Stephen E. D. Webb

Cell wall constrains lateral diffusion of plant plasma-membrane proteins

A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein–protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.

Focal adhesions are sites of integrin extension

FRET experiments model integrin conformation changes in adherent cells.

Uniform patchy and hollow rectangular platelet micelles from crystallizable polymer blends

Growing patterned rectangular objects The growth of patterned objects usually requires a template to aid the positioning of multiple materials. Qiu et al. used the seeded growth of a crystallizable block copolymer and a homopolymer to produce highly uniform rectangular structures (see the Perspective by Ballauff). Chemical etching, or dissolution, of uncross-linked regions of the rectangular structures produced perforated platelet micelles. The sequential addition of different blends and cross-linking/dissolution strategies allowed the formation of well-defined hollow rectangular micelles, which can be functionalized in a variety of ways. Science, this issue p. 697; see also p. 656 Crystallization-driven living supramolecular polymerization can selectively form rectangular objects. The preparation of colloidally stable, self-assembled materials with tailorable solid or hollow two-dimensional (2D) structures represents a major challenge. We describe the formation of uniform, monodisperse rectangular platelet micelles of controlled size by means of seeded-growth methods that involve the addition of blends of crystalline-coil block copolymers and the corresponding crystalline homopolymer to cylindrical micelle seeds. Sequential addition of different blends yields solid platelet block comicelles with concentric rectangular patches with distinct coronal chemistries. These complex nano-objects can be subject to spatially selective processing that allows their disassembly to form perforated platelets, such as well-defined hollow rectangular rings. The solid and hollow 2D micelles provide a tunable platform for further functionalization and potential for a variety of applications.

A ‘pocket guide’ to total internal reflection fluorescence

The phenomenon of total internal reflection fluorescence (TIRF) was placed in the context of optical microscopy by Daniel Axelrod over three decades ago. TIRF microscopy exploits the properties of an evanescent electromagnetic field to optically section sample regions in the close vicinity of the substrate where the field is induced. The first applications in cell biology targeted investigation of phenomena at the basolateral plasma membrane. The most notable application of TIRF is single‐molecule experiments, which can provide information on fluctuation distributions and rare events, yielding novel insights on the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell. This short review intends to provide a ‘one stop shop’ explanation of the electromagnetic theory behind the remarkable properties of the evanescent field, guide the reader through the principles behind building or choosing your own TIRF system and consider how the most popular applications of the method exploit the evanescent field properties.

Automated multidimensional single molecule fluorescence microscopy feature detection and tracking

Characterisation of multi-protein interactions in cellular networks can be achieved by optical microscopy using multidimensional single molecule fluorescence imaging. Proteins of different species, individually labelled with a single fluorophore, can be imaged as isolated spots (features) of different colour light in different channels, and their diffusive behaviour in cells directly measured through time. Challenges in data analysis have, however, thus far hindered its application in biology. A set of methods for the automated analysis of multidimensional single molecule microscopy data from cells is presented, incorporating Bayesian segmentation-based feature detection, image registration and particle tracking. Single molecules of different colours can be simultaneously detected in noisy, high background data with an arbitrary number of channels, acquired simultaneously or time-multiplexed, and then tracked through time. The resulting traces can be further analysed, for example to detect intensity steps, count discrete intensity levels, measure fluorescence resonance energy transfer (FRET) or changes in polarisation. Examples are shown illustrating the use of the algorithms in investigations of the epidermal growth factor receptor (EGFR) signalling network, a key target for cancer therapeutics, and with simulated data.

Subcellular and single-molecule imaging of plant fluorescent proteins using total internal reflection fluorescence microscopy (TIRFM)

Total internal reflection fluorescence microscopy (TIRFM) has been proven to be an extremely powerful technique in animal cell research for generating high contrast images and dynamic protein conformation information. However, there has long been a perception that TIRFM is not feasible in plant cells because the cell wall would restrict the penetration of the evanescent field and lead to scattering of illumination. By comparative analysis of epifluorescence and TIRF in root cells, it is demonstrated that TIRFM can generate high contrast images, superior to other approaches, from intact plant cells. It is also shown that TIRF imaging is possible not only at the plasma membrane level, but also in organelles, for example the nucleus, due to the presence of the central vacuole. Importantly, it is demonstrated for the first time that this is TIRF excitation, and not TIRF-like excitation described as variable-angle epifluorescence microscopy (VAEM), and it is shown how to distinguish the two techniques in practical microscopy. These TIRF images show the highest signal-to-background ratio, and it is demonstrated that they can be used for single-molecule microscopy. Rare protein events, which would otherwise be masked by the average molecular behaviour, can therefore be detected, including the conformations and oligomerization states of interacting proteins and signalling networks in vivo. The demonstration of the application of TIRFM and single-molecule analysis to plant cells therefore opens up a new range of possibilities for plant cell imaging.

Multidimensional single-molecule imaging in live cells using total-internal-reflection fluorescence microscopy

We have developed a wide-field total-internal-reflection fluorescence microscope capable of imaging single molecules in live cells, resolved in both wavelength and polarization. We show fluorescence resonance energy transfer between single pairs of fluorescent molecules bound to signaling receptors in the plasma membrane of live cells and demonstrate the importance of polarization discrimination in addition to wavelength separation.

Interaction of metastasis-inducing S100A4 protein in vivo by fluorescence lifetime imaging microscopy

Elevated levels of the calcium-binding regulatory protein, S100A4, have been shown to be causative of a metastatic phenotype in models of cancer metastasis and to be associated with reduced patient survival in breast cancer patients. Recombinant S100A4 protein interacts in vitro in a calcium-dependent manner with the heavy chain of non-muscle myosin isoform A at a protein kinase C phosphorylation site. At present, the mechanism of metastasis induction by S100A4 in vivo is almost completely unknown. The binding of S100A4 to a C-terminal recombinant fragment of non-muscle myosin heavy chain in living HeLa cells has now been shown using confocal microscopy, fluorescence lifetime imaging microscopy and time-correlated single-photon counting. The association between S100A4 and non-muscle myosin heavy chain was studied by determining fluorescence resonance energy transfer-derived changes in the fluorescence lifetime of enhanced cyan fluorescent protein fused to S100A4 in the presence of a recombinant fragment of the C-terminal region of non-muscle myosin heavy chain (rNMMHCIIA) fused to enhanced yellow fluorescent protein. There was no interaction between the non-muscle myosin heavy chain fragment and a calcium-binding-deficient mutant of S100A4 protein which has been shown to be defective in the induction of metastasis in model systems in vivo. The results demonstrate, for the first time, not only direct interaction between S100A4 and a target rNMMHCIIA in live mammalian cells, but also that the interaction between S100A4 and the non-muscle myosin heavy chain in vivo could contribute to the mechanism of metastasis induction by a high level of S100A4 protein.

Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes

Background The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging. Methodology/Principal Findings A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells. Conclusions/Significance We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470–1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging.

In Situ Visualization of Block Copolymer Self-Assembly in Organic Media by Super-Resolution Fluorescence Microscopy.

Abstract Analytical methods that enable visualization of nanomaterials derived from solution self‐assembly processes in organic solvents are highly desirable. Herein, we demonstrate the use of stimulated emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallization‐driven block copolymer (BCP) self‐assembly in organic media at the sub‐diffraction scale. Four different dyes were successfully used for single‐colour super‐resolution imaging of the BCP nanostructures allowing micelle length distributions to be determined in situ. Dual‐colour SMLM imaging was used to measure and compare the rate of addition of red fluorescent BCP to the termini of green fluorescent seed micelles to generate block comicelles. Although well‐established for aqueous systems, the results highlight the potential of super‐resolution microscopy techniques for the interrogation of self‐assembly processes in organic media.