PaCS is a novel cytoplasmic structure containing functional proteasome and inducible by cytokines/trophic factors

Abstract

A variety of ubiquitinated protein-containing cytoplasmic structures has been reported, from aggresomes to aggresome-like induced structures/sequestosomes or particle-rich cytoplasmic structures (PaCSs) that we recently observed in some human diseases. Nevertheless, the morphological and cytochemical patterns of the different structures remain largely unknown thus jeopardizing their univocal identification. Here, we show that PaCSs resulted from proteasome and polyubiquitinated protein accumulation into well-demarcated, membrane-free, cytoskeleton-poor areas enriched in glycogen and glycosaminoglycans. A major requirement for PaCS detection by either electron or confocal microscopy was the addition of osmium to aldehyde fixatives. However, by analyzing living cells, we found that proteasome chymotrypsin-like activity concentrated in well-defined cytoplasmic structures identified as PaCSs by ultrastructural morphology and immunocytochemistry of the same cells. PaCSs differed ultrastructurally and cytochemically from sequestosomes which may coexist with PaCSs. In human dendritic or natural killer cells, PaCSs were induced in vitro by cytokines/trophic factors during differentiation/activation from blood progenitors. Our results provide evidence that PaCS is indeed a novel distinctive cytoplasmic structure which may play a critical role in the ubiquitin-proteasome system response to immune, infectious or proneoplastic stimuli.

Figure 1. PaCSs and sequestosomes in HeLa cells.

(A) Several PaCSs are scattered in the cytoplasm of two cells, the larger one (boxed) is enlarged in (a1) and further in (a2) to show particle accumulation in a clear cytosolic background and selective FK1 immunogold reactivity for polyubiquitinated proteins. (B) Two small PaCSs (arrows) adjacent to a large central sequestosome in a ribosome-rich cytosol; the boxed area is enlarged (b1) 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 (b2) of a sequestosome with poorly contrasted amorphous interfibrillary material. (C) Three PaCSs surrounding a sequestosome; the larger PaCS enlarged in (c1) shows 19S proteasome immunoreactivity, which is missing in sequestosome (c2), whose thin granules are often aligned to form beaded fibrils. (D) PaCS (top) and sequestosome (bottom) in ribosome-rich cytoplasm, enlarged in (d1) and (d2), respectively, to show sequestosome p62 protein immunoreactivity and PaCS non-reactivity; ribosomes in the left lower corner of (d1). (E) 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. (F) Sparse glycogen immunoreactivity of a PaCS, enlarged (f1) to be compared with a glycogen-unreactive granular–fibrillary sequestosome (f2) of the same section. The anti-ubiquitin Z0498 antibody reacted with both PaCS (G) and sequestosome (G1).

Figure 2. PaCSs in SH-SY5Y and HL-60 cells.

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

Figure 3. Detection of PaCSs by confocal microscopy.

(A) Only weak diffuse 20S proteasome immunofluorescence was seen in control HeLa cells after standard preparation (i.e., 15 min paraformaldehyde fixation), compared to the large 20S proteasome-reactive structures visible after aldehyde–osmium fixation and paraffin-embedding (A1, immunofluorescence and phase-contrast overlay). (B) Poor FK1 reactivity (green) and a few p62-reactive (red) spots were seen in the cytoplasm of standard-prepared HeLa cells, whereas after aldehyde–osmium fixation, large FK1-positive (blue) structures appeared (B1), lacking colocalization with p62 green fluorescence despite occasional juxtaposition (inset). (C) Only scattered minute glycogen deposits appeared in standard-prepared cells, whereas large deposits were seen after aldehyde–osmium fixation (C1); large deposits of glycogen synthase were seen after methanol fixation (C2). Standard confocal microscopy of epoxomicin-treated (D) or puromycin-treated (D1) HeLa cells showed prominent FK1/20S proteasome-reactive cytoplasmic bodies. Bars, 10 µm.

Figure 4. Correlative light/electron microscopy of PaCSs.

(A, B) Direct correlation between confocal microscopy immunofluorescence and TEM in an aldehyde–osmium fixed HeLa cell. (A) 20S proteasome immunofluorescence (green) of numerous cytoplasmic bodies, projected on the corresponding TEM micrograph (A1) to show overlapping of proteasome immunofluorescence spots with cytoplasmic PaCSs; a few of which are enlarged in (a2) and further in (a3) to show their distinctive ultrastructure. (B) Combined immunofluorescence/TEM image showing several proteasome-reactive PaCSs (green) and a large proteasome-unreactive sequestosome, as shown by TEM alone in (B1); part of the sequestosome and an adhering PaCS are enlarged (b2) to show their distinctive ultrastructure. (C and D) Direct identification of metachromatic bodies with PaCSs using consecutive semithin (light microscopy)/thin (TEM) section analysis from aldehyde–osmium-fixed, resin-embedded HeLa cells. Toluidine blue staining of a semithin section shows red–violet metachromatic bodies (C), corresponding to clear areas in a consecutive TEM section (C1). Boxed areas are enlarged in insets 1 (further enlarged in c2) and 2 to magnify 20S proteasome immunogold, besides faintly contrasted PaCS-type particles. Note the weak grey–blue staining (C) and the heavier electron density (C1) of the two sequestosomes adhering to boxed PaCSs and showing granular–fibrillary ultrastructure (c2) (right upper corner). (D and D1) Several small PaCSs in the bottom cell and a larger one in the upper cell (arrowhead) heavily stained by toluidine blue (D), while three sequestosomes were lightly stained. Most of such bodies were also identified by TEM in a consecutive thin section (D1), where PaCSs appeared as clear spots and sequestosomes as areas with intermediate electron density. The largest sequestosome is enlarged in (d2) and (d3) to magnify its thin granular–fibrillary ultrastructure and p62 protein immunoreactivity.

Figure 5. PaCSs are metachromatic and chondroitin sulfate-positive bodies.

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

Figure 6. Proteasome activity in PaCSs of living cells.

(A) 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 (A1) was greatly reduced by epoxomicin treatment. (A2) No comparable fluorescent areas appeared in the cytoplasm of TED-incubated living COS-7 cells. (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 (B1) to clear spots identified as PaCSs at higher resolution, owing to their faintly contrasted barrel-like particles and selective FK1 immunoreactivity, as shown in (b2) and (b3) for the one arrowhead in (B) and (B1).

Figure 7. PaCS in human DCs.

(A) Several PaCS-filled blebs (arrow) and intracytoplasmic PaCSs; the largest of which is enlarged (a) to show barrel-like particles and sparse 19S proteasome immunoreactivity. (B) Four PaCSs, one enlarged in (b1) and further in (b2) to show particles FK1 reactivity. (C) Two PaCSs, enlarged in (c1) and (c2), showed glycogen immunoreactivity polarized on the right side; note that PaCS particles were not polarized and that glycogen immunogold deposits were frequently unrelated to them. Selective 20S proteasome reactivity of PaCS particles from another section of the same cell is shown in (c3). (c1) Note residual round islets of cytoskeleton-rich cytoplasm inside cytoskeleton-poor PaCS – an uncommon finding. (D) An untreated blood monocyte lacking cytoplasmic PaCSs. Confocal microscopy of three aldehyde–osmium-fixed DCs showed immunofluorescence for proteasome (E), FK1 (F), and chondroitin sulfate (G).

Figure 8. PaCSs in human NK cells.

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