Biology subjects

Cvjetkovic, A.

Publications and source records attributed to Cvjetkovic, A..

4 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

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↗

bioRxiv2017

A subgroup of mitochondrial extracellular vesicles discovered in human melanoma tissues are detectable in patient blood

Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted from all cells, and convey messages between cells in health and disease. However, the diversity of EV subpopulations are only beginning to be explored. Since EVs have been implicated in tumor microenvironmental communication, we started to determine the diversity of EVs specifically in this tissue. To do this, we isolated EVs directly from patient melanoma metastatic tissues. Using EV membrane isolation and mass spectrometry analysis, we discovered enrichment of mitochondrial membrane proteins in the melanoma tissue-derived EVs, compared to non-melanoma-derived EVs. Specifically, EVs positive for a combination of the two mitochondrial inner membrane proteins MT-CO2 (mitochondrial genome) and COX6c (nuclear genome) were detected in the plasma of melanoma patients, and in ovarian and breast cancer patients. Furthermore, this subpopulation of EVs, contains active mitochondrial enzymes. Our findings show that tumor tissues are enriched in EVs with mitochondrial proteins and enzymatic activity, and these EVs can be detected in blood.

cancer biology↗

bioRxiv2016

Exosomes purified from a single cell type have diverse morphology and composition

Extracellular vesicles (EVs) are produced by all known organisms and have important roles in cell communication and physiology. Exosomes are known in the literature to be small round EVs (40 to 100 nm in diameter) and are commonly purified with a serial ultracentrifugation protocol followed by density gradient floatation. Great morphological diversity has been described before regarding EVs found in body fluids such as blood plasma, breast milk and ejaculate. However, a detailed morphological analysis has never been performed on exosomes purified from a single cell type.\n\nTherefore, the aim of this study was to analyze and quantify via multiple electron microscopy techniques the morphology of exosomes purified from the human mast cell line HMC-1. The results revealed a novel spectrum of diversity in exosomes, which suggests that subpopulations of exosomes with different and specific functions might also exist. Our findings therefore argue that a new and more efficient way of defining exosome subpopulations is necessary. A system was proposed where exosomes were classified into nine different categories according to their size and shape. Three additional morphological features could also be found in exosomes regardless of their classification.\n\nThese findings show that morphological diversity is found among exosomes purified from a single cell line, similarly to what was previously observed for EVs in body fluids. This knowledge can help improving the interpretation of experimental results and widening our general understanding of the biological functions of exosomes.

cell biology↗