Biology subjects

Alvelid, J.

Publications and source records attributed to Alvelid, J..

7 recordsLinked to original sources

bioRxiv2025

Event-triggered MINFLUX microscopy: smart microscopy to catch and follow rare events

MINFLUX microscopy is a powerful microscopy method allowing for the characterization of molecular organization and dynamics with single nanometer spatial resolution and sub-hundred microseconds temporal resolution. However, acquisition times often span minutes to hours as a single fluorophore is measured at a time. Applying it to study cellular processes in living cells therefore requires careful consideration of where and when to apply MINFLUX data acquisition, a consideration where manual control limits its potential applications. Here, to overcome the limitations of acquisition speed, acquisition initiation, and data throughput, we present a smart microscopy method that uses confocal imaging as a monitoring method, runs real-time image analysis, and only applies MINFLUX data acquisition exactly where and when deemed necessary based on the analysis outcome. The method, event-triggered MINFLUX, is controlled through a custom-written and open source Python widget that automatically controls a commercial MINFLUX microscope. We apply this method to investigate molecular membrane dynamics and organization during three different cellular events: two-dimensional lipid dynamics at caveolae; three-dimensional membrane topography during dynamin-mediated endocytosis; and three-dimensional membrane fluidity and topography during budding site formation of HIV-1 proteins. Thanks to rapid event detection and minimal regions of interest the method provides data that would be unfeasible or impossible to acquire through manual control of the microscope.

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

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↗

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↗