Search

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.

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.

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 Forster 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.

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.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved heterotrimeric complex composed of α (catalytic α1 and α2) and β/γ (regulatory β1, β2, γ1, γ2, and γ3) subunits and serves as a gatekeeper of energy metabolism by responding to metabolic needs of a cell. Recent findings implicated the enzyme in a broad range of cellular functions, including cell growth, autophagy, polarity, transcription, as well as metabolism [1]. AMPK has thus emerged as a potent target for treating diseases such as cancer and diabetes. Previously, Cantley and colleagues have reported findings regarding compartmentalized signaling of AMPK using a FRET-based AMPK biosensor [2]. AMPK biosensors localized to intracellular compartments such as nucleus and cytosol have revealed that glycolysis inhibitor, 2-deoxyglucose (2-DG), increased AMPK activity in the cytosol but not in the nucleus. In contrast, ionomycin-induced influx of calcium ions caused AMPK activation in both regions. AMPK fulfills this role likely by dynamically adjusting its activity at individual compartments. These observations implied that AMPK is part of a broader signaling network involving other subcellular localizations. We recently improved the dynamic range of a series of organelle-specific AMPK biosensors [3], [4]. With the improved properties of the biosensor, we were able to visualize AMPK activity at many of the major subcellular compartments in real time. We then compared these activity profiles between AMPK-null mouse embryonic fibroblasts (5-DKO :MEFs) and genetically matched wildtype MEFs (5-WT MEFs). Under a nutrient-rich condition, AMPK exhibited various degrees of activation across the different subcellular compartments. In general, AMPK activity at the organellar membrane was significantly higher than in the cytosol or the nucleus. A constitutively active form of AMPK increased in the cytosol and nucleus but was less effective at other organellar membranes. This was further corroborated by fractionation assays revealing that endogenous AMPK was mainly localized at the organellar membranes rather than the cytosol. The emichment of AMPK in non-cytosolic compartments can be a possible mechanism by which the activation threshold is lowered for AMPK-mediated acute responses. To further examine the compartmentalized AMPK signaling, we measured AMPK activity at each organelle under nutrient-limited conditions triggered either by 2-DG treatment or glucose starvation. As expected, the activity of AMPK was versatile across different compartments to glycolytic perturbations. This suggests that the stimuli were independently processed across the compartments despite similar physiological outcomes. Furthermore, a constituent of the AMPK complexes determined their localization. AMPK complexes that solely contained al subunit tended to favor the plasma membrane, Golgi apparatus, and lysosome; complexes containing either αl or α2 subunit distributed the enzyme equally across the cytosol, endoplasmic reticulum, and mitochondria. We next developed a genetically encoded molecular inhibitor based on AMPK peptide inhibitor (AIP). By targeting the AIPs to individual compartments, we achieved inhibition of AMPK activity, for example, at the mitochondria or the Golgi apparatus without affecting activity in cytosol. In addition, we gained control over the compartment in which AMPK inhibition took place by rapid recruitment of AIP to the organelle using a chemically-inducible dimerization technique [6]. One noteworthy finding was that prolonged perturbation of AMPK signaling at the mitochondria in WT MEFs caused metabolic reprogramming by which intracellular ATP increased relative to that observed in DKO :MEFs. This suggests that localized perturbation is sufficient for inhibiting specific aspects of AMPK signaling. In the study presented here, we revealed the intricacy of compartmentalized AMPK regulation in living cells by developing and implementing genetically encoded organelle-specific molecular tools for AMPK. We unambiguously demonstrated that AMPK activity is compartmentalized. Possible future studies could explore how cells achieve spatio temporally precise AMPK activity and identify the molecules that determines AMPK activity under conditions beyond nutrient levels. Understanding the molecular mechanisms that underlie AMPK regulation could provide insights into the development of new therapeutic strategies for AMPK-related diseases.

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.