Biology subjects

Foster, H. R.

Publications and source records attributed to Foster, H. R..

7 recordsLinked to original sources

bioRxiv2026

Compartmentalized glycolysis powers ATP production in primary cilia and engages mitochondria via the phosphoenolpyruvate cycle

Primary cilia are antenna-like sensory and signaling organelles present on most mammalian cells, including glucose-sensing pancreatic {beta}-cells. Here, we show that the local energetic demands of primary cilia require the ATP-producing enzyme pyruvate kinase, with loss of PKm1, but not PKm2, impairing ciliary glycolytic flux. While the entire glycolytic machinery localizes to cilia, our data indicate that mitochondria are a critical source of phosphoenolpyruvate (PEP), the high-energy glycolytic intermediate that drives the pyruvate kinase reaction. Abolishing PCK2, the mitochondrial enzyme that generates PEP, prevents cilia from sensing not only glucose but also the amino acids glutamine and leucine. Finally, by mislocalizing glycolysis, we demonstrate that primary cilia can utilize ATP generated within the cell body when glucose is limiting. These findings indicate that primary cilia, while possessing the capacity for local ATP generation, leverage a ciliary-mitochondrial signaling axis to meet their bioenergetic needs.

cell biology↗

bioRxiv2026

ATP11A and ATP11C are plasma membrane phosphatidylserine flippases in in vitro human megakaryocytes.

Thrombotic diseases are the major worldwide cause of cardiovascular death. Platelets prevent blood loss following injury (haemostasis), but inappropriate and excessive platelet activation can lead to thrombosis. Platelet activation must be tightly controlled. Pro-coagulant platelets expose phosphatidylserine (PS), enabling coagulation complex assembly, enhancing thrombin generation and thrombosis. PS is normally restricted to the inner leaflet of the plasma membrane by flippase (aminophospholipid translocase) activity. However, the flippase protein(s) responsible for this crucial activity in platelets remains unidentified. The P4 ATPases ATP11A and ATP11C, regulated by their obligatory partner CDC50A, flip PS at the plasma membrane in a range of different cell types. To investigate platelet flippases, human induced pluripotent stem cells (hiPSCs) were forward-programmed into CD41+/CD42+ megakaryocytes, the platelet precursor. Wildtype (WT) forward-programmed megakaryocytes showed similar flippase activity to human platelets with internalisation of NBD-PS that could be inhibited by high cytosolic Ca2+ or N-ethylmaleimide (NEM). We then generated CDC50A, ATP11A or ATP11C single knockout and ATP11A/11C double knockout (DKO) hiPSCs using CRISPR-Cas9. CDC50A-KO, ATP11A-KO, ATP11C-KO and DKO hiPSC clones successfully formed CD41a+/CD42a+ mature megakaryocytes. CDC50A-KO megakaryocytes bound Annexin V when unstimulated and had no remaining NEM-sensitive flippase activity indicating the involvement of a P4-ATPase. Although ATP11A-KO and ATP11C-KO megakaryocytes had similar flippase activity to WT clones, DKO clones had inhibited NBD-PS internalisation compared to WT and had no remaining NEM-sensitive flippase activity. This indicates that the CDC50A-regulated P4-ATPases ATP11A and ATP11C act together at the megakaryocyte plasma membrane and are responsible for PS flippase activity and therefore likely responsible in human platelets.

cell biology↗

bioRxiv2025

Amino acid sensing by the α-cell mitochondrial phosphoenolpyruvate cycle regulates intracellular Ca2+ levels without impacting glucagon secretion

Pancreatic islet -cells are increasingly recognized as amino acid sensors for the organism. Building on our prior work in {beta}-cells, we sought to determine whether the mitochondrial phosphoenolpyruvate (PEP) cycle is involved in -cell amino acid sensing. Three different methods were used to probe the PEP cycle, including pyruvate kinase activators (TEPP-46), and mice with -cell specific deletion of pyruvate kinase (PKM1/2-KO) or mitochondrial PEP carboxykinase (PCK2-KO). The mitochondrial fuels glutamine/leucine antagonized alanine/arginine-stimulated Ca2+ influx and glucagon secretion under hypoglycemic conditions. Both PKM1/2 and PCK2 were required for glutamine/leucine to close KATP channels and limit amino acid-stimulated membrane depolarization. The Ca2+ response to amino acids was suppressed by pyruvate kinase activation with TEPP-46, and enhanced by -cell deletion of pyruvate kinase or PCK2 - all without changing glucagon secretion. Finally, using diazoxide/KCl to probe the pathways downstream of membrane depolarization, we identified an essential role of the PEP cycle in homeostatically restoring intracellular Ca2+ levels. In sum, the -cell mitochondrial PEP cycle senses glutamine/leucine and inhibits KATP channels similarly to {beta}-cells, while restricting amino acid-stimulated membrane depolarization and Ca2+ influx. However, defying expectations, none of the amino acids tested, including alanine/arginine, regulate glucagon secretion by modulating membrane depolarization or intracellular Ca2+. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/656009v2_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@18a29f0org.highwire.dtl.DTLVardef@1c77041org.highwire.dtl.DTLVardef@9adc1dorg.highwire.dtl.DTLVardef@e91407_HPS_FORMAT_FIGEXP M_FIG Graphical abstract-cells as amino acids sensors. Arginine, alanine, and glutamine potentiate glucagon secretion, while leucine has a suppressive effect (left). Independently of glucagon secretion, glutamine and leucine suppress alanine and arginine-stimulated Ca2+ influx via the phosphoenolpyruvate (PEP) cycle, which closes KATP channels and suppresses Vm depolarization (right). C_FIG HighlightsO_LIOur studies identify a role for the -cell PEP cycle in sensing amino acids under hypoglycemic conditions. C_LIO_LIPyruvate kinase and PCK2 are required for glutamine/leucine to close -cell KATP channel and limit membrane depolarization and Ca2+ influx. C_LIO_LIGlutamine/leucine oppose alanine/arginine-stimulated Ca2+ influx and glucagon secretion. C_LIO_LIAll of the amino acids tested regulate glucagon secretion, but none do so by modulating membrane depolarization or intracellular Ca2+ levels. C_LI

cell biology↗

bioRxiv2023

Leucine suppresses glucagon secretion from pancreatic islets by directly modulating alpha-cell cAMP

ObjectivePancreatic islets are nutrient sensors that regulate organismal blood glucose homeostasis. Glucagon release from the pancreatic -cell is important under fasted, fed, and hypoglycemic conditions, yet metabolic regulation of -cells remains poorly understood. Here, we identified a previously unexplored role for physiological levels of leucine, which is classically regarded as a {beta}-cell fuel, in the intrinsic regulation of -cell glucagon release. MethodsGcgCreERT:CAMPER and GcgCreERT:GCaMP6s mice were generated to perform dynamic, high-throughput functional measurements of -cell cAMP and Ca2+ within the intact islet. Islet perifusion assays were used for simultaneous, time-resolved measurements of glucagon and insulin release from mouse and human islets. The effects of leucine were compared with glucose and the mitochondrial fuels 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH, non-metabolized leucine analog that activates glutamate dehydrogenase), -ketoisocaproate (KIC, leucine metabolite), and methyl-succinate (complex II fuel). CYN154806 (Sstr2 antagonist), diazoxide (KATP activator, which prevents Ca2+-dependent exocytosis from , {beta}, and {delta}-cells), and dispersed -cells were used to inhibit islet paracrine signaling and identify -cell intrinsic effects. ResultsMimicking the effect of glucose, leucine strongly suppressed amino acid-stimulated glucagon secretion. Mechanistically, leucine dose-dependently reduced -cell cAMP at physiological concentrations, with an IC50 of 57, 440, and 1162 M at 2, 6, and 10 mM glucose, without affecting -cell Ca2+. Leucine also reduced -cell cAMP in islets treated with Sstr2 antagonist or diazoxide, as well as dispersed -cells, indicating an -cell intrinsic effect. The effect of leucine was matched by KIC and the glutamate dehydrogenase activator BCH, but not methyl-succinate, indicating a dependence on mitochondrial anaplerosis. Glucose, which stimulates anaplerosis via pyruvate carboxylase, had the same suppressive effect on -cell cAMP but with lower potency. Similarly to mouse islets, leucine suppressed glucagon secretion from human islets under hypoglycemic conditions. ConclusionsThese findings highlight an important role for physiological levels of leucine in the metabolic regulation of -cell cAMP and glucagon secretion. Leucine functions primarily through an -cell intrinsic effect that is dependent on glutamate dehydrogenase, in addition to the well-established -cell regulation by {beta}/{delta}-cell paracrine signaling. Our results suggest that mitochondrial anaplerosis-cataplerosis facilitates the glucagonostatic effect of both leucine and glucose, which cooperatively suppress -cell tone by reducing cAMP. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/551113v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1af2865org.highwire.dtl.DTLVardef@8508fborg.highwire.dtl.DTLVardef@10c009org.highwire.dtl.DTLVardef@1afb6e9_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LILeucine inhibits glucagon secretion from mouse and human islets C_LIO_LILeucine suppresses -cell cAMP via both direct and paracrine effects C_LIO_LIAnaplerosis via glutamate dehydrogenase is sufficient to suppress -cell cAMP C_LIO_LILeucine suppresses -cell cAMP and glucagon secretion more potently than glucose C_LI

molecular biology↗

bioRxiv2022

Nutrient-dependent regulation of β-cell KATP channels is controlled by the isoforms of pyruvate kinase and the source of phosphoenolpyruvate

Pyruvate kinase (PK) and the phosphoenolpyruvate (PEP) cycle play key roles in nutrient-stimulated KATP channel closure and insulin secretion. To identify the PK isoforms involved, we generated mice lacking {beta}-cell PKm1, PKm2, and mitochondrial PEP carboxykinase (PCK2) that generates mitochondrial PEP. Glucose metabolism generates both glycolytic and mitochondrially-derived PEP, which triggers KATP closure through local PKm1 and PKm2 signaling at the plasma membrane. Amino acids, which generate mitochondrial PEP without producing glycolytic fructose 1,6-bisphosphate to allosterically activate PKm2, signal through PKm1 to raise ATP/ADP, close KATP channels, and stimulate insulin secretion. Raising cytosolic ATP/ADP with amino acids is insufficient to close KATP channels in the absence of PK activity or PCK2, indicating that KATP channels are regulated by mitochondrially-derived PEP that provides ATP via plasma membrane-associated PK, but not via mitochondrially-derived ATP. Following membrane depolarization, the PEP cycle is also involved in an "off-switch" that facilitates KATP channel reopening and Ca2+ extrusion, as shown by PK activation experiments and {beta}-cell PCK2 deletion that prolonged Ca2+ oscillations and increased insulin secretion. In conclusion, the differential response of PKm1 and PKm2 to the glycolytic and mitochondrial sources of PEP influences the {beta}-cell nutrient response, and controls the oscillatory cycle regulating insulin secretion.

cell biology↗

bioRxiv2021

Natural transformation protein ComFA exhibits single-stranded DNA translocase activity

Natural transformation is one of the major mechanisms of horizontal gene transfer in bacterial populations and has been demonstrated in numerous species of bacteria. Despite the prevalence of natural transformation, much of the molecular mechanism remains unexplored. One major outstanding question is how the cell powers DNA import, which is rapid and highly processive. ComFA is one of a handful of proteins required for natural transformation in gram-positive bacteria. Its structural resemblance to the DEADbox helicase family has led to a long-held hypothesis that ComFA acts as a motor to help drive DNA import into the cytosol. Here, we explored the helicase and translocase activity of ComFA to address this hypothesis. We followed the DNA-dependent ATPase activity of ComFA and, combined with mathematical modeling, demonstrated that ComFA likely translocates on single-stranded DNA from 5 to 3. However, this translocase activity does not lead to DNA unwinding in the conditions we tested. Further, we analyzed the ATPase cycle of ComFA and found that ATP hydrolysis stimulates the release of DNA, providing a potential mechanism for translocation. These findings help define the molecular contribution of ComFA to natural transformation and support the conclusion that ComFA plays a key role in powering DNA uptake. ImportanceCompetence, or the ability of bacteria to take up and incorporate foreign DNA in a process called natural transformation is common in the bacterial kingdom, but understanding of the mechanism is still limited. Increasing evidence in several bacteria confirms that long, contiguous stretches of DNA are imported into cells, and yet how bacteria power processive transformation remains unclear. Our finding that ComFA, a DExD-box helicase required for competence in gram-positive bacteria, translocates on single-stranded DNA from 5 to 3, supports the long held hypothesis that ComFA may be the motor powering DNA transport during natural transformation. Moreover, ComFA may be a previously unidentified type of DExD-box helicase--one with the capability of extended translocation on single-stranded DNA.

microbiology↗

bioRxiv2020

Pyruvate kinase controls signal strength in the insulin secretory pathway

Pancreatic {beta}-cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies {beta}-cell sensing of both glycolytic and mitochondrial fuels. PK present at the plasma membrane is sufficient to close KATP channels and initiate calcium influx. Small-molecule PK activators increase {beta}-cell oscillation frequency and potently amplify insulin secretion. By cyclically depriving mitochondria of ADP, PK restricts oxidative phosphorylation in favor of the mitochondrial PEP cycle with no net impact on glucose oxidation. Our findings support a compartmentalized model of {beta}-cell metabolism in which PK locally generates the ATP/ADP threshold required for insulin secretion, and identify a potential therapeutic route for diabetes based on PK activation that would not be predicted by the {beta}-cell consensus model. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/907790v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1f6503forg.highwire.dtl.DTLVardef@e9393org.highwire.dtl.DTLVardef@fab08borg.highwire.dtl.DTLVardef@5c7c55_HPS_FORMAT_FIGEXP M_FIG C_FIG The consensus model for {beta}-cell glucose sensing supports a dominant role for OxPhos. This model doesnt fully explain the observed metabolic and electrophysiologic oscillations associated with glucose-stimulated insulin secretion. Lewandowski et al. challenge this model by mechanistically connecting the anaplerotic PEP cycle to the electrically silent triggering phase, and OxPhos to the electrically active secretory phase. Here, the allosteric recruitment of pyruvate kinase directs metabolic traffic between the two cycles and identifies potential therapeutic strategies for diabetes based on pharmacologic pyruvate kinase activation. HIGHLIGHTSO_LICompartmentalized pyruvate kinase (PK) activity underlies {beta}-cell fuel sensing C_LIO_LIMembrane-associated PK closes KATP channels and controls calcium influx C_LIO_LIBy lowering ADP, PK toggles mitochondria between OxPhos and PEP biosynthesis C_LIO_LIPharmacologic PK activation increases oscillatory frequency and amplifies secretion C_LI

cell biology↗