Pac1/LIS1 promotes an uninhibited conformation of dynein that coordinates its localization and activity

Abstract

45Cytoplasmic dynein is a minus end-directed microtubule motor that transports myriad 46 cargos in various cell types and contexts. How dynein is regulated to perform all these 47 62Cytoplasmic dynein is an enormous minus end-directed microtubule motor 63 complex that transports numerous cargoes. At first glance, this motor seems 64 exceedingly complex in terms of its architecture, size, and reliance on accessories and 65 regulators for proper activity. For instance, processive single molecule motility of human 66 dynein -itself comprised of 4 to 6 subunits -requires the 11 subunit dynactin complex 67 in addition to an adaptor that links them together 1,2 . Although yeast dynein does not 68 require dynactin for in vitro single molecule motility 3 , it does require this complex for in 69 vivo activity 4,5 . Recent studies have yielded invaluable insight into the underlying 70 reasons for the complexity of the dynein motor. For instance, the reliance on adaptors 71 (e.g., BicD2, Spindly, Hook3 1,2 ) to link dynein to dynactin ensures that cytoplasmic 72 dynein-1 -which effects motility of numerous and varied cargoes throughout the cell 73 cycle 6 -and dynactin are linked together at the right place (and presumably time) for 74 appropriate motility. Additionally, recent studies have revealed that dynactin helps to 75 orient the motor domains in a parallel manner that is conducive for motility 7 , thus 76 revealing the mechanistic basis for dynein's reliance on this large complex. Thus, the 77 complexity of this molecular motor ensures that cargoes are transported to their target 78 destinations in accordance with the needs of the cell. 79In addition to its regulation by extrinsic factors, several studies have 80 demonstrated that human dynein-1 and dynein-2 can also be auto-regulated by intra-81 complex interactions. Specifically, intermolecular interactions between the motor 82 domains have been shown to stabilize an autoinhibited conformation of human dynein 83 called the phi particle (named for its resemblance to the Greek letter) 7-10 . In the case of 84 4 dynein-2 (responsible for intraflagellar transport), the phi particle conformation -which 85 has been observed in its native context 10 -reduces its velocity, ATPase activity and 86 microtubule landing rate 9 . Similarly, the autoinhibited dynein-1 conformation has been 87 shown to reduce its microtubule landing rate and motility properties 7,11 . Moreover, unlike 88 dynein-2 which is not regulated by dynactin 12 , uninhibited dynein-1 mutants interact 89 more readily with dynactin and the adaptor BicD2 7 . 90Although it is well established that human dynein adopts the autoinhibited phi 91 particle conformation (both dynein-1 and dynein-2), it is unclear if this conformational 92 state is evolutionarily conserved. Yeast dynein is of particular interest due to two 93 notable in vitro discrepancies with human dynein. In particular, unlike human dynein, 94 yeast dynein is processive in single molecule assays without the need for other factors, 95 such as dynactin...

Lis1 activates dynein motility by modulating its pairing with dynactin

“…This finding explains the requirement for Lis1 for transport initiation in vivo 34 , 38 . Consistent with two recent reports that studied the role of Lis1 in mammalian and yeast dynein-dynactin motility 55 , 56 , we show that Lis1’s ability to promote the association of dynein with dynactin also favors the adoption of the two-motor state, which accounts for more frequent stepping and higher force generation per complex. The increased probability of recruiting two dyneins to dynactin also means that Lis1 induces more effective competition against kinesin in a tug-of-war, a result consistent with an increase in anterograde velocity observed when Lis1 is inhibited in cells 37 , 39 .…”

LIS1 promotes the formation of activated cytoplasmic dynein-1 complexes

“…We propose this based on our data showing that Open dynein has a higher affinity for Lis1 and because the structure of Phi dynein is incompatible with Lis1 binding at site ring . Recent work in Aspergillus nidulans 46 and Saccharomyces cerevisiae 47 also supports this. Second, Lis1 may favor a conformation of dynein that is primed to assemble the fully activated complex ( Fig.…”

“…Together our work suggests that Lis1 promotes the formation of human dynein/ dynactin/ activating adaptor complexes containing two dynein dimers. Experiments in human cells 18 , Drosophila embryos 24 , Xenopus extracts 39 , Aspergillus nidulans 46 , and yeast 47 showed that Lis1 is required for the interaction of dynein and dynactin with each other and/or with their cargos. Our work offers a biochemical explanation for this requirement for Lis1.…”

Dynein harnesses active fluctuations of microtubules for faster movement

“…In comparison, mammalian dynein heavy chain is auto-inhibited if it is not transporting cargos, and activation of its motility requires its association with dynactin and a cargo adaptor protein (referred to as dynein transport machinery). The formation of the dynein transport machinery is strongly favored by regulatory proteins, such as Lis1 [56][57][58] . Cargo adaptors, such as BICDR, BICD2, Hook3 and TRAK1-2, contain a long coiled-coil region that links the tail of the heavy chain to the actinlike filament of dynactin.…”

“…Consistent with its asymmetric force-dependent detachment from MT, dynein moved faster towards the minus-end compared to the plus-end under the same magnitude of force ( Figure 8b) 83,84,92 . 51,50,59,53,41,35,48,46,43,54,62,47,82,68,56,92,85,44,45,78 The average velocity (V, positive velocities are in the minus-end direction) at a given force is given by:…”