The Glycolytic Protein Phosphofructokinase Dynamically Relocalizes into Subcellular Compartments with Liquid-like Properties in vivo

Abstract

While much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. Here we built a hybrid microfluidic-hydrogel device for use in Caenorhabditis elegans to systematically examine and quantify the dynamic subcellular localization of the rate-limiting enzyme of glycolysis, phosphofructokinase-1/PFK-1.1. We determine that endogenous PFK-1.1 localizes to distinct, tissue-specific subcellular compartments in vivo. In neurons, PFK-1.1 is diffusely localized in the cytosol, but capable of dynamically forming phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in the dispersion of PFK-1.1 in the cytosol, indicating that PFK-1.1 reversibly organizes into biomolecular condensates in response to cues within the cellular environment. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Prolonged conditions of energy stress during sustained hypoxia alter the biophysical properties of PFK-1.1 in vivo, affecting its viscosity and mobility within phase-separated condensates. PFK-1.1's ability to form tetramers is critical for its capacity to form condensates in vivo, and heterologous self-association domain such as cryptochrome 2 (CRY2) is sufficient to constitutively induce the formation of PFK-1.1 condensates. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically compartmentalize in vivo to specific subcellular compartments in response to acute energy stress via multivalency as phase-separated condensates.

Presynaptic autophagy is coupled to the synaptic vesicle cycle via ATG-9

“…The absence of phosphofructokinase, like the absence of other glycolytic proteins, results in impaired synaptic vesicle endocytosis during transient hypoxia (Jang et al, 2016). Through the use of a microfluidic device that allows precise control of transient cycles of normoxia and hypoxia, we can tempo-rally control the endocytic reaction (Jang et al, 2021). We examined ATG-9 localization in the synaptic Zone 3 region (Figures 2B and 2B 00 ).…”

GIT/PIX Condensates Are Modular and Ideal for Distinct Compartmentalized Cell Signaling

“…However, enzymes are known to be concentrated at specific subcellular compartments to perform spatially defined cellular functions. Via liquid-liquid phase separation, enzymes such as GIT1 and b-Pix can autonomously form highly concentrated molecular assemblies, providing a novel mechanism for enriching limited amounts of enzymes into specific cellular regions for fast and spatially defined catalysis (Jang et al, 2019;Webb et al, 2017). Guided by the atomic structures of GIT1, b-Pix, and the GIT/b-Pix complex, we also demonstrated that phase separation-mediated GIT1/b-Pix complex condensation, instead of the classical binary interaction between GIT1 and b-Pix, is required for the enzyme complex to modulate cell migration and synapse formation.…”

Presynaptic autophagy is coupled to the synaptic vesicle cycle via ATG-9

“…The absence of phosphofructokinase, like the absence of other glycolytic proteins, results in impaired synaptic vesicle endocytosis during transient hypoxia (Jang et al, 2016). Through the use of a microfluidic device that allows precise control of transient cycles of normoxia and hypoxia, we can temporally control the endocytic reaction (Jang et al, 2020). We examined ATG-9 localization in the synaptic Zone 3 region (Figure L-L'), in which we can observe discrete and interspersed presynaptic specializations (compare to the Zone 2 region ( Figure 2B-B'), which consists of one large and continuous presynaptic area).…”

“…To inhibit oxidative phosphorylation by hypoxia, a reusable microfluidic polydimethylsiloxane (PDMS) microfluidic device was used, as described (Jang et al, 2020) while imaging ATG-9::GFP localization in pfk-1.1(gk922689);olaIs34…”