MECHANISM OF MEMBRANE FUSION

膜融合是许多细胞活动的重要过程， 胞吐作用，胞吞作用，胞内膜运输，细胞受精 和细胞分裂。 这些融合现象是由各种 刺激及其作用机制在很大程度上是未知的。 以试图 了解分子水平的基本机制，我们专注于 Ca2+是许多膜基本要求， 融合事件以及胞吐和分泌。 在早期的工作中， 我们研究了Ca 2+与特定磷脂的相互作用， synexin，并建立了体外系统，证明了一些 胞吐融合事件的性质。 在这项拨款申请中，我们重点关注一组细胞质中的功能， 钙离子结合蛋白最近被称为“膜联蛋白”，其功能尚未完全阐明。 至今已成立。 我们建议研究他们各自的能力， 使用涉及脂质体的体外模型的对照融合，分离的 胞内颗粒、内吞囊泡和倒置质膜 囊泡 使用灵敏的膜融合分析系统， 胞吐条件下，我们将检查脂质和蛋白质的作用 特异性，并确定其他细胞质因子的影响。 的 敏感的融合试验的可用性和生物化学的存在 关于蛋白质结构的信息使这个项目成为一个及时和 令人兴奋的前景。 我们的方法涉及三个不同层次的实验策略： 一：不同纯化的膜联蛋白的初步筛选， 它们能够引起特定脂质体组合物在 不同的Ca2+浓度。 第二：对于那些有希望的蛋白质 对融合试验的影响（促进或抑制），我们将进行 详细分析了与脂质双层相互作用的机制， 分子水平。 第三：为了验证他们在 在细胞水平上，我们将研究这些蛋白质促进 细胞内颗粒与倒置质膜的融合 重建胞吐事件的生理条件。 了解胞吐融合的作用具有广泛的健康相关 的影响，因为分泌特定的颗粒内容物是重要的， 宿主防御机制，以及中性粒细胞通过 血管内皮 中性粒细胞不受控制的胞吐作用可能是 参与成人呼吸窘迫综合征的肺部并发症， 心肌梗死后的粘膜损伤，也可能影响诱变。 因此，在分子水平上理解胞吐作用可能会澄清 控制生命和病理功能的机制， 嗜中性粒细胞和其他分泌细胞。

Membrane fusion is a vital process for many cellular activities such as exocytosis, endocytosis, intracellular membrane traffic, cell fertilization and cell division. These fusion phenomena are regulated by a variety of stimuli and their mechanism of action is largely unknown. In an attempt to understand the basic mechanism at the molecular level, we have focussed on the role of Ca2+, which is an essential requirement for many membrane fusion events and certainly for exocytosis and secretion. In earlier work, we have studied the interactions of Ca2+ with specific phospholipids and synexin and have established in vitro systems that demonstrate some of the properties of exocytotic fusion events. In this grant proposal, we focus on the function of a group of cytoplasmic Ca2+-binding proteins recently termed "annexins", whose function has not been established as yet. We propose to study their respective ability to control fusion using in vitro models that involve liposomes, isolated intracellular granules, endocytotic vesicles and inverted plasma membrane vesicles. Using a sensitive assay systems for membrane fusion which mimic exocytotic conditions we will examine the role of lipid and protein specificity and determine the influence of other cytoplasmic factors. The availability of sensitive fusion assays and the existence of biochemical information on the protein structure makes this project a timely and exciting prospect. Our approach involves three different levels of experimental strategy: One: an initial screening of the different purified annexins with respect to their ability to cause fusion of specific liposome compositions at various Ca2+ concentrations. Two: for the proteins that show promising effects on the fusion assays (promotion or inhibition) we will undertake a detailed analysis of the mechanism of interaction with the lipid bilayer at the molecular level. Three: in order to validate their function at the cellular level, we will study the ability of these proteins to promote fusion of intracellular granules with inverted plasma membranes under physiological conditions reconstituting exocytotic events. Understanding the role of exocytotic fusion has wide health-related implications, since secretion of specific granule contents is important in host defense mechanisms, as well as in neutrophil emigration through the vascular endothelium. Uncontrolled exocytosis by neutrophils may be involved in pulmonary complications of adult respiratory distress syndrome, mucosal damage after myocardial infarction and may also affect mutagenesis. Therefore understanding exocytosis at the molecular level may clarify the mechanisms which control both the vital and pathological function of neutrophils and other secretory cells.