Crosstalk between Yeast Cell Plasma Membrane Ergosterol Content and Cell Wall Stiffness under Acetic Acid Stress Involving Pdr18.

Acetic acid is a major inhibitory compound in several industrial bioprocesses, in particular in lignocellulosic yeast biorefineries. Cell envelope remodeling, involving cell wall and plasma membrane composition, structure and function, is among the mechanisms behind yeast adaptation and tolerance to stress. Pdr18 is a plasma membrane ABC transporter of the pleiotropic drug resistance family and a reported determinant of acetic acid tolerance mediating ergosterol transport. This study provides evidence for the impact of Pdr18 expression in yeast cell wall during adaptation to acetic acid stress. The time-course of acetic-acid-induced transcriptional activation of cell wall biosynthetic genes (FKS1, BGL2, CHS3, GAS1) and of increased cell wall stiffness and cell wall polysaccharide content in cells with the PDR18 deleted, compared to parental cells, is reported. Despite the robust and more intense adaptive response of the pdr18Δ population, the stress-induced increase of cell wall resistance to lyticase activity was below parental strain levels, and the duration of the period required for intracellular pH recovery from acidification and growth resumption was higher in the less tolerant pdr18Δ population. The ergosterol content, critical for plasma membrane stabilization, suffered a drastic reduction in the first hour of cultivation under acetic acid stress, especially in pdr18Δ cells. Results revealed a crosstalk between plasma membrane ergosterol content and cell wall biophysical properties, suggesting a coordinated response to counteract the deleterious effects of acetic acid.

Yeast adaptive response to acetic acid stress involves structural alterations and increased stiffness of the cell wall.

This work describes a coordinate and comprehensive view on the time course of the alterations occurring at the level of the cell wall during adaptation of a yeast cell population to sudden exposure to a sub-lethal stress induced by acetic acid. Acetic acid is a major inhibitory compound in industrial bioprocesses and a widely used preservative in foods and beverages. Results indicate that yeast cell wall resistance to lyticase activity increases during acetic acid-induced growth latency, corresponding to yeast population adaptation to sudden exposure to this stress. This response correlates with: (i) increased cell stiffness, assessed by atomic force microscopy (AFM); (ii) increased content of cell wall ß-glucans, assessed by fluorescence microscopy, and (iii) slight increase of the transcription level of the GAS1 gene encoding a ß-1,3-glucanosyltransferase that leads to elongation of (1→3)-ß-D-glucan chains. Collectively, results reinforce the notion that the adaptive yeast response to acetic acid stress involves a coordinate alteration of the cell wall at the biophysical and molecular levels. These alterations guarantee a robust adaptive response essential to limit the futile cycle associated to the re-entry of the toxic acid form after the active expulsion of acetate from the cell interior.

Mechanical Properties of Human Bronchial Epithelial Cells Expressing Wt- and Mutant CFTR.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). A single recessive mutation, the deletion of phenylalanine 508 (F508del), causes severe CF and resides on 70% of mutant chromosomes. Disorganization of the actin cytoskeleton has been previously reported in relation to the CF phenotype. In this work, we aimed to understand this alteration by means of Atomic Force Microscopy and Force Feedback Microscopy investigation of mechanical properties of cystic fibrosis bronchial epithelial (CFBE) cells stably transduced with either wild type (wt-) or F508del-CFTR. We show here that the expression of mutant CFTR causes a decrease in the cell's apparent Young modulus as compared to the expression of the wt protein.

Variation of Burkholderia cenocepacia cell wall morphology and mechanical properties during cystic fibrosis lung infection, assessed by atomic force microscopy.

The influence that Burkholderia cenocepacia adaptive evolution during long-term infection in cystic fibrosis (CF) patients has on cell wall morphology and mechanical properties is poorly understood despite their crucial role in cell physiology, persistent infection and pathogenesis. Cell wall morphology and physical properties of three B. cenocepacia isolates collected from a CF patient over a period of 3.5 years were compared using atomic force microscopy (AFM). These serial clonal variants include the first isolate retrieved from the patient and two late isolates obtained after three years of infection and before the patient's death with cepacia syndrome. A consistent and progressive decrease of cell height and a cell shape evolution during infection, from the typical rods to morphology closer to cocci, were observed. The images of cells grown in biofilms showed an identical cell size reduction pattern. Additionally, the apparent elasticity modulus significantly decreases from the early isolate to the last clonal variant retrieved from the patient but the intermediary highly antibiotic resistant clonal isolate showed the highest elasticity values. Concerning the adhesion of bacteria surface to the AFM tip, the first isolate was found to adhere better than the late isolates whose lipopolysaccharide (LPS) structure loss the O-antigen (OAg) during CF infection. The OAg is known to influence Gram-negative bacteria adhesion and be an important factor in B. cenocepacia adaptation to chronic infection. Results reinforce the concept of the occurrence of phenotypic heterogeneity and adaptive evolution, also at the level of cell size, form, envelope topography and physical properties during long-term infection.

A role for diacylglycerol kinase 4 in signalling crosstalk during Arabidopsis pollen tube growth.

Diacylglycerol kinases (DGKs) play a major role in the production of phosphatidic acid (PtdOH) and were implicated in endomembrane trafficking and signalling cascades. In plants, the role of DGKs is less clear, as PtdOH seems to arise mostly from phospholipase D activity. Here, we investigated the function of the Arabidopsis gene encoding DGK4, which is highly expressed in pollen. In vitro, pollen tubes from homozygous dgk4 plants showed normal morphology, but reduced growth rate and altered stiffness and adhesion properties (revealed by atomic force microscopy). In vivo, dgk4 pollen was able to fertilize wild-type ovules, but self-pollination in dgk4 plants led to fewer seeds and shorter siliques. Phenotypic analysis revealed that the dgk4 mutation affects not only the male germ line but also the vegetative tissue. DGK4-green fluorescent protein fusion imaging revealed a cytosolic localization with a slightly higher signal in the subapical or apical region. dgk4 pollen tubes were found to exhibit perturbations in membrane recycling, and lipid analysis revealed a minor increase of PtdOH concomitant with decreased phosphatidylcholine, compared with wild-type. In vitro, DGK4 was found to exhibit kinase and guanylyl cyclase activity. Quantitative PCR data revealed downregulation of genes related to actin dynamics and phosphoinositide metabolism in mutant pollen, but upregulation of the DGK6 isoform. Altogether, these results are discussed considering a role of DGK4 in signalling cross-talk.

Direct measurement of the capillary condensation time of a water nanobridge.

Water menisci wet all sorts of cavities, produce among the most intense forces at the nanoscale and play a role in many physical and chemical processes. The physical properties of these menisci are therefore relevant to understand a multitude of phenomena at the nanoscale where these are involved. Here, using a force feedback microscope, we directly measured the capillary condensation time of a water meniscus, by approaching two surfaces at different speeds and monitoring the relative position of the surfaces at the instant the meniscus is formed.

Effect of sliding friction in harmonic oscillators.

Sliding friction is ubiquitous in nature as are harmonic oscillators. However, when treating harmonic oscillators the effect of sliding friction is often neglected. Here, we propose a simple analytical model to include both viscous and sliding friction in common harmonic oscillator equations, allowing to separate these different types of dissipation. To compare this model with experimental data, a nanometric vibration was imposed on a quartz tuning fork, while an atomic force microscope tip was used to disturb its motion. We analyzed tuning fork resonance and 'ring down' experimental curves and for each case calculated the amount of sliding friction and of viscous damping, finding an agreement between the two different experiments and the model proposed.

Giant resonance tuning of micro and nanomechanical oscillators.

We present a method to tune the resonance frequency and the Q-factor of micro and nano-metric mechanical oscillators. A counteracting loop drives a capacitive force applied to the oscillator. The proportional and differential gains are used to shift the resonance frequency up to 75% and to tune the Q-factor of the oscillator, by changing its effective stiffness and damping ratio. The oscillator position is monitored in a large bandwidth with a fiber-optic based interferometer. We applied this simple operational scheme with different oscillators for modifying easily their dynamical properties. Compared to alternative methods requiring external fields, our method can either increase or decrease the resonance frequency in a frequency range much more extended. This opens up a wide range of applications, from force sensors with extremely low elastic constants but high quality factor to tunable energy harvesters or to high-frequency tuning of radio frequency filters. The control scheme can work in different media, and is then suitable to be applied to biological sensors and actuators.