Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy

The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution

Single particle trajectories reveal active endoplasmic reticulum luminal flow

The endoplasmic reticulum (ER), a network of membranous sheets and pipes, supports functions encompassing biogenesis of secretory proteins and delivery of functional solutes throughout the cell[1, 2]. Molecular mobility through the ER network enables these functionalities, but diffusion alone is not sufficient to explain luminal transport across supramicrometre distances. Understanding the ER structure–function relationship is critical in light of mutations in ER morphology-regulating proteins that give rise to neurodegenerative disorders[3, 4]. Here, super-resolution microscopy and analysis of single particle trajectories of ER luminal proteins revealed that the topological organization of the ER correlates with distinct trafficking modes of its luminal content: with a dominant diffusive component in tubular junctions and a fast flow component in tubules. Particle trajectory orientations resolved over time revealed an alternating current of the ER contents, while fast ER super-resolution identified energy-dependent tubule contraction events at specific points as a plausible mechanism for generating active ER luminal flow. The discovery of active flow in the ER has implications for timely ER content distribution throughout the cell, particularly important for cells with extensive ER-containing projections such as neurons.Wellcome Trust - 3-3249/Z/16/Z and 089703/Z/09/Z [Kaminski] UK Demential Research Institute [Avezov] Wellcome Trust - 200848/Z/16/Z, WT: UNS18966 [Ron] FRM Team Research Grant [Holcman] Engineering and Physical Sciences Research Council (EPSRC) - EP/L015889/1 and EP/H018301/1 [Kaminski] Medical Research Council (MRC) - MR/K015850/1 and MR/K02292X/1 [Kaminski

Accumulative Difference Image Protocol for Particle Tracking in Fluorescence Microscopy Tested in Mouse Lymphonodes

The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done