Biology subjects

Rauch, M.

Publications and source records attributed to Rauch, M..

3 recordsLinked to original sources

bioRxiv2021

Measuring mitochondrial electron transfer complexes in previously frozen cardiac tissue from the offspring of sow: A model to assess exercise induced mitochondrial bioenergetics changes

Measuring the mitochondrial electron transfer complex (ETC) profile from previously frozen heart tissue samples from offspring born to an exercised sow provided descriptive data about exercise-induced mitochondrial biochemical changes in heart tissue from the offspring born to the exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis. SUMMARYPreparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

biochemistry↗

bioRxiv2020

On identifying the eukaryotic N-glycosylation scramblase by activity correlation profiling

The canonical pathway of N-linked protein glycosylation in yeast and humans involves transfer of the oligosaccharide moiety from the glycolipid Glc3Man9GlcNAc2-PP-dolichol to select asparagine residues in proteins that have been translocated into the lumen of the endoplasmic reticulum (ER). Synthesis of Glc3Man9GlcNAc2-PP-dolichol occurs in two stages, producing first the key intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) on the cytoplasmic face of the ER, followed by translocation of M5-DLO across the ER membrane to the luminal leaflet where the remaining glycosyltransfer reactions occur to complete the structure. Despite its critical importance for N-glycosylation, the scramblase protein that mediates the translocation of M5-DLO across the ER membrane has not been identified. Building on our ability to recapitulate scramblase activity in large unilamellar proteoliposomes reconstituted with a crude mixture of ER membrane proteins, we developed a mass spectrometry-based activity correlation profiling approach to identify scramblase candidates in the yeast Saccharomyces cerevisiae. Curation of the activity correlation profiling data prioritized six polytopic ER membrane proteins as scramblase candidates, but reconstitution-based assays and gene disruption in the protist Trypanosoma brucei revealed, unexpectedly, that none of these proteins were necessary for M5-DLO scramblase activity. Our results instead suggest the possibility that the M5-DLO scramblase may be a protein, or protein complex, whose activity is regulated at the level of quaternary structure. This key insight will aid future attempts to identify the scramblase.

biochemistry↗

bioRxiv2020

Fast retrograde access to projection neuron circuits underlying vocal learning in songbirds

Understanding the structure and function of neural circuits underlying speech and language is a vital step towards better treatments for diseases of these systems. Songbirds, among the few animal orders that share with humans the ability to learn vocalizations from a conspecific, have provided many insights into the neural mechanisms of vocal development. However, research into vocal learning circuits has been hindered by a lack of tools for rapid genetic targeting of specific neuron populations to meet the quick pace of developmental learning. Here, we present a new viral tool that enables fast and efficient retrograde access to projection neuron populations. In zebra finches, Bengalese finches, canaries, and mice, we demonstrate fast retrograde labeling of cortical or dopaminergic neurons. We further demonstrate the suitability of our construct for detailed morphological analysis, for in vivo imaging of calcium activity, and for multicolor brainbow labeling.

neuroscience↗