Phase-separated condensates of metabolic complexes in living cells: Purinosome and glucosome

Abstract

Sequential metabolic enzymes have long been hypothesized to form multienzyme metabolic complexes to regulate metabolic flux in cells. Although in vitro biochemistry has not been much fruitful to support the hypothesis, advanced biophysical technologies have successfully resurrected the hypothesis with compelling experimental evidence. As biochemistry has always evolved along with technological advancement over the century (e.g. recombinant protein expression, sitedirected mutagenesis, advanced spectroscopic and structural biology techniques, etc), there has been growing interests in advanced imaging-based biophysical methods to explore enzymes inside living cells. In this work, we describe how we visualize two phase-separated biomolecular condensates of multienzyme metabolic complexes that are associated with de novo purine biosynthesis and glucose metabolism in living human cells and how imaging-based data are quantitatively analyzed to advance our knowledge of enzymes and their assemblies in living cells. Therefore, we envision that the framework we describe here would be the starting point to investigate other metabolic enzymes and their assemblies in various cell types with an unprecedented potential to comprehend enzymes and their network in native habitats.

Subcellular regulation of glucose metabolism through multienzyme glucosome assemblies by EGF–ERK1/2 signaling pathways

“…Then, 30 ng/ml EGF was supplemented to the Roswell Park Memorial Institute 1640 medium (RPMI 1640, Mediatech, Cat# 10-040-CV) that contains 10% dFBS. Note that FBS (Sigma, Cat# F2442) was dialyzed with Spectra Por 12 to 14,000 MWCO as previously described ( 27 , 28 ). ERK1/2 inhibitor (SCH772984, Selleckchem, Cat# S7101) and Akt inhibitor X (Sigma, Cat# 124020) were dissolved in DMSO (Sigma, Cat# 276855).…”

“…Human triple-negative breast carcinoma Hs578T (HTB-126) cell line was obtained from the American Type Culture Collection (ATCC). The cells were maintained in the RPMI 1640 medium supplemented with 10% dFBS and 50 μg/ml gentamycin sulfate (Corning, Cat# 61-098-RF) in a HeraCell CO 2 incubator (37 °C, 5% CO 2 , and 95% humidity) ( 27 ). The cells were also verified to be free of mycoplasma contamination by employing the Universal Mycoplasma Detection Kit (ATCC, Cat# 30-1012K) and authenticated by ATCC’s short tandem repeat profiling service.…”

“…on a Nikon Eclipse Ti inverted C2 confocal microscope. Wide-field imaging was carried out using a set of Z488/10-HC clean-up, HC TIRF dichroic, and 525/50-HC emission filter from Chroma Technology for mEGFP detection ( 1 , 27 ). Each small molecule was diluted with DMSO to achieve respective final concentrations of SCH772984 (0.5 μM) and Akt inhibitor X (13 μM).…”

Phase Separation Can Increase Enzyme Activity by Concentration and Molecular Organization

“…Indeed, a number of recent studies have shown that enzymatic rates can be increased by LLPS, including nucleation and assembly of actin filaments and microtubules 7,13,[21][22][23][24][25] , RNA autosplicing 26 , production of 3',5'-cyclic GMP-AMP 27 , carboxylation of ribulose-1,5-bisphosphate [28][29][30][31] and activation of Ras 32 . Given that condensates may selectively concentrate certain enzymes in a pathway and/or certain enzyme/substrate pairs, phase separation has also been invoked as a mechanism to provide specificity in signaling or metabolic networks through accelerating some reactions over others 1,33,34 . Since many substrates often compete for the same enzyme, especially in signaling networks, spatial separation could be used to bias some pathways over others.…”

Functional oscillation of a multienzyme glucosome assembly during cell cycle progression

“…Cell Culture: Human triple-negative breast carcinoma Hs578T cells (HTB-126) were obtained from the ATCC. Cells were cultured in the Roswell Park Memorial Institute 1640 (RPMI 1640, Mediatech, Cat# 10-040-CV) supplemented with 10% dialyzed fetal bovine serum (dFBS) (Sigma, Cat# F2442) (34,35) and 50 µg/ml gentamicin sulfate (Corning, Cat# 61-098-RF). Cells were then maintained in a HeraCell CO 2 incubator (37 °C, 5 % CO 2 , and 95 % humidity).…”