Components And Kinetics In Exocytosis

尽管细胞质膜（PM）中蛋白质和脂质的横向组织（聚集）对于不同的基本细胞过程至关重要，但对于支配这种聚集的组织机制存在相当大的分歧，例如，1)限制由基于细胞骨架的栅栏，2）蛋白质特异性分区成液体有序的脂筏，或3）拴系的集团ofmolecules到底层肌动蛋白细胞骨架，amongothers。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体是高度动态的，需要以比迄今为止更高的时间带宽记录单个分子，以更好地理解调节膜聚集的物理化学原理。除了生理过程之外，疾病状态的病理生理学基础越来越集中于集群。定位于宿主细胞PM的HA自发聚集，并且对于融合、病毒出芽和感染至关重要;需要所得病毒体上的高HA密度以进入下一个宿主细胞并与之融合。然而，即使这个模型系统产生相互矛盾的数据与HA的机制，脂质集群甚至没有定性的协议，脂质与HA共簇。与其他机制的蛋白质-脂质相互作用，如订购的分子进入脂筏，脂质限制蛋白质围栏，拴系的脂质运动，或缓冲固定的结合位点，我们的研究结果描述和解释空间PIP 2的分布，以及它们如何通过一个明显的动态机制随时间变化的潜在梯度，由于结合位点本身都是移动的和集群。 脂质磷脂酰肌醇4，5-二磷酸（PIP 2）在细胞质膜中形成纳米级簇;然而，决定PIP 2迁移率的过程及其空间模式尚未完全了解。使用活细胞的超分辨率成像，我们发现PIP 2与流感病毒蛋白血凝素（HA）的过表达紧密共定位并受其调节。内和附近的集群，HA和PIP 2遵循类似的空间依赖性，这可以描述由HA依赖的电位梯度; PIP 2分子移动，如果他们被吸引到簇的中心的径向力为0.079 - 0.002 pN的HAb 2细胞。PIP 2的测量聚类和动态与未修改形式的筏、系绳和栅栏模型不一致。相反，我们发现，空间PIP 2分布和它们如何随时间变化的解释，通过一个新的，据我们所知，动态机制：径向梯度的PIP 2结合位点，本身是移动的。这个模型可能是有用的了解其他生物膜域的分布显示密度梯度，同时保持其流动性。

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.