Biology subjects

Pomorski, T. G.

Publications and source records attributed to Pomorski, T. G..

3 recordsLinked to original sources

bioRxiv2026

Long-term single-molecule Ca2+ flux recordings reveal mode-switching regulation of Ca2+-ATPases

Calcium (Ca{superscript 2}) is a universal second messenger that governs processes ranging from muscle contraction and secretion to gene expression and cell fate. Ca{superscript 2}-ATPases establish and maintain steep Ca{superscript 2} gradients across intracellular membranes, yet how regulatory inputs modulate the underlying single-pump Ca{superscript 2} currents has remained inaccessible. Here we develop a non-saturating, self-regenerating single-vesicle assay that monitors over hours the zeptoampere (10-{superscript 2}{superscript 1} A) currents produced by individual Ca{superscript 2}-ATPases. In parallel, we establish a workflow to record single-molecule currents from human sarco/endoplasmic reticulum Ca{superscript 2}-ATPases (hSERCA) in native endoplasmic reticulum vesicles. Using reconstituted LMCA1, a bacterial SERCA homologue, we observe stochastic switching between minute-long pumping and inactive modes, as well as uncoupled Ca{superscript 2} leakage events that are suppressed by vanadate. Extravesicular pH controls a previously unrecognized dormant pre-activation mode that delays the onset of pumping, without measurably altering pumping rates or active-mode lifetimes. Extending the assay to endogenous hSERCA reveals delayed activation and ultraslow pumping/inactive mode-switching without detectable transprotein Ca{superscript 2} leakage. ATP and Ca{superscript 2} regulate the probability of hSERCA activation by modulating dormant-mode occupancy. Together, these results extend ultraslow mode-switching, previously observed only for proton pumps, to Ca{superscript 2}-ATPases and identify probability-gated entry into productive cycling as a distinct regulatory axis of human Ca{superscript 2}-ATPase regulation that can modulate the timing and heterogeneity of Ca{superscript 2} store refilling without changing on-cycle kinetics.

biophysics↗

bioRxiv2025

A single vesicle fluorescence microscopy platform to quantify phospholipid scrambling

Scramblases play important roles in physiology by translocating phospholipids bidirectionally across cell membranes. For example, scrambling facilitated by dimers of the Voltage-Dependent Anion Channel 1 (VDAC1) enables endoplasmic reticulum-derived phospholipids to cross the outer membrane to enter mitochondria. Precise quantification of lipid scrambling, while critical for mechanistic understanding, cannot be obtained from ensemble averaged measurements of reconstituted scramblases. Here, we describe a microscopy platform for high-throughput imaging of single vesicles reconstituted with fluorescently labeled phospholipids and heterogeneously crosslinked VDAC1 dimers. For each vesicle, we quantify size, protein occupancy and scrambling rate. Notably, we find that individual VDAC1 dimers have different activities, ranging from <100 to >10,000 lipids per second. This kinetic heterogeneity, masked in ensemble measurements, reveals that only some dimer interfaces are capable of promoting rapid scrambling, as suggested by molecular dynamics simulations. We extend our analyses to opsin, a monomeric G protein-coupled receptor scramblase, thereby demonstrating the versatility of our platform for quantifying transbilayer lipid transport and exploring its regulation.

biophysics↗

bioRxiv2021

Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders

P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with severe diseases, e.g. in ATP8B1 causing progressive familial intrahepatic cholestasis, a rare inherited disorder progressing toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report functional studies and the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 [A] resolution. We find that ATP8B1 is autoinhibited by its N- and C-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the C-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C- termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminal segment further suggests molecular communication between N- and C-termini in the autoinhibition and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A recurring (G/A)(Y/F)AFS motif of the C-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.

biochemistry↗