Biology subjects

Deerinck, T.

Publications and source records attributed to Deerinck, T..

4 recordsLinked to original sources

bioRxiv2025

Deep mapping of the endomembrane system of cerebellar Purkinje neurons

Neuronal function relies on the precise spatial organization of intracellular membrane-bounded organelles involved in anabolism and Ca2+ sequestration, such as the Golgi apparatus, mitochondria and the endoplasmic reticulum (ER), along with structures involved in catabolism, such as lysosomes. Despite their known roles in energy supply, calcium homeostasis, and proteostasis, our understanding of how the anabolism-linked organelles are structurally arranged within neurons remains incomplete. Due to the tremendous complexity in the morphologies and fine structural features and interwoven nature of these intracellular organelles, particularly the ER, our understanding of their structural organization is limited, particularly, with regard to quantitative assessments of their sites of interaction and accurate measures of their volumetric proportions inside of a single large neuron. To approach this challenge, we used serial block-face scanning electron microscopy (SBEM) to generate large-scale 3D EM volumes and electron tomography on high-pressure frozen tissue of the rodent cerebellum, including the largest cells in the vertebrate brain, the cerebellar Purkinje neuron as well as the most abundant cell type in the vertebrate brain, the much smaller cerebellar granule neuron. We reconstructed the neuronal ultrastructure of these different cell types, focusing on the ER, mitochondria and membrane contact sites, to then characterize intracellular motifs and organization principles in detail, providing a first full map to quantitatively describe a neuronal endoarchitectome. At the gross level organization, we found that the intracellular composite of organelles are cell type specific features, with specific differences between Purkinje neurons and Granule cells. At the level of fine structure, we mapped ultrastructural domains within Purkinje neurons where ER and mitochondria associate directly. In addition to cell type specific differences, we observed significant subcellular regional variation, particularly within the axon initial segment (AIS) of Purkinje neurons, where we identified ultrastructural domains with sharply contrasting distributions of ER and mitochondria. These findings suggest a finely tuned spatial organization of organelles that may underpin the distinct functional demands along the axon. We expect that our subcellular map, along with the methods developed to obtain these maps, will facilitate future studies in health, aging and disease to characterize defined features, by developing a framework for quantitative analysis of the neuronal ultrastructure.

neuroscience↗

bioRxiv2025

Development of a Charge-Resistant Embedding Media for High-Performance Serial Block Face Imaging of Cells and Tissues

Serial block-face scanning electron microscopy (SBEM) enables high-resolution 3D imaging of biological specimens but is often limited by specimen charging due to the use of non-conductive epoxy resins. While heavy metal staining and variable-pressure SEM can reduce charging, these methods compromise resolution or are only partially effective. We report a novel approach using polyethylene glycol (PEG 3350), a water-soluble, non-conductive polymer, as a doping agent to reduce specimen charging without sacrificing imaging quality. Although PEG 3350 alone lacks the mechanical and sectioning properties required for SBEM, we found it can be dissolved in standard Durcupan epoxy resin to improve charge resistance while maintaining physical integrity. Resins doped with 10% PEG 3350 demonstrated a substantial reduction in charging at 1.4-1.8 keV in charge-prone samples such as cultured HeLa cells, lung, and brain tissues, while retaining transparency and sectionability. Unlike other methods, the doped resin performs in high vacuum without compromising signal-to-noise ratio or spatial resolution. Notably, the PEG-doped resin reduced charging at voltages above the typical threshold for undoped resins, which show artifacts above 1.0 keV. Though some charging persisted in lung tissue at 2.0 keV, near-complete mitigation was achieved at slightly lower voltages. Our findings suggest that PEG-doped resins provide a promising route to minimize specimen charging in SBEM, and future studies optimizing PEG molecular weight and concentration could yield a universally charge-resistant embedding medium compatible with high-resolution imaging across diverse sample types.

cell biology↗

bioRxiv2023

Caloric restriction promotes beta cell longevity and delays aging and senescence by enhancing cell identity and homeostasis mechanisms.

Caloric restriction (CR) extends organismal lifespan and health span by improving glucose homeostasis mechanisms. How CR affects organellar structure and function of pancreatic beta cells over the lifetime of the animal remains unknown. Here, we used single nucleus transcriptomics to show that CR increases the expression of genes for beta cell identity, protein processing, and organelle homeostasis. Gene regulatory network analysis link this transcriptional phenotype to transcription factors involved in beta cell identity (Mafa) and homeostasis (Atf6). Imaging metabolomics further demonstrates that CR beta cells are more energetically competent. In fact, high-resolution light and electron microscopy indicates that CR reduces beta cell mitophagy and increases mitochondria mass, increasing mitochondrial ATP generation. Finally, we show that long-term CR delays the onset of beta cell aging and senescence to promote longevity by reducing beta cell turnover. Therefore, CR could be a feasible approach to preserve compromised beta cells during aging and diabetes.

cell biology↗

bioRxiv2021

A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpus itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 required PTPN23 but not the retromer subunits. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23. Author SummaryVpu triggers the degradation or mis-localization of proteins important to the hosts immune response. Vpu acts as an adaptor, linking cellular protein targets to the ubiquitination and membrane trafficking machinery. Vpu has been localized to various cellular membrane systems. By fusing wild type Vpu and Vpu-mutants to the enzyme ascorbate peroxidase, we defined the cellular proteome in proximity to Vpu and correlated this with the proteins location. We found that wild type Vpu is proximal to ESCRT proteins, retromer complexes, and sorting and late endosomal proteins. Functionally, we found that the Vpu-mediated degradation of the innate defense protein BST-2 required PTPN23, an ALIX-like protein, consistent with our observation of Vpus presence at the limiting membranes of multi-vesicular bodies.

microbiology↗