Kareem Elsayad

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

Fluorescence-Brillouin imaging reveals that plants regulate the mechanical properties of the extracellular matrix in response to light. Seeing mechanical properties of living cells Mechanical properties of cells and the matrix that surrounds them contribute to cell shape, control cell migration, and regulate cell growth. Elsayad et al. engineered a microscope system that integrated fluorescence emission detection with detection of a light-scattering process called the Brillouin frequency shift and called the method fluorescence emission–Brillouin scattering imaging (FBi). With this optical approach, the authors showed that the mechanical properties of live plants can be visualized at the submicrometer scale and demonstrated that this approach can be used to investigate regulatory events that alter cellular and extracellular mechanical properties of living cells within tissues. This work also revealed that the cytoplasm near the cell membrane and the extracellular matrix are regions of locally increased stiffness and showed that the sides parallel to the growth axis of an expanding plant hypocotyl, but not root, cells are “stiffer” than the sides perpendicular to the growth axis. Thus, FBi is another tool in the microscopy toolkit for exploring properties of cells and tissues. 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 β‐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. J. Comp. Neurol. 523:2161–2186, 2015.

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

Abstract 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.

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

Significance In this paper we describe a high-resolution light microscopy technique that translates spatial (position) information of fluorescent markers into spectral (color) information for improved biological imaging. By designing a thin biocompatible nanostructure on a microscope slide, we show how the distance-dependent spectral “fingerprint” of fluorophores can be used to monitor their relative distance from the nanostructure with an accuracy far beyond the resolution power of a conventional light microscope. We demonstrate the technique by studying the positions and dynamics of key proteins that play a role in cell motility. 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 ( nm) or too far ( 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.

β-Catenin–dependent mechanotransduction dates back to the common ancestor of Cnidaria and Bilateria

Significance Besides genetic regulation, mechanical forces have been identified as important cues in numerous developmental processes. Mechanical forces can activate biochemical cascades in a process called mechanotransduction. Recent studies in vertebrates and flies elucidated the role of mechanical forces for mesodermal gene expression. However, it remains unclear whether mechanotransduction is a universal regulatory mechanism throughout Metazoa. Here, we show in the sea anemone Nematostella vectensis that mechanical pressure can ectopically activate or restore brachyury expression. This mechanotransduction is dependent on β-catenin, similar to vertebrates. We propose that a regulatory feedback loop between genetic and mechanical gene activation exists during gastrulation and the β-catenin–dependent mechanotransduction is an ancient regulatory mechanism, which was present in the common ancestor of cnidarians and bilaterians. 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 β-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 β-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.

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

ABSTRACT 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.

Frequency Extrapolation of Mechanical Properties Obtained from Brillouin Scattering Measurements in Biological Samples

Brillouin Light Scattering Microspectroscopy (BLSM) is an all optical technique that can be used to yield information on the high frequency (GHz) mechanical properties of materials (Longitudinal Modulus, Shear Modulus). Results from BLSM measurements are thus distinct from those obtained using lower frequency perturbation or correlation based approaches, often yielding their interpretation or direct comparison challenging. Here we compare studies of the frequency dependence of the mechanical parameters (Shear Modulus) up to the MHz-range - derived from Fluorescence Correlation Spectroscopy (FCS) studies - to the corresponding BLSM obtained elastic parameters, for a range of biological samples.

Fluorescence enhancements and spectral modifications near the cut-off frequency of plasmonic structure

The effect of a population of fluorophores coupling to weakly bound surface plasmons in dielectric/metal/dielectric structures is investigated for the purpose of fluorescence enhancement near interfaces and live cell fluorescence surface imaging. We show theoretically and experimentally that for sufficient fluorophore concentrations near such SPP supporting structures significant enhancements in the radiative emission intensity can be observed, with a spectral modification that can be correlated to the average separation of the fluorophores from the substrate. We will discuss the theory behind the effect and some experimental results on imaging labeled proteins in the focal adhesion sites of cells.