Mechanism of microtubule severing enzymes

细胞通过微管聚合酶、解聚合酶、交联剂和切断酶的协同作用，不断地组装和拆卸其微管细胞骨架。微管切断酶痉挛蛋白和角朊蛋白在微管中产生内部断裂。它们在广泛的细胞生物学过程中起着至关重要的作用，包括神经元和非中心体微管阵列的生物发生、向光性、纺锤体缩放、染色体分离以及中心粒和纤毛数量的控制。微管切断酶的突变导致严重的神经退行性和神经发育障碍。这些酶破坏微管稳定性的机制及其对微管动力学和微管网络形态的影响尚不清楚。我们的目标是(1)了解spastin和katanin在微管分解过程中所经历的结构转变；(2)表征了微管切断反应中角朊蛋白和肌蛋白酶六聚体中ATP水解的机制，以及它们如何与微管晶格中微管蛋白二聚体去除的机械功耦合；(3)建立微管蛋白修饰对微管切断的影响；(4)表征微管切断酶对微管动力学和结构的影响；(5)全面了解与遗传性痉挛性截瘫和小头畸形相关的spastin和katanin疾病突变如何分别影响蛋白质结构和功能；(6)确定调节spastin和katanin的细胞因子。今年，我们继续使用低温电镜和单分子荧光成像技术研究微管切断酶及其调节因子的复合物结构，并确定了其通过辅助因子和翻译后修饰的调节机制。

Cells constantly assemble and disassemble their microtubule cytoskeleton through the concerted action of microtubule polymerases, depolymerases, crosslinkers and severing enzymes. Microtubule severing enzymes spastin and katanin generate internal breaks in microtubules. They are are critical in a wide range of cell biological processes including biogenesis of neuronal and non-centrosomal microtubule arrays, phototropism, spindle scaling, chromosome segregation, and control of centriole and cilia numbers. Mutations in microtubule severing enzymes cause severe neurodegenerative and neurodevelopmental disorders. The mechanism used by these enzymes to destabilize the microtubule and their effect on microtubule dynamics and the morphology of microtubule networks is still poorly understood. We aim (1) to understand the structural transitions that spastin and katanin undergo during microtubule disassembly; (2) characterize the mechanism of ATP hydrolysis in the katanin and spastin hexamers during the microtubule severing reaction and how they are coupled to the mechanical work of tubulin dimer removal from the microtubule lattice; (3) establish the effects of tubulin modifications on microtubule severing; (4) characterize the effects of microtubule severing enzymes on microtubule dynamics and architecture; (5) develop a comprehensive understanding of how spastin and katanin disease mutations associated with hereditary spastic paraplegia and microcephaly, respectively, affect protein structure and function and (6) identify cellular factors that regulate spastin and katanin. This year we have continued our studies into the structure of microtubule severing enzymes in complex with their regulators using cryo-EM and single molecule fluorescence imaging and have identified mechanisms of their regulation by accessory factors and posttranslational modifications.