Laser stimulation of the chloroplast/endoplasmic reticulum nexus in tobacco transiently produces protein aggregates (boluses) within the endoplasmic reticulum and stimulates local ER remodeling.
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Does the ER subdomain that associates with the chloroplast in vivo, hereafter referred to as the chloroplast/ER nexus, play a role in protein flow within the ER? In studies of tobacco cells either constitutively or transiently expressing ER-retained luminal, GFP-HDEL, or trans-membrane, YFP-RHD3, fluorescent fusion proteins, brief 405-nm (3-6-mW) laser stimulation of the nexus causes a qualitative difference in the movement and behavior of proteins in the ER. Photostimulating the nexus produces fluorescent protein punctate aggregates (boluses) within the lumen and membrane of the ER. The aggregation propagates through the membrane network throughout the cell, but within minutes can revert to normal, with disaggregation propagating back toward the originally photostimulated nexus. In the meantime, the ER grows and anastomoses around the chloroplast, forming a dense cisternal and tubular network. If this network is again photostimulated, bolus formation does not recur and, if the photostimulation results in photobleaching, fluorescence recovery after photobleaching occurs as it would typically in areas away from the nexus. Bolus propagation is not mediated by the actin cytoskeleton, but can be reversed by pre-conditioning the cells for 30 min with high, 40-45C, temperature (heat stress). Because it is not reversed with heat stress, the reorganization of the ER at the nexus following photostimulation is a separate event.
