Parotid gland cancer.

<p>(<b>A</b>) CCEM shows proteasome immunofluorescence (green) distributed inside cytoplasmic PaCSs while sparing the nucleus (<b>A</b>, 3,000x). The boxed area is enlarged in <b>B</b> (50,000x) to magnify parallel 20S immunogold reactivity despite loss of density of barrel-like particles. Note in <b>A</b> mitochondria and clear unreactive areas merging with PaCS areas. In <b>C</b> (3,000x) a ballooning tumour cell with remnant of a central picnotic nucleus (arrow) is almost entirely formed by a degenerating PaCS only partly reactive to proteasome fluorescence. The boxed area, taken in <b>C</b> at the transition border between its highly and poorly fluorescent parts, shows in <b>D</b> (50,000x) the disappearance of particles and 20S proteasome immunogold below the border, in parallel with immunofluorescence. Another boxed area taken from the top of the heavily fluorescent part in <i>C</i>, shows well-preserved particles and proteasome immunogold in <b>E</b> (50,000x). (<b>F</b> and <b>G</b>) TEM (5,000x) showing two neoplastic cells, one with well-preserved normal nucleus (left), the other (right) with intranuclear accumulation of PaCS-like particles and proteasome immunogold, part of which is boxed in <b>F</b> and enlarged in <b>G</b> (50,000x). Note in <b>F</b> abundant mitochondria. n, nucleolus. (<b>H</b>) Bulging neoplastic cell with a small nucleus surrounded by a rim of dense cytoplasm and immersed in a lake of PaCS-type partly proteasome-reactive, particulate material (4,000x); the boxed area is enlarged in <b>I</b> (20,000x) with islets of residual cytoplasm, one of which is further enlarged in <b>L</b> (75,000x) to show a qualitative autophagic vesicle with double membrane enclosing a collection of well-preserved immunoproteasome-reactive PaCS-type particles. Note in <b>H</b> and <b>I</b> (arrowheads) three other double membrane delimited bodies enclosing PaCS-type particles.</p

Ovarian serous papillary, cervical endometrioid, and thyroid papillary carcinomas.

<p>Juxtanuclear PaCSs in ovarian serous papillary (<b>A</b>, 10,000x), cervical endometrioid (<b>D</b>, 10,000x), and thyroid papillary carcinoma (<b>G</b>, 10,000x), enlarged in <b>B</b>, <b>E</b>, and <b>H</b> (all 25,000x) and even further in <b>C</b>, <b>F</b>, and <b>I</b> (all, 50,000x), respectively, to show their particulate ultrastructure (partially lost in <b>I</b> and, focally, in <b>F</b>) and 20S proteasome immunogold reactivity.</p

<i>H. pylori</i>-colonized non-neoplastic human gastric epithelium (A–C) and well differentiated, gland-forming, deeply invasive, gastric cancer (D–M).

<p>(<b>A</b>) Foveolar epithelium showing 20S proteasome immunofluorescence (green) of infranuclear PaCSs. Overlapping of confocal fluorescence microscopy and toluidine blue images of a semithin resin section from an aldehyde-osmium-fixed block (500x). (<b>B</b>) Juxtanuclear PaCS (asterisk) in a foveolar cell (5,000x) from an adjacent section to <b>A</b>. (<b>C</b>) High resolution (X 65,000) TEM of a boxed PaCS area in <b><i>B</i></b> to show its particulate ultrastructure and 20S proteasome immunogold reactivity. (<b>D</b>) Toluidine blue stained semithin resin section (500x); note pink stained PaCSs (arrowheads), many of which subluminal. (<b>E</b>) Correlative confocal and electron microscopy (CCEM), of a consecutive thin section of the boxed area in <i>D,</i> shows 20S proteasome immunofluorescence (green spots) on a TEM background (5,000x). The juxtaluminal PaCS (arrowhead) is enlarged in <b>F</b> (20,000x) whose boxed area is further enlarged in <b>G</b> (50,000x) to illustrate its particulate ultrastructure with 20S proteasome immunogold reactivity. The same proteasome antibody was used on each face of the gilder finder grid used for CCEM. Note several basally located PaCSs in <b>E</b>. (<b>H</b>) Another thin section (5,000x) from the same tumour block with several supranuclear PaCSs, one of which (arrowhead) is enlarged in the top link inset (18,000x) to show an inner bacterial body (for comparison, another intracellular bacterium positive for H. pylori OMPs immunogold (21,000x) is shown in the bottom right inset), while an juxtaluminal PaCS (boxed area) is enlarged in <b><i>I</i></b> (25,000x) to illustrate NOD1 immunogold reactivity. N, nucleus. (<b>L</b>) TEM (20.000x) of another PaCS in another section from the same block immunostained with <i>H. pylori</i> OMP antibodies; boxed area enlarged in M (50,000x) to better magnify the immunogold particles and barrel-like putative proteasome particles, one of which (boxed in <b>M</b>) is further enlarged in the <b>L</b> inset (200,000x) to illustrate the characteristic punctuate-aligned structure.</p

Lung large cell carcinoma, hepatocarcinoma and colon adenocarcinoma.

<p>(<b>A</b>–<b>C</b>) Juxtanuclear PaCS in a lung large cell carcinoma (<b>A</b>, 10,000x), boxed area enlarged in <b>B</b> (50,000x) whose boxed area is enlarged in <b>C</b> (75,000x) to show its particulate structure and proteasome immunogold reactivity. (<b>D</b> and <b>E</b>) Hepatocellular carcinoma showing both hyaline bodies (hb), reactive for p62 protein in <b>D</b> (30,000x) and unreactive for 19S proteasome in <b>E</b> (25,000x), and PaCS (asterisks) unreactive for p62 and reactive for 19S proteasome. r, ribosomes. Inset to <b>D</b> (450x): hyaline and Mallory bodies react for p62 protein immunoperoxidase in formalin-fixed paraffin sections. (<b>F</b>–<b>H</b>) Colon adenocarcinoma (<b>F</b>, 10,000x) with partly intracellular and partly interstitial bacteria, a cytoplasmic vacuole (v) and a PaCS (boxed) bordering the cell plasma membrane, enlarged in <b>G</b> (40,000x) and <b>H</b> (75,000x). Note partial lysis of proteasome particles, though retaining 19S proteasome reactivity, development of irregular dense bodies and an encircling peripheral membrane, suggesting transition from a PaCS to an autophagic vacuole, also enclosing ribosome-like particles (r) and a bacterium (top right in <b>G</b> and <b>H</b>) with capsulated wall typical of Gram-positive organisms.</p

Pancreatic serous microcystic adenoma.

<p>(<b>A</b>, <b>B</b>) Formalin-fixed paraffin sections; note the clear, apparently “empty” cytoplasm of most cells in <b>A</b> (hematoxylin-eosin, 1,000x) and the poor reactivity in <b>B</b> to proteasome immunofluorescence under confocal microscopy (blue: nuclei; green: proteasome; 800x). (<b>C</b>, <b>D</b>) Semithin aldehyde-osmium fixed resin sections from the same tumour as in <b>A</b> and <b>B</b>, show abundant cytoplasmic PaCSs metachromatically stained pink with toluidine blue (<b>C</b>, 1,000x) and extensively proteasome immunofluorescent under confocal microscopy (<b>D</b>, green: proteasome; 500x). (<b>E</b>) CCEM showing proteasome immunofluorescent PaCS (5,000x); the same specimen under TEM only (<b>F</b>, 5,000x), boxed area enlarged in <b>G</b> (50,000x) shows the PaCS particulate pattern with 20S proteasome immunogold reactivity. (<b>H, I</b>) TEM of another specimen of the same tumour in <b>H</b> (5,000x), boxed area enlarged in <b>I</b> (30,000x) and further enlarged in the inset (50,000x) to show PaCS particulate ultrastructure and 19S proteasome immunogold.</p

PaCSs in SH-SY5Y and HL-60 cells.

<p>(<b>A</b>) Ultrastructure of a neuroblastoma SH-SY5Y cell with characteristic PaCSs, enlarged (<b>a1</b>) to show relatively spaced particles and selective FK1 antibody immunogold. In (<b>A</b>) a neural cell process whose hillock-like origin and terminal button abutted on another cell is enlarged in (<b>a2</b>) and (<b>a3</b>), respectively. In (<b>a4</b>) a 20S proteasome-reactive PaCS from a different SH-SY5Y cell is filled with particles and surrounded by ribosomes. (<b>B</b>) Several PaCSs in an HL-60 cell; one of which (<b>b1</b>) shows FK1 immunogold; in (<b>b2</b>) ALFY reactivity of an autophagic vesicle, enlarged in (<b>b3</b>), and non-reactivity of a small PaCS (arrow), enlarged in (<b>b4</b>).</p

PaCSs are metachromatic and chondroitin sulfate-positive bodies.

<p>(<b>A</b>) Toluidine blue metachromatic bodies (arrows) corresponding to chondroitin sulfate immunofluorescent bodies (<b>a1</b>), and TEM-characterized PaCSs (<b>a2–4</b>) in consecutive aldehyde–osmium-fixed resin sections of HeLa cells. A sequestosome (arrowhead) lightly stained (<b>A</b>), unreactive for chondroitin sulfate (<b>a1</b>) and moderately electron dense (<b>a2</b>) is enlarged in (<b>a3</b>) and (<b>a4</b>) to show its distinctive ultrastructure and unreactivity for FK1 immunogold, which selectively labeled the adjacent particle-filled PaCS.</p

PaCSs and sequestosomes in HeLa cells.

<p>(<b>A</b>) Several PaCSs are scattered in the cytoplasm of two cells, the larger one (boxed) is enlarged in (<b>a1</b>) and further in (<b>a2</b>) to show particle accumulation in a clear cytosolic background and selective FK1 immunogold reactivity for polyubiquitinated proteins. (<b>B</b>) Two small PaCSs (arrows) adjacent to a large central sequestosome in a ribosome-rich cytosol; the boxed area is enlarged (<b>b1</b>) to show PaCS 20S proteasome reactivity (right) and non-reactivity of the sequestosome (left), characterized by amorphous to thinly granular material often forming short fibrils. The curved fibrils are better seen at higher magnification (<b>b2</b>) of a sequestosome with poorly contrasted amorphous interfibrillary material. (<b>C</b>) Three PaCSs surrounding a sequestosome; the larger PaCS enlarged in (<b>c1</b>) shows 19S proteasome immunoreactivity, which is missing in sequestosome (<b>c2</b>), whose thin granules are often aligned to form beaded fibrils. (<b>D</b>) PaCS (top) and sequestosome (bottom) in ribosome-rich cytoplasm, enlarged in (<b>d1</b>) and (<b>d2</b>), respectively, to show sequestosome p62 protein immunoreactivity and PaCS non-reactivity; ribosomes in the left lower corner of (<b>d1</b>). (<b>E</b>) High resolution micrograph of PaCS particles reactive for 20S proteasome (10 nm gold) and FK1 (5 nm gold) antibodies. Some particles were aligned end-on to form 40-nm-long cylinders. (<b>F</b>) Sparse glycogen immunoreactivity of a PaCS, enlarged (<b>f1</b>) to be compared with a glycogen-unreactive granular–fibrillary sequestosome (<b>f2</b>) of the same section. The anti-ubiquitin Z0498 antibody reacted with both PaCS (<b>G</b>) and sequestosome (<b>G1</b>).</p

Proteasome activity in PaCSs of living cells.

<p>(<b>A</b>) Proteasome chimotrypsin-like activity shown by TED peptide cleavage in living HeLa cells concentrated in cytoplasmic bodies resembling PaCSs in size and intracellular distribution, and (<b>A1</b>) was greatly reduced by epoxomicin treatment. (<b>A2</b>) No comparable fluorescent areas appeared in the cytoplasm of TED-incubated living COS-7 cells. (<b>B</b>) TED-induced proteasome fluorescent bodies in two living HeLa cells under confocal microscopy corresponding in an aldehyde–osmium-fixed resin TEM section of the same cells (<b>B1</b>) to clear spots identified as PaCSs at higher resolution, owing to their faintly contrasted barrel-like particles and selective FK1 immunoreactivity, as shown in (<b>b2</b>) and (<b>b3</b>) for the one arrowhead in (<b>B</b>) and (<b>B1</b>).</p

PaCSs in human NK cells.

<p>(<b>A</b>) IL-15-treated human NK cell showing several small PaCSs; one of which is enlarged in (<b>a1</b>) and further in (<b>a2</b>) to show barrel-like particles with FK1 (5 nm gold) and 20S proteasome (10 nm gold) immunoreactivity; (<b>a3</b>) a PaCS-filled bleb. (<b>B</b>) Part of a mixed lytic granule, enlarged in (<b>b1</b>), showing in its vesicular component both barrel-like particles (some of which had FK1 and/or 20S proteasome immunoreactivity) and unreactive vesicles (arrowheads). Note in the upper part of (<b>B</b>) and (<b>b1</b>) the unreactive dense core of the lytic granule. For comparison, a multivesicular body, unreactive for both FK1 and 20S proteasome antibodies, is shown in (<b>b2</b>), from another NK cell in the same section as in (<b>A</b>) and (<b>B</b>).</p