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At all levels in biology, form and function are closely interdependent. Mitochondria have particularly attracted attention since their altered morphology is often observed under pathophysiological conditions including heart failure and Alzheimer’s disease. Despite the physiological significance, assessing the direct causal relationship between mitochondrial morphology and function has been out of reach, primarily due to limitations of the existing experimental technologies such as optical tweezers and atomic force microscopy in manipulating the morphology of this submicron length-scale entity deep inside living cells. By engineering an actin regulator, ActA, derived from Listeria monocytogenes , and coupling it with protein dimerization schemes, we first established molecular tools collectively termed ActuAtor that can trigger actin polymerization to exert constrictive forces at subcellular locations of choice in a rapidly inducible manner. The ActuAtor-mediated forces drove striking movement and/or deformation of target intracellular structures including mitochondria, Golgi apparatus, and nucleus, as well as non-membrane-bound biomolecular condensates such as stress granules. We then implemented ActuAtor in functional assays of mitochondria, uncovering that fragmented mitochondria are more susceptible to degradation, while none of the other essential functions tested like ATP synthesis are morphology dependent. The modular and genetically-encoded features of ActuAtor should enable its applications in de novo studies of the interplay between form and function at various intracellular and subcellular spaces.

SummaryMechanical force underlies fundamental cell functions such as division, migration and differentiation. While physical probes and devices revealed cellular mechano-responses, how force is translated inside cells to exert output functions remains largely unknown, due to the limited techniques to manipulate force intracellularly. By engineering an ActA protein, an actin nucleation promoting factor derived fromListeria monocytogenes, and implementing this in protein dimerization paradigms, we developed a molecular tool termed ActuAtor, with which actin polymerization can be triggered at intended subcellular locations to generate constrictive force in a rapidly inducible manner. The ActuAtor operation led to striking deformation of target intracellular structures including mitochondria, Golgi apparatus, nucleus, and non-membrane-bound RNA granules. Based on functional analysis before and after organelle deformation, we found the form-function relationship of mitochondria to be generally marginal. The modular design and genetically-encoded nature enable wide applications of ActuAtor for studies of intracellular mechanobiology processes.

Non-membrane bound, hydrogel-like entities, such as RNA granules, nucleate essential cellular functions through their unique physico-chemical properties. However, these intracellular hydrogels have not been as extensively studied as their extracellular counterparts, primarily due to technical challenges in probing these materials in situ. Here, by taking advantage of a chemically inducible dimerization paradigm, we developed iPOLYMER, a strategy for rapid induction of protein-based hydrogels inside living cells. A series of biochemical and biophysical characterizations, in conjunction with computational modeling, revealed that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that behaves as a size-dependent molecular sieve. We studied several properties of the gel and functionalized it with RNA binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. Therefore, we here demonstrate that iPOLYMER presents a unique and powerful approach to synthetically reconstitute hydrogel-like structures including RNA granules in intact cells.

Non-membrane bound, hydrogel-like entities, such as RNA granules, nucleate essential cellular functions through their unique physico-chemical properties. However, these intracellular hydrogels have not been as extensively studied as their extracellular counterparts, primarily due to technical challenges in probing these materialsin situ.Here, by taking advantage of a chemically inducible dimerization paradigm, we developed iPOLYMER, a strategy for rapid induction of protein-based hydrogels inside living cells. A series of biochemical and biophysical characterizations, in conjunction with computational modeling, revealed that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that behaves as a size-dependent molecular sieve. We studied several properties of the gel and functionalized it with RNA binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. Therefore, we here demonstrate that iPOLYMER presents a unique and powerful approach to synthetically reconstitute hydrogel-like structures including RNA granules in intact cells.