Jeannine Lherminier

Lateral reorganization of plasma membrane is involved in the yeast resistance to severe dehydration.

In this study, we investigated the kinetic and the magnitude of dehydrations on yeast plasma membrane (PM) modifications because this parameter is crucial to cell survival. Functional (permeability) and structural (morphology, ultrastructure, and distribution of the protein Sur7-GFP contained in sterol-rich membrane microdomains) PM modifications were investigated by confocal and electron microscopy after progressive (non-lethal) and rapid (lethal) hyperosmotic perturbations. Rapid cell dehydration induced the formation of many PM invaginations followed by membrane internalization of low sterol content PM regions with time. Permeabilization of the plasma membrane occurred during the rehydration stage because of inadequacies in the membrane surface and led to cell death. Progressive dehydration conducted to the formation of some big PM pleats without membrane internalization. It also led to the modification of the distribution of the Sur7-GFP microdomains, suggesting that a lateral rearrangement of membrane components occurred. This event is a function of time and is involved in the particular deformations of the PM during a progressive perturbation. The maintenance of the repartition of the microdomains during rapid perturbations consolidates this assumption. These findings highlight that the perturbation kinetic influences the evolution of the PM organization and indicate the crucial role of PM lateral reorganization in cell survival to hydric perturbations.

Dynamic changes in the subcellular distribution of the tobacco ROS-producing enzyme RBOHD in response to the oomycete elicitor cryptogein

Highlight text The oomycete elicitor cryptogein triggers the relocation of RBOHD from intracellular compartments to the plasma membrane in tobacco cells. This suggests that intracellular trafficking is a potential determinant of RBOHD activity.

Immunocytological detection of salivary mucins (MUC5B) on the mucosal pellicle lining human epithelial buccal cells

The mucosal pellicle is defined as the protein film adsorbed onto oral mucosa. This study aimed at characterizing the ultrastructure of human epithelial buccal cells and localizing salivary mucins MUC5B, a major constituent of the mucosal pellicle. Cells were sampled from the buccal surface and prepared for Transmission Electron Microscopy using high‐pressure freezing/cryosubstitution followed by immunogold labelling of MUC5B. Morphologically, cells were visualized as typical cells of the superficial layer of a squamous nonkeratinized epithelium with a partly degraded plasma membrane. The outer surface of the plasma membrane was lined with a biological material of medium electron density. MUC5B were detected in the extracellular space, and particularly in the vicinity of the plasma membrane, sometimes onto fibrils protruding from the membrane. This area was, therefore, considered as constituting the mucosal pellicle, which appeared as a mixed film of both salivary and epithelial components. The distribution of gold particles suggested that the surface of the pellicle was not uniform, and that the film thickness could reach up to 100 nm. This work showed the feasibility of visualizing and characterizing the mucosal pellicle directly on human epithelial buccal cells sampled in a noninvasive manner. Microsc. Res. Tech. 77:453–457, 2014.

Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells.

The effects of changes in plasma membrane (PM) sterol lateral organization and availability on the control of signaling pathways have been reported in various animal systems, but rarely assessed in plant cells. In the present study, the pentaene macrolide antibiotic filipin III, commonly used in animal systems as a sterol sequestrating agent, was applied to tobacco cells. We show that filipin can be used at a non-lethal concentration that still allows an homogeneous labeling of the plasma membrane and the formation of filipin-sterol complexes at the ultrastructural level. This filipin concentration triggers a rapid and transient NADPH oxidase-dependent production of reactive oxygen species, together with an increase in both medium alkalinization and conductivity. Pharmacological inhibition studies suggest that these signaling events may be regulated by phosphorylations and free calcium. By conducting FRAP experiments using the di-4-ANEPPDHQ probe and spectrofluorimetry using the Laurdan probe, we provide evidence for a filipin-induced increase in PM viscosity that is also regulated by phosphorylations. We conclude that filipin triggers ligand-independent signaling responses in plant cells. The present findings strongly suggest that changes in PM sterol availability could act as a sensor of the modifications of cell environment in plants leading to adaptive cell responses through regulated signaling processes.

The membrane-associated MUC1 improves adhesion of salivary MUC5B on buccal cells. Application to development of an in vitro cellular model of oral epithelium.

OBJECTIVES The mucosal pellicle is a thin layer of salivary proteins, mostly MUC5B mucins, anchored to epithelial oral cells. This pellicle is involved in protection of oral mucosae against abrasion, pathogenic microorganisms or chemical xenobiotics. The present study aimed at studying the involvement of MUC1 in mucosal pellicle formation and more specifically in salivary MUC5B binding using a cell-based model of oral epithelium. DESIGN MUC1 mRNAs were not detected in TR146 cells, and therefore a stable cell line named TR146/MUC1 expressing this protein was developed by transfection. TR146 and TR146/MUC1 were incubated with human saliva in order to evaluate retention of MUC5B by epithelial cells. RESULTS The cell surface of both TR146 and TR146/MUC1 was typical of a squamous non-keratinized epithelium, with the presence of numerous microplicae. After incubation for 2h with saliva diluted in culture medium (1:1) and two washes with PBS, saliva deposits on cells appeared as a loose filamentous thin network. MUC5B fluorescent immunostaining evidenced a heterogeneous lining of confluent cell cultures by this salivary mucin but with higher fluorescence on TR146/MUC1 cells. Semi-quantification of MUC5B bound to cells confirmed a better retention by TR146/MUC1, evaluated by Dot Blot (+34.1%, p<0.05) or by immunocytochemistry (+44%, p<0.001). CONCLUSION The membrane-bound mucin MUC1 is a factor enhancing the formation of the mucosal pellicle by increasing the binding of salivary MUC5B to oral epithelial cells. An in vitro model suitable to study specifically the function and properties of the mucosal pellicle is proposed.