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Neuronal calcium signals propagating by simple diffusion and reaction with mobile and stationary buffers are limited to cellular microdomains. The distance intracellular calcium signals can travel may be significantly increased by means of calcium-induced calcium release from internal calcium stores, notably the endoplasmic reticulum. The organelle, which can be thought of as a cell-within-a-cell, is able to sequester large amounts of cytosolic calcium ions via SERCA pumps and selectively release them into the cytosol through ryanodine receptor channels leading to the formation of calcium waves. In this study, we set out to investigate the basic properties of such dendritic calcium waves and how they depend on the three parameters dendrite radius, ER radius and ryanodine receptor density in the endoplasmic membrane. We demonstrate that there are stable and abortive regimes for calcium waves, depending on the above morphological and physiological parameters. In stable regimes, calcium waves can travel across long dendritic distances, similar to electrical action potentials. We further observe that abortive regimes exist, which could be relevant for spike-timing dependent plasticity, as travel distances and wave velocities vary with changing intracellular architecture. For some of these regimes, analytic functions could be derived that fit the simulation data. In parameter spaces, that are non-trivially influenced by the three-dimensional calcium concentration profile, we were not able to derive such a functional description, demonstrating the mathematical requirement to model and simulate biochemical signaling in three-dimensional space.
Many cellular processes are regulated via pH, and maintaining the pH of different organelles is crucial for cell survival. A pH-sensitive GFP variant, the so-called pHluorin, has proven to be a valuable tool to study the pH of the cytosol, mitochondria and other organelles in vivo. We found that the fluorescence intensity of Endoplasmic Reticulum (ER)-targeted pHluorin in the yeast Saccharomyces cerevisiae was very low and barely showed pH sensitivity, probably due to misfolding in the oxidative environment of the ER. We therefore developed a superfolder variant of pHluorin which enabled us to monitor pH changes in the ER and the cytosol of S. cerevisiae in vivo. The superfolder pHluorin variant is likely to be functional in cells of different organisms as well as in additional compartments that originate from the secretory pathway like the Golgi apparatus and pre-vacuolar compartments, and therefore has a broad range of possible future applications.
The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.