Scope: Lysosomal storage disorders, like Niemann Pick type C disease, are characterized by abnormal cholesterol accumulation in lysosomes (LYS). Mutations in either NPC1, a large membrane spanning protein in late endosomes, or of NPC2, a small soluble protein in the lumen of LYS, cause cholesterol and sphingolipid accumulation in the endocytic and lysosomal pathway of skin, spleen, liver and neurons of affected patients [1-2]. The strong accumulation of these lipids causes aberration and extension of LYS into so-called lysosomal storage organelles (LSOs). Both, NPC1 and NPC2 have been implicated in cholesterol egress from the LYS/LSOs, but the intracellular transport routes of cholesterol to and from these organelles are not known. We aim for determining the intracellular trafficking routes of cholesterol in NPC disease fibroblasts compared to cells from healthy donors. To this end, we track the intrinsically fluorescent cholesterol analog dehydroergosterol (DHE) by ultraviolet optimized wide field (UV-WF) microscopy [3-4].
Materials and Methods: DHE allows for live-cell imaging of sterol redistribution in pulse-chase experiments, which is not possible with stains like filipin. Endocytic pathways in normal and NPC2-fibroblasts were specifically labelled with the red fluorescent probes rhodamine-dextran to visualize LYS/LSOs and the infrared dye, Alexa647-Tf, to identify recycling endosomes (REs). Cells pre-stained with these organelle markers were pulse-labeled with DHE exclusively in the plasma membrane (PM) and chased for various times (i.e., kept at 37 degree in a suitable incubator to allow for sterol trafficking from the PM), followed by multi-color UV-WF imaging using a 63x objective and EMCCD camera. For reliable and automated data analysis, we developed an efficient image analysis software solution as plugin to ImageJ.
Algorithm and Analysis: Our ImageJ-based plugin quantifies sterol content selectively and separately for the PM, LYS/LSOs and REs using the two organelle markers as reference. First, cells are separated from the background in the DHE channel using a simple intensity thresholding. Subsequently, our plugin generates a binary mask from the images of the respective organelle marker after dynamic intensity thresholding (DIT). We found that cell-to-cell variation of marker uptake can compromise simple intensity thresholding, while in DIT, the organelle-associated intensity of Rh-Dextran or Alexa647-Tf becomes weighted by the respective mean value of total cellular fluorescence of the organelle markers. The binary masks are separately applied to the DHE image and individual organelle spots are identified and labeled in our plugin using flood-filling. Sterol intensity per organelle can be correlated with endosome size etc. [5-7]. The plugin quantifies the fluorescent sterol inside PM, LYS/LSOs or REs compared to the fluorescent sterol in total cell. The program also takes into account apparent sterol intensity in the organelles originating from the overlaying PM as well as eventual overlap area between the LYS/LSOs and the REs.
Niemann Pick disease type C, DHE, NPC2, PM, LYS/LSOs, REs, DIT, Healthy fibroblasts, Niemann Pick C2 disease fibroblasts
Ahmed Shah Mehadi received his M.Sc. in Electrical Engineering from Blekinge Institute of Technology, Sweden and did his project work in Karolinska Institutet, Sweden. He worked as a professional software developer in Blueguards AB, Sweden ( http://www.blueguards.com). Currently, he is working on his PhD on image processing applied to membrane trafficking studies at the Department of Biochemistry and Molecular Biology, University of Southern Denmark (http://www.sdu.dk/staff/asme) under the supervision of Daniel Wüstner. Research interest includes bioimaging, computer vision, computation biology, bioinformatics.
Presenting author: Ahmed Shah Mehadi
Organisation: University of Southern Denmark, Department of Biochemistry and Molecular Biology, 5230 Odense M, Odense, Denmark
co-authors: Frederik Wendelboe Lund, Daniel Wüstner (Supervisor)