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First published online 8 April 2008
doi: 10.1242/jcs.017665

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Dynamics of an F-actin aggresome generated by the actin-stabilizing toxin jasplakinolide

¹ Departament de Biologia Cel·lular i Anatomia Patològica, Facultat de Medicina, Universitat de Barcelona, E-08036 Barcelona, Spain
² Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, E-08036 Barcelona, Spain
³ Institut de Nanociència i Nanotecnologia (IN2UB), Universitat de Barcelona, E-08036 Barcelona, Spain
⁴ Laboratorio de Biología Celular, Centro de Investigación Príncipe Felipe and CIBERER, E-46013 Valencia, Spain

View larger version (97K): [in this window] [in a new window]   Fig. 1. Actin-cytoskeleton disruption by actin toxins is reversible: formation of different F-actin aggregates and/or inclusion bodies. (A-D) Vero cells (A) were incubated with LtB (500 nM for 45 minutes; B), MyB (100 nM for 60 minutes; C) or Jpk (500 nM for 45 minutes; D). (E-J) To examine the reversibility, actin toxins were washed out from the culture medium and cells were left to recover for different durations (–LtB, E,F; –MyB, G,H; –Jpk, I,J). Cells were stained with TRITC-phalloidin for F-actin (A-J, red), with DAPI for nuclei (G-J, blue) or with anti--tubulin antibodies for the centrosome (inset in I, green). (E,G) Arrowheads indicate juxtanuclear accumulations of F-actin punctae. Scale bar: 10 µm.

View larger version (97K): [in this window] [in a new window]   Fig. 2. Formation and clearance of the single large FAG produced by the continuous presence of Jpk in the culture medium. (A-L) Vero cells (control, A) were treated with low concentrations of Jpk (50 nM), and at different times were fixed and stained with TRITC-phalloidin. Examples of F-actin punctae (B-D,G-K, arrowheads), F-actin amorphous aggregates (B-E,G-I, arrows) and the FAG (F) are shown. Inset in D is an enlargement of the circled area, to better visualize F-actin punctae and F-actin amorphous aggregate structures. Scale bar: 10 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 3. Organelle association and ultrastructure of the FAG. (A) 3D model of the FAG and the Golgi complex in Vero cells co-stained with TRITC-phalloidin (red) and anti-giantin (green) antibodies. (B) Co-staining with FITC-phalloidin (green) and the mitochondrial marker MitoTracker (red). (C-G) Ultrastructure of the FAG. The FAG is invariably surrounded by mitochondria (C,D), autophagic vacuoles (D), lysosomes (G), the Golgi complex (D,G) and intermediate filaments (arrowheads in G). In some FAGs (E), there are regions containing highly ordered F-actin or actin bundles (F, enlargement of the boxed area shown in E). Autophagic vacuoles (a), lysosome (l), Golgi cisternae (g), peri-Golgi COPI-coated transport carriers (arrows in G) and electron-dense material in the FAG (asterisks in D) are indicated. Scale bars: 10 µm (B), 3 µm (A,C), 300 nm (D,E) and 100 nm (F,G).

View larger version (157K): [in this window] [in a new window]   Fig. 4. Molecular composition of the FAG in different cell types assessed by immunofluorescence. (A-J) Vero cells (A-F) and mouse hippocampal neurons (G-J) were treated with Jpk (50 nM for 6 hours and 500 nM for 24 hours, respectively), fixed and double-stained with TRITC-phalloidin (red) and with a variety of antibodies against different cytoskeleton and/or cytoskeleton-associated proteins and neuronal markers (indicated in each panel; green). Scale bar: 10 µm.

View larger version (115K): [in this window] [in a new window]   Fig. 5. Microtubule involvement in the formation and location of the FAG. (A-C) Vero cells were treated with NZ (30 µM for 6 hours; A, +NZ) or TX (30 µM for 6 hours; A, +TX) alone or subsequently co-incubated with Jpk (50 nM for 6 hours; B,C). (B) In NZ+Jpk-treated cells, no FAG was formed. Instead, numerous F-actin punctae and F-actin amorphous aggregates were dispersed throughout the cytoplasm (also see inset). (C) Similar results were obtained in TX+Jpk-treated cells (also see inset). (D,E) When NZ and TX were washed-out but Jpk remained (D and E, respectively), a FAG was formed. (F) When cells were incubated with Jpk (50 nM for 6 hours) and then with NZ (30 µM for 6 hours), the structural integrity of the FAG (left panel) remained unaltered despite the disruption of microtubules (right panel). (A-F) Cells were stained with anti-β-tubulin antibodies (A and right panel in F) or TRITC-phalloidin (B-E and left panel in F). Scale bars: 10 µm.

View larger version (107K): [in this window] [in a new window]   Fig. 6. Lysosomal distribution and function during the formation and/or clearance of the FAG. (A-C) Vero cells were treated with Jpk (+Jpk, 50 nM) for different time periods, as indicated in the panels. Cells were double-stained with TRITC-phalloidin (red) and anti-LAMP2 antibodies (green). Lysosomes in cells containing a FAG show either a uniform distribution or accumulate around the FAG (arrow in B). At 48 hours after Jpk treatment, an increase in lysosomal staining was observed despite the absence of a FAG. (D-F) Vero cells were treated with Jpk (+Jpk, 50 nM for 6 hours) and then co-incubated with bafilomycin A1 (Baf, 100 mM; +Jpk +Baf; D,E) or pepstatin A (Pep, 10 µM; +Jpk +Pep; F). Notice that the dysfunction of the lysosomal activity prolonged the lifespan of the FAG. (E) Enlargement of the boxed area in D, in which the co-staining with TRITC-phalloidin and anti-LAMP2 antibodies revealed that Baf treatment traps lysosomes in an F-actin net (asterisks) that is structurally different from the FAG (arrowhead). Scale bars: 10 µm.

View larger version (114K): [in this window] [in a new window]   Fig. 7. Autophagy in cells containing a FAG. (A) Vero cells were transfected with LC3-GFP plasmid and cultured in medium without FBS, but containing pepstatin A (Pep, 10 µM) plus E-64-d (10 µM). (B) LC3-GFP accumulated around the FAG in transfected cells cultured in complete medium without lysosomal inhibitors. (C) Representative experiment of an immunoblot using anti-LC3 antibody in cells treated with (to induce FAG formation) or without Jpk (+/–Jpk) and grown in medium with or without (4 hours) serum (+/–FBS) in the presence of lysosomal inhibitors. The positions of endogenous LC3-I and LC3-II are indicated. Fold increases in the ratios of LC3-II to tubulin (calculated as described in the Materials and Methods) from three independent experiments were 2.4±0.1 (Jpk–/FBS–), 3.8±0.3 (Jpk+/FBS–), 1.0 (control, Jpk–/FBS+) and 2.4±0.2 (Jpk+/FBS+). All differences were statistically significant at P0.005 using the Student's t-test. (D) Ultrastructure of a FAG-containing cell (the FAG is limited by broken line), in which autophagic vacuoles appeared tightly associated with the FAG. (E) MDC-containing vesicles accumulated around the FAG. (F,G) During clearance of the FAG after Jpk removal (–Jpk for 16 or 24 hours), the number of MDC-containing vesicles and lysosomes (stained with MDC and/or anti-LAMP2 antibodies) increased and were associated with F-actin punctae and F-actin amorphous aggregates (arrowheads in G). (F-I) Rpc (200 nM) added after Jpk removal accelerated the dissolution of the FAG (compare H with F and I with G). Inset in H shows the tight association and partial colocalization of F-actin punctae (red) with lysosomes and/or MDC-containing vesicles (green and blue, respectively). (J) The number of autophagic vacuoles increased during the dissolution of the FAG (–Jpk for 36 hours). (K-M) Some autophagic vacuoles contained fragments of microfilaments arranged in parallel (arrow in K; high magnification in L; M is an enlargement of the boxed area in L). Scale bars: 10 µm (A,B,E-I), 1 µm (J), 200 nm (D), 100 nm (K), 30 nm (L), 10 nm (M).

View larger version (44K): [in this window] [in a new window]   Fig. 8. Proteasomes and proteasomal function in the FAG. (A,B) Vero cells containing a FAG (red) co-stained with anti-FK2 antibodies to show (poly)ubiquitylated proteins (A, green) or with anti-C9 antibodies to reveal the presence of proteasomes (B, green), indicating that the FAG contains proteasomes but not polyubiquitylated proteins. (C-F) FAG clearance was delayed in cells treated with lactacystin (Lac; compare E with C and F with D). (G) Proteasome activity measured in clarified lysates from untreated Vero cells (control, c), from cells treated with lactacystin (+Lac, 10 µM), or from cells treated with Jpk (+Jpk, 50 nM) for different times. Results are the mean ± s.d. from three independent experiments. Differences from control significant at P0.01 (**) and P0.001 (***). Scale bar: 10 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 9. Cells can generate segregated aggresomes with different molecular compositions. Vero cells were transfected with a GFP-huntingtin mutant plasmid (GFP-httm). (A) At early expression times of GFP-httm (4 hours), a diffuse cytoplasmic staining was seen (green). (B,C) Subsequent treatment with Jpk (+Jpk, 50 nM) produced F-actin amorphous aggregates and a FAG (red) as long as the GFP-httm aggresome was also forming (green). (D,E) After longer Jpk treatment, the FAG is fragmented in F-actin amorphous aggregates and F-actin punctae (D, red) until its complete clearance (E), but the GFP-httm aggresome (E, green) remained in the cytoplasm. (F) Notice that the two aggresomes, one containing GFP-httm (green) and the other F-actin (red), constantly remain morphologically and molecularly segregated, even when cells were submitted to various Jpk pulses (also see supplementary material Fig. S1). Scale bar: 10 µm.

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