Dynamic behavior of lasp‐1 and dynamin‐2 regulated by epidermal growth factor and forskolin

Abstract

Lasp‐1 is a nebulin family actin‐binding phosphoprotein and is expressed in a variety of epithelial cells. In gastric parietal cells, lasp‐1 is localized to the apical and basolateral membrane, sites enriched in F‐actin, and it is serine‐phosphorylated upon histamine stimulation. Affinity columns with lasp‐1 as bait bind to dynamin‐2 (dyn‐2) and clathrin from parietal cell lysates, suggesting that lasp‐1 may play a role in endocytosis. We have expressed a fusion protein of enhanced Green Fluorescent Protein and lasp‐1 (eGFP‐lasp‐1) in HeLa cells to characterize the dynamic behavior of lasp‐1 and its role in endocytosis by live‐cell imaging. Imaging of eGFP‐lasp‐1 revealed that it is targeted to filopodia and lamellipodia in HeLa cells, especially after cells have been incubated with epidermal growth factor. Lasp‐1‐containing structures resembling actin comet tails are also observed, often in spatiotemporal proximity to structures resembling large endocytotic vesicles or macropinosomes. Incubation of cells with forskolin results in a significantly lower level of cortical membrane activity of eGFP‐lasp‐1. However, in cells co‐expressing untagged lasp‐1 and eGFP‐dyn‐2, forskolin treatment results in recruitment of eGFP‐dyn‐2 to these large vesicles. These data suggest that lasp‐1, possibly together with dyn‐2, may regulate the formation or transport of these large membrane vesicles.