Nanomechanical characterization by double-pass force-distance mapping

Cataloged from PDF version of article.We demonstrate high speed force–distance mapping using a double-pass scheme. The topography is measured in tapping mode in the first pass and this information is used in the second pass to move the tip over the sample. In the second pass, the cantilever dither signal is turned off and the sample is vibrated. Rapid (few kHz frequency) force–distance curves can be recorded with small peak interaction force, and can be processed into an image. Such a double-pass measurement eliminates the need for feedback during force–distance measurements. The method is demonstrated on self-assembled peptidic nanofibers

Peroxisomes, lipid droplets, and endoplasmic reticulum "hitchhike" on motile early endosomes

This is the final version of the article. Available from the publisher via the DOI in this record.Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3- and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell.This work was supported by the Portuguese Foundation for Science and Technology and FEDER/COMPETE (SFRH/BD/73532/2010 to S.C. Guimaraes) and CRUP/Treaty of Windsor (ACÇÕES INTEGRAD AS 2009, B-33/09 to G. Steinberg and M. Schuster). G. Steinberg acknowledges support from the Biotechnology and Biological Sciences Research Counc

Interfiber interactions alter the stiffness of gels formed by supramolecular self-assembled nanofibers

Cataloged from PDF version of article.Molecular self-assembly is a powerful technique for developing novel nanostructures by using noncovalent interactions such as hydrogen bonding, hydrophobic, electrostatic, metal-ligand, p-p and van der Waals interactions. These interactions are highly dynamic and are often delicate due to their relatively weak nature. However, a sufficient number of these weak interactions can yield a stable assembly. In this work, we studied the mechanical properties of self-assembled peptide amphiphile nanostructures in the nanometre and micrometre scale. Hydrogen bonding, hydrophobic and electrostatic interactions promote self-assembly of peptide amphiphile molecules into nanofibers. Bundles of nanofibers form a three-dimensional network resulting in gel formation. The effect of the nanofiber network on the mechanical properties of the gels was analyzed by AFM, rheology and CD. Concentration and temperature dependent measurements of gel stiffness suggest that the mechanical properties of the gels are determined by a number of factors including the interfiber interactions and mechanical properties of individual nanofibers. We point out that the divergence in gel stiffness may arise from the difference in strength of interfiber bonds based on an energetic model of elastic rod networks, along with continuum mechanical models of bundles of rods. This finding differs from the results observed with traditional polymeric materials. Understanding the mechanisms behind the viscoelastic properties of the gels formed by self-assembling molecules can lead to development of new materials with controlled stiffness. Tissue engineering applications can especially benefit from these materials, where the mechanical properties of the extracellular matrix are crucial for cell fate determination. © The Royal Society of Chemistry 2011

CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species

Mechanical forces in plant tissue matrix orient cell divisions via microtubule stabilization.

Plant morphogenesis relies exclusively on oriented cell expansion and division. Nonetheless, the mechanism(s) determining division plane orientation remain elusive. Here, we studied tissue healing after laser-assisted wounding in roots of Arabidopsis thaliana and uncovered how mechanical forces stabilize and reorient the microtubule cytoskeleton for the orientation of cell division. We identified that root tissue functions as an interconnected cell matrix, with a radial gradient of tissue extendibility causing predictable tissue deformation after wounding. This deformation causes instant redirection of expansion in the surrounding cells and reorientation of microtubule arrays, ultimately predicting cell division orientation. Microtubules are destabilized under low tension, whereas stretching of cells, either through wounding or external aspiration, immediately induces their polymerization. The higher microtubule abundance in the stretched cell parts leads to the reorientation of microtubule arrays and, ultimately, informs cell division planes. This provides a long-sought mechanism for flexible re-arrangement of cell divisions by mechanical forces for tissue reconstruction and plant architecture

A sensor kinase controls turgor-driven plant infection by the rice blast fungus

The blast fungus Magnaporthe oryzae gains entry to its host plant by means of a specialized pressure-generating infection cell called an appressorium, which physically ruptures the leaf cuticle. Turgor is applied as an enormous invasive force by septin-mediated reorganization of the cytoskeleton and actin-dependent protrusion of a rigid penetration hypha. However, the molecular mechanisms that regulate the generation of turgor pressure during appressorium-mediated infection of plants remain poorly understood. Here we show that a turgor-sensing histidine–aspartate kinase, Sln1, enables the appressorium to sense when a critical turgor threshold has been reached and thereby facilitates host penetration. We found that the Sln1 sensor localizes to the appressorium pore in a pressure-dependent manner, which is consistent with the predictions of a mathematical model for plant infection. A Δsln1 mutant generates excess intracellular appressorium turgor, produces hyper-melanized non-functional appressoria and does not organize the septins and polarity determinants that are required for leaf infection. Sln1 acts in parallel with the protein kinase C cell-integrity pathway as a regulator of cAMP-dependent signalling by protein kinase A. Pkc1 phosphorylates the NADPH oxidase regulator NoxR and, collectively, these signalling pathways modulate appressorium turgor and trigger the generation of invasive force to cause blast disease

Peptide nanofiber scaffolds for multipotent stromal cell culturing

Self-assembled peptide nanofibers are versatile materials providing suitable platforms for regenerative medicine applications. This chapter describes the use of peptide nanofibers as extracellular matrix mimetic scaffolds for two-dimensional (2D) and three-dimensional (3D) multipotent stromal cell culture systems and procedures for in vitro experiments using these scaffolds. Preparation of 2D and 3D peptide nanofiber scaffolds and cell culturing procedures are presented as part of in vitro experiments including cell adhesion, viability, and spreading analysis. Analysis of cellular differentiation on peptide nanofiber scaffolds is described through immunocytochemistry, qRT-PCR, and other biochemical experiments towards osteogenic and chondrogenic lineage. © Springer Science+Business Media New York 2013

Anti-Apoptotic Machinery Protects the Necrotrophic Fungus Botrytis cinerea from Host-Induced Apoptotic-Like Cell Death during Plant Infection

Necrotrophic fungi are unable to occupy living plant cells. How such pathogens survive first contact with living host tissue and initiate infection is therefore unclear. Here, we show that the necrotrophic grey mold fungus Botrytis cinerea undergoes massive apoptotic-like programmed cell death (PCD) following germination on the host plant. Manipulation of an anti-apoptotic gene BcBIR1 modified fungal response to PCD-inducing conditions. As a consequence, strains with reduced sensitivity to PCD were hyper virulent, while strains in which PCD was over-stimulated showed reduced pathogenicity. Similarly, reduced levels of PCD in the fungus were recorded following infection of Arabidopsis mutants that show enhanced susceptibility to B. cinerea. When considered together, these results suggest that Botrytis PCD machinery is targeted by plant defense molecules, and that the fungal anti-apoptotic machinery is essential for overcoming this host-induced PCD and hence, for establishment of infection. As such, fungal PCD machinery represents a novel target for fungicides and antifungal drugs

ATG8ylation of vacuolar membrane protects plants against cell wall damage

Vacuoles are essential for cellular metabolism and growth and the maintenance of internal turgor pressure. They sequester lytic enzymes, ions and secondary metabolites that, if leaked into the cytosol, could lead to cell death. Despite their pivotal roles, quality control pathways that safeguard vacuolar integrity have remained elusive in plants. Here we describe a conserved vacuolar quality control pathway that is activated upon cell wall damage in a turgor-pressure-dependent manner. Cell wall perturbations induce a distinct modification-ATG8ylation-on the vacuolar membrane (tonoplast) that is regulated by the V-ATPase and ATG8 conjugation machinery. Genetic disruption of tonoplast ATG8ylation impairs vacuolar integrity, leading to cell death. Together, our findings reveal a homeostatic pathway that preserves vacuolar integrity upon cell wall damage.We acknowledge funding from the Austrian Academy of Sciences, the Austrian Science Fund (FWF, P32355, P34944, ESP 580), the Austrian Science Fund (FWF‐SFB F79), the Vienna Science and Technology Fund (WWTF, LS17‐047, LS21‐009), a European Research Council Grant (project no. 101043370), the Vienna International Postdoctoral Program (VIP2) and a Marie Curie Fellowship to J.J. and J.C.D.L.C. (project no. 847548) and M.S. (project no. 101107472). M.B. and J.S. are funded by the European Research Council (ERC CoG Catch, project no. 101000981). Open access funding provided by Research Institute of Molecular Pathology (IMP) / Institute of Molecular Biotechnology (IMBA)/ Gregor Mendel Institute of Molecular Plant Biology (GMI).Peer reviewe