The microtubule-based motor dynein generates pulling forces for centrosome centration and mitotic spindle positioning in animal cells. How the essential dynein activator dynactin regulates these functions of the motor is incompletely understood. Here, we dissect the role of dynactin's microtubule binding activity, located in the p150 CAP-Gly domain and an adjacent basic patch, in the C. elegans zygote. Analysis of p150 mutants engineered by genome editing suggests that microtubule tip tracking of dynein-dynactin is dispensable for targeting the motor to the cell cortex and for generating robust cortical pulling forces. Instead, mutations in p150's CAP-Gly domain inhibit cytoplasmic pulling forces responsible for centration of centrosomes and attached pronuclei. The centration defects are mimicked by mutations of α-tubulin's C-terminal tyrosine, and both p150 CAP-Gly and tubulin tyrosine mutants decrease the frequency of early endosome transport from the cell periphery towards centrosomes during centration. Our results suggest that p150 GAP-Gly domain binding to tyrosinated microtubules promotes initiation of dynein-mediated organelle transport in the dividing one-cell embryo, and that this function of p150 is critical for generating cytoplasmic pulling forces for centrosome centration.

Cytokinesis is the last step of cell division that physically partitions the mother cell into two daughter cells. Cytokinesis requires the assembly and constriction of a contractile ring, a circumferential array of filamentous actin (F-actin), non-muscle myosin II motors (myosin), and actin-binding proteins that forms at the cell equator. Cytokinesis is accompanied by long-range cortical flows from regions of relaxation toward regions of compression. In the C. elegans one-cell embryo, it has been suggested that anterior-directed cortical flows are the main driver of contractile ring assembly. Here, we use embryos co-expressing motor-dead and wild-type myosin to show that cortical flows can be severely reduced without major effects on contractile ring assembly and timely completion of cytokinesis. Fluorescence recovery after photobleaching in the ingressing furrow reveals that myosin recruitment kinetics are also unaffected by the absence of cortical flows. We find that myosin cooperates with the F-actin crosslinker plastin to align and compact F-actin bundles at the cell equator, and that this cross-talk is essential for cytokinesis. Our results thus argue against the idea that cortical flows are a major determinant of contractile ring assembly. Instead, we propose that contractile ring assembly requires localized concerted action of motor-competent myosin and plastin at the cell equator.

To take full advantage of fast-acting temperature-sensitive mutations, thermal control must be extremely rapid. We developed the Therminator, a device capable of shifting sample temperature in ~17 s while simultaneously imaging cell division in vivo. Applying this technology to six key regulators of cytokinesis, we found that each has a distinct temporal requirement in the Caenorhabditis elegans zygote. Specifically, myosin-II is required throughout cytokinesis until contractile ring closure. In contrast, formin-mediated actin nucleation is only required during assembly and early contractile ring constriction. Centralspindlin is required to maintain division after ring closure, although its GAP activity is only required until just prior to closure. Finally, the chromosomal passenger complex is required for cytokinesis only early in mitosis, but not during metaphase or cytokinesis. Together, our results provide a precise functional timeline for molecular regulators of cytokinesis using the Therminator, a powerful tool for ultra-rapid protein inactivation.

The molecular motor dynein concentrates at the kinetochore region of mitotic chromosomes in animals to accelerate spindle microtubule capture and to control spindle checkpoint signaling. In this study, we describe the molecular mechanism used by the Rod-Zw10-Zwilch complex and the adaptor Spindly to recruit dynein to kinetochores in Caenorhabditis elegans embryos and human cells. We show that Rod's N-terminal β-propeller and the associated Zwilch subunit bind Spindly's C-terminal domain, and we identify a specific Zwilch mutant that abrogates Spindly and dynein recruitment in vivo and Spindly binding to a Rod β-propeller-Zwilch complex in vitro. Spindly's N-terminal coiled-coil uses distinct motifs to bind dynein light intermediate chain and the pointed-end complex of dynactin. Mutations in these motifs inhibit assembly of a dynein-dynactin-Spindly complex, and a null mutant of the dynactin pointed-end subunit p27 prevents kinetochore recruitment of dynein-dynactin without affecting other mitotic functions of the motor. Conservation of Spindly-like motifs in adaptors involved in intracellular transport suggests a common mechanism for linking dynein to cargo.

The kinetochore is a dynamic multi-protein assembly that forms on each sister chromatid and interacts with microtubules of the mitotic spindle to drive chromosome segregation. In animals, kinetochores without attached microtubules expand their outermost layer into crescent and ring shapes to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. Kinetochore expansion is an example of protein co-polymerization, but the mechanism is not understood. Here, we present evidence that kinetochore expansion is driven by oligomerization of the Rod-Zw10-Zwilch (RZZ) complex, an outer kinetochore component that recruits the motor dynein and the SAC proteins Mad1-Mad2. Depletion of ROD in human cells suppresses kinetochore expansion, as does depletion of Spindly, the adaptor that connects RZZ to dynein, although dynein itself is dispensable. Expansion is also suppressed by mutating ZWILCH residues implicated in Spindly binding. Conversely, supplying cells with excess ROD facilitates kinetochore expansion under otherwise prohibitive conditions. Using the C. elegans early embryo, we demonstrate that ROD-1 has a concentration-dependent propensity for oligomerizing into micrometer-scale filaments, and we identify the ROD-1 β-propeller as a key regulator of self-assembly. Finally, we show that a minimal ROD-1-Zw10 complex efficiently oligomerizes into filaments in vitro. Our results suggest that RZZ's capacity for oligomerization is harnessed by kinetochores to assemble the expanded outermost domain, in which RZZ filaments serve as recruitment platforms for SAC components and microtubule-binding proteins. Thus, we propose that reversible RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

In mitosis, the molecular motor dynein is recruited to kinetochores by the Rod-Zw10-Zwilch complex (RZZ) and Spindly to control spindle assembly checkpoint (SAC) signaling and microtubule attachment. How the ubiquitous dynein co-factors Lis1 and NudE contribute to these functions remains poorly understood. Here, we show that the C. elegans NudE homolog NUD-2 is dispensable for dynein- and LIS-1-dependent mitotic spindle assembly in the zygote. This facilitates functional characterization of kinetochore-localized NUD-2, which is recruited by the CENP-F-like proteins HCP-1 and HCP-2 independently of RZZ-Spindly and dynein-LIS-1. Kinetochore dynein levels are reduced in Δnud-2 embryos, and, as occurs upon RZZ inhibition, loss of NUD-2 delays the formation of load-bearing kinetochore-microtubule attachments and causes chromatin bridges in anaphase. Survival of Δnud-2 embryos requires a functional SAC, and kinetochores without NUD-2 recruit an excess of SAC proteins. Consistent with this, SAC signaling in early Δnud-2 embryos extends mitotic duration and prevents high rates of chromosome mis-segregation. Our results reveal that both NUD-2 and RZZ-Spindly are essential for dynein function at kinetochores, and that the gain in SAC strength during early embryonic development is relevant under conditions that mildly perturb mitosis.

All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo.

The mechanism(s) through which mammalian kinase MELK promotes tumorigenesis is not understood. We find that the C. elegans orthologue of MELK, PIG-1, promotes apoptosis by partitioning an anti-apoptotic factor. The C. elegans NSM neuroblast divides to produce a larger cell that differentiates into a neuron and a smaller cell that dies. We find that in this context, PIG-1 MELK is required for partitioning of CES-1 Snail, a transcriptional repressor of the pro-apoptotic gene egl-1 BH3-only. pig-1 MELK is controlled by both a ces-1 Snail- and par-4 LKB1-dependent pathway, and may act through phosphorylation and cortical enrichment of nonmuscle myosin II prior to neuroblast division. We propose that pig-1 MELK-induced local contractility of the actomyosin network plays a conserved role in the acquisition of the apoptotic fate. Our work also uncovers an auto-regulatory loop through which ces-1 Snail controls its own activity through the formation of a gradient of CES-1 Snail protein.

The dynein-2 motor complex drives retrograde intraflagellar transport (IFT), playing a pivotal role in the assembly and functions of cilia. However, the mechanisms that regulate dynein-2 motility remain poorly understood. Here, we identify the Caenorhabditis elegans WDR60 homologue, WDR-60, and dissect the roles of this intermediate chain using genome editing and live imaging of endogenous dynein-2/IFT components. We find that loss of WDR-60 impairs dynein-2 recruitment to cilia and its incorporation onto anterograde IFT trains, reducing retrograde motor availability at the ciliary tip. Consistent with this, we show that fewer dynein-2 motors power WDR-60-deficient retrograde IFT trains, which move at reduced velocities and fail to exit cilia, accumulating on the distal side of the transition zone. Remarkably, disrupting the transition zone's NPHP module almost fully restores ciliary exit of underpowered retrograde trains in wdr-60 mutants. This work establishes WDR-60 as a major contributor to IFT, and the NPHP module as a roadblock to dynein-2 passage through the transition zone.