sciMET-cap: High-throughput single-cell methylation analysis with a reduced sequencing burden.

DNA methylation is a key component of the mammalian epigenome, playing a regulatory role in development, disease, and other processes. Robust, high-throughput single-cell DNA methylation assays are now possible (sciMET); however, the genome-wide nature of DNA methylation results in a high sequencing burden per cell. Here, we leverage target enrichment with sciMET to capture sufficient information per cell for cell type assignment using substantially fewer sequence reads (sciMET-cap). Sufficient off-target coverage further enables the production of near-complete methylomes for individual cell types. We characterize sciMET-cap on human PBMCs and brain (middle frontal gyrus).

GLUT1 is associated with sphingolipid-organized, cholesterol-independent domains in L929 mouse fibroblast cells.

Glucose is a preferred metabolite in most mammalian cells, and proper regulation of uptake is critical for organism homeostasis. The glucose transporter 1 (GLUT1) is responsible for glucose uptake in a wide variety of cells and appears to be regulated in a tissue specific manner. Therefore, a better understanding of GLUT1 regulation within its various cellular environments is essential for developing therapeutic strategies to treat disorders associated with glucose homeostasis. Previous findings suggest that plasma membrane subdomains called lipid rafts may play a role in regulation of GLUT1 uptake activity. While studying this phenomenon in L929 mouse fibroblast cells, we observed that GLUT1 associates with a low density lipid microdomain distinct from traditionally-defined lipid rafts. These structures are not altered by cholesterol removal with methyl-ß-cyclodextrin and lack resistance to cold Triton X-100 extraction. Our data indicate that the GLUT1-containing membrane microdomains in L929 cells, as well as GLUT1's basal activity, are instead sphingolipid-dependent, being sensitive to both myriocin and sphingomyelinase treatment. These microdomains appear to be organized primarily by their lipid composition, as disruption of the actin cytoskeleton or microtubules does not alter the association of GLUT1 with them. Furthermore, the association of GLUT1 with these microdomains appears not to require palmitoylation or glycosylation, as pharmacologic inhibition of these processes had no impact on GLUT1 density in membrane fractions. Importantly, we find no evidence that GLUT1 is actively translocated into or out of low density membrane fractions in response to acute activation in L929 cell.

Mitochondria, ER, and nuclear membrane defects reveal early mechanisms for upper motor neuron vulnerability with respect to TDP-43 pathology.

Insoluble aggregates containing TDP-43 are widely observed in the diseased brain, and defined as "TDP-43 pathology" in a spectrum of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease and ALS with frontotemporal dementia. Here we report that Betz cells of patients with TDP-43 pathology display a distinct set of intracellular defects especially at the site of nuclear membrane, mitochondria and endoplasmic reticulum (ER). Numerous TDP-43 mouse models have been generated to discern the cellular and molecular basis of the disease, but mechanisms of neuronal vulnerability remain unknown. In an effort to define the underlying causes of corticospinal motor neuron (CSMN) degeneration, we generated and characterized a novel CSMN reporter line with TDP-43 pathology, the prp-TDP-43A315T-UeGFP mice. We find that TDP-43 pathology related intracellular problems emerge very early in the disease. The Betz cells in humans and CSMN in mice both have impaired mitochondria, and display nuclear membrane and ER defects with respect to TDP-43 pathology.