The Michnick Lab

Research Statement - Michnick, Stephen W.

Overview

A central goal of our research program over the next five-ten years will be to continue our efforts to understand the structure and dynamics of protein-protein interaction networks, now extending to non-membrane organelles, their roles in generating forces and controlling chemical processes and their implications in neurodegenerative diseases. In a separate but related theme, we continue our research on biochemical mechanisms of signal processing in response to the environment and molecular mechanisms of cellular “memory”.

Principles and function of non-membranous organelles in organization of living cells

Over a century ago colloidal phase-separation of matter was recognized as a fundamental organizing principle of cells. The properties of such phase-separated bodies (“liquid droplets” or “non-membrane organelles”) present challenges to our understanding of cellular networks and opportunities for understanding molecular interaction networks, their evolution, physiological roles and insights into disease origins. This has inspired us to explore new questions that follow from our interests in the organization of matter in living cells (Bergeron-Sandoval et al., Cell, 2016; Landry et al., Cell, 2013; Tarassov et al. Science, 2008).

Discovering compositions and functions of non-membrane organelles

We’ve known since we performed the first study of the in vivo protein interactome of the yeast Saccharomyces cerevisiae that proteins are mostly not organized into complexes as we would have expected. Phase separation of proteins into liquid droplets could explain some or much of this non-complex protein interactome. We will apply our expertise in large-scale screening and quantitative imaging to map out the liquid droplet protein interactome of yeast. Our main aim will be to develop heuristic rules and perhaps physical principles to explain how unique droplets are formed from intrinsically unstructured proteins separated from their cellular milieu. The results could provide a first glimpse of the physical principles that have governed the evolution of non-membranous organelles and may reveal unforeseen cellular functions governed by liquid-liquid phase separation of protein.

Clathrin-mediated endocytosis (CME) is a liquid droplet-driven process

Among the consequences of droplet formation is the possibility that the interfacial tension of droplets and their milieu could be a source of energy necessary for the large-scale reorganization of matter. CME is critical to the homeostasis of membrane proteins, to morphogenesis and to uptake of nutrients and other molecules from outside the cell. Yet we don’t know how this process initiates. Our central hypothesis is that CME is driven by interfacial tension between a protein liquid droplet formed at the site of CME on the plasma membrane and cytosol, creating the force needed to drive membrane invagination. We are applying super-resolution microscopy to follow CME evolution, and with Paul François (McGill), we are testing a predictive model for droplet-mediated membrane invagination. If our hypothesis is correct, these studies could open up a new avenue to investigating mechanisms of generation of all forms of membrane-borne vesicles.

Liquid droplet-driven transcriptional memory

Localization of chromatin to the nuclear periphery has been demonstrated to underlie transcriptional memory of environmental stimuli that can persist for several cell cycles. Several inducible genes in yeast relocate to the nuclear envelope (NE) when activated. We hypothesize that the relocalization of active gene loci to NE is driven by a phase separation of specific locus-binding proteins to form liquid droplets that interact with the nuclear membrane. To test this hypothesis, we will employ the same strategies used to study CME and apply them to the INO1 gene locus. If the hypothesis is validated, it will constitute the first example of a phase-separated structure regulating the transcription of an inducible gene. The same principles tested in this project may apply to other gene loci as well as other cases where chromatin reorganization occurs in yeast and other eukaryotes, such as during replication.

Polyvalent molecules drive normal or pathological behaviors of non-membrane organelles

Behaviors of phase-separating proteins may be modulated by ionic polymers, including polyphosphates and polyamines, and these molecules may also affect the pathogenesis of neurodegenerative diseases. We will test the hypothesis that these polymers stabilize or destabilize known non-membrane organelles called processing (P)-bodies and stress granules, thus altering their homeostasis. Polyamine abundance decreases with age, and this may affect interactions of proteins implicated in neurodegenerative diseases with liquid droplets, potentially contributing to the pathogenic effects of these molecules.

Memory and chance in environmental sensing

Much like human knowledge, information in simple single-cell organisms about the environment is retained, erased and even passed from one generation to the next. Intelligence emerges from the dynamic and self-organizing principles that govern liquid phase-separation of proteins in living cells. We have developed experimental strategies and conceptual tools to understand these principles and have applied them to study self-organization of molecular networks in cells and their consequences to cell-fate decision processes. We are now expanding our efforts to study molecular organizing principles and their implications in single cell and trans-generational memory. Similar molecular memory mechanisms likely exist in higher organisms, including processes in healthy individuals such as differentiation of stem cells and imprinting of memory, and pathological processes such as protein aggregation in neurodegenerative disease or nutrient-seeking strategies of cancer cells.

A cell-fate switch governed by phase-separation of mating-versus cell division-driving machineries

We are exploring the molecular mechanisms generating history-dependent behavior during nutrient or pheromone sensing in budding yeast and investigating environments that favor such hysteresis. We aim, using quantitative imaging and other approaches, to quantify the inheritance of "knowledge" of previous environments from mother to daughter cells.

Further, we are determining epigenetic and signaling mechanisms of hysteresis, the environmental conditions under which hysteresis can be actively erased, and the mechanism of erasure. Our goal is to generate a system-level view of how the past influences cellular decision-making and, as such, how memory shapes individuality. One such approach we have already used to discover a hypersensitive mechanism for mate selection (Malleshaiah et al. Nature, 2010) and delay or acceleration of the cell cycle in response to nutrients (Messier et al., Cell, 2013).

What do others say about our research

Jensen, L. J. and Bork, P. Not comparable, but complementary. Science, 2008. 322: p. 56-7.

Kast, J. Making connections for life: an in vivo map of the yeast interactome. HFSP J, 2008. 2 (5): p. 244-50.

Secko, D. Canvassing Protein Complexes. The Scientist, 2008. 22: p. 55.

Kaganman, I. Live-cell map quest. Nature Methods, 2008. 5: p. 582.

Graham-Rowe, D. Finding "Hidden" Drug Effects: A new technique could reveal the benefits and drawbacks of experimental drugs earlier in the discovery process. MIT Technology Review, 2006.

Borman, S. Detecting Drugs 'Influence: Technique reveals compounds' direct effects on protein signaling interactions. Chemical and Engineering News, 2006. p. 11.

Abraham, R. T. Signalomic signatures enlighten drug profiling. Nature Chem Biol, 2006. 2: p. 295-6.

Perks, B. Biochemists reveal hidden drug effects. Chemistry World, 2006.

Kapoor, T. Faculty of 1000 Evaluation.2006.

Marcotte, E. M. Assembling a jigsaw puzzle with 20,000 parts. Genome Biol., 2003. 2: p. 323.

Forst, C. V. Using a systems biological approach to develop novel therapeutics. Pharmacogenomics, 2002. 3, 12-4.

Brock, A., and Huang, S. Proteomics in context. Trends Biotechnology, 2001. 19 (11): p. 435-6.

Brenner, C.; Langridge, P. and Crews, C. Faculty of 1000 Evaluation. 2001.

Ostermeier, M. and Benkovic, S. J. Finding Cinderella's slipper-proteins that fit. Nature Biotechnology, 1999. 19: p. 639-40.

Mayeux, P. En l'absence d'hormone, le récepteur de l'érythropoïétine est déjà un dimère. Médecine/Sciences, 1999. 15: p. 1049-50.

Jiang, G. and Hunter, T. Receptor signaling: when dimerization is not enough. Curr. Biol., 1999. 9 (15): p. R568-71.