Hypertonic conditions trigger transient plasmolysis, growth arrest and blockage of transporter endocytosis in Aspergillus nidulans and Saccharomyces cerevisiae

Abstract

By using Aspergillus nidulans strains expressing functional GFP-tagged transporters under hypertonic conditions, we noticed the rapid appearance of cortical, relatively static, fluorescent patches (0.5–2.3 μm). These patches do not correspond to transporter microdomains as they co-localize with other plasma membrane-associated molecules, such as the pleckstrin homology (PH) domain and the SsoA t-Snare, or the lipophilic markers FM4-64 and filipin. In addition, they do not show characteristics of lipid rafts, MCCs or other membrane microdomains. Deconvoluted microscopic images showed that fluorescent patches correspond to plasma membrane invaginations. Transporters remain fully active during this phenomenon of localized plasmolysis. Plasmolysis was however associated with reduced growth rate and a dramatic blockage in transporter and FM4-64 endocytosis. These phenomena are transient and rapidly reversible upon wash-out of hypertonic media. Based on the observation that block in endocytosis by hypertonic treatment altered dramatically the cellular localization of tropomyosin (GFP-TpmA), although it did not affect the cortical appearance of upstream (SlaB-GFP) or downstream (AbpA-mRFP) endocytic components, we conclude that hypertonicity modifies actin dynamics and thus acts indirectly on endocytosis. This was further supported by the effect of latrunculin B, an actin depolymerization agent, on endocytosis. We show that the phenomena observed in A. nidulans also occur in Saccharomyces cerevisiae, suggesting that they constitute basic homeostatic responses of ascomycetes to hypertonic shock. Finally, our work shows that hypertonic treatments can be used as physiological tools to study the endocytic down-regulation of transporters in A. nidulans, as non-conditional genetic blocks affecting endocytic internalization are lethal or severely debilitating.

Keywords::

• Fungi
• patches
• plasma membrane invaginations
• microdomain/actin dynamics

Acknowledgements

We are extremely grateful to Dr Areti Pantazopoulou for critical discussions, her help in microscopy and for performing the deconvolution analysis of images at CSIC/CIB (Madrid), and to Dr Miguel Angel Peňalva (CSIC/CIB) for critical discussions and sharing molecular markers and strains. We thank the students Andreas Pavlides and Fivos Borbolis (Faculty of Biology, University of Athens) for preliminary experiments in initial stages of this work. We thank Prof. Margarida Casal for her generous hospitality at her lab in Universidade do Minho (Braga, Portugal), where part of this work was carried out during a sabbatical of GD and Erasmus visits of MK and VB. We also thank Mrs Goretti for her technical help with the confocal microscope at the Medical School of Universidade do Minho and Dr Berl Oakley (Ohio State University) for the GFP-TpmA strain, Prof. R. Haguenauer-Tsapis (Institut Jacques Monod, Paris VII) and Assoc. Prof. S. Paiva (Universidade do Minho) for the Fur4p-GFP and Jen1p-GFP strains, respectively. We also thank Dr Sotiris Amillis for help in making the Figures and Dr Herlander Azevedo (Universidade do Minho), Prof. Basil Galatis and Ass. Prof. Panagiotis Apostolakos (Faculty of Biology, University of Athens) for helpful discussions. Finally, we thank Prof. Claudio Scazzocchio (Imperial College) for comments on the manuscript and the unknown referee who suggested to us an important recent publication relevant to this work. C.G was supported by I.K.Y.

Declaration of interest: This work was carried out through a minimal support from Athens University and the Erasmus program.