Hypertonic conditions trigger transient plasmolysis, growth arrest and blockage of transporter endocytosis inAspergillus nidulansandSaccharomyces 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.

The arrestin‐like protein ArtA is essential for ubiquitination and endocytosis of the UapA transporter in response to both broad‐range and specific signals

“…Calcofluor white (Sigma‐Aldrich, Life Science Chemilab SA) staining was performed according to Bitsikas et al . (). For endocytosis, uric acid (UA) or ammonium l ‐(+)‐tartrate (NH 4 + ) were added at concentrations of 0.5 mM or 10–20 mM respectively for 2 h before observation.…”

“…Staining with FM4-64 and CMAC (7-amino-4-chloromethyl coumarin) (Life Technologies, Molecular Probes, Invitrogen, Antisel SA) was according to Gournas et al (2010). Calcofluor white (Sigma-Aldrich, Life Science Chemilab SA) staining was performed according to Bitsikas et al (2010). For endocytosis, uric acid (UA) or ammonium L-(+)-tartrate (NH4 + ) were added at concentrations of 0.5 mM or 10-20 mM respectively for 2 h before observation.…”

Early endocytosis as a key to understanding mechanisms of plasma membrane tension regulation in filamentous fungi

“…Previously they were credited with the function of regulating PMT due to membrane reservation but now such a function is questionable. In hyperosmotic conditions, they deepen to inside the yeast cell (Moseley, 2018;Appadurai et al, 2020 (Amir et al, 1995;Torralba et al, 1998;Heath, Steinberg, 1999;Lew et al, 2004;Peñalva, 2005;Lew, 2010Lew, , 2019Bitsikas et al, 2011;Walker, White, 2017). e) Some model species from Saprolegniaceae and Peronosporaceae (Gupta, Heath, 1997;Heath, Steinberg, 1999;Chitcholtan, Garrill, 2005;Walker et al, 2006).…”

Plasmolysis: Loss of Turgor and Beyond

“…This structure forms a solid shell encasing the osmo-sensitive and membrane-covered protoplast. Consequently, plasmolysis occurs in walled cell types ranging from bacteria, to fungi and finally plants [ 1 , 2 , 3 , 4 ]. When large molecules with a size above the cell wall exclusion limit are used as osmotica (e.g., polyethylene glycols with a MW above 20 kDa) [ 5 ], then hyperosmolarity induces the collapse of the entire cell wall—plasma membrane continuum in a phenomenon known as cytorrhysis [ 6 ].…”