β2-m amyloid fibrils are endocytosed into endosomes/lysosomes, leading to the disruption of their membranes.

<p>Representative electron micrographs of HIG-82 cells taken as described in Materials and Methods. HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer for 6 hrs (A), or 100 μg/ml β2-m fibrils for 2 hrs (B, C) or 6 hrs (D-F) as described in Materials and Methods. The inset in (B) is a higher magnification of the box. (B, D) HIG-82 cells were covered with amyloid fibrils. Note that a part of the plasma membrane invaginated and fused to form an endocytic vesicle containing amyloid fibrils (inset in B). (C, E) Many endosomes/lysosomes were filled with amyloid fibrils, and some endosomal/lysosomal membranes were disrupted by intravesicular fibrils. (F) Nuclear deformation, shrinkage, and chromatin condensation at the nuclear rim were also observed. The scale bars are 5 μm long in A, B, D and F and 1 μm long in C and E.</p

β2-m amyloid fibrils induce apoptosis of HIG-82 cells as measured by the TUNEL assay.

<p>After HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, TUNEL assay was performed as described in Materials and Methods. (A) The representative fluorescence images of TUNEL and DAPI double staining. The original magnification was x100. (B) The percentage of apoptotic cells to total cells. Data were presented as a dot plot of the ratios of five independent experiments with the mean value. Statistical analysis was performed by Mann-Whitney U-test. *P < 0.05.</p

β2-m amyloid fibrils are internalized and sorted to lysosomes.

<p>HIG-82 cells incubated with Ham’s F12 medium containing vehicle buffer, 10 μg/ml β2-m monomer, or 10 μg/ml β2-m fibrils for 12 hrs were stained for lysosomes (red), β2-m (green), and nuclei (blue), and observed with the confocal laser microscope as described in Materials and Methods. When the cells were incubated with fibrils (right column), green fluorescence indicating β2-m fibrils were observed inside the cells in a granular pattern, as well as on the surface of the cells. Importantly, some green-colored granules containing β2-m fibrils were merged with red-colored lysosomes. The scale bars are 10 μm long.</p

Endocytosed β2-m amyloid fibrils leak from endosomes/lysosomes into the cytosol.

<p>Representative electron micrographs of HIG-82 cells incubated with Ham’s F12 medium containing 100 μg/ml β2-m fibrils for 6 hrs as described in Materials and Methods. Images were taken as described in Materials and Methods. (D-F) Higher magnifications of the boxes in A-C, respectively. Note that the endocytosed amyloid fibrils leaked from endosomal/lysosomal vesicles into the cytosol (A, D), and some fibrils were found adjacent to mitochondria (B, C, E, F). The scale bars are 500 nm long in A-C and 200 nm long in D-F.</p

β2-m amyloid fibrils have no effect on artificial plasma membranes.

<p>(A) A representative light micrograph of large unilamellar vesicles (LUVs) containing carboxyfluorescein prepared as described in Materials and Methods. They were less than 50 μm in diameter. The scale bars are 50 μm long. After LUVs were incubated with β2-m fibrils or r-β2-m monomer (final 0 or 100 μg/ml), or Triton X-100 as a positive control (final 2%) for 15 min (B) or 1 day (C), the fluorescence was measured as described in Materials and Methods. (B, C) β2-m amyloid fibrils did not significantly destruct artificial plasma membranes of LUVs. Statistical analysis was performed by Student’s unpaired t-test. *P < 0.05 vs. positive control.</p