Biology subjects

Testa, I.

Publications and source records attributed to Testa, I..

13 recordsLinked to original sources

bioRxiv2026

Single-particle tracking reveals neuronal activity-dependent shuttling of ARC/ARG3.1 protein between cytoplasmic clusters and the nucleus

The activity-regulated cytoskeleton-associated protein (ARC/ARG3.1) is a key regulator of synaptic plasticity and has both synaptic and nuclear functions. ARC is known to undergo nuclear import and export, yet the dynamic transport behavior of individual ARC particles remains unknown. Using live-cell single-particle tracking, we directly visualize ARC nucleocytoplasmic transport and shuttling in primary hippocampal neurons. Synaptic activation by chemical long-term potentiation (cLTP) treatment increases shuttling behavior and reveals a previously underappreciated organization of ARC within the neuronal cell body cytoplasm, characterized by perinuclear ARC clusters. Disruption of the N-terminal ARC oligomerization motif markedly reduced both perinuclear cluster formation and nucleocytoplasmic shuttling. Together, these findings reveal an activity-dependent relationship between ARC self-assembly, perinuclear organization, and nucleocytoplasmic trafficking, providing a potential mechanism for coordinating the synaptic and nuclear functions of ARC during neuronal plasticity.

neuroscience↗

bioRxiv2025

Nanobinders for Synaptotagmin 1 enable the analysis of synapticvesicle dynamics in rodent and human models.

Synaptic neurotransmission is a critical hallmark of brain activity and one of the first processes to be affected in neural diseases. Monitoring this process, and in particular synaptic vesicle recycling, in living cells has been instrumental in unraveling mechanisms responsible for neurotransmitter release. However, currently available reporters suffer from major limitations such large probe size or lack of suitability for human neurons, hampering the understanding of human synaptic pathophysiology. Here we describe the NbLumSyt1 toolkit, a panel of nanobody-based affinity probes targeting the luminal domain of the synaptic vesicle protein Synaptotagmin 1 (Syt1). These new tools enable quantitative, non-invasive imaging and functional interrogation of synaptic transmission in human neurons, with unprecedented precision, versatility and cost efficiency, in technologies ranging from fixed-and live-cell super-resolution imaging to electron microscopy and mass spectrometry. Overall, NbLumSyt1 nanobinders provide a valuable platform for human synaptic physiology and pathophysiology, benefiting fundamental neuroscience and translational efforts to study and develop treatments for brain-related disorders.

neuroscience↗

bioRxiv2025

All-Optical Strategies to Minimize Photo-Bleaching in Reversibly Switchable Fluorescent Proteins

Photo-bleaching is a general hurdle of fluorescence-based techniques that becomes even more severe in high- resolution microscopy that relies on prolonged, focused and complex illumination sequences. Strategies to reduce photo-bleaching require chemical modifications of the cell media, which often stave off physiological cellular conditions. Here, we outline an all-optical strategy to minimize photo-bleaching in reversibly switching fluorescent proteins (RSFPs), a class of probes used in several super-resolution and protein-multiplexing imaging techniques. By identifying the photobleaching pathways, we developed novel imaging schemes to increase the number of ON- OFF photo-switching cycles based on a designed modulation of the on-switching light or a co-irradiation with red- shifted light. By rationalizing the photo-cycle, we expand multiplexing strategies with RSFPs to high- spatiotemporal resolutions while maintaining the accuracy and recording longer time-lapse imaging of sub-cellular structures with both confocal microscopy and parallelized RESOLFT nanoscopy.

biophysics↗

bioRxiv2024

Structural diversity of Arc oligomers within the excitatory synapse

The Activity-Regulated Cytoskeleton-Associated protein (Arc), is pivotal to mediate plasticity responses in neuronal cells. In vitro studies suggest its ability to form high- and low-order oligomers, which are involved in neuronal trafficking. Despite its important functions, no direct observation of Arc oligomers in cells has been presented due to its highly regulated spatiotemporal expression, the small size of the structures, the lack of appropriate labelling strategies and the background associated to free diffusing cytosolic proteins. Here, we apply super resolution microscopy to observe Arc oligomeric states in cellular environment with focus on the excitatory synapse. In cells, we provide the first evidence of Arc high-order oligomers; we uncovered intermolecular interactions of Arc, its tendency to form liquid condensates and interaction with lipid bilayers. Arc high-order oligomers affect AMPA receptor surface levels. Together, our observations suggest a model by which Arc oligomerization mediates plasma membrane negative curvature favoring AMPA receptors endocytosis.

neuroscience↗

bioRxiv2024

Most axonal mitochondria in cortical pyramidal neuronslack mitochondrial DNA and consume ATP

In neurons of the mammalian central nervous system (CNS), axonal mitochondria are thought to be indispensable for supplying ATP during energy-consuming processes such as neurotransmitter release. We have previously shown that glutamatergic excitatory cortical pyramidal neurons (CPNs) are characterized by a striking compartmentalization of mitochondrial structure. Axonal mitochondria are maintained at a small uniform size ([~]1 microns length) by high levels of MFF-dependent fission, whereas dendritic mitochondria form a highly fused network of mitochondria throughout the entire dendritic arbor(1, 2). Here, we tested whether these striking structural differences reflect functional and molecular compartmentalization. Using four independent techniques, we reveal that in CPNs as well as in two subtypes of GABAergic inhibitory cortical interneurons and neuromodulatory dopaminergic neurons of the substantia nigra, the majority of axonal, but not dendritic, mitochondria lack mitochondrial DNA (mtDNA) in vivo. We also show that axonal mitochondria are depleted in both mtDNA-encoded mRNAs and proteins compared to dendritic mitochondria. Timelapse imaging establishes that most mitochondria entering nascent dendritic and axonal processes from the soma are small and lack mtDNA. Using dynamic, optical imaging of genetically encoded sensors for ATP and pH targeted to the mitochondrial matrix, we demonstrate that in axons of CPNs, but not in their dendrites, mitochondrial F1F0-ATP synthase (Complex V) functions in a reverse way, hydrolyzing ATP and extruding H+ out of the matrix to maintain mitochondrial membrane potential. Our results indicate that in neurons of the mammalian CNS, the majority of axonal mitochondria lack mtDNA and likely do not play a major role in ATP generation, despite playing other functions such as regulation of neurotransmission via presynaptic Ca2+ buffering(1, 3-5).

neuroscience↗

bioRxiv2021

Event-triggered STED imaging

The observation of protein organization during cellular signalling calls for imaging methods with increased spatial and temporal resolution. STED nanoscopy can access dynamics of nanoscale structures in living cells. However, the available number of recordable frames is often limited by photo-bleaching. Here, we present an automated method, event-triggered STED, which instantly (< 40 ms) images synaptic proteins with high spatial and temporal resolution (~30 nm, 2.5 Hz) in small regions upon and at the site of local calcium sensing.

biophysics↗

bioRxiv2021

Identification of electroporation sites in the complex lipid organization of the plasma membrane

The plasma membrane of a biological cell is a complex assembly of lipids and membrane proteins, which tightly regulate transmembrane transport. When a cell is exposed to a strong electric field, the membrane integrity becomes transiently disrupted by formation of transmembrane pores. This phenomenon, termed electroporation, is already utilized in many rapidly developing applications in medicine including gene therapy, cancer treatment, and treatment of cardiac arrythmias. However, the molecular mechanisms of electroporation are not yet sufficiently well understood; in particular, it is unclear where exactly pores form in the complex organization of the plasma membrane. In this study we combine coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis to identify how formation of pores depends on the local lipid organization. We show that pores do not form homogeneously across the membrane, but colocalize with domains that have specific features, the most important being high density of polyunsaturated lipids. We further show that knowing the lipid organization is sufficient to reliably predict poration sites with machine learning. However, by analysing poration kinetics with Bayesian survival analysis we then show that poration does not depend solely on local lipid arrangement, but also on membrane mechanical properties and the polarity of the electric field. Finally, we discuss how the combination of atomistic and coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis can guide the design of future experiments and help us to develop an accurate description of plasma membrane electroporation on the whole-cell level. Achieving this will allow us to shift the optimization of electroporation applications from blind trial-and-error approaches to mechanistic-driven design.

biophysics↗

bioRxiv2021

ExSTED microscopy reveals contrasting functions of dopamine and somatostatin CSF-c neurons along the central canal

The spatial location of cerebrospinal fluid contacting (CSF-c) neurons enables important regulatory homeostatic functions regarding pH and motion control. Their intricate organization, facing the central canal and extending across the spinal cord, in relation to specific subtypes is poorly understood. This calls for imaging methods with a high spatial resolution (5-10 nm) to resolve the synaptic and ciliary compartments of each individual cell to elucidate their signalling pathways and enough throughput to dissect the cellular organization. Here, light-sheet and expansion microscopy resolved the persistent ventral and lateral organization of dopamine and somatostatin CSF-c neuronal types. The number of somatostatin-containing dense core vesicles, resolved by STED microscopy, was shown to be markedly reduced upon each exposure to alkaline or acidic pH inhibiting any movement as part of a homeostatic response. Their cilia symmetry was unravelled by ExSTED as sensory in contrast with the motile one found in the dopaminergic ph insensitive neurons. This novel experimental workflow elucidates the functional role of CSF-c neuron subtypes in situ paving the way for further spatial and functional cell type classification.

neuroscience↗

bioRxiv2020

Three dimensional parallelized RESOLFT nanoscopy for volumetric live cell imaging

The volumetric architecture of organelles and molecules inside cells can only be investigated with microscopes featuring sufficiently high resolving power in all three spatial dimensions. Current methods suffer from severe limitations when applied to live cell imaging such as long recording times and/or photobleaching. By introducing a novel optical scheme to switch reversibly switchable fluorescent molecules, we demonstrate volumetric nanoscopy of living cells with resolution below 100 nm in 3D, large field of view and minimal illumination intensities (W-kW/cm2).

biophysics↗

bioRxiv2019

Pulsed electric fields create pores in the voltage sensors of voltage-gated ion channels

Pulsed electric fields are increasingly used in medicine to transiently increase the cell membrane permeability via electroporation, in order to deliver therapeutic molecules into the cell. One type of events that contributes to this increase in membrane permeability is the formation of pores in the membrane lipid bilayer. However, electrophysiological measurements suggest that membrane proteins are affected as well, particularly voltage-gated ion channels (VGICs). The molecular mechanisms by which the electric field could affects these molecules remain unidentified. In this study we used molecular dynamics (MD) simulations to unravel the molecular events that take place in different VGICs when exposing them to electric fields mimicking electroporation conditions. We show that electric fields induce pores in the voltage-sensor domains (VSDs) of different VGICs, and that these pores form more easily in some channels than in others. We demonstrate that poration is more likely in VSDs that are more hydrated and are electrostatically more favorable for the entry of ions. We further show that pores in VSDs can expand into so-called complex pores, which become stabilized by lipid head-groups. Our results suggest that such complex pores are considerably more stable than conventional lipid pores and their formation can lead to severe unfolding of VSDs from the channel. We anticipate that such VSDs become dysfunctional and unable to respond to changes in transmembrane voltage, which is in agreement with previous electrophyiological measurements showing a decrease in the voltage-dependent transmembrane ionic currents following pulse treatment. Finally, we discuss the possibility of activation of VGICs by submicrosecond-duration pulses. Overall our study reveals a new mechanism of electroporation through membranes containing voltage-gated ion channels. Statement of SignificancePulsed electric fields are often used for treatment of excitable cells, e.g., for gene delivery into skeletal muscles, ablation of the heart muscle or brain tumors. Voltage-gated ion channels (VGICs) underlie generation and propagation of action potentials in these cells, and consequently are essential for their proper function. Our study reveals the molecular mechanisms by which pulsed electric fields directly affect VGICs and addresses questions that have been previously opened by electrophysiologists. We analyze VGICs characteristics, which make them prone for electroporation, including hydration and electrostatic properties. This analysis is easily transferable to other membrane proteins thus opening directions for future investigations. Finally, we propose a mechanism for long-lived membrane permeability following pulse treatment, which to date remains poorly understood.

biophysics↗

bioRxiv2019

Tiled STED Imaging of Extended Sample Regions

Stimulated Emission Depletion (STED) nanoscopy has become one of the most used nanoscopy techniques over the last decade. However, most recordings are done in specimen regions no larger than 10-30 x 10-30 {micro}m2 due to aberrations, instability and manual mechanical stages. Here, we demonstrate automated STED nanoscopy of extended sample regions up to 0.5 x 0.5 mm2 by using a back-aperture-stationary beam scanning system. The setup allows up to 80-100 x 80-100 {micro}m2 field of view (FOV) with uniform spatial resolution, a mechanical stage allowing sequential tiling to record larger sample areas, and a feedback system keeping the sample in focus at all times. Taken together, this allows automated recording of theoretically unlimited-sized sample areas and volumes, without compromising the achievable spatial resolution and image quality.

biophysics↗

bioRxiv2018

Growth-driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus

All living cells must deal with protein aggregation, which can occur as a result of experiencing stress. In the bacteria Escherichia coli and Mycobacterium smegmatis, aggregates collect at the cell poles and are retained over consecutive cell divisions only in the daughter cell that inherits the old pole, resulting in aggregation-free progeny within a few generations. Here we have studied the in vivo kinetics of aggregate formation and clearance following heat and antibiotic stress in Caulobacter crescentus, which divides by a pre-programmed asymmetric cell cycle. Unexpectedly, we find that aggregates do not preferentially collect at the cell poles, but form as multiple distributed foci throughout the cell volume. Time-lapse microscopy revealed that under moderate stress, the majority of protein aggregates are short-lived and rapidly dissolved by the major chaperone DnaK and the disaggregase ClpB. Severe stress or genetic perturbation of the protein quality machinery results in long-lived protein aggregates, which individual cells can only clear by passing on to their progeny. Importantly, these persistent aggregates are neither collected at the old pole over multiple generations nor inherited exclusively by the old pole-inheriting stalked cell, but instead are partitioned between both daughter cells during successive division events in the same ratio. Our data indicate that this symmetric mode of aggregate inheritance is driven by the elongation and division of the growing mother cell. In conclusion, our study revealed a new pattern of aggregate inheritance in bacteria.

microbiology↗

bioRxiv2018

Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems

The theoretically unlimited spatial resolution of fluorescence nanoscopy often comes at the expense of time, contrast and increased dose of energy for recording. Here, we developed MoNaLISA, for Molecular Nanoscale Live Imaging with Sectioning Ability, a nanoscope capable of imaging structures at a scale of 45-65 nm within the entire cell volume at low light intensities (W-kW/cm2). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined in a novel way to gain fluorescence ON-OFF switching cycles and image contrast. By maximizing the detected photon flux MoNaLISA enables prolonged (40-50 frames) and large (50 x 50 m2) recordings at 0.3-1.3 Hz with enhanced optical sectioning ability. We demonstrated the general use of our approach by 4D imaging of organelles and fine structures in epithelial human cells, colonies of mouse embryonic stem cells, brain cells, and organotypic tissues.

biophysics↗