Proper chromosome segregation requires dynamic regulation of kinetochore-microtubule attachments throughout mitosis. Multiple kinetochore proteins display microtubule-binding activity, yet how exactly these proteins are spatially and temporally regulated is unclear. Cytoplasmic linker-associating proteins (CLASPs) are present in the outer kinetochore, in close proximity to microtubule ends, and are required for mitosis. Here, we test whether phosphorylation of CLASP2 during mitosis serves as a mechanism to regulate kinetochore-microtubule interactions and the fidelity of chromosome segregation. We show that cyclin-dependent kinase 1 (Cdk1) and glycogen synthase kinase beta; (GSK3beta;)-dependent mitotic phosphorylation of CLASP2 within its microtubule end-binding domain inhibits its microtubule end-binding activity but does not affect CLASP2 kinetochore binding. Deregulation of CLASP2 phosphorylation does not affect initial chromosome congression, but weakens kinetochore-microtubule attachments. In the presence of endogenous CLASP2, phosphorylation-deficient CLASP2 increases average interkinetochore distance, while phosphomimetic CLASP2 decreases average interkinetochore distance measurements. Together, these results suggest that CLASP2 microtubule binding at the kinetochore is required for stable, tension-generating kinetochore-microtubule attachments. Furthermore, cells expressing CLASP2 phosphomutants have abnormal kinetochore dynamics and in some cells, kinetochore pairs flip about the metaphase plate, suggesting absent or imbalanced microtubule attachments. Expression of either nonphosphorylatable or phosphomimetic CLASP2 fails to rescue depletion of CLASP2 and ultimately leads to an increase in lagging chromosomes. Together, these results characterize specific phosphorylation sites in CLASP2 that negatively regulate microtubule binding and present a novel mechanism of Cdk1 and GSK3beta;-mediated control of kinetochore-microtubule interactions.

Here we find that the nematode C. elegans makes goal-oriented turns while foraging and can maintain a working memory of sensory activation prior to the execution of a turn. This information is integrated with body posture to localize appetitive stimuli. We construct a virtual-reality whole-brain imaging and neural perturbation system, and with it we find that this working memory is implemented by the coupled oscillation of two distributed neural motor command complexes. One complex decouples from motor output after sensory evidence accumulation and initiates turn execution; the second gates this process with a reliable time constant. We propose that the function of working memory via internalization of motor oscillations could represent the evolutionary origin of internal neural processing i.e. thought, and a foundation of higher cognition.

Epithelial remodeling, in which apical-basal polarized cells switch to a migratory phenotype, plays essential roles in development and disease of multicellular organisms. How cytoskeleton dynamics, especially microtubule dynamics, are controlled and contribute to epithelial remodeling in a more physiological three-dimensional (3D) environment is not understood. We optimize confocal live-cell imaging to analyze microtubule function and dynamics during 3D epithelial remodeling of polarized Madin-Darby kidney epithelial cells that undergo a partial epithelial-to-mesenchymal transition (pEMT) in response to hepatocyte growth factor (HGF). We found that cellular extensions at the basal surface of HGF-treated cysts are densely packed with microtubules. Computational tracking of EB1-2xEGFP showed large numbers of microtubules growing persistently from the apical domain into these extensions and an increase in microtubule growth rate in response to HGF. We tested the role of microtubule plus-end-tracking protein (+TIP) complexes in 3D epithelial remodeling by depleting cells of EB1, an adaptor protein that mediates recruitment of other +TIP proteins to growing microtubule plus ends. In EB1-depleted cells, microtubules displayed rapid lateral and retrograde movements demonstrating that EB1 is required to stabilize and organize microtubules in HGF-induced extensions. EB1-depleted cysts formed shorter, more branched, extensions suggesting that EB1 is required for productive HGF-induced extension outgrowth. Analysis of cell-matrix interactions and F-actin dynamics revealed that control extensions progressively pulled on and deformed the extracellular matrix (ECM) typically with one F-actin-rich protrusion near the cell tip with cell-matrix adhesions that turned over in a coordinated fashion. In contrast, EB1-depleted cells produced multiple highly dynamic protrusions with nascent adhesions that were uncoordinated, mislocalized and did not productively engage the matrix. As a result EB1-depleted extensions rapidly protruded, retracted and changed direction. Finally we show that trafficking of VAMP3-positive vesicles to the protrusion tip is disrupted in EB1-depleted cells. Together these data suggest that EB1-mediated organization of the MT cytoskeleton and associated vesicle delivery to the tip of HGF-induced extensions are likely required to coordinate cell-matrix adhesion and protrusion dynamics during 3D epithelial remodeling. It will be important in the future to investigate a broader role for EB1-mediated +TIP complexes in other normal and disease states of epithelial remodeling.