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

Many microRNAs (miRNAs) are expressed with high spatiotemporal specificity during organismal development, with some being limited to rare cell types, often embedded in complex tissues. Yet, most miRNA profiling efforts remain at the tissue and organ levels. To overcome challenges in accessing the microRNomes from tissue-embedded cells, we had previously developed mime-seq (miRNome by methylation-dependent sequencing), a technique in which cell-specific miRNA methylation in C. elegans and Drosophila enabled chemo-selective sequencing without the need for cell sorting or biochemical purification. Here, we present mime-seq 2.0 for profiling miRNAs from specific mouse cell types. We engineered a chimeric RNA methyltransferase that is tethered to Argonaute protein and efficiently methylates miRNAs at their 3'-terminal 2'-OH in mouse and human cell lines. We also generated a transgenic mouse for conditional expression of this methyltransferase, which can be used to direct methylation of miRNAs in a cell type of choice. We validated the use of this mouse model by profiling miRNAs from B cells and bone marrow plasma cells.

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.

Targeting surface voids to counter membrane disorders in lipointoxication-related diseases.

Saturated fatty acids (SFA), which are abundant in the so-called western diet, have been shown to efficiently incorporate within membrane phospholipids and therefore impact on organelle integrity and function in many cell types. In the present study, we have developed a yeast-based two-step assay and a virtual screening strategy to identify new drugs able to counter SFA-mediated lipointoxication. The compounds identified here were effective in relieving lipointoxication in mammalian ß-cells, one of the main targets of SFA toxicity in humans. In vitro reconstitutions and molecular dynamics simulations on bilayers revealed that these molecules, albeit according to different mechanisms, can generate voids at the membrane surface. The resulting surface defects correlate with the recruitment of loose lipid packing or void-sensing proteins required for vesicular budding, a central cellular process that is precluded under SFA accumulation. Taken together, the results presented here point at modulation of surface voids as a central parameter to consider in order to counter the impacts of SFA on cell function.

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae.

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1∆ deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1∆ mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.

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 ubiquinones. 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 hem1Delta mutant accumulated a large amount of squalene, which was stored almost exclusively in cytoplasmic lipid particles/droplets. Interestingly, a strain bearing a hem1Delta deletion in a dga1Delta lro1Delta are1Delta are2Delta quadruple mutant background (QMhem1Delta), which is devoid of the classical storage lipids, triacylglycerols and steryl esters, and lacks lipid particles, accumulated squalene at similar amounts as the hem1Delta mutant in a wild type background. In QMhem1Delta, however, increased amounts of squalene were found in cellular membranes, especially in microsomes. The fact that QMhem1Delta 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.