How lipids and proteins cooperate to form functional membrane domains in cells remains a major unanswered question in cell biology. The lipid raft hypothesis has become a widely studied model for understanding this process. Rafts have been implicated in normal cellular functions ranging from membrane trafficking to cell signaling. They have also have been linked to a number of diseases and are thought to represent a major site for pathogen entry into cells. Current models propose that raft domains normally exist as nanoscale compositional fluctuations at steady state in cells, but can be stabilized to form functional rafts. However, how stabilized rafts assemble and function remains unclear. In this application, we propose to address these fundamental questions using the non-toxic membrane binding B-subunit of cholera toxin (CTxB) as a model. CTxB is a homopentamer that contains 5 binding sites for its glycolipid receptor, ganglioside GM1, itself a raft-associated molecule. Give its ability to cluster multiple GM1 molecules simultaneously, CTxB is widely regarded to serve as a raft crosslinker that builds stabilized raft domains. Moreover, the entry of cholera toxin int cells is thought to depend importantly on its targeting to raft-dependent, clathrin-independent endocytic pathways. However, the mechanisms by which these CTxB- stabilized domains form, how this process is regulated in cells, and how stabilized rafts are preferentially sorted into clathrin-independent endocytic carriers remain poorly understood. Here, we propose to investigate these questions though the following aims: 1) to test the hypothesis that CTxB must cluster raft-associated glycolipids to assemble stabilized raft domains; 2) to determine how toxin-stabilized rafts interact with the actin cytoskeleton; and 3) to determine if stabilized raft play an active role in targeting CTxB for uptake by clathrin independent endocytosis by mechanically deforming membranes. Successful completion of these aims will provide mechanistic insights into proteins and lipids cooperate to form stabilized rafts, how raft assembly is regulated by the cytoskeleton, and the role of stabilized rafts in endocytosis.
Many pathogens, including the bacterial toxin that causes cholera, gain entry into cells by binding to lipid components of the plasma membrane and inducing the formation of complexes that must be internalized by cells in specific ways to cause disease. The properties of the complexes formed by these lipid-binding toxins are not well understood, but are thought to rely importantly on the ability of the toxins to bind certain types f lipids, as well as to bind multiple lipids simultaneously. Here, we will study how these complexes assemble and how they manipulate cell membranes to enable toxins to hijack cells.
| Raghunathan, Krishnan; Foegeding, Nora J; Campbell, Anne M et al. (2018) Determinants of Raft Partitioning of the Helicobacter pylori Pore-Forming Toxin VacA. Infect Immun 86: |
| Saito-Diaz, Kenyi; Benchabane, Hassina; Tiwari, Ajit et al. (2018) APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway. Dev Cell 44:566-581.e8 |
| Kenworthy, Anne K (2018) Bigger Isn't Always Better: Bulking Up Impedes Receptor Internalization. Biophys J 114:1255-1256 |
| Raghunathan, Krishnan; Kenworthy, Anne K (2018) Dynamic pattern generation in cell membranes: Current insights into membrane organization. Biochim Biophys Acta Biomembr 1860:2018-2031 |
| Simunovic, Mijo; Manneville, Jean-Baptiste; Renard, Henri-François et al. (2017) Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Proteins. Cell 170:172-184.e11 |
| Chandrasekaran, Ramyavardhanee; Kenworthy, Anne K; Lacy, D Borden (2016) Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis. PLoS Pathog 12:e1006070 |
| Klaus, Colin James Stockdale; Raghunathan, Krishnan; DiBenedetto, Emmanuele et al. (2016) Analysis of diffusion in curved surfaces and its application to tubular membranes. Mol Biol Cell 27:3937-3946 |
| Han, Bing; Copeland, Courtney A; Kawano, Yumeko et al. (2016) Characterization of a caveolin-1 mutation associated with both pulmonary arterial hypertension and congenital generalized lipodystrophy. Traffic 17:1297-1312 |
| Tiwari, Ajit; Copeland, Courtney A; Han, Bing et al. (2016) Caveolin-1 is an aggresome-inducing protein. Sci Rep 6:38681 |
| Raghunathan, Krishnan; Wong, Tiffany H; Chinnapen, Daniel J et al. (2016) Glycolipid Crosslinking Is Required for Cholera Toxin to Partition Into and Stabilize Ordered Domains. Biophys J 111:2547-2550 |
Showing the most recent 10 out of 15 publications
