Dendritic cell aggresome-like induced structures are dedicated areas for ubiquitination and storage of newly synthesized defective proteins

Abstract

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control antigen processing and presentation. One of these mechanisms is the sorting of polyubiquitinated proteins in large cytosolic aggregates called dendritic cell aggresome-like induced structures (DALIS). DALIS formation and maintenance are tightly linked to protein synthesis. Here, we took advantage of an antibody recognizing the antibiotic puromycin to follow the fate of improperly translated proteins, also called defective ribosomal products (DRiPs). We demonstrate that DRiPs are rapidly stored and protected from degradation in DALIS. In addition, we show that DALIS contain the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E225K, and the COOH terminus of Hsp70-interacting protein ubiquitin ligase. The accumulation of these enzymes in the central area of DALIS defines specific functional sites where initial DRiP incorporation and ubiquitination occur. Therefore, DCs are able to regulate DRiP degradation in response to pathogen-associated motifs, a capacity likely to be important for their immune functions.

Figure 1.

Puromycin-containing proteins are targeted to DALIS during DC maturation. (A) Maturing DCs (8 h LPS) were treated with 0.1–5.0 μg/ml of puromycin for 30 min before 1% Triton X-100 extraction and fixation. DCs were stained for puromycin (left) and ubiquitinated proteins (Ub-proteins, right), and were visualized by confocal microscopy. Puromycin is detected in DALIS (arrows). Note that the highest dose of puromycin (5 μg/ml) induced de novo formation of aggregates. (B) Top: co-treatment with 100 μM cycloheximide (CHX) abolishes puromycin incorporation in DALIS (arrow), which is solely due to newly synthesized puromycin-containing proteins. Middle: in immature DCs, no DALIS formation is observed, neither is accumulation of puromycin-containing proteins. Bottom: puro-proteins incorporated into DALIS resist sequential biochemical extraction with 1% Triton X-100, 100 μg/ml Dnase I, and 2 M NaCl. (C) Immature (No LPS) or maturing DCs (8 h LPS) were treated with 0.1–5.0 μg/ml puromycin for 30 min. DCs were submitted to biochemical extraction, and puromycin-containing proteins present in the final insoluble pellet were analyzed by immunoblot. Bar, 10 μm.

Figure 2.

Puromycin-labeled proteins are rapidly incorporated in the central area of DALIS by a mechanism independent of microtubules or actin filaments. (A) Puromycin-labeled protein (green) incorporation into DALIS (red, arrows) is visualized as soon as 5 min after puromycin was added. (B) Maturing DCs were treated with 0.5 μg/ml puromycin for 15 min. After extraction with 1% Triton X-100, nucleus (N) and DALIS (arrows) were visualized by DIC or by immunofluorescence. Puro-protein (green) incorporation takes place right in the center of DALIS labeled by Ub-proteins (red), as illustrated by the yellow overlap in the combined xy, xz, and yz view. The inset displays a higher magnification view of a DALIS by DIC. (C) Disruption of microtubules with nocodazole before the puromycin pulse had no effect on puro-protein (green) incorporation in DALIS or on interaction between DALIS (red), as illustrated by the combined xy, xz, and yz view. (D) Disruption of actin network (stained by phalloidin in green, control on the left) with latrunculin B (Lat B) for 10 min before a 30-min puromycin pulse neither disrupted DALIS (Ub-proteins, red, center) nor prevented the incorporation of puro-proteins (green, right). Bars, 10 μm for all panels.

Figure 3.

DALIS are motile and constantly incorporate ubiquitin. Using a retroviral infection system, bone marrow progenitors expressing GFP-tagged ubiquitin (GFP-Ub) were differentiated in DCs. (A) In MHC class II positive (red) maturing DCs, GFP-Ub (green) is targeted to DALIS (arrows) labeled with FK2 antibody (blue) as shown by confocal microcopy. (B) Video microscopy performed over 2 h demonstrates that in DCs visualized by DIC, GFP-Ub labeled DALIS (green, arrow) are motile and can fuse together. Large aggregates (arrowhead) are in general less motile. Bar, 2 μm. (C) FRAP was performed on DALIS. Representative images obtained before and after photobleaching are presented sequentially. The mean fluorescence intensity measurement (max = 240) plotted against time (real data in red and correlation curve in blue) is shown on the right. 50% fluorescence was recovered in 2 min, and 90% after 18 min.

Figure 4.

DRiPs are ubiquitinated and stored in DALIS. (A) maturing DCs (8 h LPS) were pulsed with 1 μg/ml puromycin together with ³⁵S-labeled Pro-Mix (puro + ³⁵S) for 30 min and chased for the indicated time. Alternatively, ³⁵S was added alone for 30 min at the indicated time during the puromycin chase (puro → ³⁵S). Puromycin clearance efficiency during the chase was monitored by autoradiography of 1% Triton X-100 soluble material (left) and by autoradiography of puromycin-labeled protein immunoprecipitation material (right). (B) Maturing DCs were pulsed with 1 μg/ml puromycin and chased for the indicated time. Puromycin-labeled proteins were visualized by confocal microscopy after detergent extraction. Even after 16 h of chase, puromycin-labeled proteins are detected in DALIS (arrows). (C) The maximal fluorescence intensity of puromycin-labeled proteins visualized by confocal microscopy was established for a statistically relevant number of DALIS (15 < n < 20) and was plotted against time of puromycin chase. (D) Proteasome inhibition (1 μM epoxomicin) effect on puromycin-labeled (gray bars) and polyubiquitinated (black bars) proteins stored in DALIS was estimated by the same fluorescence intensity measurement method (*, P < 0.005). (E) In the same experiment as above, puromycin-containing proteins were detected in DALIS-enriched fractions by immunoblot. The formation of high mol wt ladders, characteristic of polyubiquitination, could be observed with time, and this independently of proteasome inhibition (Epox). (F) After 16 h of chase, proteasome inhibition clearly prevented the loss of puromycin-labeled material from DALIS-enriched fraction, suggesting that DRiP degradation takes place at this late stage of maturation.

Figure 5.

Regulation and DALIS targeting of the ubiquitin- conjugation enzyme system during DC maturation. (A) The E1 ubiquitin-activating enzyme (left) is present (arrow) in the central area of DALIS (Ub-proteins, right). (B) E1 expression strongly increased during the first 8 h of DC maturation without any change in its solubility, as monitored by immunoblot on soluble (top) and DALIS-enriched (bottom) fraction. (C) The ubiquitin-conjugating enzyme E2_25K (left) is present (arrow) in DALIS (Ub-proteins, right). (D) E2_25K expression increased during the first 8 h of DC maturation without any change in its solubility, as monitored by immunoblot on soluble (top) and DALIS-enriched (bottom) fraction. (E) The E3 ubiquitin ligase CHIP (left) is present (arrow) in the central area of some DALIS (Ub-proteins, right) and absent from others (arrowheads). (F) During DC maturation, expression of CHIP (as detected by immunoblot) increases in DALIS-enriched fraction (bottom), but not in Triton X-100 soluble fraction (top). Bars, 10 μm for all panels.