Biology subjects

Miner, G. E.

Publications and source records attributed to Miner, G. E..

8 recordsLinked to original sources

bioRxiv2026

APOE interacts with COX-2 on lipid droplets to modulate inflammatory lipid signaling

Alzheimers Disease (AD) is the leading cause of dementia worldwide. Expression of the E4 variant of apolipoprotein E (APOE) greatly increases individuals risk of developing AD. In response to lipogenesis in astrocytes, APOE can escape secretion and traffic to the cytoplasmic surface of lipid droplets (LDs), but protein interactors of APOE at the LD were unknown. Here we find that LD-localized APOE physically interacts with the inflammatory lipid signaling enzyme cyclooxygenase-2 (COX-2). Like APOE, COX-2 can avoid the secretory pathway and traffic to LDs in response to lipogenesis. APOE3, but not APOE4, increases COX-2 localization to LDs. Computational modeling, microscopy-based assays, and targeted lipidomics reveal that APOE3 promotes while APOE4 suppresses COX-2 enzymatic activity at LDs and intracellular prostaglandin production. This work identifies a novel and targetable protein-protein interaction of APOE and provides a mechanistic link between APOE4 and dysregulated inflammatory lipid signaling.

cell biology↗

bioRxiv2023

Contact-FP: a dimerization-dependent fluorescent protein toolkit for visualizing membrane contact site dynamics

Membrane contact sites (MCSs) are sites of close apposition between two organelles used to exchange ions, lipids, and information. Cells respond to changing environmental or developmental conditions by modulating the number, extent, or duration of MCSs. Because of their small size and dynamic nature, tools to study the dynamics of MCSs in live cells have been limited. Dimerization-dependent fluorescent proteins (ddFPs) targeted to organelle membranes are an ideal tool for studying MCS dynamics because they reversibly interact to fluoresce specifically at the interface between two organelles. Here, we build on previous work using ddFPs as sensors to visualize the morphology and dynamics of MCSs. We engineered a suite of ddFPs called Contact-FP that targets ddFP monomers to lipid droplets (LDs), the endoplasmic reticulum (ER), mitochondria, peroxisomes, lysosomes, plasma membrane, caveolae, and the cytoplasm. We show that these probes correctly localize to their target organelles. Using LDs as a test case, we demonstrate that Contact-FP pairs specifically localize to the interface between two target organelles. Titration of LD-mitochondria ddFPs revealed that these sensors can be used at high concentrations to drive MCSs or can be titrated down to minimally perturb and visualize endogenous MCSs. We show that Contact-FP probes can be used to: (1) visualize LD-mitochondria MCS dynamics, (2) observe changes in LD-mitochondria MCS dynamics upon overexpression of PLIN5, a known LD-mitochondrial tether, and (3) visualize two MCSs that share one organelle simultaneously (e.g., LD-mitochondria and LD-ER MCSs). Contact-FP probes can be optimized to visualize MCSs between any pair of organelles represented in the toolkit.

cell biology↗

bioRxiv2022

Quantitative Proteomic Profiling of Murine Embryonic Heart Development Reveals a Role for the Mevalonate Pathway in Cardiomyocyte Proliferation

Defining the molecular mechanisms that govern heart development is essential for identifying the etiology of congenital heart disease. Here, quantitative proteomics was used to measure temporal changes in the cardiac proteome at eight critical stages of murine embryonic heart development. Global temporal profiles of the over 7,300 identified proteins uncovered signature cardiac protein interaction networks that linked protein dynamics with molecular pathways. Using this integrated dataset, we identified and established a functional role for the mevalonate pathway in the regulation of embryonic cardiomyocyte proliferation and cell signaling. Overall, our proteomic datasets are an invaluable resource for studying molecular events that regulate embryonic heart development and contribute to congenital heart disease.

developmental biology↗

bioRxiv2022

Perilipin 5 interacts with Fatp4 at membrane contact sites to promote lipid droplet-to-mitochondria fatty acid transport

Cells adjust their metabolism by remodeling membrane contact sites that channel metabolites to different fates. Lipid droplet (LD)-mitochondria contacts change in response to fasting, cold exposure, and exercise. However, their function and mechanism of formation have remained controversial. We focused on perilipin 5 (Plin5), an LD protein that tethers mitochondria, to probe the function and regulation of LD-mitochondria contacts. We demonstrate that efficient LD-to-mitochondria fatty acid (FA) trafficking and {beta}-oxidation during starvation of myoblasts requires both phosphorylation of Plin5 and an intact Plin5 mitochondrial tethering domain. We further identified the acyl-CoA synthetase, Fatp4 (ACSVL4) as a novel mitochondrial interactor of Plin5. The C-terminal domains of Plin5 and Fatp4 constitute a minimal protein interaction capable of inducing organelle contacts. Our work suggests that starvation leads to phosphorylation of Plin5, lipolysis, and subsequent channeling of FAs from LDs to Fatp4 on mitochondria for conversion to fatty-acyl-CoAs and subsequent oxidation.

cell biology↗

bioRxiv2020

Reciprocal regulation of vacuolar calcium transport and V-ATPase activity, and the effects of Phosphatidylinositol 3,5-bisphosphate

Yeast vacuoles are acidified by the V-ATPase, a protein complex comprised of the membrane embedded VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme is required for the transfer of H+ into the vacuole lumen for acidification. The assembly of the V1-VO holoenzyme is stabilized by the lipid phosphatidylinositol 3,5-bisphospate (PI(3,5)P2) made by the PI3P 5-kinase Fab1/PIKfyve. The absence of PI(3,5)P2 leads to the dissociation of the V1 complex from the membrane. Separately, PI(3,5)P2 has been shown to modulate Ca2+ transport across the vacuole membrane during fission and fusion. Here we examined whether the regulation of H+ and Ca2+ by PI(3,5)P2 are interdependent. We show that modulating extraluminal Ca2+ concentrations inhibit V-ATPase activity. As extraluminal CaCl2 levels are raised, the activity of H+ pumping is reduced. Conversely, chelating free Ca2+ with EGTA stimulated vacuole acidification. Not only did Ca2+ levels affect H+ translocation, we also show that blocking V-ATPase activity inhibited Ca2+ transport into the vacuole lumen. Together, these data illustrate that Ca2+ transport and V-ATPase regulation are interconnected through the modulation of vacuolar lipid profiles. Summary StatementHere we show that Ca2+ and H+ transport across the vacuole membrane is reciprocally regulated and that it is linked to the production of Phosphatidylinositol 3,5-bisphoshpate.

biochemistry↗

bioRxiv2019

Copper Blocks V-ATPase Activity and SNARE Complex Formation to Inhibit Yeast Vacuole Fusion

The accumulation of Copper in organisms can lead to altered functions of various pathways, and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg+, Cd2+, and Cu2+ are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations. Excess copper ions can subsequently be transported into the vacuole lumen by an unknown mechanism. Transport across membranes requires the reduction of Cu2+ to Cu+. Labile copper ions can interact with membranes to alter fluidity, lateral phase separation and fusion. Here we found that CuCl2 potently inhibited vacuole fusion by blocking SNARE pairing. This was accompanied by the inhibition of V-ATPase H+ pumping. Deletion of the vacuolar reductase Fre6 had no effect on the inhibition of fusion by copper. This suggests that that Cu2+ is responsible for the inhibition of vacuole fusion and V-ATPase function. This notion is supported by the differential effects chelators. The Cu2+-specific chelator TETA rescued fusion, whereas the Cu+-specific chelator BCS had no effect on the inhibited fusion.

biochemistry↗

bioRxiv2019

Phosphatidylinositol 3,5-Bisphosphate regulates Ca2+ Transport During Yeast Vacuolar Fusion through Activation of the Ca2+ ATPase Pmc1

The transport of Ca2+ across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles during hyperosmotic shock become fragmented through fission events that require Ca2+ efflux of their luminal stores through the TRP channel Yvc1. This requires the production of the lipid PI(3,5)P2 by Fab1. Ca2+ is also released during vacuole fusion upon trans-SNARE complex assembly, however, the role of PI(3,5)P2 remains unclear. Here we demonstrate that elevated PI(3,5)P2 levels abolish Ca2+ efflux during fusion, indicating that PI(3,5)P2 has opposing effects on Ca2+ transport in fission versus fusion. Notably, Ca2+ efflux was enhanced when PI(3,5)P2 levels were reduced. Importantly, the effect of PI(3,5)P2 on Ca2+ flux was independent of Yvc1. Rather, the effect was dependent on the Ca2+ pump Pmc1. Vacuoles lacking Pmc1 were resistant to the effects of PI(3,5)P2, while those lacking Yvc1 remained sensitive. Furthermore altering PI(3,5)P2 levels affects the interactions of Pmc1 with the Vo component Vph1 and the R-SNARE Nyv1. We now propose a model in which elevated PI(3,5)P2 activates continued Pmc1 function to prevent the accumulation of released extraluminal Ca2+. SummaryDuring osmotic stress PI(3,5)P2 triggers Ca2+ release from vacuoles. Here we show PI(3,5)P2 stimulates Ca2+ uptake by vacuoles during fusion, illustrating that it has a dual role in Ca2+ transport.

biochemistry↗

bioRxiv2018

Phosphatidylinositol 3,5-Bisphosphate Regulates Yeast Vacuole Fusion at the Transition between trans-SNARE Complex Formation and Hemifusion

Phosphoinositides (PIs) regulate myriad cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phos-phatidylinositol 3,5-bisphosphate [PI(3,5)P2] on vacuole fusion remains unknown. PI(3,5)P2 is made by the PI3P 5-kinase Fab1/PIKfyve and has been characterized as a regulator of vacuole fission during hyperosmotic shock where it interacts with the TRP family Ca2+ channel Yvc1. Here we demonstrate that exogenously added dioctanoyl (C8) PI(3,5)P2 abolishes homotypic vacuole fusion. This effect was not linked to interactions with Yvc1, as fusion was equally affected using yvc1{Delta} vacuoles. Thus, the effects of C8-PI(3,5)P2 on fusion versus fission operate through distinct mechanisms. Further testing showed that C8-PI(3,5)P2 inhibited vacuole fusion after the formation of trans-SNARE pairs. Although SNARE complex formation was unaffected we found that C8-PI(3,5)P2 strongly inhibited hemifusion. Overproduction of endogenous PI(3,5)P2 by the fab1T2250A hyperactive kinase mutant also inhibited at the hemifusion stage, bolstering the model in which PI(3,5)P2 inhibits fusion when present elevated levels. Taken together, this work identifies a novel function for PI(3,5)P2 as a negative regulator of vacuolar fusion. Moreover, it suggests that this lipid acts as a molecular switch between fission and fusion.

biochemistry↗