Phase separation is an emerging organizing principle for intracellular biology. Processes that are now understood to exploit phase separation include storage of genetic material, gene expression, ribosome synthesis, signaling, stress response, and metabolism. While each phase-separating system has unique features, there are universal themes relevant to all such systems, including regulation of the phase boundary, the dynamics of mixing within and between condensates, and the interactions of condensates with their surroundings. To uncover general principles regarding these common themes, we focus on a well-suited model organism and system: the genetically tractable alga Chlamydomonas reinhardtii and its pyrenoid, a non-membrane bound, phase-separated organelle responsible for efficient carbon fixation. The pyrenoid offers many practical advantages: 1. its phase separation is driven by two well- characterized components, the rigid enzyme complex Rubisco and the flexible linker protein EPYC1, via a known specific binding interface; 2. the pyrenoid?s in vivo liquidity is reproduced in vitro with no energy source; 3. in vivo assembly/disassembly is controllable by external cues; and 4. the pyrenoid is singular, large, and stable enough to systematically investigate its functional interactions with other cellular components. Based on these advantages, the pyrenoid has already proven to be a source of many discoveries including the ability of a flexible multivalent linker to condense a rigid component, inheritance of non-membrane bound organelles by fission, specific recruitment via a conserved binding motif, and a magic-number effect. The key universal questions we will address with this system are: What is the role of the valence, strength, and spacing of the interacting motifs in determining condensate properties? How does the stability of small oligomers control phase boundaries? What keeps condensates in a liquid state? How do cells control the number, size, and location of condensates, including their relation to other cellular structures? Our approach will closely integrate theory and experiment, as providing fundamental answers to these questions requires a multidisciplinary approach that places specific data within a broad theoretical framework. We anticipate that our focus on underlying biophysical mechanisms will facilitate generalizability of our results to a wide range of phase-separated intracellular systems.

Public Health Relevance

It is becoming clear that many cellular components are localized to non-membrane-bound intracellular compartments and this organization is crucial for functions including molecular processing, storage, signaling, and metabolism. Dysfunctions in the organization of non-membrane-bound compartments are implicated in human diseases including cancer and amyotrophic lateral sclerosis (ALS). Our study will reveal general principles governing the assembly, internal properties, and structural integration of non-membrane-bound compartments within cells, with impacts on our understanding of their roles in health and disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Modeling and Analysis of Biological Systems Study Section (MABS)
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Lyster, Peter
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Princeton University
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United States
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