Mechanism of microtubule severing enzymes

细胞通过微管聚合酶、解聚酶、交联剂和切割酶的协同作用不断地组装和拆卸它们的微管细胞骨架。 微管切断酶spastin和katanin在微管中产生内部断裂。它们在广泛的细胞生物学过程中至关重要，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。 微管切割酶的突变导致严重的神经退行性和神经发育障碍。这些酶使微管不稳定的机制及其对微管动力学和微管网络形态的影响仍然知之甚少。我们的目标是：（1）了解微管分解过程中spastin和katanin的结构转变;（2）描述微管切割反应中katanin和spastin六聚体中ATP水解的机制，以及它们如何与微管蛋白二聚体从微管晶格中去除的机械功耦合;（3）建立微管蛋白修饰对微管切割的影响;（4）描述微管切割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痉挛素和katanin疾病突变如何分别影响蛋白质结构和功能;（6）鉴定调节痉挛素和katanin的细胞因子。 今年，我们继续我们的研究微管切断酶的结构与他们的监管机构使用cryo-EM和单分子荧光成像复杂，并确定了他们的调节机制，由辅助因子和翻译后修饰。具体地，我们发现谷氨酰化和甘氨酰化是拮抗性变阻器，甘氨酰化保护微管免于切断（Szczesna等人，2022年）。Katanin对α和β-微管蛋白尾部的谷氨酰化表现出分级和不同的反应，并且这些反应是组合作用的。α-尾上的谷氨酰化是纯刺激性的，而β-尾上的谷氨酰化是由katanin六聚体中心孔识别谷氨酰化尾决定的双相反应。元件远端卡他宁AAA核心正义-微管蛋白酪氨酸，和去酪氨酸下调切断。这种使katanin能够读取多个微管蛋白修饰输入的多价微管识别解释了体内观察，并说明了效应器如何整合微管蛋白编码信号以在细胞中产生不同的功能结果。

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. Specifcially, we discovered that glutamylation and glycylation are antagonistic rheostats with glycylation protecting microtubules from severing (Szczesna et al., 2022). Katanin exhibits graded and divergent responses to glutamylation on the alpha and beta-tubulin tails, and these act combinatorially. Glutamylation on the alpha-tail is purely stimulatory, while glutamylation on the beta-tail elicits a biphasic response dictated by the recognition of the glutamylated tail by the katanin hexamer central pore. Elements distal to the katanin AAA core sense -tubulin tyrosination, and detyrosination downregulates severing. This multivalent microtubule recognition that enables katanin to read multiple tubulin modification inputs explains in vivo observations and illustrates how effectors can integrate tubulin code signals to produce diverse functional outcomes in cells.