Hormone-regulated expansins: Expression, localization, and cell wall biomechanics in Arabidopsis root growth.

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young's modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fourier-transform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.

PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing.

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.

Leaky and waveguide modes in biperiodic holograms.

This study details a theoretical analysis of leaky and waveguide modes in biperiodic all-dielectric holograms. By tuning diffraction orders and subsequently confining local density of optical states at two distinct resonance wavelengths, we present a new class of highly sensitive refractive index biosensing platforms that are capable of resolving 35.5 to 41.3 nm/RIU of spectral shift for two separate biological analytes.

Direct measurement of protein-protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells.

Protein-protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein-protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein-protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein-protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein-protein interactions at DNA damage sites.

Recent progress and current opinions in Brillouin microscopy for life science applications.

Many important biological functions and processes are reflected in cell and tissue mechanical properties such as elasticity and viscosity. However, current techniques used for measuring these properties have major limitations, such as that they can often not measure inside intact cells and/or require physical contact-which cells can react to and change. Brillouin light scattering offers the ability to measure mechanical properties in a non-contact and label-free manner inside of objects with high spatial resolution using light, and hence has emerged as an attractive method during the past decade. This new approach, coined "Brillouin microscopy," which integrates highly interdisciplinary concepts from physics, engineering, and mechanobiology, has led to a vibrant new community that has organized itself via a European funded (COST Action) network. Here we share our current assessment and opinion of the field, as emerged from a recent dedicated workshop. In particular, we discuss the prospects towards improved and more bio-compatible instrumentation, novel strategies to infer more accurate and quantitative mechanical measurements, as well as our current view on the biomechanical interpretation of the Brillouin spectra.

Optical imaging spectroscopy for plant research: more than a colorful picture.

Optical imaging is a routine and indispensable tool in plant research. Here we review different emerging spectrally resolved optical imaging approaches and the wealth of information they can be used to obtain pertaining to the underlying chemistry, structure and mechanics of plants.

Brillouin Light Scattering Microspectroscopy for Biomedical Research and Applications: introduction to feature issue.

There has been a marked revival of interest in brillouin light scattering spectroscopy/microscopy over the last decade in regards to applications related to all optically studying the mechanical problems associated with systems of biological and medical interest. This revival has been driven by advancements in spectrometer design, together with mounting evidence of the critical role that mechanical properties can play in biological processes as well as the onset of diverse diseases. This feature issue contains a series of papers spanning some of the latest developments in the field of Brillouin light scattering spectroscopy and microscopy as applied to systems of biomedical interest.

Hydration and nanomechanical changes in collagen fibrils bearing advanced glycation end-products.

Accumulation of advanced glycation end-products (AGEs) in biological tissues occurs as a consequence of normal ageing and pathology. Most biological tissues are composed of considerable amounts of collagen, with collagen fibrils being the most abundant form. Collagen fibrils are the smallest discernible structural elements of load-bearing tissues and as such, they are of high biomechanical importance. The low turnover of collagen cause AGEs to accumulate within the collagen fibrils with normal ageing as well as in pathologies. We hypothesized that collagen fibrils bearing AGEs have altered hydration and mechanical properties. To this end, we employed atomic force and Brillouin light scattering microscopy to measure the extent of hydration as well as the transverse elastic properties of collagen fibrils treated with ribose. We find that hydration is different in collagen fibrils bearing AGEs and this is directly related to their mechanical properties. Collagen fibrils treated with ribose showed increased hydration levels and decreased transverse stiffness compared to controlled samples. Our results show that BLS and AFM yield complementary evidence on the effect of hydration on the nanomechanical properties of collagen fibrils.

Probing Mechanical Properties in Biology Using Brillouin Microscopy.

Brillouin microscopy can be used to map the mechanical properties of samples in a noncontact and label-free manner, with potential applications in cell biology. Here, we provide an overview of the underlying principles and technology as well as the current challenges and outlook.

Simultaneous dual-channel imaging to quantify interdependent protein recruitment to laser-induced DNA damage sites.

Fluorescence microscopy in combination with the induction of localized DNA damage using focused light beams has played a major role in the study of protein recruitment kinetics to DNA damage sites in recent years. Currently published methods are dedicated to the study of single fluorophore/single protein kinetics. However, these methods may be limited when studying the relative recruitment dynamics between two or more proteins due to cell-to-cell variability in gene expression and recruitment kinetics, and are not suitable for comparative analysis of fast-recruiting proteins. To tackle these limitations, we have established a time-lapse fluorescence microscopy method based on simultaneous dual-channel acquisition following UV-A-induced local DNA damage coupled with a standardized image and recruitment analysis workflow. Simultaneous acquisition is achieved by spectrally splitting the emitted light into two light paths, which are simultaneously imaged on two halves of the same camera chip. To validate this method, we studied the recruitment of poly(ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) glycohydrolase (PARG), proliferating cell nuclear antigen (PCNA) and the chromatin remodeler ALC1. In accordance with the published data based on single fluorophore imaging, simultaneous dual-channel imaging revealed that PARP1 regulates fast recruitment and dissociation of PARG and that in PARP1-depleted cells PARG and PCNA are recruited with comparable kinetics. This approach is particularly advantageous for analyzing the recruitment sequence of fast-recruiting proteins such as PARP1 and ALC1, and revealed that PARP1 is recruited faster than ALC1. Split-view imaging can be incorporated into any laser microirradiation-adapted microscopy setup together with a recruitment-dedicated image analysis package.

ß-Catenin-dependent mechanotransduction dates back to the common ancestor of Cnidaria and Bilateria.

Although the genetic regulation of cellular differentiation processes is well established, recent studies have revealed the role of mechanotransduction on a variety of biological processes, including regulation of gene expression. However, it remains unclear how universal and widespread mechanotransduction is in embryonic development of animals. Here, we investigate mechanosensitive gene expression during gastrulation of the starlet sea anemone Nematostella vectensis, a cnidarian model organism. We show that the blastoporal marker gene brachyury is down-regulated by blocking myosin II-dependent gastrulation movements. Brachyury expression can be restored by applying external mechanical force. Using CRISPR/Cas9 and morpholino antisense technology, we also show that mechanotransduction leading to brachyury expression is ß-catenin dependent, similar to recent findings in fish and Drosophila [Brunet T, et al. (2013) Nat Commun 4:1-15]. Finally, we demonstrate that prolonged application of mechanical stress on the embryo leads to ectopic brachyury expression. Thus, our data indicate that ß-catenin-dependent mechanotransduction is an ancient gene regulatory mechanism, which was present in the common ancestor of cnidarians and bilaterians, at least 600 million years ago.

Axicon-based Bessel beams for flat-field illumination in total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy (TIRF-M) provides low-invasive high-contrast surface imaging with optical sectioning of typically 100-200 nm. Thus, TIRF-M has become an established tool for imaging surfaces, including cell membranes. For TIRF-M, a homogenous evanescent field of excitation over the whole field of view is generally desired for quantitative microscopy; however, this is not necessarily straightforward to generate with Gaussian beams. In recent years, several improvements on TIRF-M have been developed that have addressed non-uniform scattering fringes and other artifacts. Here, we introduce a cost-effective TIRF setup with a very low degree of complexity and no moving parts, which provides a flattop-like excitation profile. The setup uses a tunable laser ring zoom focus system to generate a full 360° TIRF illumination. Two axicon lenses and one focus lens allow for generation and control of the ring diameter to tune the TIRF excitation angle. We show that 360° laser illumination in combination with a radial polarizer will generate an evanescent Bessel-beam excitation field that exhibits a flattop intensity over an extended part of the field of view, and demonstrate the advantages of this axicon-based Bessel beam illumination for live-cell imaging.

A novel non-canonical PIP-box mediates PARG interaction with PCNA.

Poly(ADP-ribose) glycohydrolase (PARG) regulates cellular poly(ADP-ribose) (PAR) levels by rapidly cleaving glycosidic bonds between ADP-ribose units. PARG interacts with proliferating cell nuclear antigen (PCNA) and is strongly recruited to DNA damage sites in a PAR- and PCNA-dependent fashion. Here we identified PARG acetylation site K409 that is essential for its interaction with PCNA, its localization within replication foci and its recruitment to DNA damage sites. We found K409 to be part of a non-canonical PIP-box within the PARG disordered regulatory region. The previously identified putative N-terminal PIP-box does not bind PCNA directly but contributes to PARG localization within replication foci. X-ray structure and MD simulations reveal that the PARG non-canonical PIP-box binds PCNA in a manner similar to other canonical PIP-boxes and may represent a new type of PIP-box. While the binding of previously described PIP-boxes is based on hydrophobic interactions, PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Our data explain the mechanism of PARG-PCNA interaction through a new PARG PIP-box that exhibits non-canonical sequence properties but a canonical mode of PCNA binding.

Mapping the subcellular mechanical properties of live cells in tissues with fluorescence emission-Brillouin imaging.

Extracellular matrices (ECMs) are central to the advent of multicellular life, and their mechanical properties are modulated by and impinge on intracellular signaling pathways that regulate vital cellular functions. High spatial-resolution mapping of mechanical properties in live cells is, however, extremely challenging. Thus, our understanding of how signaling pathways process physiological signals to generate appropriate mechanical responses is limited. We introduce fluorescence emission-Brillouin scattering imaging (FBi), a method for the parallel and all-optical measurements of mechanical properties and fluorescence at the submicrometer scale in living organisms. Using FBi, we showed that changes in cellular hydrostatic pressure and cytoplasm viscoelasticity modulate the mechanical signatures of plant ECMs. We further established that the measured "stiffness" of plant ECMs is symmetrically patterned in hypocotyl cells undergoing directional growth. Finally, application of this method to Arabidopsis thaliana with photoreceptor mutants revealed that red and far-red light signals are essential modulators of ECM viscoelasticity. By mapping the viscoelastic signatures of a complex ECM, we provide proof of principle for the organism-wide applicability of FBi for measuring the mechanical outputs of intracellular signaling pathways. As such, our work has implications for investigations of mechanosignaling pathways and developmental biology.

The Expression of Tubb2b Undergoes a Developmental Transition in Murine Cortical Neurons.

The development of the mammalian brain requires the generation, migration, and differentiation of neurons, cellular processes that are dependent on a dynamic microtubule cytoskeleton. Mutations in tubulin genes, which encode for the structural subunits of microtubules, cause detrimental neurological disorders known as the tubulinopathies. The disease spectra associated with different tubulin genes are overlapping but distinct, an observation believed to reflect functional specification of this multigene family. Perturbation of the ß-tubulin TUBB2B is known to cause polymicrogyria, pachygyria, microcephaly, and axon guidance defects. Here we provide a detailed analysis of the expression pattern of its murine homolog Tubb2b. The generation and characterization of BAC-transgenic eGFP reporter mouse lines has revealed that it is highly expressed in progenitors and postmitotic neurons during cortical development. This contrasts with the 8-week-old cortex, in which Tubb2b expression is restricted to macroglia, and expression is almost completely absent in mature neurons. This developmental transition in neurons is mirrored in the adult hippocampus and the cerebellum but is not a universal feature of Tubb2b; its expression persists in a population of postmitotic neurons in the 8-week-old retina. We propose that the dynamic spatial and temporal expression of Tubb2b reflects specific functional requirements of the microtubule cytoskeleton.

Spectrally coded optical nanosectioning (SpecON) with biocompatible metal-dielectric-coated substrates.

Fluorescence nanosectioning within a submicron region above an interface is desirable for many disciplines in the life sciences. A drawback, however, to most current approaches is the a priori need to physically scan a sculptured point spread function in the axial dimension, which can be undesirable for optically sensitive or highly dynamic samples. Here we demonstrate a fluorescence imaging approach that can overcome the need for scanning by exploiting the position-dependent emission spectrum of fluorophores above a simple biocompatible nanostructure. To achieve this we have designed a thin metal-dielectric-coated substrate, where the spectral modification to the total measured fluorescence can be used to estimate the axial fluorophore distribution within distances of 10-150 nm above the substrate with an accuracy of up to 5-10 nm. The modeling and feasibility of the approach are verified and successfully applied to elucidate nanoscale adhesion protein and filopodia dynamics in migrating cells. It is likely that the general principle can find broader applications in, for example, single-molecule studies, biosensing, and studying fast dynamic processes.

Photounbinding of calmodulin from a family of CaM binding peptides.

BACKGROUND: Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding. METHODOLOGY/PRINCIPAL FINDINGS: We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked. CONCLUSIONS/SIGNIFICANCE: The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner.

Defining a superlens operating regime for imaging fluorescent molecules.

It has been shown that thin metal-based films can at certain frequencies act as planar near-field lenses for certain polarization components. A desirable property of such "lenses" is that they can also enhance and focus some large transverse spatial frequency components which contain sub-diffraction limit details. Over the last decade there has been much work in optimizing designs to reduce effects (such as material losses and surface roughness) that are detrimental to image reconstruction. One design that can reduce some of these undesirable effects, and which has received a fair amount of attention recently, is the stacked metal-dielectric superlens. Here we theoretically explore the imaging ability of such a design for the specific purpose of imaging a fluorescent dye (the common bio-marker GFP) in the vicinity of the superlens surface. Our calculations take into consideration the interaction (damping) of an oscillating electric dipole with the metallic layers in the superlens. We also assume a Gaussian frequency distribution spectrum for the dipole. We treat the metallic-alloy and dielectric-alloy layers separately using an appropriate effective medium theory. The transmission properties are evaluated via Transfer matrix (-matrix) calculations that were performed in the MatLab and MathCad environments. Our study shows that it is in principle possible to image fluorescent molecules using a simple bilayer planar superlens. We find that optimal parameters for such a superlens occur when the peak dipole emission-frequency is slightly offset from the Surface Plasmon resonance frequency of the metal-dielectric interfaces. The best resolution is obtained when the fluorescent molecules are not too close (>/ approximately 10 nm) or too far (>/approximately 30 nm) from the superlens surface. The realization and application of a superlens with the specified design is possible using current nanofabrication techniques. When combined with e.g. a sub-wavelength grating structure (such as in the far-field superlens design previously proposed [1]) or a fast near-field scanning probe, it could provide a means for fast fluorescent imaging with sub-diffraction limit resolution.