Biology subjects

Stamou, D.

Publications and source records attributed to Stamou, D..

7 recordsLinked to original sources

bioRxiv2026

LAT condensation gates PLCγ1 activation via bimodal LAT phosphorylation

T cells can respond to even a single molecular binding event of antigen to a TCR. A key step in the TCR signaling pathway that definitively exhibits this single molecule response is the initiation of calcium influx by activation of PLC{gamma}1 in the LAT protein condensate. Here, we describe detailed kinetic measurements examining how protein condensation of LAT regulates activation of PLC{gamma}1 using a reconstituted membrane system. The results reveal that membrane recruitment of PLC{gamma}1 is tightly controlled by the LAT phosphorylation state, with no measurable independent recruitment to PIP2 or PIP3 lipids via the PLC{gamma}1 PH domains. We further observe PLC{gamma}1 is rapidly activated by membrane-associated kinase upon recruitment, irrespective of the LAT condensation state. These studies also revealed a crosstalk mechanism in which the TEC family kinases responsible for PLC{gamma}1 activation also phosphorylate LAT. This interaction establishes a positive feedback loop in LAT phosphorylation, mediated through LAT condensation, which drives a bimodal LAT phosphorylation response to TCR activation. Kinetic modeling reveals how this LAT phosphorylation response can cooperatively gate PLC{gamma}1 activation from a single TCR. These results suggest the LAT condensate facilitates both signal amplification and noise suppression in PLC{gamma}1 activation through a bimodal switch affecting LAT phosphorylation. Significance StatementT cells are sensitive sensors capable of detecting and responding to trace amounts of foreign antigen. Understanding how they achieve such sensitivity while maintaining accurate antigen discrimination remains a key challenge. Here, through detailed kinetic measurements of PLC{gamma}1 activation, we identify a cross reactivity in which kinases responsible for PLC{gamma}1 phosphorylation also phosphorylate LAT. This creates a bimodal switch controlling LAT phosphorylation levels, which gates PLC{gamma}1 activation from single TCR signals. We suggest this mechanism plays a key role in the signal amplification and noise suppression required for T cells to detect single antigen molecules.

biophysics↗

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

Pharmacological inhibition of V-ATPase targets mode-switching but not the proton transport cycle

Vacuolar-type adenosine triphosphatases (V-ATPases) are rotary proton pumps that establish proton gradients across cellular membranes1,2. Their pharmacological inhibition is currently under active investigation as a therapeutic strategy for cancer, infectious diseases, and autophagy-related disorders3,4. However, the molecular mechanism underlying V-ATPase inhibition remains poorly understood. Based on ensemble average measurements, it is widely assumed that inhibitors suppress activity by slowing the catalytic transport cycle and reducing proton transport rates5-7. Here, we tested this popular notion by directly measuring single-molecule proton pumping in the presence of three potent V-ATPase inhibitors: bafilomycin A1, concanamycin A, and diphyllin. Although all compounds abolish proton gradients in a canonical concentration-dependent manner (IC50 of 0.2 nM, 0.6 nM, and 41 nM, respectively), they leave the proton transport rate of active V-ATPases essentially unchanged. Instead, inhibitors modulate the reversible switching kinetics between ultralong-lived active (pumping) and inactive modes. Distinct inhibitors modulate mode lifetimes in a mode-specific and differentially efficient manner, altering the probability of the pump being in the active mode. Given that mode-switching has been documented across diverse primary8,9 and secondary10-13 active transporters, our results suggest a novel strategy for therapeutic intervention that targets mode occupancy rather than the canonical transport cycle.

biophysics↗

bioRxiv2024

Multimodal intrinsic activation of GPCRs in ultrastable plasma membrane nanodomains

G protein-coupled receptors (GPCRs) mediate many physiological functions and are key targets in drug development1-3. A long-held tenet of molecular pharmacology is that GPCRs can spontaneously sample preexisting active conformations. This concept is pivotal to our understanding of ligand pharmacology4, however, direct evidence supporting it has only been obtained with reconstituted receptors5-12. Here, we introduce a method for quantitatively imaging the intrinsic activation probability of GPCRs directly at the plasma membrane of live cells, utilizing fluorescent conformational biosensors13,14. Our findings unveil a remarkable spatial multimodality in intrinsic activation probability, with a significant majority (up to 99%) of plasma membrane-expressed receptors showing negligible spontaneous activation. In contrast, the remaining minority of receptors exhibits spontaneous activation up to 22-fold higher than previously estimated. Experiments and theoretical calculations revealed that receptors diffuse into and out of ultralong-lived ([~]5 minutes) nanodomains where the local membrane curvature allosterically enhances activation in the absence and presence of ligands. Extensive testing across five prototypic GPCRs indicates spatial nanoscale multimodality is ubiquitous, but varying in magnitude depending on the receptor and cell type. Upending conventional wisdom, this study reveals that drug efficacy is not a constant number but a spatiotemporal function {varepsilon} (x, y, z, t) whose properties define and multiplex the signaling potency and efficacy of ternary complexes of GPCRs and likely other plasma membrane-receptors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/582451v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e4680borg.highwire.dtl.DTLVardef@16aaebcorg.highwire.dtl.DTLVardef@f5df52org.highwire.dtl.DTLVardef@18a5ea_HPS_FORMAT_FIGEXP M_FIG C_FIG GPCR spontaneous activation and intrinsic efficacy are not uniform across the plasma membrane but exhibit ultralong-lived spatial multimodality. Spatial variations in the curvature and composition of the plasma membrane, lead to the emergence of ultralong-lived nanodomains with contrasting physicochemical properties that allosterically regulate GPCR conformations. This results in a multimodal landscape of intrinsic efficacy{epsilon} (x, y, z, t) that ultimately governs cell signaling. XY scalebar: 500 nm. Z-range: 100 nm.

biophysics↗

bioRxiv2022

Regulation of the mammalian-brain V-ATPase via ultra-slow mode-switching

Summary paragraphVacuolar-type adenosine triphosphatases (V-ATPases)1-3 are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases4,5. They hydrolyze ATP to establish electrochemical proton gradients for a plethora of cellular processes1,3. In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by ~1 V-ATPase molecule per synaptic vesicle6,7. To shed light into this bona fide single-molecule biological process, we investigated electrogenic proton pumping by single mammalian-brain V-ATPases, using individual synaptic vesicles fused with immobilized liposomes. We show V-ATPases do not pump continuously in time, as hypothesized by observing the rotation of bacterial homologs8 and assuming strict ATP/proton coupling. Instead, they stochastically switch between three novel ultra-long-lived proton-pumping, inactive, and proton-leaky modes. Upending conventional wisdom, direct observation of pumping revealed that physiologically relevant concentrations of ATP do not regulate the intrinsic pumping rate. Instead, ATP regulates V-ATPase activity via the switching probability of the proton-pumping mode. In contrast, electrochemical proton gradients regulate the pumping rate and the switching of the pumping and inactive modes. This work reveals and emphasises the mechanistic and biological importance of mode-switching in protein regulation.

biophysics↗

bioRxiv2021

WASP integrates substrate topology and cell polarity to guide neutrophil migration

To control their shape and movement, cells leverage nucleation promoting factors (NPFs) to regulate when and where they polymerize actin. Here we investigate the role of the immune-specific NPF WASP during neutrophil migration. Endogenously-tagged WASP localizes to substrate-induced plasma membrane deformations. Super-resolution imaging of live cells reveals that WASP preferentially enriches to the necks of these substrate-induced membrane invaginations, a distribution that could support substrate pinching. Unlike other curvature-sensitive proteins, WASP only enriches to membrane deformations at the cell front, where it controls Arp2/3 complex recruitment and actin polymerization. Despite relatively normal migration on flat substrates, WASP depletion causes defects in topology sensing and directed migration on textured substrates. WASP therefore both responds to and reinforces cell polarity during migration. Surprisingly, front-biased WASP puncta continue to form in the absence of Cdc42. We propose that WASP integrates substrate topology with cell polarity for 3D guidance by selectively polymerizing actin around substrate-induced membrane deformations at the leading edge. A misregulation of WASP-mediated contact guidance could provide insight into the immune disorder Wiskott-Aldrich syndrome.

cell biology↗

bioRxiv2019

WAVE complex self-organization templates lamellipodial formation

How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. For example, why does the WAVE complex build lamellipodia, the broad sheet-like protrusions that power cell migration, whereas the homologous actin regulator N-WASP forms spiky finger-like actin networks? N-WASP is known to oligomerize into focal condensates that generate an actin finger. In contrast, the WAVE complex exhibits the linear distribution needed to generate an actin sheet. This linear organization of the WAVE complex could either arise from interactions with the actin cytoskeleton or could represent an ability of the complex to self-organize into a linear template. Using super-resolution microscopy, we find that the WAVE complex forms higher-order linear oligomers that curve into 270 nanometer-wide ring structures in the absence of actin polymer. These rings localize to the necks of membrane invaginations, which display saddle point geometries with positive curvature in one axis and negative curvature in the orthogonal axis. To investigate the molecular mechanism of saddle curvature enrichment, we show that the WAVE complex and IRSp53, a membrane curvature-sensitive protein, collaborate to recognize saddle curvature that IRSp53 cannot sense alone. This saddle preference for the WAVE complex could explain emergent cell behaviors, such as expanding and self-straightening lamellipodia as well as the ability of endothelial cells to recognize and seal transcellular holes. Our work highlights how partnering protein interactions enable complex shape sensing and how feedback between cell shape and actin regulators yields self-organized cell morphogenesis.

cell biology↗