Assembly and dynamics of macromolecular protein complexes at the membrane-microtubule nexus during cell division

细胞分裂的精确时空调控是植物生长和形态发生的基础。植物细胞骨架由肌动蛋白丝和微管组成，在细胞分裂过程中起着重要作用。在G2到M的转变过程中，皮层间期微管重组成一个宽的带，称为前期带（PPB），在未来的分裂位点环绕细胞。PPB在前期后期解体。它的位置信息在整个细胞分裂过程中通过极化膜结构域维持，称为皮质分裂区（CDZ）。新细胞壁的形成是由纺锤体分离染色体后在细胞中心开始的。它是由一种双极性微管阵列，作为囊泡传递和细胞板形成的支架而促进的。膜质体离心膨胀并引导生长的细胞板到达CDZ，在那里它最终与母体壁融合产生一个新的交叉壁。不同的微管相关蛋白（MAPs）组调节PPB和纺锤体形成、CDZ的建立、层质体动力学以及微管和膜间室之间的连接。一些遗传学、细胞生物学和生理学的研究已经深入了解了单个map和膜相关蛋白在细胞板形成过程中微管组织和膜动力学中的作用。然而，不同的map如何相互作用，以及微管阵列如何在CDZ、细胞板和囊泡传递过程中与膜协调，仍然是难以捉摸的。我们在模式植物拟南芥中的研究发现，植物特异性的IQ67结构域（IQD）蛋白是一种新的map，可以作为大分子复合物组装的细胞支架。我们发现IQD家族成员通过控制PPB的形成和cdz驻留蛋白的募集来确定分裂面定位。各自的IQD蛋白分别定位于CDZ和细胞板、PPB和phragmoplast的膜和微管上，并通过与其同名的IQ67结构域相邻的不同基序和结构域与CDZ、phragmoplast和细胞板定位的蛋白相互作用。IQ67结构域包含多个功能性钙调蛋白（Calmodulin, CaM）结合基序，表明iqd在钙信号传导中的作用。然而，iqd如何或是否在微管细胞骨架上促进cam介导的钙信号传导仍是未知的。本研究旨在结合细胞生物学、遗传学和蛋白质生物化学，阐明（cam调控的）iqd组装复合物在片质体和细胞板上的动力学和组成。这种结合的方法将加深我们对微管组织的调控及其与膜的联系的理解。因此，我们的策略提供了一个机制框架来揭示细胞分裂及以后大分子蛋白组装信号整合的原理。

Precise spatio-temporal regulation of cell division is fundamental for plant growth and morphogenesis. The plant cytoskeleton, comprised of actin filaments and microtubules, plays essential roles during cell division. During G2 to M transition, cortical interphase microtubules reorganize into a broad band, termed preprophase band (PPB) that encircles the cell at the future division site. The PPB disassembles at late prophase. Its positional information is maintained throughout cell division by a polarized membrane domain, referred to as cortical division zone (CDZ). The formation of a new cell wall is initiated at the cell center after separation of chromosomes by the spindle apparatus. It is facilitated by the phragmoplast, a bipolar array of microtubules that serves as scaffold for vesicle delivery and cell plate formation. The phragmoplast expands centrifugally and guides the growing cell plate to the CDZ, where it eventually fuses with the maternal wall to generate a new crosswall. Distinct sets of microtubule-associated proteins (MAPs) regulate PPB and spindle formation, CDZ set up, phragmoplast dynamics and the connection between microtubules and membrane compartments. Several genetic, cell biological, and physiological studies have provided insights into the role of individual MAPs and membrane-associated proteins in microtubule organization and membrane dynamics during cell plate formation. How different MAPs interact with each other, and how microtubule arrays are coordinated with membranes at the CDZ, cell plate and during vesicle delivery, however, remains elusive. Our work in the model plant Arabidopsis thaliana identified plant-specific IQ67 Domain (IQD) proteins as novel MAPs with proposed roles as cellular scaffolds for macromolecular complex assembly. We showed that members of the IQD family determine division plane positioning by controlling PPB formation and recruitment of CDZ-resident proteins. The respective IQD proteins localize to membranes and microtubules at the CDZ and cell plate, and at the PPB and phragmoplast, respectively, and interact with several CDZ-, phragmoplast- and cell plate-localized proteins via distinct motifs and domains adjacent to its eponymous IQ67 domain. The IQ67 domain harbors multiple functional Calmodulin (CaM) binding motifs pointing to a role of IQDs in calcium signaling. How or if IQDs contribute to CaM-mediated calcium signaling at the microtubule cytoskeleton, however, is still unknown.This proposal aims at elucidating the (CaM-regulated) dynamics and composition of IQD-assembled complexes at the phragmoplast and cell plate by combining cell biology, genetics and protein biochemistry. The combined approach will deepen our understanding of the regulation of microtubule organization and its connection with membranes. Thereby, our strategy offers a mechanistic framework to unravel principles governing signal integration at macromolecular protein assemblies during cell division and beyond.