Components And Kinetics In Exocytosis

尽管蛋白质和脂类在细胞膜(PM)中的横向组织(聚集)对不同的基本细胞过程至关重要，但对于控制这种聚集的组织机制仍存在相当大的分歧，例如，1)基于细胞骨架的围栏限制，2)蛋白质特定的分配到液体有序的脂筏中，或3)分子群与潜在的肌动蛋白细胞骨架捆绑在一起。对组织原理的机械理解仍然难以捉摸的一个原因是，这种纳米级分子组装是高度动态的，需要在比迄今可能的更高的时间带宽上记录单个分子，以更好地理解调节膜聚集的物理化学原理。除了生理过程外，疾病状态的病理生理学基础越来越多地集中在聚集性上。HA自发地定位于宿主细胞群的PM，对融合、病毒萌发和感染至关重要；所产生的病毒粒子上需要高HA密度才能进入下一个宿主细胞并与其融合。然而，即使这个模型系统产生了关于脂类与HA聚集机制的相互矛盾的数据，对于哪些脂类与HA共聚甚至没有定性的一致。与蛋白质-脂质相互作用的其他机制不同，如分子有序进入脂筏、蛋白质栅栏限制脂类、脂类运动的拴系或固定结合位点的缓冲，我们的发现描述并解释了PIP2的空间分布以及它们如何通过一种明显的动态机制在时间上发生变化，这是由于结合位点本身既是移动的又是聚集的。 脂质磷脂酰肌醇4，5-二磷酸(PIP2)在细胞膜上形成纳米团簇，然而，决定PIP2迁移率的过程及其空间模式并不完全清楚。利用活细胞的超分辨成像，我们发现PIP2与流感病毒蛋白血凝素(HA)的过度表达紧密地共存并受到其调控。在簇内和簇附近，HA和PIP2遵循相似的空间依赖关系，这可以用依赖于HA的势梯度来描述；在HBA2细胞中，PIP2分子的运动就像它们被0.079 0.002 pN的径向力吸引到簇的中心一样。测量到的PIP2的聚集性和动力学与未修改的筏子、系绳和栅栏模型的形式不一致。相反，我们发现PIP2的空间分布及其随时间的变化可以通过一种新的动力学机制来解释：PIP2结合位点的径向梯度本身是可移动的。这个模型可能有助于理解其他生物膜结构域，这些结构域的分布在保持迁移率的同时表现出密度梯度。

Although the lateral organization of proteins and lipids (clustering) in the cell plasma membrane (PM) is crucial to diverse fundamental cellular processes, there is considerable disagreement on the organizational mechanisms that govern such clustering, e.g., 1) confinement by cytoskeleton-based fences, 2) protein-specific partitioning into liquid-ordered lipid rafts, or 3) tethering of groups ofmolecules to the underlying actin cytoskeleton, amongothers. One reason a mechanistic understanding of the organizing principles has remained elusive is that such nanoscale molecular assemblies are highly dynamic, requiring recordings of individual molecules at higher temporal bandwidth than hitherto possible to gain a better understanding of the physicochemical principles that regulate membrane clustering. In addition to physiological processes, the pathophysiological basis of disease states is increasingly focused on clusters. HA localized to the PM of host cells clusters spontaneously and is crucial for fusion, viral budding, and infection; high HA density on resultant virions is needed for entry into and fusion with the next host cell. Yet even this model system generates conflicting data on the mechanism of lipid clustering with HAthere is not even qualitative agreement as to which lipids cocluster with HA. In contrast to other mechanisms of protein-lipid interactions such as ordering of molecules into lipid rafts, lipid confinement by protein fences, tethering of lipid motion, or buffering by fixed binding sites, our findings describe and explain spatial PIP2 distributions and how they change in time via a distinctly dynamic mechanism a potential gradient due to binding sites that are themselves both mobile and clustered. The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using super-resolution imaging of living cells, we find that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA-dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.079 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained via a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.