Molecular Basis of Dynamic Localization of Class-I Myosins

I 类肌球蛋白动态定位的分子基础

• 批准号: 8939824
• 负责人: EDWARD D KORN
• 金额: $ 75.07万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939824
• 关键词: Acidic Region; Actins; Binding; Binding Sites; Biological Models; Cell division; Cell membrane; Cells; Charge; Complex; DNA; Dictyostelium; Dictyostelium discoideum; Embryonic Development; F-Actin; Fluorescence Microscopy; Genetic Transcription; Glutamine; Goals; Head; Hearing; Human; Immune response; Laboratories; Membrane; Microfilaments; Molecular; Monitor; Motor; Myosin ATPase; Myosin Type I; Myosin Type II; Neoplasm Metastasis; Neurons; Null Lymphocytes; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Protein Binding; Pseudopodia; Regulation; Research; Scaffolding Protein; Site; Tail; Vision; Wound Healing; Yeasts; base; cancer cell; cell motility; glycylproline; mutant; non-muscle myosin; organelle movement

Dictyostelium myosin IB (DMIB) is a typical long-tail class-I myosin with a short (compared to conventional Class II myosins) non-helical tail that contains a basic-hydrophobic membrane-binding region (BH-site), a Gly-Pro-Gln region that binds F-actin, and a Src-homology 3 (SH3) domain. The BH-site binds to membrane regions where acidic phospholipids are concentrated based solely on net negative charge, irrespective of the composition of the phospholipids, in contrast to other myosin-Is that bind preferentially to phosphatidylinositol bisphosphate (PIP2). The Gly-Pro-Gln region binds F-actin in the presence or absence of ATP unlike the ATP-sensitive actin-binding site in the motor domain. Actin waves are self-propagating, membrane-associated F-actin complexes that move from the rear to the front of Dictyostelium cells providing the actin required to advance the leading edge of motile cells and formation of pseudopodia. DMIB, CARMIL (a scaffolding protein that binds Arp2/3) and Arp2/3 (which initiates actin filament branching) are known components of actin waves. The Gerisch lab has postulated that DMIB may be involved in the association of actin waves with the plasma membrane and, through its SH3-domain, bind to CARMIL which would bind Arp2/3. We have investigated the molecular basis of the association of DMIB with actin waves by co-expressing GFP-tagged wild-type and mutant DMIB constructs with RFP-tagged lifeact in DMIB-null cells and monitoring the dynamic localization of DMIB and F-actin by fluorescence microscopy. We find that DMIB is not required for wave formation (i.e. DMIB-null cells form waves) possibly because there are two other long-tailed myosins Is in Dictyostelium. We find that both the membrane-binding BH-site and the actin-binding Gly-Pro-Gln region in the DMIB tail are required for binding DMIB to waves. The motor domain (head) of DMIB is not required for association of DMIB with actin waves but the actin-binding site in the head strengthens the association and stabilizes waves. We conclude that DMIB contributes to anchoring actin waves to the plasma membrane by the binding of the BH-site to acidic phospholipids in the plasma membrane and the binding of the Gly-Pro-Gln region to F-actin in the wave.

网骨藻肌球蛋白IB（DMIB）是典型的长尾I类肌球蛋白，具有短的（与常规II类肌球蛋白相比）非螺旋尾部，其包含碱性疏水膜结合区（BH位点）、结合F-肌动蛋白的Gly-Pro-Gln区和Src-同源3（SH 3）结构域。 BH-位点结合到膜区域，其中酸性磷脂仅基于净负电荷而浓缩，与磷脂的组成无关，这与优先结合到磷脂酰肌醇二磷酸（PIP 2）的其他肌球蛋白-I相反。 Gly-Pro-Gln区域在ATP存在或不存在下结合F-肌动蛋白，这与运动域中的ATP敏感肌动蛋白结合位点不同。 肌动蛋白波是自传播的、膜相关的F-肌动蛋白复合物，其从网骨藻细胞的后部移动到前部，提供推进运动细胞的前缘和伪足形成所需的肌动蛋白。 DMIB、CARMIL（一种结合Arp 2/3的支架蛋白）和Arp 2/3（启动肌动蛋白丝分支）是肌动蛋白波的已知成分。 Gerisch实验室推测DMIB可能参与肌动蛋白波与质膜的结合，并通过其SH 3结构域与CARMIL结合，CARMIL将结合Arp 2/3。 我们研究了DMIB与肌动蛋白波的关联的分子基础，通过在DMIB空细胞中共表达GFP标记的野生型和突变体DMIB构建体与RFP标记的lifeact，并通过荧光显微镜监测DMIB和F-肌动蛋白的动态定位。 我们发现，DMIB是不需要波的形成（即DMIB-空细胞形成波）可能是因为有两个其他长尾肌球蛋白是在网骨藻。 我们发现，DMIB尾部的膜结合BH位点和肌动蛋白结合Gly-Pro-Gln区域都是将DMIB与波结合所必需的。 DMIB的运动域（头部）不需要DMIB与肌动蛋白波的关联，但头部中的肌动蛋白结合位点加强了关联并稳定了波。 我们得出结论，DMIB有助于锚定肌动蛋白波的质膜的BH-网站的结合，在质膜中的酸性磷脂和结合的Gly-Pro-Gln区域的F-肌动蛋白的波。