Biology subjects

Pedaci, F.

Publications and source records attributed to Pedaci, F..

7 recordsLinked to original sources

bioRxiv2026

Single-Cell Electrophysiology Reveals Verapamil's Disruption of Bacterial Membrane Energetics

Verapamil, a clinically used calcium channel blocker, enhances the activity of several tuberculosis antibiotics, but its mechanism of action and physiological effects on bacteria remain unresolved. A central debate concerns whether verapamil primarily inhibits efflux pumps or disrupts membrane energetics. Here, we use Escherichia coli as a model system to quantify single-cell and population-level physiological responses to verapamil with high temporal resolution. Real-time measurements of the rotational speed of individual flagellar motors, a single-cell proxy for the proton motive force (PMF), reveal a heterogeneous response to verapamil: treated cells exhibit either a dose-dependent gradual decrease in PMF, or a rapid collapse of PMF. Although loss of the outer-membrane efflux channel TolC increases growth inhibition by verapamil, it does not alter the rapid PMF disruptions observed at the single-cell level, suggesting that efflux contributes to long-term susceptibility but not to the initial PMF disruption. Independent assays of population-level motility, pH, and membrane-integrity suggest that verapamil may selectively dissipate the electrical component of PMF while leaving intracellular pH largely unchanged. A minimal electrical circuit model captures both steady-state and dynamic behavior. Together, these findings demonstrate that verapamil rapidly and reversibly perturbs bacterial membrane energetics through a mechanism distinct from classical protonophores, helping to reconcile conflicting interpretations of its activity and clarifying how membrane effects may interact with efflux inhibition during antibiotic potentiation.

biophysics↗

bioRxiv2025

Deep learning-based photo-identification for non-invasive monitoring of animal populations: Application to penguins and tortoises

We describe DL-ID, a deep learning-based framework for the individual identification of animals from images with higher accuracy and characteristics that make it more robust in challenging conditions, such as field studies, than widely used algorithms. This is demonstrated through testing with small datasets involving two species that exhibit distinctive morphological features: Humboldt penguins (Spheniscus humboldti Meyen, 1834) and Hermanns tortoises (Testudo hermanni Gmelin, 1789). We define a confidence index based on entropy within the model that significantly improves handling of the open set recognition problem, offering a new way to discriminate unknown individuals. DL-IDs accuracy of 87%, even with as few as four images per class, contrasts with the usual practice in deep learning of building large datasets to assess performance. The model outperforms traditional photo-identification methods like Wild-ID and I3S Pattern, offering a significant advancement for research and conservation efforts. Its efficiency and adaptability indicate its potential in real-time monitoring, opening new possibilities for wildlife conservation. Instead of designing a complex new deep learning model, we focused on adapting existing methods to address a relevant ecological problem. Our approach effectively tackles issues like limited training data and recognizing new individuals in field studies. Notably, it performs well even with small datasets, making it particularly useful for data-limited ecological research. This makes our approach a valuable step forward in applying deep learning to ecological studies.

ecology↗

bioRxiv2024

Insertion of fluorescent proteins near the plug domain of MotB generates functional stator complex.

Many bacteria swim by the rotation of the bacterial flagellar motor (BFM). The BFM is powered by proton translocation across the inner membrane through the hetero-heptameric MotA5MotB2 protein complex. Two periplasmic domains of MotB are critical in activating BFM rotation: (1) the peptidoglycan binding (PGB) domain that anchors MotB in the peptidoglycan layer and (2) the plug domain that modulates the proton flow. Existing cytoplasmic fluorescent probes have been shown to negatively affect motor rotation and switching. Here we inserted a fluorescent probe in the periplasm near the plug of MotB in an attempt to circumvent issues with cytoplasmic probes and for possible use in observing the mechanism of plug-based regulation of proton flow. We inserted green fluorescent protein (GFP) and iLOV, a fluorescent version of the light-oxygen-voltage (LOV) domain, in four periplasmic locations in MotB. Insertions near the plug retained motility but showed limited fluorescence for both fluorophores. Additional short, flexible glycine-serine (GS) linkers improved motility but did not improve brightness. Further optimization is necessary to improve the fluorescence of these periplasmic probes.

microbiology↗

bioRxiv2023

Spatio-temporal dynamics of the proton motive force on single bacterial cells

Electrochemical gradients established across biological membranes are fundamental in the bioenergetics of all forms of life. In bacteria, the proton motive force (PMF), the electrochemical potential associated to protons, powers an impressive array of fundamental processes, from ATP production to motility. While far from equilibrium, it has classically been considered homeostatic in time and space. Yet, recent experiments have revealed rich temporal dynamics at the single cell level and functional spatial dynamics at the scale of multicellular communities. Lateral segregation of supramolecular respiratory complexes begs the question of whether spatial heterogeneity of the PMF exists even at the single cell level. By using a light-activated proton pump as a spatially and temporally modulatable source, and the bacterial flagellar motor as a local electro-mechanical gauge, we both perturb and probe the PMF on single cells. Using global perturbations, we resolve temporal dynamics on the ms time scale and observe an asymmetrical capacitive response of the cell. Using localized perturbations, we find that the PMF is rapidly homogenized along the entire cell, faster than proton diffusion can allow. Instead, the electrical response can be explained in terms of electrotonic potential spread, as found in passive neurons and described by cable theory. This implies a global coupling between PMF sources and consumers in the bacterial membrane, excluding a sustained spatial heterogeneity while allowing for fast temporal dynamics. SignificanceStoring energy in the form of a proton gradient across a membrane is a fundamental feature of living systems. In mitochondria, spatial compartmentalization separates electrically distinct regions. In bacteria, it is unclear how this energy reservoir, the proton motive force, behaves at the single cell level: can it be heterogeneous in space as in mitochondria? How fast can it change in time? Using a light-driven proton pump and the flagellar motor as a local electro-mechanical gauge, we find that the bacterial proton motive force can change in a few tens of milliseconds, and that it is instantaneously homogenized along the membrane. This electrophysiological response is surprisingly similar to electrotonic voltage spread in passive neurons.

biophysics↗

bioRxiv2018

Kinetic analysis methods applied to single motor protein trajectories

Molecular motors convert chemical or electrical energy into mechanical displacement, either linear or rotary. Under ideal circumstances, single-molecule measurements can spatially and temporally resolve individual steps of the motor, revealing important properties of the underlying mechanochemical process. Unfortunately, steps are often hard to resolve, as they are masked by thermal noise. In such cases, details of the mechanochemistry can nonetheless be recovered by analyzing the fluctuations in the recorded traces. Here, we expand upon existing statistical analysis methods, providing two new avenues to extract the motor step size, the effective number of rate-limiting chemical states per translocation step, and the compliance of the link between the motor position and the probe particle. We first demonstrate the power and limitations of these methods using simulated molecular motor trajectories, and we then apply these methods to experimental data of kinesin, the bacterial flagellar motor, and F1-ATPase.

biophysics↗

bioRxiv2017

A catch-bond drives stator mechanosensitivity in the Bacterial Flagellar Motor

The bacterial flagellar motor (BFM) is the rotary motor which powers the swimming and swarming of many motile bacteria. The torque is provided by stator units, ion motive force powered ion channels known to assemble and disassemble dynamically in the BFM. This turnover is mechano-sensitive, with the number of engaged units dependent upon the viscous load experienced by the motor through the flagellum. However, the molecular mechanism driving BFM mechano-sensitivity is unknown. Here we directly measure the kinetics of arrival and departure of the stator units in individual wild-type motors via analysis of high-resolution recordings of motor speed, while dynamically varying the load on the motor via external magnetic torque. Obtaining the real-time stator stoichiometry before and after periods of forced motor stall, we measure both the number of active stator units at steady-state as a function of the load and the kinetic association and dissociation rates, by fitting the data to a reversible random sequential adsorption model. Our measurements indicate that BFM mechano-sensing relies on the dissociation rate of the stator units, which decreases with increasing load, while their association rate remains constant. This implies that the lifetime of an active stator unit assembled within the BFM increases when a higher force is applied to its anchoring point in the cell wall, providing strong evidence that a catch-bond mechanism can explain the mechano-sensitivity of the BFM.

biophysics↗

bioRxiv2017

Impact of fluorescent protein fusions on the bacterial flagellar motor

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects. Such insight, however, is still lacking for many applications of fluorescent fusions. This is particularly relevant in the study of the internal dynamics of motor protein complexes, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the bacterial flagellar motor (BFM) complex have previously been used to successfully unveil the internal subunit dynamics of the motor. Here we report the effects of three different fluorescent proteins fused to the stator, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry in the complex remained unaffected. MotB fusions decreased the rotation-direction switching frequency of single motors and induced a novel BFM behavior: a bias-dependent asymmetry in the speed attained in the two rotation directions. All these effects could be mitigated by the insertion of a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions on BFM dynamics and their alleviation--new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.\n\nAuthor summaryMuch of what is known about the biology of proteins was discovered by fusing them to fluorescent proteins that allow detection of their location. But the label comes at a cost: the presence of the tag can alter the behavior of the protein of interest in unforeseen, yet biologically relevant ways. These side effects limit the depth to which fluorescent proteins can be used to probe protein function. One of the systems that has been successfully studied with fluorescent fusions for which these effects have not been addressed are dynamic protein complexes that carry out mechanical work. We examined how fluorescent proteins fused to a component of the bacterial flagellar motor complex impacts its function. Our findings show that the fusion proteins altered biologically relevant dynamical properties of the motor, including induction of a novel mechanical behavior, and demonstrate an approach to alleviate this. These results advance our ability to dissect the bacterial flagellar motor, and the internal dynamics of protein complexes in general, with fluorescent fusion proteins while causing minimal perturbation.

biophysics↗