Soenmez, A. | bioRxiv Science

bioRxiv2017

FLIM-based pH measurements reveal incretin-induced rejuvenation of aged insulin secretory granules.

Insulin is stored in dense-core secretory granules (SGs) and is released from beta cells in two distinct phases upon glucose stimulation. Newly synthesized insulin SGs are secreted preferentially, but the underlying mechanism of this phenomenon remains elusive. The relationship of SG age with their intraluminal pH is of particular interest: proinsulin conversion by prohormone convertases follows the acidification of immature SGs by the vacuolar proton-translocating ATPase (v-ATPase). v-ATPases may also participate in the formation of the fusion pore for SG exocytosis, with intraluminal alkalinization inhibiting membrane fusion. Previous studies examined the luminal pH of SGs on a population level only. Here we measured the pH-dependent lifetime changes of eCFP fused to the ICA512-RESP18 homology domain to assess for the first time the luminal pH of individual age-defined SGs in insulinoma INS-1 cells by fluorescence-lifetime imaging microscopy. We show that 2-4-hour-old young SGs have a pH of ~5.5, while 26-28-hour-old SGs have a pH of ~6.2. Remarkably, the GLP-1 receptor agonist Exendin-4 prompted the re-acidification of old SGs in a glutamate-dependent fashion, while it did not affect the pH of young SGs. This study demonstrates that insulin SGs change their pH over time - a change that is reversible by insulin secretagogues. Hence, it provides novel insight into the mechanisms accounting for aging and exocytosis of SGs and suggests that their rejuvenation may be exploited to enhance insulin secretion in diabetes.

cell biology↗

bioRxiv2017

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array \"waves\", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

genomics↗