Biology subjects

Gemin, O.

Publications and source records attributed to Gemin, O..

4 recordsLinked to original sources

bioRxiv2024

EasyGrid: A versatile platform for automated cryo-EM sample preparation and quality control

Imaging biological macromolecules in their native state with single-particle cryo-electron microscopy (cryo-EM) or in situ cryo-electron tomography (cryo-ET) requires optimized approaches for the preparation and vitrification of biological samples. Here, we describe EasyGrid, a versatile technology enabling systematic, tailored and advanced sample preparation for cellular and structural biology. This automated, standalone platform combines in-line plasma treatment, microfluidic dispensing, blot-less sample spreading, jet-based vitrification and on-the-fly grid quality control using light interferometry to streamline cryo-EM sample optimization. With EasyGrid, we optimized grid preparation for different purified macromolecular complexes and subsequently determined their structure with cryo-EM. We also demonstrated how the platform allows better vitrification of large, mammalian cells compared to standard plunge-freezing. Automated sample preparation with EasyGrid establishes an advanced, high-throughput platform for both single-particle cryo-EM and cellular cryo-ET sample preparation.

molecular biology↗

bioRxiv2023

Hibernating ribosomes tether to mitochondria as an adaptive response to cellular stress during glucose depletion

Cell survival under nutrient-deprived conditions relies on cells ability to adapt their organelles and to rewire their metabolic pathways. In the fission yeast Schizosaccharomyces pombe, nutrient depletion is an unfavorable condition for protein synthesis and triggers a response characterized by mitochondrial fragmentation and the sequestration of cytosolic ribosomes on mitochondria. The molecular mechanism underlying ribosomal sequestration remains elusive. In this study, we performed time-lapse in situ cryo-electron tomography and cryo-electron microscopy complemented by biochemical experiments to elucidate the molecular details of this adaptive response. Our analysis indicate that upon glucose depletion protein synthesis is halted, causing ribosomes to enter an inactive state characterized by a conformational change that obstructs the peptidyl transferase center. Our in situ experiments reveal the presence of oligomeric arrays of hibernating ribosomes tethered to the mitochondrial surface. Surprisingly, ribosomes bind to the outer mitochondrial membrane via the small ribosomal subunit, an interaction facilitated by the ribosomal protein RACK1-orthologue Cpc2. Our experiments show that ribosome tethering is important for cell survival under glucose depletion conditions. This study broadens our understanding of the cellular adaptations triggered by nutrient scarcity and the underlying molecular mechanisms that regulate cell quiescence.

molecular biology↗

bioRxiv2021

Identification of a stereotypic molecular arrangement of glycine receptors at native spinal cord synapses

Precise quantitative information about the molecular architecture of synapses is essential to understanding the functional specificity and downstream signaling processes at specific populations of synapses. Glycine receptors (GlyRs) are the primary fast inhibitory neurotransmitter receptors in the spinal cord and brainstem. These inhibitory glycinergic networks crucially regulate motor and sensory processes. Thus far the nanoscale organization of GlyRs underlying the different network specificities has not been defined. Here, we have quantitatively characterized the molecular arrangement and ultra-structure of glycinergic synapses in spinal cord tissue using quantitative super-resolution correlative light and electron microscopy (SR-CLEM). We show that endogenous GlyRs exhibit equal receptor-scaffold occupancy and constant packing densities of about 2000 GlyRs m-2 at synapses across the spinal cord and throughout adulthood, even though ventral horn synapses have twice the total copy numbers, larger postsynaptic domains and more convoluted morphologies than dorsal horn synapses. We demonstrate that this stereotypic molecular arrangement is maintained at glycinergic synapses in the oscillator mouse model of the neuromotor disease hyperekplexia despite a decrease in synapse size, indicating that the molecular organization of GlyRs is preserved in this hypomorph. We thus conclude that the morphology and size of inhibitory postsynaptic specializations rather than differences in GlyR packing determine the postsynaptic strength of glycinergic neurotransmission in motor and sensory spinal cord networks.

molecular biology↗

bioRxiv2020

Morphologically constrained modeling of spinous inhibition in the somato-sensory cortex

Pyramidal neurons are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light-electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 pyramidal neurons of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually-innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient than shaft inhibition and that it can decouple voltage and calcium signaling, potentially impacting synaptic plasticity.

neuroscience↗