Measurement of Lysosome Positioning by Shell Analysis and Line Scan

Abstract

Lysosomes are membrane-bound organelles that degrade diverse biomolecules and regulate a multitude of other essential processes including cell growth and metabolism, signaling, plasma membrane repair and infection. Such diverse functions of lysosomes are highly coordinated in space and time and are therefore tightly coupled to the directional transport of the organelles within the cytoplasm. Thus, robust quantitative assessments of lysosome positioning within the cell provide a valuable tool for researchers interested in understanding these multifunctional organelles. Here, we present point-by-point methodology to measure lysosome positioning by two straight forward and widely used techniques: shell analysis and line scan.

Keywords: ImageJ analysis; Line scan; Lysosome distribution; Lysosome positioning measurements; Lysosome transport; Organelle positioning; Shell analysis.

Fig. 1

Determining the shell gap width for shell analysis. (a) Maximum intensity projections of z-stack confocal fluorescence micrographs of HeLa cells transfected with plasmid vectors expressing GFP, GFP-RILP (a lysosomal dynein adaptor that causes lysosomal clustering in the juxtanuclear area) or GFP-KIF1A (a kinesin that moves lysosomes towards the cell periphery). The cells were fixed and immunostained for the lysosomal marker LAMP1 and nuclei were labeled with DAPI. (b) A series of manually drawn lines extending from the edge of the nuclei to cell edges were applied to the images in a. The average line length was determined to be 7.8 μm and used to compute the gap width of 1.6 μm for downstream shell analysis (bottom). Red “X” in a and b indicates a narrow cell that cannot be accurately measured by shell analysis and is excluded. Scale bars = 10 μm

Fig. 2

Shell analysis workflow. (a) Maximum intensity projections of z-stack confocal fluorescence micrographs of HeLa cells from Fig. 1a, with manually traced cell edges (ROIs) in white dashed lines. (b) Cells and their ROIs from a showing only the lysosomal channel, to which a threshold has been applied to eliminate background. Asterisk denotes a cell that is used as an example in c. Red “X” in a and b indicates a narrow cell that cannot be accurately measured by shell analysis and is excluded. (c) Example cell showing iterative concentric shrinking of an ROI and measurement of lysosomal area within each ROI. To shrink each ROI by the gap width of 1.6 μm determined in Fig. 1, the ImageJ command “Enlarge” is used to enter “−1.6 μm”. Lysosomal area is measured, and the steps are repeated 5 times to generate measurements of the area covered by lysosomal signal within 5 shells (right). (d) Example quantification of the area of lysosomal signal within each ROI such as in c, to determine the lysosomal area per shell and the fraction of total lysosomal area per shell. For the purposes of shell analysis, Area units can be arbitrary as long as all images are acquired with the same scaling. Note that due to the embedded metadata in our images, the Area unit here is square microns. In the absence of metadata, ImageJ reports square pixels for Area. (e) Data as in d collected across 14 cells per condition for each of the three conditions indicated, are plotted in 2 different ways. The graph on the left shows data for each shell as means ± SEM. The box and whiskers plot on the right shows a line at the median and the minimum and maximum values. One-way ANOVA with Dunnett’s multiple comparisons test was applied to determine significance, ***p < 0.001. Scale bars = 10 μm

Fig. 3

Line scan analysis. (a) Maximum intensity projections of z-stack confocal fluorescence micrographs of HeLa cells from Fig. 1a, with manually traced cell edges (ROIs) in white dashed lines. Line segments (yellow lines), 30 pixels wide (yellow dashed lines), were drawn starting at the nucleus and ending at the cell periphery. Scale bars = 10 μm. (b) Positional information corresponding to the maximum intensity of the lysosome (LAMP1) channel was recorded as a fractional distance along the line segment (distance reading at the intensity maximum divided by total line segment distance) for 14 cells per condition. The mean fractional distance ± SD of lysosomes is plotted for each condition. Statistical significance was calculated using One-way ANOVA with Tukey’s multiple comparisons test (n = 14, n.s. means not significant, ***p < 0.001). Blue box highlights average position along the line scan occupied by the nucleus. (c) Average lysosome position plotted as line graphs. Relative intensity (lysosomal intensity at each pixel divided by the sum of all intensity values for the line segment) along 24 ± 0.2 μm line segments from six cells per condition were recorded. The mean relative intensity (lines) ± SEM (vertical bars) of lysosome signal was plotted against the line distance (in μm). Blue box highlights average position along the line scan occupied by the nucleus

Fig. 4

Methods for automating image analysis in ImageJ/Fiji according to analysis complexity and user software proficiency