Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin 7) and Dynein-Dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The corona\'s building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we report near-complete depletion of RZZS and DD from kinetochores after depletion of CENP-E and the outer kinetochore protein KNL1. With inhibited MPS1, CENP-E, which we show binds directly to RZZS, is required to retain kinetochore RZZS. An RZZS phosphomimetic mutant bypasses this requirement. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo.

Multiple microtubule-directed activities concentrate on chromosomes during mitosis to ensure their accurate distribution to daughter cells. These activities include couplers and dynamics regulators localized at the kinetochore, the specialized microtubule interface built on centromeric chromatin, as well as motor proteins recruited to kinetochores and to mitotic chromatin. Here, we describe an in vivo reconstruction approach in which the effect of removing the major microtubule-directed activities on mitotic chromosomes is compared to the selective presence of individual activities. This approach revealed that the kinetochore dynein module, comprised of the minus end-directed motor cytoplasmic dynein and its kinetochore-specific adapters, is sufficient to biorient chromosomes and to remodel outer kinetochore composition following microtubule attachment; by contrast, the kinetochore dynein module is unable to support chromosome congression. The chromosome-autonomous action of kinetochore dynein, in the absence of the other major microtubule-directed factors on chromosomes, rotates and orients a substantial proportion of chromosomes such that their sister chromatids attach to opposite spindle poles. In tight coupling with orientation, the kinetochore dynein module drives removal of outermost kinetochore components, including the dynein motor itself and spindle checkpoint activators. The removal is independent of the other major microtubule-directed activities and kinetochore-localized protein phosphatase 1, suggesting that it is intrinsic to the kinetochore dynein module. These observations indicate that the kinetochore dynein module has the ability coordinate chromosome biorientation with attachment state-sensitive remodeling of the outer kinetochore that facilitates cell cycle progression.

The microtubule minus-end-directed motility of cytoplasmic dynein 1 (dynein), arguably the most complex and versatile cytoskeletal motor, is harnessed for diverse functions, such as long-range organelle transport in neuronal axons and spindle assembly in dividing cells. The versatility of dynein raises a number of intriguing questions, including how is dynein recruited to its diverse cargo, how is recruitment coupled to activation of the motor, how is motility regulated to meet different requirements for force production and how does dynein coordinate its activity with that of other microtubule-associated proteins (MAPs) present on the same cargo. Here, these questions will be discussed in the context of dynein at the kinetochore, the supramolecular protein structure that connects segregating chromosomes to spindle microtubules in dividing cells. As the first kinetochore-localized MAP described, dynein has intrigued cell biologists for more than three decades. The first part of this Review summarizes current knowledge about how kinetochore dynein contributes to efficient and accurate spindle assembly, and the second part describes the underlying molecular mechanisms and highlights emerging commonalities with dynein regulation at other subcellular sites.

Chromosomal instability (CIN) plays a key role in the carcinogenesis of several human cancers and can be related to the deregulation of core components of the spindle assembly checkpoint (SAC) including BUBR1 protein kinase. These proteins have been related to tumor development and poor survival rates in human patients with oral squamous cell carcinoma (OSCC). To investigate the expression of the SAC proteins BUBR1, BUB3 and SPINDLY and also Ki-67 in canine OSCC, we performed an immunohistochemical evaluation in 60 canine OSCCs and compared them with clinical and pathological variables. BUBR1, Ki-67, BUB3 and SPINDLY protein expressions were detected in all cases and classified as with a high-expression extent score in 31 (51.7%) cases for BUBR1, 33 (58.9%) cases for BUB3 and 28 (50.9%) cases for SPINDLY. Ki-67 high expression was observed in 14 (25%) cases. An independent prognostic value for BUBR1 was found, where high BUBR1 expression was associated with lower survival (p = 0.012). These results indicate that BUBR1 expression is an independent prognostic factor in these tumors, suggesting the potential use for clinical applications as a prognostic biomarker and also as a pharmacological target in canine OSCC.

SPINDLY is involved in some aspects of plant development. However, the nature of this protein as an O-fucosyltransferase was recently discovered. In this study, we show that SPINDLY (SPY) interacts with CPN20 in yeast two-hybrid and split-luc assays, and the interaction is promoted by ABA. CPN20 is a chloroplast-localized co-chaperonin that negatively regulates ABAR-mediated ABA signaling. By using Electron Transfer Dissociation-MS/MS analysis, two O-fucosylation sites, e.g., 116th and 119th threonines, were detected in ectopically expressed CPN20 in mammalian cells and in Arabidopsis. The O-fucosylation at both threonine residues was confirmed by in vitro peptide O-fucosylation assay. We further show that CPN20 accumulates in the chloroplast of spy mutants, suggesting that SPY negatively regulates CPN20 localization in the chloroplast. In vivo protein degradation assay along with CPN20 localization behavior suggest that import of CPN20 into the chloroplast is negatively regulated by SPY. Genetic analysis shows that ABA insensitive phenotypes of spy-3 in terms of seed germination and early seedling development are partially suppressed by the cpn20 mutation, suggesting that CPN20 acts downstream of SPY in this ABA signaling pathway and that there may exist other pathways in parallel with CPN20. Collectively, the above data support the notion that the O-fucosylation of CPN20 by SPY fine-tunes ABA signaling in Arabidopsis.

Since its inception, proximity-dependent biotin identification (BioID), an in vivo biochemical screening method to identify proximal protein interactors, has seen extensive developments. Improvements and variants of the original BioID technique are being reported regularly, each expanding upon the existing potential of the original technique. While this is advancing our capabilities to study protein interactions under different contexts, we have yet to explore the full potential of the existing BioID variants already at our disposal. Here, we used BioID2 in an innovative manner to identify and map domain-specific protein interactions for the human Ku70 protein. Four HEK293 cell lines were created, each stably expressing various BioID2-tagged Ku70 segments designed to collectively identify factors that interact with different regions of Ku70. Historically, although many interactions have been mapped to the C-terminus of the Ku70 protein, few have been mapped to the N-terminal von Willebrand A-like domain, a canonical protein-binding domain ideally situated as a site for protein interaction. Using this segmented approach, we were able to identify domain-specific interactors as well as evaluate advantages and drawbacks of the BioID2 technique. Our study identifies several potential new Ku70 interactors and validates RNF113A and Spindly as proteins that contact or co-localize with Ku in a Ku70 vWA domain-specific manner.

Spindly is a dynein adaptor involved in chromosomal segregation during cell division. While Spindly\'s N-terminal domain binds to the microtubule motor dynein and its activator dynactin, the C-terminal domain (Spindly-C) binds its cargo, the ROD/ZW10/ZWILCH (RZZ) complex in the outermost layer of the kinetochore. In humans, Spindly-C binds to ROD, while in C. elegans Spindly-C binds to both Zwilch (ZWL-1) and ROD-1. Here, we employed various biophysical techniques to characterize the structure, dynamics and interaction sites of C. elegans Spindly-C. We found that despite the overall disorder, there are two regions with variable α-helical propensity. One of these regions is located in the C-terminal half and is compact; the second is sparsely populated in the N-terminal half. The interactions with both ROD-1 and ZWL-1 are mostly mediated by the same two sequentially remote disordered segments of Spindly-C, which are C-terminally adjacent to the helical regions. The findings suggest that the Spindly-C binding sites on ROD-1 in the ROD-1/ZWL-1 complex context are either shielded or conformationally weakened by the presence of ZWL-1 such that only ZWL-1 directly interacts with Spindly-C in C. elegans.

To maintain genome stability, chromosomes must be equally distributed among daughter cells at the end of mitosis. The accuracy of chromosome segregation requires sister-kinetochores to stably attach to microtubules emanating from opposite spindle poles. However, initial kinetochore-microtubule interactions are able to turnover so that defective attachment configurations that typically arise during early mitosis may be corrected. Growing evidence supports a role for the RZZ complex in preventing the stabilization of erroneous kinetochore-microtubule attachments. This inhibitory function of RZZ toward end-on attachments is relieved by DYNEIN-mediated transport of the complex as chromosomes congress and appropriate interactions with microtubules are established. However, it remains unclear how DYNEIN is antagonized to prevent premature RZZ removal. We recently described a new mechanism that sheds new light on this matter. We found that POLO kinase phosphorylates the DYNEIN adaptor SPINDLY to promote the uncoupling between RZZ and DYNEIN. Elevated POLO activity during prometaphase ensures that RZZ is retained at kinetochores to allow the dynamic turnover of kinetochore-microtubule interactions and prevent the stabilization of erroneous attachments. Here, we discuss additional interpretations to explain a model for POLO-dependent regulation of the RZZ-SPINDLY-DYNEIN module during mitosis.

Accurate chromosome segregation in mitosis requires sister kinetochores to bind to microtubules from opposite spindle poles. The stability of kinetochore-microtubule attachments is fine-tuned to prevent or correct erroneous attachments while preserving amphitelic interactions. Polo kinase has been implicated in both stabilizing and destabilizing kinetochore-microtubule attachments. However, the mechanism underlying Polo-destabilizing activity remains elusive. Here, resorting to an RNAi screen in Drosophila for suppressors of a constitutively active Polo mutant, we identified a strong genetic interaction between Polo and the Rod-ZW10-Zwilch (RZZ) complex, whose kinetochore accumulation has been shown to antagonize microtubule stability. We find that Polo phosphorylates Spindly and impairs its ability to bind to Zwilch. This precludes dynein-mediated removal of the RZZ from kinetochores and consequently delays the formation of stable end-on attachments. We propose that high Polo-kinase activity following mitotic entry directs the RZZ complex to minimize premature stabilization of erroneous attachments, whereas a decrease in active Polo in later mitotic stages allows the formation of stable amphitelic spindle attachments. Our findings demonstrate that Polo tightly regulates the RZZ-Spindly-dynein module during mitosis to ensure the fidelity of chromosome segregation.

Elucidation of the genetic control of rice architecture is crucial due to the global demand for high crop yields. Rice architecture is a complex trait affected by plant height, tillering, and panicle morphology. In this study, principal component analysis (PCA) on 8 typical traits related to plant architecture revealed that the first principal component (PC), PC1, provided the most information on traits that determine rice architecture. A genome-wide association study (GWAS) using PC1 as a dependent variable was used to isolate a gene encoding rice, SPINDLY (OsSPY), that activates the gibberellin (GA) signal suppression protein SLR1. The effect of GA signaling on the regulation of rice architecture was confirmed in 9 types of isogenic plant having different levels of GA responsiveness. Further population genetics analysis demonstrated that the functional allele of OsSPY associated with semidwarfism and small panicles was selected in the process of rice breeding. In summary, the use of PCA in GWAS will aid in uncovering genes involved in traits with complex characteristics.