Is Wortmannin-Induced Reorganization of the trans-Golgi Network the Key to Explain Charasome Formation?

Abstract

Wortmannin, a fungal metabolite and an inhibitor of phosphatidylinositol-3 (PI3) and phosphatidylinositol-4 (PI4) kinases, is widely used for the investigation and dissection of vacuolar trafficking routes and for the identification of proteins located at multivesicular bodies (MVBs). In this study, we applied wortmannin on internodal cells of the characean green alga Chara australis. Wortmannin was used at concentrations of 25 and 50 μM which, unlike in other cells, arrested neither constitutive, nor wounding-induced endocytosis via coated vesicles. Wortmannin caused the formation of "mixed compartments" consisting of MVBs and membranous tubules which were probably derived from the trans-Golgi network (TGN) and within these compartments MVBs fused into larger organelles. Most interestingly, wortmannin also caused pronounced changes in the morphology of the TGNs. After transient hypertrophy, the TGNs lost their coat and formed compact, three-dimensional meshworks of anastomosing tubules containing a central core. These meshworks had a size of up to 4 μm and a striking resemblance to charasomes, which are convoluted plasma membrane domains, and which serve to increase the area available for transporters. Our findings indicate that similar mechanisms are responsible for the formation of charasomes and the wortmannin-induced reorganization of the TGN. We hypothesize that both organelles grow because of a disturbance of clathrin-dependent membrane retrieval due to inhibition of PI3 and/or PI4 kinases. This leads to local inhibition of clathrin-mediated endocytosis during charasome formation in untreated cells and to inhibition of vesicle release from the TGN in wortmannin-treated cells, respectively. The morphological resemblance between charasomes and wortmannin-modified TGN compartments suggests that homologous proteins are involved in membrane curvature and organelle architecture.

Keywords: Chara australis; charasome; endocytosis; multivesicular body; trans-Golgi network; wortmannin.

Figure 1

Effect of wortmannin on internalization of AM1-44 in internodal cells of Chara australis. (A) Chloroplast-free window for imaging endosomal organelles (DIC) (B) Relative numbers of AM1-44-stained organelles in control cells, in cells treated with 25 μM wortmannin (WM) for 2.5 h, in cells recovering from this treatment for 2 h in artificial fresh water (WMr) and in cells treated with 50 μM wortmannin for 2.5 h are compared in box-and-whisker plots. Shown are median values with upper and lower quartiles (boxes), whiskers indicating the 10th and 90th percentiles, and outliers (dots) with n = between 5 and 17. Differences between median values of control and treated cells are significant (asterisks; one way analysis of variance, P = 0.001). (C–H) AM1-44-stained organelles in cells treated with 0.5% DMSO (C), with 25 μM wortmannin for 2.5 h (D), with 25 μM wortmannin for 2.5 h followed by 2 h “recovery” in artificial fresh water (E), with 50 μM wortmannin for 2.5 h (F,G is the corresponding DIC image), and with 50 μM wortmannin for 2.5 h, followed by 7 d recovery in artificial fresh water (H, compare with F). Arrows indicate enlarged organelles in response to wortmannin treatment; C, chloroplast. Bars are 50 μm (A) and 10 μm (C–H).

Figure 2

Immunofluorescence with antibodies against endosomal markers of control cells (A,D), of internodal cells treated with 50 μM wortmannin for 2 h (B,C,E,F) and corresponding histograms (G,H). (A,B) Immunofluorescence with an antibody against CaARA7, a marker for MVBs, shows small punctate organelles in untreated cells and larger compartments (B) in wortmannin-treated cells. (C) Double immunofluorescence with anti-CaARA7 and anti-OsSCAMP1, a marker of TGNs suggests that groups of small MVBs intermingle with TGN membranes. (D–F) Immunofluorescence with anti-OsSCAMP1. The antibody recognizes abundant small organelles with various shapes in the endoplasm of an untreated cell (D). In wortmannin-treated cells, large SCAMP1-positive aggregates (E) and large, globular organelles (F) are present. (G,H) The histograms show the size distributions of SCAMP1-positive organelles in solvent-treated cells, and in cells treated with 50 μM wortmannin for 2 h. Bars are 10 μm.

Figure 3

Effect of wortmannin on MVBs in Chara internodal cells. Cells were treated with 50 μM wortmannin for 2 h (A,B,E), and with 25 μM for 30 min followed by 2 h recovery (F). Images of multivesicular bodies (C,D) are from untreated cells. (A) Cross-section through the chloroplast (C)-containing cortex near the plasma membrane (arrow head), and the endoplasm located between cortex and vacuole (V). Wortmannin does not affect the fine structure of chloroplasts, Golgi bodies (G), glycosomes (g), mitochondria (M), and endoplasmic reticulum (ER). Note also that the TGN visible in this area has a normal shape and morphology. (B) The charasome in a wortmannin-treated cell is similar to those in control cells. Thick arrows indicate the openings of charasome tubules to the cell wall space. The thin arrow points to a coated vesicle, probably released from the smooth plasma membrane (arrow heads). (C,D) MVBs in untreated branchlet internodal cells are small and variable in shape. (E) In wortmannin-treated cells, MVBs form large clusters with intertwining membrane tubules. The white arrow indicates fusion between two MVBs, and the black arrows point to continuities between MVBs and membrane tubules. Arrow heads indicate tubules within MVBs. The arrow in the inset points to a cross-sectioned tubule or vesicle with a central core. (F) Large MVBs in a cell recovering from wortmannin-treatment. Bars are 1 μm (A), 500 nm (E,B,F), and 250 nm (C,D and inset in E).

Figure 4

Effect of wortmannin on TGNs in Chara internodal cells. Cells were treated with 50 μM wortmannin for 2 h (A–F), and with 25 μM for 30 min, followed by a recovery for 2 h (G). (A,B) Enlarged TGN near a Golgi body (G) in a wortmannin-treated cell (C, chloroplast; ER, endoplasmic reticulum). The higher magnification in (B) shows smooth and coated tubules, and vesicles which occasionally contain a central core (arrow heads). Enlarged regions with amorphous content are indicated by arrows. (C,D) Wortmannin-modified TGN located in the endoplasm between a Golgi body (G) and a nucleus (N). Arrow heads in the enlarged detail (D) indicate tubules with cross- or longitudinal sectioned central core. Note uniform diameter of tubules and absence of coated regions. (E) Charasomes (black asterisks), mitochondria (M), and wortmannin-modified TGNs (white asterisks) squeezed between cell wall (CW) and chloroplast (C). (F) Higher magnification of a charasome (black asterisk) and a wortmannin-modified compact TGN (white asterisk) in the cortex. Arrows indicate openings of the charasome and the TGN to the cytoplasmic space, respectively. Arrow heads indicate tubules or vesicles with central core. g, glycosome; M, mitochondrion. (G) Huge TGN complex in a cell recovering from wortmannin-treatment. g, glycosome; V, vacuole. Arrows point to enlarged areas with electron dense granular material. Bars are 1 μm (A,C,E,G) and 500 nm (B,D,F).

Figure 5

Effect of wortmannin on wound response in Chara internodal cells. (A–D) DIC images of healed puncture wounds in a DMSO-treated cell (control; A,B), and in a cell treated with 50 μM wortmannin (C,D). The wound plug (P), consisting of vacuolar inclusions, is covered by a cellulosic wound wall (arrows in B,D). (E–H) Healed UV-induced wounds in a control cell (E,F), and in a cell treated with 50 μM wortmannin before and after wounding (G,H). (E,G) are DIC images and (F,H) are the corresponding images of callose visualized after staining with sirofluor. Bars are 50 μm (A,C,E–H) and 10 μm (B,D).

Figure 6

Effect of wortmannin on charasome degradation in Chara internodal cells. (A) AM1-44 stained charasomes at the acid region of a cell exposed to standard light/dark conditions for 8 days (before treatment). (B) AM1-44 stained charasomes in a DMSO-treated cell after 8 days incubation in darkness (control). (C) AM1-44 stained charasomes in a wortmannin (WM)-treated cell after 8 days incubation in darkness. (D) Maximum charasome area fractions in control and wortmannin-treated cells are compared in box-and-whisker plots. Shown are median values with upper and lower quartiles (boxes), whiskers indicating the 10th and 90th percentiles and outliers (dots). Values are based on data obtained from 12 to 16 cells. Differences between median values of control and 0.4 μM wortmannin treated cells are significant (asterisk; P = 0.003, Kruskal–Wallis one way analysis of variance on ranks). (E) Table showing the survival rate and pH banding activity of the cells analyzed in (D). Bars are 10 μm.

Figure 7

Effect of wortmannin on charasome formation in Chara internodal cells. (A) AM1-44 stained charasomes in a cell which was dark-incubated for 2 weeks. (B) AM1-44 stained charasomes at the acid region of a control cell exposed to standard light conditions for 8 days. (C) AM1-44 stained charasomes in a cell treated with 0.2 μM wortmannin (WM) under standard light conditions for 8 days. (D) Maximum charasome area fractions in control and in wortmannin-treated cells are compared in box-and-whisker plots. Shown are median values with upper and lower quartiles (boxes), whiskers indicating the 10th and 90th percentiles and outliers (dots). Values are based on data obtained from 15 to 17 cells. Differences between median values of control cells and cells treated with 0.2 μM wortmannin are significant (asterisk; P = 0.003, Kruskal–Wallis one way analysis of variance on ranks). (E) Table showing the survival rate and pH banding activity of the cells analyzed in (D). Bars are 10 μm.

Figure 8

Schematic summary showing the formation of a wortmannin-modified TGN and its similarity with a charasome. The charasome at the left side of the image is neither growing, nor degrading, as suggested by the absence of coated pits. The periplasmic space of the charasome contains a filamentous core, and is continuous with the cell wall (CW). Treatment of cells with wortmannin neither arrests formation of endosomes (constitutive endocytosis) from smooth plasma membrane, nor production of Golgi vesicles, both required for TGN formation. The “normal” TGNs are involved in the production of various vesicles (including glycosomes, g) designed for exocytosis. A filamentous core is occasionally visible in TGN tubules and TGN-derived vesicles. Other TGNs (probably those equipped with a specific set of PI3Ps and/or PI4Ps) are much more affected by wortmannin. They lose the capacity to pinch off vesicles, eventually enlarge, and finally form compact charasome-like structures where a filamentous core is visible in all tubules. Only approximately drawn to scale.