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.

Proteomic analysis of a mosquito host cell response to persistent Wolbachia infection

“…Recent evidence suggests that, in a green alga, formation of these vesicles may be reduced by treatment with the phosphatidylinositol kinase inhibitor, wortmannin [63]. We note, however, that application of wortmannin to C/ w Str1 cells can be complicated by its short half-life in culture media (8–13 minutes), and off-target effects such as induction of nitric oxide production and protein phosphorylation (http://www.enzolifesciences.com/BML-ST415/wortmannin/).…”

Pathways for external alkalinization in intact and in microwounded Chara cells are differentially sensitive to wortmannin

“…A recent study revealed that the light-induced pH bands in Chara cells disappear after the treatment with an inhibitor of phosphoinositide 3-kinases (PI-3 kinase), wortmannin, 13 indicating the involvement of phosphorylated phosphoinositides in the generation of pH bands. Phosphoinositides have an important regulatory role in cell physiology.…”

“…The appearance of tubular structures reminiscent of charasomes after the inhibition of lipid phosphorylation by wortmannin indirectly suggests the rearrangement of lipid material trafficking and possible disturbance of the charasome-mediated proton extrusion. 13 Since the pH banding pattern in resting cells is coordinated with the patterns of photosynthetic activity and charasome frequency, the influence of wortmannin on photosynthetic activity is likely.…”

The Manifold Actions of Signaling Peptides on Subcellular Dynamics of a Receptor Specify Stomatal Cell Fate

“…Thus, ERL1-YFP predominantly resides on the MVB. This is further confirmed by a pharmacological approach using Wortmannin (Wm), a fungal drug that can cause fusion of MVBs by inhibiting phosphatidylinositol-3 (PI3) and phosphatidylinositol-4 (PI4) kinases (Foissner et al, 2016). The Wm application on Arabidopsis seedlings resulted in the formation of typical ring-like Wm bodies marked by both ERL1-YFP and RFP-Ara7 (Figure 1C).…”