Abstract
Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) is a type II PI-3-kinase that has been implicated in several important membrane transport and signaling processes. We previously found that overexpression of PI3K-C2alpha inhibits clathrin-mediated membrane trafficking and induces proliferation of novel clathrin-coated structures within the cytoplasm. Using fluorescently tagged fusions of PI3K-C2alpha and clathrin, we explored the behavior of these structures in intact cells. Both proteins are present in the structures, and using rapid image acquisition and fluorescence photoactivation probes, we find that they exhibit localized, rapid mobility (5-20 microm/s). The movement is micro-tubule-based as revealed by use of inhibitors, and PI3K-C2alpha accumulates on microtubules rapidly and reversibly following cytoplasmic acidification, which also blocks movement. Dynactin mediates the movement of these clathrin-PI3K-C2alpha structures, since disruption of dynactin function by overexpression of its p50 subunit also inhibits movement. Finally, immunoprecipitation experiments reveal an interaction between endogenous PI3K-C2alpha and dynactin subunits. Together, these results reveal a molecular linkage between PI3K-C2alpha and the microtubule motor machinery, with implications for membrane trafficking in intact cells.
Highlights
Clathrin-coated membranes are ubiquitous in all eukaryotic cells
PI3K-C2␣ Induces Cytoplasmic Clathrin-coated Structures with Rapid Movement—Our previous work has shown that overexpression of PI3K-C2␣ inhibits clathrin-mediated endocytosis and membrane trafficking (27)
This is readily apparent in single maximum projection images (Fig. 1A) formed by combining stacks of 15 images captured at 36-ms intervals from cells expressing GFP-clathrin alone or cells expressing both GFP-clathrin and PI3KC2␣
Summary
Clathrin-coated membranes are ubiquitous in all eukaryotic cells. They play key roles in physiological processes, such as receptor-mediated endocytosis and other forms of vesicular transport (reviewed in Refs. 1 and 2), and derangements of the clathrin membrane transport system have been implicated in many pathological states (3–5). It is accompanied by the appearance of numerous intracellular CCSs. To learn more about the nature of these novel structures, we investigated their behavior in live COS1 cells transiently expressing both PI3K-C2␣ and GFP-clathrin.
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