Plasma Membrane Area Increases with Spread Area by Exocytosis of a GPI-anchored Protein Compartment

Abstract

The role of plasma membrane (PM) area as a critical factor during cell motility is poorly understood, mainly due to an inability to precisely follow PM area dynamics. To address this fundamental question, we developed static and dynamic assays to follow exocytosis, endocytosis, and PM area changes during fibroblast spreading. Because the PM area cannot increase by stretch, spreading proceeds by the flattening of membrane folds and/or by the addition of new membrane. Using laser tweezers, we found that PM tension progressively decreases during spreading, suggesting the addition of new membrane. Next, we found that exocytosis increases the PM area by 40-60% during spreading. Reducing PM area reduced spread area, and, in a reciprocal manner, reducing spreadable area reduced PM area, indicating the interconnection between these two parameters. We observed that Golgi, lysosomes, and glycosylphosphatidylinositol-anchored protein vesicles are exocytosed during spreading, but endoplasmic reticulum and transferrin receptor-containing vesicles are not. Microtubule depolymerization blocks lysosome and Golgi exocytosis but not the exocytosis of glycosylphosphatidylinositol-anchored protein vesicles or PM area increase. Therefore, we suggest that fibroblasts are able to regulate about half of their original PM area by the addition of membrane via a glycosylphosphatidylinositol-anchored protein compartment.

The Plasma Membrane Flattens Out to Fuel Cell-Surface Growth during Drosophila Cellularization

“…Although microvilli could be disassembled by apical endocytosis during cellularization (our data and Fabrowski et al, 2013), the resulting endosomes make no clear contribution to furrow ingression (Fabrowski et al, 2013), consistent with our results that endocytosis is not required for the transfer of membrane from microvilli to furrows. Instead, drawing on lessons from filopodia, phagocytic cups and the leading edge of motile cells (Gauthier et al, 2009; Ji et al, 2008; Keren et al, 2008; Masters et al, 2013; Mogilner and Rubinstein, 2005; Raucher and Sheetz, 2000), we suggest that high plasma membrane tension, generated by the pulling forces of furrow ingression, could be sufficient to limit and/or stall actin polymerization at microvillar tips (Tyska and Mooseker, 2002). In this way, furrow ingression itself would act as the signal to both promote microvillar disassembly and prevent assembly of new microvilli.…”

Modification of Cellular Cholesterol Content Affects Traction Force, Adhesion and Cell Spreading

“…7 In addition to endocytosis, exocytosis has also been shown as a critical component of the cell spreading process, by aiding to increase the plasma membrane area. 17 Others have suggested that cholesterol and sphingolipid-rich microdomains of alveolar epithelial type II cells serve as functional platforms during exocytosis. 9 When these cells were depleted of cholesterol, exocytosis was inhibited.…”

Membrane Supply and Demand Regulates F-Actin in a Cell Surface Reservoir

“…Plasma membrane tension is a measure of the deformability of the membrane (Diz-Muñoz et al, 2013). Delivery of more membrane via exocytosis would make the membrane easier to deform (Gauthier et al, 2012; 2009), therefore reducing resistance to actin monomer addition at filament plus ends abutting the membrane (Diz-Muñoz et al, 2013; Keren et al, 2008; Mogilner and Oster, 1996; Mogilner and Rubinstein, 2005; Theriot and Mitchison, 1991). Thus, during cellularization, membrane supply via exocytosis may create a mechanically permissive environment for actin polymerization, which with the right complement of F-actin elongation factors (e.g.…”

“…In addition to scaffolds, reservoir regulation is likely coupled to membrane trafficking, since endo- and exocytosis control the actual amount of membrane at cell surfaces (Gauthier et al, 2012; 2009). Endo- and exocytosis make significant and indisputable contributions to cell surface homeostasis and cell shape change, with demonstrated roles in many of the same events as reservoirs (Gauthier et al, 2011; Groulx et al, 2007; Lecuit and Wieschaus, 2000; Masters et al, 2013).…”