Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic

Abstract

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 membranes1,2. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphateand phosphatidylinositol-4-phosphate mark distinct endosomal compartments2,3. 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.

Mutations in Auxilin cause parkinsonism via impaired clathrin-mediated trafficking at the Golgi apparatus and synapse

“…Our results provide the first evidence for neuronal dysfunction in a 35 murine model for Auxilin mutations and demonstrate a novel role for Auxilin in the regulation and 36 trafficking of CCVs derived from the trans-Golgi network (TGN). We show mechanistically that 37 mutations in Auxilin are loss of function through diminished interaction with clathrin even though 38 interactions with clathrin adaptor proteins were retained. These data reveal important insights that 39 expand the landscape of vesicle trafficking defects linked to PD, opening up novel pathways that are 40 relevant for therapeutic development in this disorder, as well as illuminating the basic function and 41 biology of Auxilin in CCV activity.…”

“…In addition, Auxilin interacts with the 240 clathrin lattice via clathrin binding boxes and with HSC70 via its J-domain ( Figure ? ?A) [11,[35][36][37]. 241 The subsequent stimulation of the ATPase activity of HSC70 by Auxilin then acts to drive the 242 uncoating of the clathrin coat [11][12][13][38][39][40].…”

Receptor‐Free Signaling at Curved Cellular Membranes

“…[106,107] • Advantages: Improved temporal and spatial resolution compared to confocal microscopy. Upon imaging, the cell is reconstructed in 3D.…”

NERDSS: a non-equilibrium simulator for multibody self-assembly at the cellular scale

“…CME is an essential pathway used by all eukaryotes for transport across the plasma membrane. A wealth of biochemical 20 , structural 21 , and in vivo imaging data [22][23][24] is thus available, but predicting cargo uptake by CME remains remarkably difficult because of the complexity of the pathway. CME is a stochastic process that depends on membrane mechanics, enzymatic reactions, and the stoichiometry of dozens of distinct components.…”

“…Our simulations also show how potently the membrane can act to regulate assembly simply through reduction of dimensionality (3D to 2D) 18 . With NERDSS, we are able to predict how the dephosphorylation of essential plasma membrane lipids, which occurs at sites of vesicle budding 22 , can de-stabilize clathrin-coated structures and drive their disassembly.…”