Characterization and membrane targeting of the Spir/formin action nucleator complex

Spir/formin 作用成核剂复合物的表征和膜靶向

• 批准号: 170438694
• 负责人: Professor Dr. Eugen Kerkhoff
• 金额: --
• 依托单位: Labor Molekulare Zellbiology (Kerkhoff Lab)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/170438694?language=en
• 关键词: Characterization; membrane; targeting; Spir; formin

Spir proteins are the founding members of a novel class of actin nucleation factors, which initiate actin polymerization by binding of actin monomers to one or multiple Wiskott-Aldrich syndrome protein (WASp) homology 2 (WH2) domains. In mammalian cells Spir proteins are targeted towards intracellular membranes by a modified FYVE zinc finger motif and an adjacent potential Rab GTPase binding sequence, the Spir-box. Although a role for mammalian Spir-1 in vesicle transport processes of the exocytic pathway and the transport beyond early endosomes has been described, the function and regulation of the actin organizers at vesicular membranes is unknown. One way Spir function is regulated is by a direct interaction with the distinct actin nucleation factors of the formin subgroup of formins. The interaction is mediated by the Spir KIND domain and the formin Spir interaction (FSI) sequence. Several members of the formin superfamily have been shown to contain at the corresponding position an autoregulatory peptide (DAD, diaphanous autoregulatory domain), that is responsible for binding of a N-terminal autoinhibitory domain (DID, diaphanous inhibitory domain), which causes an autoinhibited backfolded conformation of the formin proteins. It is intriguing to speculate that the FSI sequence acts like a DAD domain and mediates an autoinhibited conformation, which can be released by a competing interaction with the Spir KIND domain. By addressing the question if Spir proteins are autoregulated, we found that the N-terminal Spir KIND domain can pull down the Spir C-terminal part from cell lysates. An autoinhibitory backfolding mechanism including KIND/Spir-CT could regulate both, the membrane targeting and actin nucleation activity. In order to address the regulation of the Spir/formin complex in the context of subcellular localization and actin nucleation, we propose experiments to functionally reconstitute the Spir/formin actin nucleator complex at model membrane systems. We will employ the most recent advances in fluorescence microscopy and lipid membrane technologies, to learn outside of the overwhelming complexity of a cell, how Spir proteins are targeted towards membranes and if and how membrane targeting influences the nucleation activity of the Spir/formin complex. We will address questions such as the membrane docking and lipid specificity of the Spir FYVE module, the potential interaction of Spir with Rab GTPases, a possible dimerization of Spir proteins at membranes and the influence of membrane targeting or dimerization on actin nucleation activity. By incorporating the formin subgroup proteins into our studies, we will analyse how these processes are influenced by the Spir/formin interaction.

Spir蛋白是一类新的肌动蛋白成核因子的创始成员，其通过将肌动蛋白单体结合到一个或多个Wiskott-Aldrich综合征蛋白（WASp）同源2（WH 2）结构域来启动肌动蛋白聚合。在哺乳动物细胞中，Spir蛋白通过修饰的FYVE锌指基序和相邻的潜在Rab GT3结合序列Spir盒靶向细胞内膜。尽管哺乳动物Spir-1在胞吐途径的囊泡运输过程和早期内体以外的运输中的作用已经被描述，但囊泡膜上肌动蛋白组织者的功能和调节尚不清楚。调节Spir功能的一种方式是通过与formin的α亚组的不同肌动蛋白成核因子的直接相互作用。该相互作用由Spir KIND结构域和Spir相互作用（FSI）序列介导。已经显示出在相应的位置处包含自身调节肽（DAD，透明的自身调节结构域）的几个成员，其负责N-末端自身抑制结构域（DID，透明的抑制结构域）的结合，这引起了自身抑制的反向折叠构象的蛋白质。有趣的是，推测FSI序列的作用类似于DAD结构域，并介导自抑制构象，其可以通过与Spir KIND结构域的竞争性相互作用而释放。通过解决Spir蛋白是否被自动调节的问题，我们发现N-末端Spir KIND结构域可以从细胞裂解物中拉下Spir C-末端部分。包括KIND/Spir-CT的自抑制性回折机制可以调节膜靶向和肌动蛋白成核活性。为了解决调节的Spir/actin复合物的上下文中的亚细胞定位和肌动蛋白成核，我们提出的实验，功能重建的Spir/actin成核剂复合物在模型膜系统。我们将采用荧光显微镜和脂质膜技术的最新进展，以了解细胞的压倒性复杂性之外，Spir蛋白如何靶向膜，以及膜靶向是否以及如何影响Spir/Lipid复合物的成核活性。我们将解决的问题，如膜对接和脂质特异性的Spir FYVE模块，潜在的相互作用的Spir与Rab GTPases，一个可能的二聚化的Spir蛋白在膜和膜靶向或二聚化肌动蛋白成核活性的影响。通过将Spir亚组蛋白纳入我们的研究，我们将分析这些过程是如何受到Spir/Spir相互作用的影响。