Sequential metabolic enzymes in glucose metabolism have long been hypothesized to form multienzyme complexes that regulate glucose flux in living cells. However, it has been challenging to directly observe these complexes and their functional roles in living systems. In this work, we have used wide-field and confocal fluorescence microscopy to investigate the spatial organization of metabolic enzymes participating in glucose metabolism in human cells. We provide compelling evidence that human liver-type phosphofructokinase 1 (PFKL), which catalyzes a bottleneck step of glycolysis, forms various sizes of cytoplasmic clusters in human cancer cells, independent of protein expression levels and of the choice of fluorescent tags. We also report that these PFKL clusters colocalize with other rate-limiting enzymes in both glycolysis and gluconeogenesis, supporting the formation of multienzyme complexes. Subsequent biophysical characterizations with fluorescence recovery after photobleaching and FRET corroborate the formation of multienzyme metabolic complexes in living cells, which appears to be controlled by post-translational acetylation on PFKL. Importantly, quantitative high-content imaging assays indicated that the direction of glucose flux between glycolysis, the pentose phosphate pathway, and serine biosynthesis seems to be spatially regulated by the multienzyme complexes in a cluster-size-dependent manner. Collectively, our results reveal a functionally relevant, multienzyme metabolic complex for glucose metabolism in living human cells.
The organization of metabolic multienzyme complexes has been hypothesized to benefit metabolic processes and provide a coordinated way for the cell to regulate metabolism. Historically, their existence has been supported by various in vitro techniques. However, it is only recently that the existence of metabolic complexes inside living cells has come to light to corroborate this long-standing hypothesis. Indeed, subcellular compartmentalization of metabolic enzymes appears to be widespread and highly regulated. On the other hand, it is still challenging to demonstrate the functional significance of these enzyme complexes in the context of the cellular milieu. In this review, we discuss the current understanding of metabolic enzyme complexes by primarily focusing on central carbon metabolism and closely associated metabolic pathways in a variety of organisms, as well as their regulation and functional contributions to cells.
The resolution of asphaltene nanoscience is becoming increasingly important for a variety of purposes. One key molecular attribute of the asphaltenes is the size distribution of their polycyclic aromatic hydrocarbons (PAHs). Comparison of measured spin singletÀsinglet absorption and emission transitions with exhaustive molecular orbital (MO) calculations on 523 PAHs indicates that asphaltene PAHs have a population centroid of ∼7 fused rings. To further test this understanding of asphaltene PAHs, it is desirable to consider the dynamics of triplet states. Nevertheless, triplet-state spectroscopy is complex, especially on polydisperse materials such as asphaltenes. For validation, we compare simple expectations for asphaltenes against both experimental and theoretical results. Measurements were conducted on crude oil and asphaltene samples of dramatically different heavy end content to identify specific transitions being investigated. Experimental results include spectra at several wavelengths, lifetimes in the presence and absence of molecular oxygen, and temperature effects. Specifically, we use classic techniques [Horrocks and Wilkinson, Proc. R. Soc. London A 1968, 306, 257À273] to measure tripletÀtriplet spectra for crude oils and asphaltenes. These are compared with corresponding MO calculations. Again, using classic methods [Guzeman et al., J. Chem. Soc. Faraday Trans. 1973, 69, 708À720], quenching effects of asphaltene triplet states by molecular oxygen are measured and compared with simple diffusion expectations. The temperature dependence provides further stringent testing. Spectral comparisons versus crude oil composition rule out significant spectral contributions from free radicals. Simple expectations regarding triplet-state spectroscopy of asphaltenes and crude oils apply and corroborate previous conclusions from singlet-state spectroscopy of crude oils and asphaltenes. The data herein are consistent with asphaltene PAHs being relatively large (e.g., 7 fused rings); this, in turn, is consistent with the predominance of a single PAH per asphaltene molecule (the "island" molecular architecture). Smaller PAHs dominate the triplet transitions for the crude oil samples and optical wavelengths used herein.
Dendritic spines are the primary excitatory postsynaptic sites that act as subcompartments of signaling. Ca 2þ is often the first and most rapid signal in spines. Downstream of calcium, the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway plays a critical role in the regulation of spine formation, morphological modifications, and ultimately, learning and memory. Although the dynamics of calcium are reasonably well-studied, calcium-induced cAMP/PKA dynamics, particularly with respect to frequency modulation, are not fully explored. In this study, we present a well-mixed model for the dynamics of calcium-induced cAMP/PKA dynamics in dendritic spines. The model is constrained using experimental observations in the literature. Further, we measured the calcium oscillation frequency in dendritic spines of cultured hippocampal CA1 neurons and used these dynamics as model inputs. Our model predicts that the various steps in this pathway act as frequency modulators for calcium, and the high frequency of calcium input is filtered by adenylyl cyclase 1 and phosphodiesterases in this pathway such that cAMP/PKA only responds to lower frequencies. This prediction has important implications for noise filtering and long-timescale signal transduction in dendritic spines. A companion manuscript presents a three-dimensional spatial model for the same pathway.
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 how AMPK activity is regulated at different subcellular locations for precise spatiotemporal control over metabolism is unclear. Here we present a sensitive, single-fluorophore AMPK activity reporter (ExRai AMPKAR), which reveals distinct kinetic profiles of AMPK activity at the mitochondria, lysosome, and cytoplasm. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) results in slower AMPK activity at lysosomes but does not affect the response amplitude at lysosomes or mitochondria, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further identify a mechanism for AMPK activity in the nucleus, which results from cytoplasmic to nuclear shuttling of AMPK. Thus, ExRai AMPKAR enables illumination of the complex subcellular regulation of AMPK signaling.
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