MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures
Fig 6
Identification of lysosomes and multivesicular bodies using the OPTICS algorithm.
(A) Top: APP clusters were identified as “valleys” between RD “peaks” exceeding a static RD value (dotted line), and Bottom: LAMP1 clusters identified using a hierarchal segmentation approach. Select clusters are highlighted by colored region of the RD plots (left) and are circled by equivalent colored lines in the GSDM images (right). (B) A full hierarchal segmentation of the red LAMP1 cluster from panel A, demonstrating the nested hierarchal structure typical of multivesicular bodies. Insert plots the position of individual LAMP1 molecules within the cluster; colored circles correspond to putative intraluminal vesicles identified by the equivalent colored regions in the segmented RD plot, red arrow indicates where the RD plot joins the larger RD plot from panel A. (C) Comparison of APP cluster size and the association of APP clusters with LAMP1. (D) Quantification of APP density in LAMP1 clusters identified by OPTICS and separated based on whether the APP was associated with MVB-like, small lysosomal, or LAMP1-negative vesicles. (E) Quantification of APP density in the total (Bounding MVB) and intraluminal vesicle (ILV) portions of the MVB-like LAMP1 clusters. All images are representative of three experiments, a minimum of 4 images per experiment. Data are presented as mean ± SEM, scale bars are 500 nm (A) and 150 nm (B). * = p < 0.05 compared to MVB-like, n.s. = not significantly different.