Aurora A orchestrates entosis by regulating a dynamic MCAK-TIP150 interaction

Entosis, a ceU-in-ceU process, has been implicated in the formation of aneuploidy associated with an aberrant cell division control. Microtubule plus-end-tracking protein TI P150 facilitates the loading of MCAK onto the microtubule plus ends and orchestrates micro- tubule plus-end dynamics during cell division. Here we show that TIP150 cooperates with MCAK to govern entosis via a regulatory cir- cuitry that involves Aurora A-mediated phosphorylation of MCAK. Our biochemical analyses show that MCAK forms an intra-molecular association, which is essential for TIP150 binding. Interestingly, Aurora A-mediated phosphorylation of MCAK modulates its intra-mo- lecular association, which perturbs the MCAK-TI P150 interaction in vitro and inhibits entosis in vivo. To probe if MCAK-TIP150 inter- action regulates microtubule plasticity to affect the mechanical properties of ceUs during entosis, we used an optical trap to measure the mechanical rigidity of live MCF7 ceils. We find that the MCAK cooperates with TIP150 to promote microtubule dynamics and modulate the mechanical rigidity of the cells during entosis. Our results show that a dynamic interaction of MCAK-TI P150 orchestrated by Aurora A-mediated phosphorylation governs entosis via regulating microtubule plus-end dynamics and cell rigidity. These data reveal a previously unknown mechanism of Aurora A regulation in the control of microtubule plasticity during ceU-in-ceU pro- cesses.     

Kinesin as an Electrostatic Machine

Kinesin and related motor proteins utilize ATP fuel to propel themselves along the external surface of microtubules in a processive and directional fashion. We show that the observed step-like motion is possible through time-varying charge distributions furnished by the ATP hydrolysis cycle while the static charge configuration on the microtubule provides the guide for motion. Thus, while the chemical hydrolysis energy induces appropriate local conformational changes, the motor translational energy is fundamentally electrostatic. Numerical simulations of the mechanical equations of motion show that processivity and directionality are direct consequences of the ATP-dependent electrostatic interaction between the different charge distributions of kinesin and the microtubule.     

HX-MS2 for high performance conformational analysis of complex protein states

Water-mediated hydrogen exchange (HX) processes involving the protein main chain are sensitive to structural dynamics and molecular interactions. Measuring deuterium uptake in amide bonds provides information on conformational states, structural transitions and binding events. Increasingly, deuterium levels are measured by mass spectrometry (MS) from proteolytically generated peptide fragments of large molecular systems. However, this bottom-up method has limited spectral capacity and requires a burdensome manual validation exercise, both of which restrict analysis of protein systems to generally less than 150 kDa. In this study, we present a bottom-up HX-MS² method that improves peptide identification rates, localizes high-quality HX data and simplifies validation. The method combines a new peptide scoring algorithm (WUF, weighted unique fragment) with data-independent acquisition of peptide fragmentation data. Scoring incorporates the validation process and emphasizes identification accuracy. The HX-MS² method is illustrated using data from a conformational analysis of microtubules treated with dimeric kinesin MCAK. When compared to a conventional Mascot-driven HX-MS method, HX-MS² produces two-fold higher α/β-tubulin sequence depth at a peptide utilization rate of 74%. A Mascot approach delivers a utilization rate of 44%. The WUF score can be constrained by false utilization rate (FUR) calculations to return utilization values exceeding 90% without serious data loss, indicating that automated validation should be possible. The HX-MS² data confirm that N-terminal MCAK domains anchor kinesin force generation in kinesin-mediated depolymerization, while the C-terminal tails regulate MCAK-tubulin interactions.     

A kinesin-1 variant reveals motor-induced microtubule damage in cells

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