Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexesCytoplasmic dynein is a molecular motor that transports a large variety of cargoes (e.g., organelles, messenger RNAs, and viruses) along microtubules over long intracellular distances. The dynactin protein complex is important for dynein activity in vivo, but its precise role has been unclear. Here, we found that purified mammalian dynein did not move processively on microtubules in vitro. However, when dynein formed a complex with dynactin and one of four different cargo-specific adapter proteins, the motor became ultraprocessive, moving for distances similar to those of native cargoes in living cells. Thus, we propose that dynein is largely inactive in the cytoplasm and that a variety of adapter proteins activate processive motility by linking dynactin to dynein only when the motor is bound to its proper cargo.
Electron cryo-microscopy structure of the mechanotransduction channel NOMPCTyrosination of α‐tubulin controls the initiation of processive dynein–dynactin motilityBRD4 Coordinates Recruitment of Pause Release Factor P-TEFb and the Pausing Complex NELF/DSIF To Regulate Transcription Elongation of Interferon-Stimulated GenesMira C. Patel, Maxime Debrosse, Matthew D. Smith et al.|Molecular and Cellular Biology|2013 RNA polymerase II (Pol II) and the pausing complex, NELF and DSIF, are detected near the transcription start site (TSS) of many active and silent genes. Active transcription starts when the pause release factor P-TEFb is recruited to initiate productive elongation. However, the mechanism of P-TEFb recruitment and regulation of NELF/DSIF during transcription is not fully understood. We investigated this question in interferon (IFN)-stimulated transcription, focusing on BRD4, a BET family protein that interacts with P-TEFb. Besides P-TEFb, BRD4 binds to acetylated histones through the bromodomain. We found that BRD4 and P-TEFb, although not present prior to IFN treatment, were robustly recruited to IFN-stimulated genes (ISGs) after stimulation. Likewise, NELF and DSIF prior to stimulation were hardly detectable on ISGs, which were strongly recruited after IFN treatment. A shRNA-based knockdown assay of NELF revealed that it negatively regulates the passage of Pol II and DSIF across the ISGs during elongation, reducing total ISG transcript output. Analyses with a BRD4 small-molecule inhibitor showed that IFN-induced recruitment of P-TEFb and NELF/DSIF was under the control of BRD4. We suggest a model where BRD4 coordinates both positive and negative regulation of ISG elongation.
Disease-associated mutations in human BICD2 hyperactivate motility of dynein–dynactinWalter Huynh, Ronald D. Vale|The Journal of Cell Biology|2017 gene have been identified in patients with a dominant form of spinal muscular atrophy, but how these mutations cause disease is unknown. To investigate this question, we have developed in vitro motility assays with purified DDB and BICD2's membrane vesicle partner, the GTPase Rab6a. Rab6a-GTP, either in solution or bound to artificial liposomes, released BICD2 from an autoinhibited state and promoted robust dynein-dynactin transport. In these assays, BICD2 mutants showed an enhanced ability to form motile DDB complexes. Increased retrograde transport by BICD2 mutants also was observed in cells using an inducible organelle transport assay. When overexpressed in rat hippocampal neurons, the hyperactive BICD2 mutants decreased neurite growth. Our results reveal that dominant mutations in BICD2 hyperactivate DDB motility and suggest that an imbalance of minus versus plus end-directed microtubule motility in neurons may underlie spinal muscular atrophy.