Disordered Motifs and Domains in Cell Control - October 11-15, 2014

Disordered Motifs and Domains in Cell Control

Tuesday Speaker Abstracts

Phase Separation of a Disordered Protein in the Formation of Membrane-less Organelles Patrick Farber 1 , Timothy J. Nott 3 , Ashok Sekhar 2 , Evangelia Petsalakis 3 , Eden Fussner-Dupas 3 , David Bazett-Jones 1 , Andrew Baldwin 4 , Lewis Kay 2,1 , Julie Forman-Kay 1 . 1 Hospital For Sick Children, Toronto, Canada, 2 University of Toronto, Toronto, ON, Canada, 3 Mount Sinai Hospital, Toronto, ON, Canada, 4 Oxford, Oxford, United Kingdom. Intrinsically disordered proteins and regions (IDPs/IDRs), which do not have stable secondary and tertiary structure, are capable of adopting different structural states. Many IDPs/IDRs populate conformationally heterogeneous monomeric states or engage in discrete interactions with other proteins, leading to folding upon binding or retaining significant disorder in the bound state. Others are involved in large-scale association having different degrees of order, from more defined fibers, to variably networked gels and to disordered liquid demixed states or droplets. These latter have been suggested to provide the physical basis for cellular membrane-less organelles such as the nucleolus and RNA granules. We have studied the N-terminal disordered region of Ddx4, an RNA DEAD-box helicase that is essential for formation of a class of membrane-less organelles termed nuage or germ granules functioning in spermatogenesis. When expressed in HeLa cells, the protein forms spherical, micron-sized, liquid-like cellular organelles. In vitro , it phase separates to form droplets with similar morphological and dynamic properties to the organelles observed in cells. Phase separation is sensitive to salt, pointing to the importance of electrostatic interactions. The sequence features of the disordered N-terminus of Ddx4 underlying phase separation include clustering of charged residues into blocks of net positive and negative charge, with over- representation of FG/GF pairs and RG/GR pairs within the positive blocks. Perturbations of these properties disrupt phase separation, pointing to multi-valent cation-pi interactions playing an important role. Solution NMR data are consistent with a disordered liquid-like state. The transient sampling of multi-valent interactions in self-association of Ddx4 extends previous observations of dynamic multi-valent interactions in discrete complexes of IDPs/IDRs. The insights obtained from these biophysical studies of Ddx4 will be valuable for developing a general understanding of the biogenesis and disassembly of membrane-less cellular organelles. Assembly and Functions of mRNP Granules in Eukaryotic Cells Roy Parker 1,2 , Ross Buchan 3 , Saumya Jain 3 , David Protter 2 , Briana Van Treeck 2 , Robert Walters 2 , Carolyn Decker 1,2 , Jennifer Garcia 2 , Sarah Mitchell 2 , Siddharth Shukla 2 . 2 University of Colorado, Boulder, CO, USA, 3 University of Arizona, Tucson, AZ, USA. 1 Howard Hughes Medical Institute, Chevy Chase, MD, USA, Stress granules and P-bodies are conserved cytoplasmic aggregates of non-translating mRNPs implicated in the regulation of mRNA translation and decay, and are related to RNP granules in embryos, neurons and in some neurodegenerative diseases. In a genetic screen using bakers yeast, we identified 125 genes that affect the dynamics of P-bodies and/or stress granules including multiple components of the vacuolar ATPase, THO/TREX and prefoldin complexes. Analyses of additional mutants, including Cdc48 alleles, provide evidence that stress granules can be targeted to the vacuole by autophagy. An area of current research is understanding the mechanisms that drive stress granule and P-body assembly and clearance and how that affects mRNA function.

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