Lucocq, J. M. | bioRxiv Science

bioRxiv2021

CLEMSite, a software for automated phenotypic screens using light microscopy and FIB-SEM

Correlative light and electron microscopy (CLEM) combines two imaging modalities, balancing out the limits of one technique with the other. In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume acquisition at the ultrastructural level. We present a toolset for adherent cultured cells that enables tracking and finding cell regions previously identified in light microscopy, in the FIB-SEM along with automatic acquisition of high-resolution volume datasets. We detect a grid pattern in both modalities (LM and EM), which identifies common reference points. The novel combination of these techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscopes field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5 {micro}m accuracy, while iterating between different targets and implementing unattended data acquisition. Our results demonstrate that executing high throughput volume acquisition in electron microscopy is possible.

cell biology↗

bioRxiv2018

In Troyer Syndrome Spartin loss induces Complex I impairments and alters pyruvate metabolism

Growth delay and retardation are complex phenotypes which can results by a range of factors including genetics variants. We identified a novel homozygous frameshift mutation, c.892dupA, in SPART gene, in two brothers with short stature and psychomotor retardation, born from healthy consanguineous parents. Mutations in SPART are the cause of Troyer syndrome, an autosomal recessive form of spastic paraplegia resulting in muscle weakness, short stature and cognitive defects. SPART encodes for Spartin, a protein with different cellular functions, such as endosomal trafficking and mitochondrial stability.\n\nWe evaluated the effects of Spartin loss by transiently silencing SPART in human neural stem cells (hNSCs) and by generating an SH-SY5Y cell line model carrying the c.892dupA mutation via CRISPR/Cas9. In both models, we observed an altered neuronal growth and an increase in neurite outgrowth. In the SH-SY5Y cell line carrying the c.892dupA mutation, Spartin absence led to an altered distribution of mitochondria, and to a severe decrease in the NADH-dehydrogenase activity of mitochondrial Complex I. These impairments determined an energetic failure with a decrease in ATP synthesis due to a halt in mitochondrial oxidative phosphorylation, increased reactive oxygen species production, and alteration in intracellular Ca2+ homeostasis. Transient re-expression of Spartin in mutant cells restored an intracellular Ca2+ level. Mutant cells presented a significant increase in extracellular pyruvate, which may result from increased glycolysis due to impaired Complex I activity. Consistently, Spartin loss led to an over-activation of Signal Transducer and Activator of Transcription 3 (STAT3) factor, a key regulator of glycolysis.\n\nThese data demonstrate that Spartin loss leads to a profound bioenergetics imbalance with defective OXPHOS activity, and this altered metabolism might underlie Troyer syndrome and neurodevelopmental delays.

cell biology↗