Single Cell Organelles Research Articles

Porin 1 Modulates Autophagy in Yeast.

Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which grow in size and further expand to form autophagosomes, engulfing cellular material to be degraded by fusion with lysosomes (vacuole in yeast). Autophagosomal membranes do not bud from a single cell organelle, but are generated de novo. Several lipid sources for autophagosomal membranes have been identified, but the whole process of their generation is complex and not entirely understood. In this study, we investigated how the mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), affects autophagy in yeast. We show that POR1 deficiency reduces the autophagic capacity and leads to changes in vacuole and lipid homeostasis. We further investigated whether limited phosphatidylethanolamine (PE) availability in por1∆ was causative for reduced autophagy by overexpression of the PE-generating phosphatidylserine decarboxylase 1 (Psd1). Altogether, our results show that POR1 deficiency is associated with reduced autophagy, which can be circumvented by additional PSD1 overexpression. This suggests a role for Por1 in Psd1-mediated autophagy regulation.

Real-time monitoring of NADPH levels in living mammalian cells using fluorescence-enhancing protein bound to NADPHs

Nicotinamide adenine nucleotide phosphate (NADPH) has been known to be involved in the multiple pathways of cell metabolism. However, conventional quantification assays for NADPH have required breaking down the cell membranes of around one million cells per assay, and monitoring NADPH flux in living cells has been limited by a few available tools. Here, we visualized NADPH levels in human cervical cancer cells HeLa using metagenome-derived blue fluorescent protein (mBFP), which specifically binds to NADPH and enhances the intrinsic fluorescence of NADPH up to 10-fold when imaged by two-photon microscopy to reduce photodamage. Adding an oxidizing agent such as diamide to HeLa cells that expressed mBFP led to an immediate decrease of intracellular NADPH depending on glucose availability in culture media. Furthermore, inhibiting glucose-6-phosphate dehydrogenase (G6PD) in the pentose phosphate pathway with dehydroandrosterone (DHEA) and knockdown of G6PD transcripts gradually decreased NADPH when diamide was added to living cells. These results demonstrate that introducing a bacterial mBFP gene into mammalian cells is a straightforward approach to monitoring intracellular NADPH flux in real time at the single-cell level. Moreover, this strategy can be expanded to tracking the spatio-temporal changes in NADPH even in single-cell organelles such as mitochondria and chloroplasts, which will allow us to more precisely assess the efficacy of biochemically or biophysically metabolic perturbations in animal and plant cells.

Increase in function during growth of a single cell organelle.

The gradient diver method has been adapted to measure photosynthesis during the early development of a single zygote of the multicellular green alga Ulva mutabilis Føyn. The cells have only one chloroplast, and the results indicate that during growth of the chloroplast the rate of O(2)-production increases linearly in the early part of the cell cycle but remains constant or decreases at the end of the cycle.