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First published online 25 October 2005
doi: 10.1242/jcs.02633

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Distribution and dynamics of Lamp1-containing endocytic organelles in fibroblasts deficient in BLOC-3

Department of Human Genetics, University of California, Los Angeles, CA 90095, USA

View larger version (46K): [in a new window]   Fig. 1. Quantitative analysis of the perinuclear clustering of endogenous Lamp1 from wild-type and BLOC-deficient fibroblasts. (A-C) Low-magnification fluorescence images of cells stained with anti-Lamp1 were acquired using a fluorescence microscope equipped with a digital camera. Images were processed to calculate areas (in pixels) and mean fluorescence intensities (in arbitrary units) of (A) `whole cell', (B) `nucleus' and (C) `nucleus + perinuclear region', which were drawn (yellow lines) by an operator who was unaware of the identity of the sample. Bar, 20 µm. (D) Plot of the ratio between background-corrected, mean fluorescence values of the regions defined as perinuclear (i.e. `nucleus + perinuclear region' minus `nucleus') and cytoplasmic (i.e. `whole cell' minus `nucleus') as a function of the ratio between the areas of `nucleus + perinuclear region' and `whole cell'. Filled circles correspond to values derived from the cell shown in A-C. Open circles correspond to the analysis of another cell in which Lamp1 staining was relatively less concentrated in the perinuclear area. For each cell, the perinuclear clustering index (PCI) was calculated as the slope obtained by linear regression of the data within the range of relative area sizes 0.15-0.25 (gray in D), multiplied by –1 to render positive values. (E) PCI of immortalized skin fibroblast lines derived from mutant murine strains deficient in BLOC-1 (pallid), BLOC-2 (cocoa) and BLOC-3 (pale ear), as well as from a wild-type control strain. For each cell line, the PCI of 20 randomly selected cells was averaged. Bars represent mean±standard error of three independent cell lines per strain. *P<0.05 (ANOVA followed by Tukey's test).

View larger version (87K): [in a new window]   Fig. 2. Localization of LAMP1-GFP fusion protein expressed in transfected murine fibroblasts. Immortalized skin fibroblasts derived from wild-type (A-I) or pale ear (J-O) mice were transfected with a plasmid encoding human LAMP1 fused to GFP (A,D,G,J,M and green in C,F,I,L,O). One day after transfection, the cells were fixed/permeabilized and stained with a species-specific antibody to murine Lamp1 (B,K and red in C,L) or an antibody to EEA1 (H and red in I). Alternatively, cells were allowed to internalize Texas-Red-conjugated dextran, followed by a 4-hour chase period to ensure specific labeling of mature lysosomes (E,N and red in F,O). Bar, 20 µm. The top-left region of panels A-C, which displays staining of endogenous Lamp1 but not of LAMP1-GFP, corresponds to part of an untransfected cell. Magnified views of selected areas are shown in the insets.

View larger version (57K): [in a new window]   Fig. 3. High levels of LAMP1-GFP overexpression elicit aggregation of late endocytic compartments. Immortalized skin fibroblasts from wild-type (A-H) and pale ear (I-L) mice were transfected with plasmids encoding LAMP1 fused to either GFP or its non-dimerizing mutant form (mGFP). One day after transfection, cells were fixed/permeabilized and stained with the 1D4B antibody to mouse Lamp1 (B,D,H,J). In some experiments, cells were first allowed to internalize Texas-Red-conjugated dextran (F,L), transfected with the LAMP1-GFP-encoding plasmid, cultured for one day in medium without dextran, and then fixed and processed for fluorescence microscopy. Arrows indicate cells expressing high levels of LAMP1-GFP or LAMP-mGFP, as judged by their GFP fluorescence signal (A,C,E,G,I,K). Asterisks in A and B denote a transfected cell expressing LAMP1-GFP levels comparable with those of the cells shown in Fig. 2. Bar, 20 µm. The inset in F shows a high-resolution view of one of the dextran clusters as obtained by confocal microscopy, suggesting that the dextran-positive compartments have aggregated but not fused with each other.

View larger version (66K): [in a new window]   Fig. 4. Initial image (A) and examples of trajectories (B) observed in a representative time-lapse fluorescence microscopy experiment using wild-type murine fibroblasts expressing LAMP1-GFP. Arrowheads indicate the approximate initial positions of selected LAMP1-GFP-positive compartments that traveled significant effective distances during the course of the experiment. Bars, 20 µm.

View larger version (43K): [in a new window]   Fig. 5. Quantitative analyses of maximum speed (A,D), effective distance traveled during a 98-second period (B,E), and average speed of movements inferred to occur on microtubules based on instant speeds of at least 0.78 µm/second (C,F), obtained by time-lapse fluorescence microscopy analyses of LAMP1-GFP-positive organelles in fibroblasts derived from wild-type (open bars in A-C, open circles in D-F) and pale ear (black bars in A-C, filled circles in D-F) murine strains. Three independent fibroblast lines were analyzed per strain. For each fibroblast line, 10 cells expressing low LAMP1-GFP levels were examined by time-lapse fluorescence microscopy, and the movement of 30 randomly selected, LAMP1-GFP-positive organelles per cell was tracked and analyzed quantitatively. (A-C) Aggregate distributions of values obtained for 900 LAMP1-GFP-positive organelles (30 organelles x 10 cells x 3 fibroblast lines) per strain. Numbers on the x-axis denote bin limits; the last bin on the right includes all values greater than the upper limit of the previous bin, and the first bin on the left of C includes all values smaller than 0.8 µm/second. For comparison, the distributions are also included of maximum speed (A) and effective distance (B) of 120 LAMP1-GFP positive organelles (30 organelles x 4 cells) from wild-type fibroblasts that had been treated with nocodazole to disrupt microtubules (gray bars). (D-F) Median values per cell were plotted as individual data points, where the position on the y-axis represents the median value per cell and the position on the x-axis is arbitrarily chosen to group the data of the 10 cells analyzed for each fibroblast line. Median values obtained for cells expressing the LAMP1-mGFP variant construct and corresponding to two independent fibroblast lines derived from wild-type (open diamonds) and pale ear (filled diamonds) strains are also represented. Horizontal lines represent median values per data group. Statistical analysis of the data represented in D-F was performed using an ANOVA model upon logarithmic transformation, as described under Materials and Methods. The indicated P values correspond to the significance of `genotype' (i.e. pale ear versus wild-type) as an explanatory variable. Exclusion of the data group highlighted by arrows in D and E from the ANOVA analysis did not modify the conclusion that the differences between wild-type and pale ear cells were statistically significant.

View larger version (28K): [in a new window]   Fig. 6. Distributions of percentages of time in which 900 LAMP1-GFP-positive organelles within wild-type (open bars) or pale ear (closed bars) fibroblasts moved at instant speeds of 0.78 µm/second or faster, in any direction (A), towards the nucleus (B), or away from the nucleus (C). Insets, comparable distributions obtained for 600 organelles labeled with the LAMP1-mGFP variant construct expressed in wild-type (open bars) or pale ear (closed bars) fibroblasts. Numbers on the x-axis denote bin limits; the last bin on the right of each panel includes all values greater than 24%.

View larger version (23K): [in a new window]   Fig. 7. Distributions of the number of events during a 98-second period in which the instant speed of each of 900 LAMP1-GFP-positive organelles within wild-type (open bars) or pale ear (closed bars) fibroblasts increased from less than 0.78 µm/second to reach or exceed this threshold speed. Insets, comparable distributions obtained for 600 organelles labeled with the LAMP1-mGFP variant construct within wild-type or pale ear cells. Numbers on the x-axis denote bin limits; the last bin on the right includes all values greater than 14.