Isolation of synaptic vesicles from genetically engineered cultured neurons

Background Synaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures. New method Here, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. Results We show that ∼108 plated cortical neurons allow isolation of suitable SV amounts for functional analysis and imaging. We found that SVs isolated from cultured neurons have neurotransmitter uptake comparable to that of SVs isolated from intact cortex. Using total internal reflection fluorescence (TIRF) microscopy, we visualized an exogenous SV-targeted marker protein and demonstrated the high efficiency of SV modification. Comparison with existing methods Obtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations. Conclusions These results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts

Mime-seq 2.0: a method to sequence microRNAs from specific mouse cell types

<p>These files contain the raw sequencing data (demultiplexed, unaligned BAM files or FASTQ files) collected as part of following manuscript: "Mime-seq 2.0: a method to sequence microRNAs from specific mouse cell types.". Included are oxidized and unoxidized small RNA sequencing libraries collected from RKO cells (human cell line, timecourse for methyltransferase expression), M. musculus B cells (splenic, CD43 depleted; methyltransferase under Cd79a-Cre driver), M. Musculus Plasma cells (sorted, methyltransferase under Bhlha15-Cre driver). </p><p> </p><p>B cell experiment (<strong>Cd79aCre</strong>) samples:</p><ul><li>unox_HenT6B/HenT6B</li><li>ox_HenT6B/HenT6B</li><li>unox_HenT6B/HenT6B Cd79a-Cre</li><li>ox_HenT6B/HenT6B Cd79a-Cre</li><li>unox_HenT6B/+ Cd79a-Cre</li><li>ox_HenT6B/+ Cd79a-Cre</li></ul><p>Plasma cell titration experiment (<strong>Bhlha15Cre</strong>) samples:</p><ul><li>unox_100</li><li>ox_100</li><li>unox_1</li><li>ox_1</li><li>unox_01</li><li>ox_01</li><li>unox_001</li><li>ox_001</li></ul><p>RKO timecourse (<strong>RKO</strong>) samples:</p><ul><li>unox_0h</li><li>ox_0h</li><li>unox_12h</li><li>ox_12h</li><li>unox_48h</li><li>ox_48h</li><li>unox1_wt</li><li>ox1_wt</li><li>unox2_wt</li><li>ox2_wt</li></ul&gt

Effect of Lipid Particle Biogenesis on the Subcellular Distribution of Squalene in the Yeast Saccharomyces cerevisiae*

Squalene belongs to the group of isoprenoids and is a precursor for the synthesis of sterols, steroids, and ubiquinons. In the yeast Saccharomyces cerevisiae, the amount of squalene can be increased by variation of growth conditions or by genetic manipulation. In this report, we show that a hem1Δ mutant accumulated a large amount of squalene, which was stored almost exclusively in cytoplasmic lipid particles/droplets. Interestingly, a strain bearing a hem1Δ deletion in a dga1Δlro1Δare1Δare2Δ quadruple mutant background (QMhem1Δ), which is devoid of the classical storage lipids, triacylglycerols and steryl esters, and lacks lipid particles, accumulated squalene at similar amounts as the hem1Δ mutant in a wild type background. In QMhem1Δ, however, increased amounts of squalene were found in cellular membranes, especially in microsomes. The fact that QMhem1Δ did not form lipid particles indicated that accumulation of squalene solely was not sufficient to initiate proliferation of lipid particles. Most importantly, these results also demonstrated that (i) squalene was not lipotoxic under the conditions tested, and (ii) organelle membranes in yeast can accommodate relatively large quantities of this non-polar lipid without compromising cellular functions. In summary, localization of squalene as described here can be regarded as an unconventional example of non-polar lipid storage in cellular membranes