Spatiotemporal compartmentation of calcium dynamics is critical for neuronal function, particularly in postsynaptic spines. This exquisite level of Ca2+ compartmentalization is achieved through the storage and release of Ca2+ from various intracellular organelles particularly the endoplasmic reticulum (ER) and the mitochondria. Mitochondria and ER are established storage organelles controlling Ca2+ dynamics in neurons. Mitochondria also generate a majority of energy used within postsynaptic spines to support the downstream events associated with neuronal stimulus. Recently, high resolution microscopy has unveiled direct contact sites between the ER and the mitochondria (MERCs), which directly channel Ca2+ release from the ER into the mitochondrial membrane. In this study, we develop a computational 3D reaction-diffusion model to investigate the role of MERCs in regulating Ca2+ and ATP dynamics. This spatiotemporal model accounts for Ca2+ oscillations initiated by glutamate stimulus of metabotropic and ionotropic glutamate receptors and Ca2+ changes in four different compartments: cytosol, ER, mitochondria, and the MERC microdomain. Our simulations predict that the organization of these organelles and inter-organellar contact sites play a key role in modulating Ca2+ and ATP dynamics.We further show that the crosstalk between geometry (mitochondria and MERC) and metabolic parameters (cytosolic ATP hydrolysis, ATP generation) influences the neuronal energy state. Our findings shed light on the importance of organelle interactions in predicting Ca2+ dynamics in synaptic signaling. Overall, our model predicts that a combination of MERC linkage and mitochondria size is necessary for optimal ATP production in the cytosol.

AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But whether AMPK activity is regulated at different subcellular locations to provide precise spatial and temporal control over metabolism is unclear. Genetically encoded AMPK activity reporters (AMPKAR) have provided a window into spatial AMPK activity, but the limited dynamic range of current AMPKARs hinders detailed study. To monitor the dynamic activity of AMPK with high sensitivity, we developed a single-fluorophore AMPK activity reporter (ExRai AMPKAR) that exhibits an excitation ratiometric fluorescence change upon phosphorylation by AMPK, with over 3-fold greater response compared to previous AMPKARs. Using subcellularly localized ExRai AMPKAR, we found that the activity of AMPK at the lysosome and mitochondria are differentially regulated. While different activating conditions, irrespective of their effects on ATP, robustly yet gradually increase mitochondrial AMPK activity, lysosomal AMPK activity accumulates with much faster kinetics. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) resulted in slower AMPK activity at lysosomes but did not affect the response amplitude at either location, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further discovered AMPK activity in the nucleus, which resulted from LKB1-mediated cytoplasmic activation of AMPK followed by nuclear shuttling. Thus, a new, sensitive reporter for AMPK activity, ExRai AMPKAR, in complement with mathematical and biophysical methods, captured subcellular AMPK activity dynamics in living cells and unveiled complex regulation of AMPK signaling within subcellular compartments.

Spatiotemporal compartmentation of calcium dynamics is critical for neuronal function, particularly in postsynaptic spines. This exquisite level of Ca2+ compartmentalization is achieved through the storage and release of Ca2+ from various intracellular organelles particularly the endoplasmic reticulum (ER) and the mitochondria. Mitochondria and ER are established storage organelles controlling Ca2+ dynamics in neurons. Mitochondria also generate a majority of energy used within postsynaptic spines to support the downstream events associated with neuronal stimulus. Recently, high resolution microscopy has unveiled direct contact sites between the ER and the mitochondria, which directly channel Ca2+ release from the ER into the mitochondrial membrane. In this study, we develop a computational 3D reaction-diffusion model to investigate the role of MERCs in regulating Ca2+ and ATP dynamics. This spatiotemporal model accounts for Ca2+ oscillations initiated by glutamate stimulus of metabotropic and ionotropic glutamate receptors and Ca2+ changes in four different compartments: cytosol, ER, mitochondria, and the MERC microdomain. Our simulations predict that the organization of these organelles and differential distribution of key Ca2+ channels such as IP3 receptor and ryanodine receptor modulate Ca2+ dynamics in response to different stimuli. We further show that the crosstalk between geometry (mitochondria and MERC) and metabolic parameters (cytosolic ATP hydrolysis, ATP generation) influences the cellular energy state. Our findings shed light on the importance of organelle interactions in predicting Ca2+ dynamics in synaptic signaling. Overall, our model predicts that a combination of MERC linkage and mitochondria size is necessary for optimal ATP production in the cytosol.

AMP activated protein kinase (AMPK) is a heterotrimeric protein responsible for moderating cellular energy homeostasis. In cancer, AMPK has been implicated as a context‐dependent tumor promoter or suppressor. To illuminate spatiotemporal regulation of AMPK, which is critical for context dependent roles of AMPK, we developed a single‐fluorophore excitation‐ratiometric AMPK activity reporter (ExRai AMPKAR), which exhibits greater sensitivity and dynamic range compared to our previous AMPK biosensor. We used pharmacological perturbations to activate AMPK through both nucleotide‐dependent (2‐deoxyglucose) and ‐independent (MK‐8722) mechanisms, finding context‐dependent AMPK activity throughout the cell and fast AMPK activity dynamics around membranous organelles. A 3D computational model suggests high AMPK activity around organelles such as the lysosome are due to active transport across the lysosomal membrane. Furthermore, we observed robust AMPK activation in the nucleus following various pharmacological stimulation. To further investigate the mechanism regulating nuclear AMPK activity, we used fluorescence recovery after photobleaching experiments which showed that a catalytic subunit of AMPK, AMPK‐2, had no obvious change in subcellular location following pharmacological stimulation. Nuclear AMPK activity is enhanced upon LKB1 expression in LKB1‐null cells, indicating this important upstream regulator of AMPK is involved in nuclear AMPK activity. Altogether, using ExRai AMPKAR with complimentary computational and biophysical methods, we have characterized subcellular AMPK activity around membranous organelles, and unveiled a resident population of AMPK in the nucleus, which could be acutely controlled to provide fast regulation of transcription. Beyond the current study, our new AMPK activity reporter has the potential to provide further insight into localized AMPK activity in many cellular contexts.

AMP activated protein kinase (AMPK) is a heterotrimeric kinase essential to the maintenance of cellular energy homeostasis. AMPK has downstream substrates throughout the cell, and spatially compartmentalized AMPK activity is hypothesized to confer specificity to AMPK signaling. However, the specific spatial organization of AMPK activity and the underlying regulatory mechanisms are not clear. To monitor the compartmentalized activity of AMPK, we developed a single‐fluorophore excitation‐ratiometric AMPK activity reporter (ExRai AMPKAR) which exhibits an excitation wavelength change when phosphorylated by AMPK, providing a specific readout of AMPK activity in single living cells. Using subcellularly localized ExRai AMPKAR, we observed distinct AMPK activity dynamics at the lysosome and mitochondria. In complement with computational modeling approaches, we discovered that the kinetics of local ATP depletion influence AMPK activity at the mitochondria. In the nucleus, the occurrence and regulation of AMPK activity has been controversial. Remarkably, using nuclear‐localized ExRai AMPKAR we found AMPK activity robustly accumulates in the nucleus following pharmacological stimulation, which relies on the ability of AMPK to shuttle between the cytosol and the nucleus. Through the development of a new reporter for AMPK activity, ExRai AMPKAR, with computational and biophysical methods, we have characterized subcellular AMPK dynamics and unveiled a mechanism for nuclear AMPK activity. This work provides a greater understanding of the compartmentalized regulation of AMPK signaling networks within the cell.