Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions.

Some protein components of intracellular non-membrane-bound entities, such as RNA granules, are known to form hydrogels in vitro. The physico-chemical properties and functional role of these intracellular hydrogels are difficult to study, primarily due to technical challenges in probing these materials in situ. Here, we present iPOLYMER, a strategy for a rapid induction of protein-based hydrogels inside living cells that explores the chemically inducible dimerization paradigm. Biochemical and biophysical characterizations aided by computational modelling show that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that acts as a size-dependent molecular sieve. We functionalize these polymers with RNA-binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. These results show that iPOLYMER can be used to synthetically reconstitute the nucleation of biologically functional entities, including RNA granules in intact cells.

ActuAtor, a Listeria-inspired molecular tool for physical manipulation of intracellular organizations through de novo actin polymerization.

Form and function are often interdependent throughout biology. Inside cells, mitochondria have particularly attracted attention since both their morphology and functionality are altered under pathophysiological conditions. However, directly assessing their causal relationship has been beyond reach due to the limitations of manipulating mitochondrial morphology in a physiologically relevant manner. By engineering a bacterial actin regulator, ActA, we developed tools termed "ActuAtor" that inducibly trigger actin polymerization at arbitrary subcellular locations. The ActuAtor-mediated actin polymerization drives striking deformation and/or movement of target organelles, including mitochondria, Golgi apparatus, and nucleus. Notably, ActuAtor operation also disperses non-membrane-bound entities such as stress granules. We then implemented ActuAtor in functional assays, uncovering the physically fragmented mitochondria being slightly more susceptible to degradation, while none of the organelle functions tested are morphology dependent. The modular and genetically encoded features of ActuAtor should enable its application in studies of the form-function interplay in various intracellular contexts.

Cellular Application of Genetically Encoded Sensors and Impeders of AMPK.

Unraveling the spatiotemporal dynamics of 5'-AMP-activated protein kinase (AMPK) signaling is necessary to bridge the gap between nutrient signaling and downstream function. Three genetically encoded Förster Resonance Energy Transfer (FRET)-based AMPK biosensors are available yielding insight into how AMPK-derived signal propagates throughout a cell in response to particular inputs. These findings, together with accumulating evidence obtained from biochemical techniques, promise to give a holistic understanding of the AMPK signaling. In this protocol, we describe the procedures and materials required for imaging intracellular AMPK activity in an organelle-specific manner, with a focus on ABKAR, a FRET-based biosensor. In addition, we introduce a novel AMPK inhibitor peptide that allows us to inhibit AMPK activity at specific subcellular compartments.

Opening the conformation is a master switch for the dual localization and phosphatase activity of PTEN.

Tumor suppressor PTEN mainly functions at two subcellular locations, the plasma membrane and the nucleus. At the plasma membrane, PTEN dephosphorylates the tumorigenic second messenger PIP3, which drives cell proliferation and migration. In the nucleus, PTEN controls DNA repair and genome stability independently of PIP3. Whereas the concept that a conformational change regulates protein function through post-translational modifications has been well established in biology, it is unknown whether a conformational change simultaneously controls dual subcellular localizations of proteins. Here, we discovered that opening the conformation of PTEN is the crucial upstream event that determines its key dual localizations of this crucial tumor suppressor. We identify a critical conformational switch that regulates PTEN's localization. Most PTEN molecules are held in the cytosol in a closed conformation by intramolecular interactions between the C-terminal tail and core region. Dephosphorylation of the tail opens the conformation and exposes the membrane-binding regulatory interface in the core region, recruiting PTEN to the membrane. Moreover, a lysine at residue 13 is also exposed and when ubiquitinated, transports PTEN to the nucleus. Thus, opening the conformation of PTEN is a key mechanism that enhances its dual localization and enzymatic activity, providing a potential therapeutic strategy in cancer treatments.

Compartmentalized AMPK signaling illuminated by genetically encoded molecular sensors and actuators.

AMP-activated protein kinase (AMPK), whose activity is a critical determinant of cell health, serves a fundamental role in integrating extracellular and intracellular nutrient information into signals that regulate various metabolic processes. Despite the importance of AMPK, its specific roles within the different intracellular spaces remain unresolved, largely due to the lack of real-time, organelle-specific AMPK activity probes. Here, we present a series of molecular tools that allows for the measurement of AMPK activity at the different subcellular localizations and that allows for the rapid induction of AMPK inhibition. We discovered that AMPKα1, not AMPKα2, was the subunit that preferentially conferred spatial specificity to AMPK, and that inhibition of AMPK activity at the mitochondria was sufficient for triggering cytosolic ATP increase. These findings suggest that genetically encoded molecular probes represent a powerful approach for revealing the basic principles of the spatiotemporal nature of AMPK regulation.