Here we identify human Kinesin-5, Kif11/HsEg5, as a cellular target of Zika protease. We show that Zika NS2B-NS3 protease targets several sites within the motor domain of HsEg5 irrespective of motor binding to microtubules. The native integral ER-membrane protease triggers mitotic spindle positioning defects and a prolonged metaphase delay in cultured cells. Our data support a model whereby loss of function of HsEg5 is mediated by Zika protease and is spatially restricted to the ER-mitotic spindle interface during mitosis. The resulting phenotype is distinct from the monopolar phenotype that typically results from uniform inhibition of HsEg5 by RNAi or drugs. In addition, our data reveal novel interorganelle interactions between the mitotic apparatus and the surrounding reticulate ER network. Given that Kif11 is haplo-insufficient in humans, and reduced dosage results in microcephaly, we propose that Zika protease targeting of HsEg5 may be a key event in the etiology of Zika syndrome microcephaly.
Zika virus infection can result in profound fetal deficits, such as microcephaly and blindness. Here we identify Kif11/Kinesin-5, as a cellular target of Zika protease with both in vitro and in vivo assays. We show that soluble Zika NS2-3 protease chimera targets several sites within the motor domain of HsEg5 either when free in solution or rigor-bound to microtubules. We tested two different forms of the protease in human cells. First, we find that soluble Zika protease chimera is cytotoxic and eventually leads to cell death. Second, the native integral ERmembrane-associated protease is not only better tolerated by human cells, but also imparts unusual mitotic spindle positioning defects in cultured cells and a prolonged metaphase delay. Our data suggests a model whereby interactions at the spatially restricted ER-mitotic spindle interface bring the protease into the proximity of the pool of mitotic spindle-associated HsEg5. The resulting phenotype is distinct from the monopolar phenotype that typically results from downregulation of HsEg5 due to genetic knock-down or small molecule inhibition. Our results support the idea that not all knockouts are the same; nonuniform down-regulation of protein function can result in unanticipated phenotypes. We postulate that the mitotic spindle mobility phenotype that we observe can prematurely terminate cell lineages that are dependent on asymmetric cell division, such as neuronal lineages. Additionally, our data raise questions about novel inter-organelle communication between the mitotic apparatus and the surrounding reticulate ER network. Finally, given that Kif11 is a monogenic haploinsufficient locus linked to microcephaly, we propose that a potential direct connection between this kinesin and the protease may be a cornerstone in the subsequent pathogenesis of microcephaly.
network model, we show that such motor-driven networks with high concentrations of Arp2/3 complexes show inhibited dynamics because of the saturation of nucleation sites on actin filaments by the Arp2/3 complexes, while low Arp2/ 3 concentrations aggravate contractility of the networks with hallmarks of short contraction time and small actin clusters. At intermediate Arp2/3 concentrations, sudden collapses of actin clusters in the networks, or ''avalanches'', occur. We have implemented graph theory to quantify the higher-order organization among actomyosin networks, powerful to visualize the hierarchy of the complex networks as well as to extract unprecedented insights on the dynamics of actomyosin networks that can be validated experimentally.
Human Kinesin‐5 (Eg5) has a large number of known allosteric inhibitors that disrupt its mitotic function. Small‐molecule inhibitors of Eg5 are candidate anti‐cancer agents and important probes for understanding the cellular function. Here we show that Eg5 is capable of more than one type of microtubule interaction, and these activities can be controlled by allosteric agents. While both monastrol and S‐trityl‐L‐cysteine (STC) inhibit Eg5 motility, our data reveal an unexpected ability of these loop5 targeting inhibitors to differentially control a novel Eg5 microtubule depolymerizing activity.
Remarkably, small molecule loop5 effectors are able to independently modulate discrete functional interactions between the motor and microtubule track. We establish that motility can be uncoupled from the microtubule depolymerase activity and argue that loop5‐targeting inhibitors of Kinesin‐5 should not all be considered functionally synonymous. Also, the depolymerizing activity of the motor does not contribute to the genesis of monopolar spindles during allosteric inhibition of motility, but instead reveals a new function. We propose that, in addition to its canonical role in participating in the construction of the three‐dimensional mitotic spindle structure, Eg5 also plays a distinct role in regulating the dynamics of individual microtubules, and thereby impacts the density of the mitotic spindle.
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