Search

Very fast CRISPR on demand Numerous efforts have been made to improve the temporal resolution of CRISPR-Cas9–mediated DNA cleavage to the hour time scale. Liu et al. developed a Cas9 system that achieved genome-editing manipulation at the second time scale (see the Perspective by Medhi and Jasin). Part of the guide RNA is chemically caged, allowing the Cas9-guide RNA complex to bind at a specific genomic locus without cleavage until activation by light. This fast CRISPR system achieves genome editing at high temporal resolution, enabling the study of early molecular events of DNA repair processes. This system also has high spatial resolution at short time scales, allowing editing of one genomic allele while leaving the other unperturbed. Science , this issue p. 1265 ; see also p. 1180

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.

Physiological Ca2+ signalling in T lymphocytes and other cells depends on the STIM-ORAI pathway of store-operated Ca2+ entry. STIM1 and STIM2 are Ca2+ sensors located in the endoplasmic reticulum (ER) membrane, with ER-luminal domains that monitor cellular Ca2+ stores and cytoplasmic domains that gate ORAI channels in the plasma membrane. The STIM ER-luminal domain dimerizes or oligomerizes upon dissociation of Ca2+, but the mechanism transmitting activation to the STIM cytoplasmic domain has not been defined. Here we demonstrate, using Tb3+–acceptor energy transfer, that dimerization of STIM1 ER-luminal domains can initiate an extensive conformational change in murine STIM1 cytoplasmic domains. The conformational change, triggered by apposition of the predicted coiled-coil 1 (CC1) regions, releases the ORAI-activating domains from their interaction with the CC1 regions and allows physical extension of the STIM1 cytoplasmic domain across the gap between ER and plasma membrane to communicate with ORAI channels.

The rapid, inducible, reversible modulation of molecular activities by dual CID systems. Rapamycin (Rapa) treatment induces relocation of FRB-POI to the GAIs-FKBP-C2(LACT)-labeled plasma membrane and activates POI-dependent signaling event. A subsequent GA3-AM treatment induces second relocation of a whole GAIs-FKBP-C2(LACT)/rapamycin/FRB-POI complex from the plasma membrane to the Tom20-GID1-labeled mitochondria and leads to termination of POI-dependent signaling.

Physiological Ca2+ signalling in T lymphocytes and various other mammalian cells depends on the STIM-ORAI pathway of store-operated Ca2+ entry. STIM1 and STIM2 are Ca2+ sensors located in the endoplasmic reticulum (ER) membrane, with ER-luminal domains that monitor cellular Ca2+ stores and cytoplasmic domains that gate ORAI channels in the plasma membrane. Physiological stimulation_ through the T cell receptor, the Fce receptor of mast cells, or various G protein-coupled receptors in other cells_ initiates a sequence of ER Ca2+ depletion, dimerization or oligomerization of the STIM luminal domain, and targeting of STIM to ER-plasma membrane junctions. STIM at ER-plasma membrane junctions recruits and directly activates the ORAI channel. Here we demonstrate that dimerization of STIM1 ER-luminal domains triggered by dissociation of Ca2+ initiates an extensive conformational change in STIM1 cytoplasmic domains that involves apposition of the predicted coiled-coil 1 (CC1) regions, physical extension of the STIM1 cytoplasmic domain, and increased exposure of the STIM1 polybasic C-terminal tail. Together these conformational changes promote interaction of the STIM1 C-terminal region with the plasma membrane, the first essential step for communication of STIM1 with ORAI calcium channels in the plasma membrane.