Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated

摘要

class="head no_bottom_margin" id="sec1title">IntroductionCytoplasmic dynein-1 (dynein) associates with dynactin to form an efficient microtubule motor that transports cargo to the minus end of microtubules and organizes the internal components of eukaryotic cells. Disruption of this 2.4-megadalton machine disperses the Golgi network (), blocks transport between organelles (), and leaves viruses stuck at the cell periphery (). In addition, dynein and dynactin are required during cell division for spindle formation and correct chromosome alignment (). Dynein must therefore be carefully regulated to ensure the correct timing and location of motor activation.In cells, most dynein is diffuse in the cytoplasm, with only a small fraction on microtubules (). This prevents dynein from inappropriately saturating microtubules or traveling unnecessarily and ensures there is a pool of dynein ready to transport cargos when required. The switch of dynein and dynactin from diffuse to actively transporting cargo is controlled at many levels. It can be driven both in vitro () and in vivo () by clustering motors and influenced by targeting dynein/dynactin to specific post-translationally modified microtubules (, ) or the microtubule plus ends (, ). The switch is also controlled at the level of the dynein/dynactin machinery itself. Whereas isolated human dynein is weakly processive in vitro (), it can be activated to move over long distances (>500 nm) by binding to dynactin and a cargo-specific adaptor protein such as BICD2 (, ) or Hook3 (, href="#bib31" rid="bib31" class=" bibr popnode">Olenick et al., 2016, href="#bib43" rid="bib43" class=" bibr popnode">Schroeder and Vale, 2016). This binding stimulates processive movement by increasing the run length, velocity (href="#bib26" rid="bib26" class=" bibr popnode">McKenney et al., 2014, href="#bib41" rid="bib41" class=" bibr popnode">Schlager et al., 2014), and force output (href="#bib3" rid="bib3" class=" bibr popnode">Belyy et al., 2016) of individual motors.Dynein consists of two motor domains that are responsible for ATP hydrolysis and force production and a tail region that holds them together. It is unclear why dynein is only weakly processive on its own and how it is activated by dynactin and cargo adaptors. There is some evidence that dynein processivity is directly inhibited by the C-terminal ∼300 amino acids of the motor domain (href="#bib29" rid="bib29" class=" bibr popnode">Nicholas et al., 2015). Another theory is that inhibition is due to the tail region folding back to contact the motor domains until cargo binds (href="#bib3" rid="bib3" class=" bibr popnode">Belyy et al., 2016, href="#bib25" rid="bib25" class=" bibr popnode">Markus et al., 2009). A similar inhibition mechanism is used by cytoskeletal motors in the kinesin (href="#bib18" rid="bib18" class=" bibr popnode">Kaan et al., 2011) and myosin families (href="#bib14" rid="bib14" class=" bibr popnode">Hammer and Sellers, 2011). Alternatively, it has been proposed that dynein is auto-inhibited by self-dimerization of its motor domains (href="#bib51" rid="bib51" class=" bibr popnode">Torisawa et al., 2014). This form of dynein is referred to as the phi-particle because of its resemblance to the Greek letter phi (φ) (href="#bib1" rid="bib1" class=" bibr popnode">Amos, 1989). Activation was suggested to result from a shift in the equilibrium of dynein conformations toward an open form in which the motor domains are separated. In support of this, forced separation of isolated dynein motor domains can increase motor activity (href="#bib51" rid="bib51" class=" bibr popnode">Torisawa et al., 2014). However, these studies were performed on artificially dimerized dynein motors lacking the tail region. It is therefore not clear whether the tail contributes to inhibition or what role the phi-particle plays in the context of the whole dynein complex.In this study, we set out to determine whether the phi-particle contributes to dynein auto-inhibition and how dynein is activated by dynactin. We use cryoelectron microscopy (cryo-EM) to determine the structure of the phi-particle. We show how the motor domains self-dimerize and are locked in a conformation with weak affinity for microtubules. Disrupting the motor dimer by structure-based mutagenesis drives dynein into an open form with increased affinity for microtubules and dynactin. Surprisingly, we discover that the open form of dynein is also inhibited. We use a combination of 2D analysis of EM images and a 3D cryo-EM structure of the whole dynein/dynactin machinery to explain how dynactin overcomes this inhibition and directly reorients the motor domains to make dynein processive. Finally, we show that disrupting the phi-particle in cells causes mis-localization and mitotic defects, supporting a physiological role for the phi-particle in dynein regulation.