Conformational transitions of the Spindly adaptor underlie its interaction with Dynein and Dynactin

Abstract

Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end–directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein–Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein–Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein–Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.

Microtubule binding-induced allostery promotes LIS1 dissociation from dynein prior to cargo transport

“…Human proteins (LIS1-SNAP, or motor domains) were expressed and purified from insect cells (ExpiSf9 cells; Life Technologies) as previously described with minor modifications 3,7,20,69 . Briefly, 4 ml of ExpiSf9 cells at 2.5 × 10 6 cells/ml, which were maintained in ExpiSf CD Medium (Life Technologies), were transfected with 1-9 µg of bacmid DNA using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions. 4-8 days following transfection, the cells were pelleted, and 1-2 ml of the resulting supernatant (P1) was used to infect ∼150 ml of ExpiSf9 cells (5 × 10 6 cells/ml).…”

“…Briefly, 4 ml of ExpiSf9 cells at 2.5 × 10 6 cells/ml, which were maintained in ExpiSf CD Medium (Life Technologies), were transfected with 1-9 µg of bacmid DNA using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions. 4-8 days following transfection, the cells were pelleted, and 1-2 ml of the resulting supernatant (P1) was used to infect ∼150 ml of ExpiSf9 cells (5 × 10 6 cells/ml). Approximately 65 hours later, the cells were harvested (2000 x g, 20 min), washed with human dynein lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 1 mM DTT, 0.1 mM Mg-ATP, 1 mM PMSF; note ATP was omitted for LIS1 purification), pelleted again (1810 x g, 20 min), and resuspended in an equal volume of same.…”

“…Processive dynein motility requires that it first associate with its activating complex dynactin, and a cargo adaptor that link dynein to dynactin and a variety of cargoes 2,3 . Recent studies have revealed additional levels of regulation that include autoinhibitory mechanisms intrinsic to the dynein and dynactin complexes, and also for some of the cargo adaptors 2,[4][5][6][7][8] . For example, dynein exists in an autoinhibited conformational state referred to as the 'phi' particle (due to its similarity to the Greek letter) that restricts its ability to bind to dynactin and the cargo adaptor 7,8 .…”

A mechanism that integrates microtubule motors of opposite polarity at the kinetochore corona

“…Spindly contains various sequence elements implicated in its function and regulation as a DD adaptor (Figure 4E). In previous work, we reported that Spindly may exist in an autoinhibited conformation (Figure 4-Supplement 1A) and proposed that an unknown kinetochore trigger promotes a conformational change required for efficient binding of Spindly to DD at the kinetochore (d'Amico et al, 2022). The Spindly motif, identified in Spindly and other DD adaptors (Figure 4E), promotes an interaction with the pointed-end (PE) subcomplex of Dynactin (Gama et al, 2017;Gassmann et al, 2010).…”

“…In our previous work, we also described mutants of Spindly that overcome autoinhibition and bind PE (d'Amico et al, 2022). These mutants, however, were not recruited effectively to kinetochores, presumably because the mutated residues (in the 276-306 region of Spindly) also affected the interaction of Spindly with an unknown kinetochore receptor (Barisic et al, 2010;d'Amico et al, 2022). We therefore scanned additional mutants to identify a separation-of-function mutant that would relieve Spindly autoinhibition without affecting kinetochore recruitment.…”