The Ninth q-bio Summer School - Albuquerque: Journal Club 1

Lecture slides Media:Freeman_Journal_Club_final.pdf‎

Current Trends in Single Particle Tracking and Superresolution Microscopy

This journal club will introduce the concepts behind single particle tracking and superresolution imaging and present examples of how they can be used to address important biological questions. We will discuss current papers that use these techniques to understand the spatial dynamics of signaling proteins and the stoichiometry of protein-protein interactions on the cell membrane. For those who cannot attend on Monday, this journal club will be repeated on Tuesday

Papers to be discussed will be:

Freeman, S.A., V. Jaumouillé, K. Choi, B.E. Hsu, H.S. Wong, L. Abraham, M.L. Graves, D. Coombs, C.D. Roskelley, R. Das, S. Grinstein, and M.R. Gold. 2015. Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor. Nat Commun. 6.

Abstract: Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.

Nan, X., T.M. Tamgüney, E.A. Collisson, L.-J. Lin, C. Pitt, J. Galeas, S. Lewis, J.W. Gray, F. McCormick, and S. Chu. 2015. Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway. Proceedings of the National Academy of Sciences. 112:7996-8001.

Abstract Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.