Bioorthogonal Conjugation-Assisted Purification Method for Profiling Cell Surface Proteome.

Surface biotinylation has been widely adapted in profiling the cellular proteome associated with the plasma membrane. However, the workflow is subject to interference from the cytoplasmic biotin-associated proteins that compete for streptavidin-binding during purification. Here we established a bioorthogonal conjugation-assisted purification (BCAP) workflow that utilizes the Staudinger chemoselective ligation to label and isolate surface-associated proteins while minimizing the binding of endogenous biotin-associated proteins. Label-free quantitative proteomics demonstrated that BCAP is efficient in isolating cell surface proteins with excellent reproducibility. Subsequently, we applied BCAP to compare the surface proteome of proliferating and senescent mouse embryonic fibroblasts (MEFs). Among the results, EHD2 was identified and validated as a novel protein that is enhanced at the cell surface of senescent MEFs. We expect that BCAP will have broad applications in profiling cell surface proteomes in the future.

Hypertonicity induces mitochondrial extracellular vesicles (MEVs) that activate TNF-α and ß-catenin signaling to promote adipocyte dedifferentiation.

BACKGROUND: Recent studies demonstrated that elevated osmolarity could induce adipocyte dedifferentiation, representing an appealing procedure to generate multipotent stem cells. Here we aim to elucidate the molecular mechanisms that underlie osmotic induction of adipocyte reprogramming. METHODS: To induce dedifferentiation, the 3T3-L1 or SVF adipocytes were cultured under the hypertonic pressure in 2% PEG 300 medium. Adipocyte dedifferentiation was monitored by aspect ratio measurement, Oil Red staining and qPCR to examine the morphology, lipid droplets, and specific genes of adipocytes, respectively. The osteogenic and chondrogenic re-differentiation capacities of dedifferentiated adipocytes were also examined. To investigate the mechanisms of the osmotic stress-induced dedifferentiation, extracellular vesicles (EVs) were collected from the reprograming cells, followed by proteomic and functional analyses. In addition, qPCR, ELISA, and TNF-α neutralizing antibody (20 ng/ml) was applied to examine the activation and effects of the TNF-α signaling. Furthermore, we also analyzed the Wnt signaling by assessing the activation of ß-catenin and applying BML-284, an agonist of ß-catenin. RESULTS: Hypertonic treatment induced dedifferentiation of both 3T3-L1 and the primary stromal vascular fraction (SVF) adipocytes, characterized by morphological and functional changes. Proteomic profiling revealed that hypertonicity induced extracellular vesicles (EVs) containing mitochondrial molecules including NDUFA9 and VDAC. Functionally, the mitochondrial EVs (MEVs) stimulated TNF-α signaling that activates Wnt-ß-catenin signaling and adipocyte dedifferentiation. Neutralizing TNF-α inhibited hypertonic dedifferentiation of adipocytes. In addition, direct activation of Wnt-ß-catenin signaling using BML-284 could efficiently induce adipocyte dedifferentiation while circumventing the apoptotic effect of the hypertonic treatment. CONCLUSIONS: Hypertonicity prompts the adipocytes to release MEVs, which in turn enhances the secretion of TNF-α as a pro-inflammatory cytokine during the stress response. Importantly, TNF-α is essential for the activation of the Wnt/ß-catenin signaling that drives adipocyte dedifferentiation. A caveat of the hypertonic treatment is apoptosis, which could be circumvented by direct activation of the Wnt/ß-catenin signaling using BML-284.

Applying real-time monitoring of circadian oscillations in adult mouse brain slices to study communications between brain regions.

This protocol combines a protective cutting method to prepare various brain slices from adult mice and real-time monitoring of circadian oscillations to measure circadian rhythmicity in various brain slices. This protocol can be applied to studies of how brain damages affect local circadian clocks and subsequent circadian variations in nearby areas. Further functional analyses with in vivo systems will determine whether these circadian variations are detrimental or beneficial to the brain. For complete details on the use and execution of this protocol, please refer to Huang et al. (2020).

Demyelination Regulates the Circadian Transcription Factor BMAL1 to Signal Adult Neural Stem Cells to Initiate Oligodendrogenesis.

Circadian clocks are endogenous oscillators that generate cell-autonomous rhythms that govern cellular processes and are synchronized by external cues in the local macro- and micro-environments. Demyelination, a common brain pathology with variable degrees of recovery, changes the microenvironment via damaged myelin and activation of glial cells. How these microenvironmental changes affect local circadian clocks and with what consequences is mostly unknown. Here, we show that within demyelinating lesions, astrocyte circadian clocks produce the Wnt inhibitors SFRP1 and SFRP5. Unexpectedly, SFRP1 and SFRP5 signal to the subventricular zone (SVZ) to reduce the circadian transcription factor BMAL1. This sequence of events causes adult neural stem cells in the SVZ to differentiate into oligodendrocyte lineage cells, which are then supplied to demyelinated lesions. Our findings show that circadian clocks in demyelinating lesions respond to microenvironmental changes and communicate with the SVZ to enhance a natural repair system of spontaneous remyelination.