargoes of dynein, which include membrane-bound organelles, RNAs, protein complexes and viruses. A central challenge in the field is to understand how a single motor can transport such a diverse array of cargoes and how this process is regulated. The molecular basis by which each cargo is linked to dynein and its cofactor dynactin has started to emerge. Of particular importance for this process is a set of coiled-coil proteins - activating adaptors - that both recruit dynein-dynactin to their cargoes and activate dynein motility.
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Project description: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 nuclear distribution (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.
| S-EPMC2586032 | biostudies-literature
Project description:Cytoplasmic dynein is the primary minus-end-directed microtubule motor protein in animal cells, performing a wide range of motile activities, including transport of vesicular cargos, mRNAs, viruses, and proteins. Lissencephaly-1 (LIS1) is a highly conserved dynein-regulatory factor that binds directly to the dynein motor domain, uncoupling the enzymatic and mechanical cycles of the motor and stalling dynein on the microtubule track. Dynactin, another ubiquitous dynein-regulatory factor, releases dynein from an autoinhibited state, leading to a dramatic increase in fast, processive dynein motility. How these opposing activities are integrated to control dynein motility is unknown. Here, we used fluorescence single-molecule microscopy to study the interaction of LIS1 with the processive dynein-dynactin-BicD2N (DDB) complex. Surprisingly, in contrast to the prevailing model for LIS1 function established in the context of dynein alone, we found that binding of LIS1 to DDB does not strongly disrupt processive motility. Motile DDB complexes bound up to two LIS1 dimers, and mutational analysis suggested that LIS1 binds directly to the dynein motor domains during DDB movement. Interestingly, LIS1 enhanced DDB velocity in a concentration-dependent manner, in contrast to observations of the effect of LIS1 on the motility of isolated dynein. Thus, LIS1 exerts concentration-dependent effects on dynein motility and can synergize with dynactin to enhance processive dynein movement. Our results suggest that the effect of LIS1 on dynein motility depends on both LIS1 concentration and the presence of other regulatory factors such as dynactin and may provide new insights into the mechanism of LIS1 haploinsufficiency in the neurodevelopmental disorder lissencephaly.
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Project description:Cytoplasmic dynein is a microtubule motor vital for cellular organization and division. It functions as a ~4-megadalton complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognize diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as lissencephaly-1 (LIS1) protein (LIS1) remain unclear. In this study, we determined the cryo-electron microscopy structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. This structure reveals the molecular basis of interactions occurring during dynein activation. We show how JIP3 activates dynein despite its atypical architecture. Unexpectedly, LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Our data suggest that LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.
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Project description:Neurodegenerative disease in humans and mice can be caused by mutations affecting the microtubule motor dynein or its biochemical regulator, dynactin, a multiprotein complex required for dynein function (1-4). A single amino acid change, G59S, in the conserved cytoskeletal-associated protein glycine-rich (CAP-Gly) domain of the p150(glued) subunit of dynactin can cause motor neuron degeneration in humans and mice, which resembles ALS (2, 5-8). The molecular mechanism by which G59S impairs the function of dynein is not understood. Also, the relevance of the CAP-Gly domain for dynein motility has not been demonstrated in vivo. Here, we generate a mutant that is analogous to G59S in budding yeast, and show that this mutation produces a highly specific phenotype related to dynein function. The effect of the point mutation is identical to that of complete loss of the CAP-Gly domain. Our results demonstrate that the CAP-Gly domain has a critical role in the initiation and persistence of dynein-dependent movement of the mitotic spindle and nucleus, but it is otherwise dispensable for dynein-based movement. The need for this function appears to be context-dependent, and we speculate that CAP-Gly activity may only be necessary when dynein needs to overcome high force thresholds to produce movement.
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