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 β-glucans, assessed by fluorescence microscopy, and (iii) slight increase of the transcription level of the GAS1 gene encoding a β-1,3-glucanosyltransferase that leads to elongation of (1→3)-β-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.

Summary 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.

Atomic force microscopy ͑AFM͒ and micro-x-ray diffraction are combined to investigate nanostructures during in situ indentation. This technique allows the determination of elastic properties of individual nanoscale objects, particularly here SiGe/ Si͑001͒ self-assembled islands. Using this novel technique it was possible to select a specific island, align it in the microfocused beam, and apply a pressure onto it, using the AFM tip. Simultaneously, the x-ray diffuse scattering map from the island and the surrounding substrate was recorded in order to probe the lattice parameter change during indentation. An elastic reduction of the island lattice parameter of up to 0.6% was achieved.

The force between two interacting particles as a function of distance is one of the most fundamental curves in science. In this regard, Atomic Force Microscopy (AFM) represents the most powerful tool in nanoscience but with severe limits when it is to probe attractive interactions with high sensitivity. The Force Feedback Microscope (FFM) described here, removes from AFM the well known jump to contact problem that precludes the complete exploration of the interaction curve and the study of associated energy exchanges. The FFM makes it possible to explore tip-surface interactions in the entire range of distances with a sensitivity better than 1 pN. FFM stands out as a radical change in AFM control paradigms. With a surprisingly simple arrangement it is possible to provide the AFM tip with the right counterforce to keep it fixed at any time. The counterforce is consequently equal to the tip-sample force. The force, force gradient and damping are simultaneously measured independently of the tip position. This permits the measurement of energy transfer in thermodynamic transformations. Here we show some FFM measurement examples of the complete interaction force curve and in particular that the FFM can follow the nucleation of a water bridge by measuring the capillary attractive force at all distances, without jump to contact despite the large attractive capillary force. Real time combination of the measured parameters will lead to new imaging modalities with chemical contrast in different environments.As stated by Feynman in his lectures [1], the force versus the distance between two interacting atoms is of the utmost importance in science being at the basis of our understanding of interactions between two objects. From the sharp repulsive regime felt at very short distances, a daily manifestation of the Pauli repulsion principle, the interaction extends for many tens or hundreds of nanometers in a (usually) long attractive regime. In this regime different sources (electrostatic, magnetic, chemical, capillary, van der Waals) at different scales intervene, and the quantitative measurement of the interaction over the entire span is essential for the understanding of the underlying mechanisms. This is well reflected by the wealth of AFM experimental activity in different environments and thermodynamical conditions covering physics, mechanics, biology, chemistry and soft condensed matter. However, despite the great successes obtained by AFM [2, 3,4,5] and Surface Force Apparatus (SFA) [6,7], up to now there is no instrument that can systematically and directly provide in real time the full force curve at nanoscale and at the pN levelas a real unambiguous experimental result. The SFA integrates over microareas, while in AFM, most of the time, an uncontrolled and irreversible dive of the nanotip onto the surface prevents direct and immediate access to the interaction in a large portion of the attractive regime. This jump to contact intervenes as soon as the force gradient overcomes the stiffness of soft AFM microlevers us...

Metal ions are well known modulators of protein aggregation and are key players in Alzheimer’s Disease, being found to be associated to pathologic protein deposits in diseased brains. Therefore, understanding how metals influence amyloid aggregation is critical in establishing molecular mechanisms that underlie disease onset and progression. Here, we report data on the interaction of full-length human Tau protein with calcium and zinc ions, evidencing that Tau self-assembly is differently regulated, depending on the type of bound metal ion. We established that Tau binds 4 Zn2+ and 1 Ca2+ per monomer while using native mass spectrometry analysis, without inducing order or substantial conformational changes in the intrinsically disordered Tau, as determined by structural analysis using circular dichroism and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopies. However, Tau aggregation is found to proceed differently in the calcium- and -zinc bound forms. While the rate of aggregation, as determined from thioflavin-T (ThT) fluorescence kinetics, is highly increased in both cases, the reaction proceeds via different mechanisms, as evidenced by the absence of the lag phase in the reaction of zinc-bound Tau. Monitoring Tau aggregation using native mass spectrometry indeed evidenced a distinct distribution of Tau conformers along the reaction, as confirmed by dynamic light scattering analysis. We propose that such differences arise from zinc binding at distinct locations within the Tau sequence that prompt both the rapid formation of seeding oligomers through interactions at high affinity sites within the repeat domains, as well as amorphous aggregation, through low affinity interactions with residues elsewhere in the sequence, including at the fuzzy coat domain.