Journal Article

Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic

Gaudin, Raphael, Kirchhausen, Tom, Capraro, Benjamin R., Marsland III, Robert, Skillern, Wesley, Song, Eli, Wang, Weiming, Ma, Minghe, Upadhyayula, Srigokul, Dang, Song, He, Kangmin

2017

Overview

‘Coincidence-detecting’ phosphoinositide sensors are used to study changes in the phosphoinositide lipid species found in membranes during the development and maturation of endocytic clathrin-coated vesicles. Changing composition of cell membranes The traffic within cells is busy. At any given time, many vesicles bud off the membrane of one organelle and travel to fuse with another membrane elsewhere. Which characteristics identify the donor and acceptor membranes is an intriguing question. The answer seems to be the lipid and protein composition of the membranes, specifically the presence and relative abundance of the seven species of phosphoinositide lipids, as well as GTP-bound GTPases. Tom Kirchhausen and colleagues describe a new generation of phosphoinositide sensors. They used these sensors to study the phosphoinositide composition of clathrin-associated membranes, which are involved in the process of endocytosis. The findings provide information on how the composition of the membrane changes from the time it is coated with clathrin to form pits, to when the pits close into vesicles, and, once the vesicles bud off, to when they lose their clathrin coating and fuse with endosomes. Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes 1 , 2 . Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments 2 , 3 . It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe ‘coincidence-detecting’ sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 14 / 14/63 / 631/1647/1888 / 631/80/313/1461 / 9/10 / Analysis / Animals / Auxilins - metabolism / Cell Line / Cell Membrane - chemistry / Cell Membrane - metabolism / Chlorocebus aethiops / Clathrin - metabolism / Clathrin-Coated Vesicles - chemistry / Clathrin-Coated Vesicles - metabolism / Coated Pits, Cell-Membrane - chemistry / Coated Pits, Cell-Membrane - metabolism / COS Cells / Endocytosis / Endosomes - chemistry / Endosomes - metabolism / Humanities and Social Sciences / Humans / letter / Life Sciences / multidisciplinary / Phosphatidylinositol 4,5-Diphosphate - metabolism / Phosphatidylinositol phosphates / Phosphatidylinositol Phosphates - metabolism / Phosphatidylinositols - analysis / Phosphatidylinositols - chemistry / Phosphatidylinositols - metabolism / Phosphoric Monoester Hydrolases - metabolism / Phosphorylation / Phosphotransferases - metabolism / rab5 GTP-Binding Proteins - metabolism / Receptor-mediated endocytosis / Science