SpatTrack: An Imaging Toolbox for Analysis of Vesicle Motility and Distribution in Living Cells

Abstract

The endocytic pathway is a complex network of highly dynamic organelles, which has been traditionally studied by quantitative fluorescence microscopy. The data generated by this method can be overwhelming and its analysis, even for the skilled microscopist, is tedious and error-prone. We developed SpatTrack, an open source, platform-independent program collecting a variety of methods for analysis of vesicle dynamics and distribution in living cells. SpatTrack performs Endocytosis of proteins from the cell surface and subsequent sorting of internalized cargo in endosomes is a complex and highly dynamic process. Traditionally, endocytosis has been studied by subcellular fractionation and by transmission electron microscopy after suitable labeling of endocytic ligands (1,2). In parallel, fluorescence microscopy has evolved as a major tool to study the endocytic pathway. This is a consequence of the high specificity and sensitivity of fluorescence and the relative ease of labeling endocytic cargo with small fluorescent probes.Moreover, only fluorescence studies of the endocytic pathway bear the potential of measuring biophysical properties of the endosome, like pH or concentration of ions like calcium and chloride (3,4). Ligands of endocytic receptors have several fates once arrived in the endosome; they can recycle to the cell surface from early sorting or recycling endosomes, like transferrin after releasing iron into the endosomal lumen (5). They can become targeted to the trans-Golgi network in a retrograde trafficking route, as furin or several plant toxins (5,6). Ligands can be also 1406 www.traffic.dk

Live‐cell imaging of new polyene sterols for improved analysis of intracellular cholesterol transport

“…C4P and E4Pac were imaged either in the same channel or in the blue (DAPI) channel using the Leica filter A-cube of excitation: 360 nm (20-nm bandpass); dichroic: 400-nm; and emission: 425-nm longpass. The same filter cube is often used for imaging the sterol-binding polyene antibiotic filipin (Lund et al, 2014). TopFluor-cholesterol was imaged on the same system using a standard fluorescein filter set [470-nm, (20-nm bandpass) excitation filter, 510-nm longpass dichromatic filter and 537-nm (23-nm bandpass) emission filter].…”

Analysis of cholesterol export from endo-lysosomes by Niemann Pick C2 protein using combined fluorescence and X-ray microscopy

“…2, 3 and 9). We observed previously an increased contribution of active transport to the mobility of NPC2 containing vesicles over the course of cholesterol efflux 21 . This active transport will shift the steady state distribution of LE/LYSs towards the PM thereby accelerating further sterol exchange and cellular release ( Fig.…”

“…Supporting that notion, we measured a selective release of DHE over fluorescent NPC2 on a per-vesicle basis suggesting that NPC2 is largely retained in LE/LYSs during sterol efflux. Previously, we reported that LE/LYSs containing Alexa546-NPC2 became increasingly mobile and moved preferentially by active transport in the course of lysosomal cholesterol mobilization 21 . Increased motility, repositioning and tubulation of LE/LYSs have been recently reported as being intimately linked to restoration of LE/LYS function in lysosomal storage diseases 57,58 .…”

“…This active transport will shift the steady state distribution of LE/LYSs towards the PM thereby accelerating further sterol exchange and cellular release ( Fig. 9; Step 4) 21 . The initially hindered but accelerating sterol efflux kinetics ( Fig.…”

“…Using spatial statistical analysis of the determined endosome positions 21 , we observed that LE/LYSs enriched in DHE were initially clustered but reallocated to the cell periphery during NPC2 mediated sterol efflux ( Fig.6A-D). This result is in accordance with earlier studies reporting a direct effect of the endo-lysosomal cholesterol content on the spatial location of these organelles 16,17 .…”