Components And Kinetics In Exocytosis

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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.