Michelle A. Baird

Improving FRET dynamic range with bright green and red fluorescent proteins

A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.

A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.

Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms

Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells.

Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics

Adding to the super-resolution arsenal Structured illumination microscopy (SIM) uses light intensities that are orders of magnitude lower than other super-resolution methods. SIM is also far faster over cellular-sized fields of view. Li et al. used two approaches to improve the resolution of SIM to allow live cell imaging of dynamic cellular processes, including endocytosis and cytoskeleton remodeling. The contrast in performance between SIM and other techniques is due to a few key differences. Defining the practical resolution at the limited signal-to-noise ratios necessary for live cell imaging will require better imaging metrics. Science, this issue 10.1126/science.aab3500 Super-resolution imaging of fast dynamic processes in living cells is facilitated by improvements to structured illumination microscopy. INTRODUCTION Various methods of super-resolution (SR) fluorescence microscopy have the potential to follow the dynamic nanoscale interactions of specific macromolecular assemblies in living cells. However, this potential is often left unfulfilled, either owing to the method’s inability to follow these processes at the speeds dictated by nature or because they require intense light that can substantially perturb the very physiology one hopes to study. An exception is structured illumination microscopy (SIM), which can image live cells far faster and with orders of magnitude less light than required for other SR approaches. However, SIM’s resolution is usually limited to only a twofold gain beyond conventional optical microscopes, or ~100 nm with visible light. RATIONALE We endeavored to find ways to extend SIM to the sub-100-nm regime while retaining, to the greatest extent possible, the advantages that make it the preferred SR method for live-cell imaging. Our first solution used an ultrahigh numerical aperture (NA) lens and total internal reflection fluorescence (TIRF) to achieve 84-nm resolution at subsecond acquisition speeds over hundreds of time points in multiple colors near the basal plasma membrane. Our second exploited the spatially patterned activation of a recently developed, reversibly photoswitchable fluorescent protein to reach 45- to 62-nm resolution, also at subsecond acquisition, over ∼10 to 40 time points. RESULTS We used high-NA TIRF-SIM to image the dynamic associations of cortical filamentous actin with myosin IIA, paxillin, or clathrin, as well as paxillin with vinculin and clathrin with transferrin receptors. Thanks to the combination of high spatial and temporal resolution, we were able to measure the sizes of individual clathrin-coated pits through their initiation, growth, and internalization. We were also able to relate pit size to lifetime, identify and characterize localized hot spots of pit generation, and describe the interaction of actin with clathrin and its role in accelerating endocytosis. With nonlinear SIM by use of patterned activation (PA NL-SIM), we monitored the remodeling of the actin cytoskeleton and the dynamics of caveolae at the cell surface. By combining TIRF-SIM and PA NL-SIM for two-color imaging, we followed the dynamic association of actin with α-actinin in expanding filopodia and membrane ruffles and characterized shape changes in and the transport of early endosomes. Last, by combining PA NL-SIM with lattice light sheet microscopy, we observed, in three dimensions and across the entire volume of whole cells, the dynamics of the actin cytoskeleton, the fusion and fission of mitochondria, and the trafficking of vesicles to and from the Golgi apparatus, each at axial resolution fivefold better than that of conventional widefield microscopy. In addition, through direct experimental comparisons, we demonstrated that the resolution for our methods is comparable with or better than other SR approaches yet allowed us to image at far higher speeds, and for far longer durations. To understand why this is so, we developed a detailed theoretical model showing that our methods transmit the information encoded in spatial frequencies beyond the diffraction limit with much greater strength than do other alternatives and hence require far fewer photons emitted from the specimen, using far less intense light. CONCLUSION High-NA TIRF-SIM and PA NL-SIM fill an unmet need for minimally invasive tools to image live cells in the gap between the 100-nm resolution traditionally associated with SIM and the sub-60-nm regime of protein-specific structural imaging served by single-molecule localization microscopy. Two approaches for improved live-cell imaging at sub-100-nm resolution. (Left) Association of cortical actin (purple) with clathrin-coated pits (green), the latter seen as rings (inset) at 84-nm resolution via a combination of total internal reflection fluorescence and structured illumination microscopy at ultrahigh numerical aperture (high-NA TIRF-SIM). (Right) Progression of resolution improvement across the actin cytoskeleton of a COS-7 cell, from conventional, diffraction-limited TIRF (220-nm resolution), to TIRF-SIM (97-nm resolution), and nonlinear SIM based on the patterned activation of a reversibly photoswitchable fluorescent protein (PA NL-SIM, 62 nm resolution). (Left and right represent single frames from time-lapse movies over 91 and 30 frames, respectively. Scale bars, 2 μm (left); 3 μm (right). Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions

Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin’s interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function.

A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells

A contractile actomyosin meshwork at the top of a cell is mechanically coupled to dorsal actin fibers that are anchored via focal adhesions to the cell surface, generating a counterbalanced adhesion/contraction system that drives cell shape changes.

NMII Forms a Contractile Transcellular Sarcomeric Network to Regulate Apical Cell Junctions and Tissue Geometry

Nonmuscle myosin II (NMII) is thought to be the master integrator of force within epithelial apical junctions, mediating epithelial tissue morphogenesis and tensional homeostasis. Mutations in NMII are associated with a number of diseases due to failures in cell-cell adhesion. However, the organization and the precise mechanism by which NMII generates and responds to tension along the intercellular junctional line are still not known. We discovered that periodic assemblies of bipolar NMII filaments interlace with perijunctional actin and α-actinin to form a continuous belt of muscle-like sarcomeric units (∼400-600 nm) around each epithelial cell. Remarkably, the sarcomeres of adjacent cells are precisely paired across the junctional line, forming an integrated, transcellular contractile network. The contraction/relaxation of paired sarcomeres concomitantly impacts changes in apical cell shape and tissue geometry. We show differential distribution of NMII isoforms across heterotypic junctions and evidence for compensation between isoforms. Our results provide a model for how NMII force generation is effected along the junctional perimeter of each cell and communicated across neighboring cells in the epithelial organization. The sarcomeric network also provides a well-defined target to investigate the multiple roles of NMII in junctional homeostasis as well as in development and disease.

mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities

Recent advances in fluorescence microscopy have extended the spatial resolution to the nanometer scale. Here, we report an engineered photoconvertible fluorescent protein (pcFP) variant, designated as mMaple, that is suited for use in multiple conventional and super-resolution imaging modalities, specifically, widefield and confocal microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy. We demonstrate the versatility of mMaple by obtaining super-resolution images of protein organization in Escherichia coli and conventional fluorescence images of mammalian cells. Beneficial features of mMaple include high photostability of the green state when expressed in mammalian cells and high steady state intracellular protein concentration of functional protein when expressed in E. coli. mMaple thus enables both fast live-cell ensemble imaging and high precision single molecule localization for a single pcFP-containing construct.

Fixation-resistant photoactivatable fluorescent proteins for CLEM

Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5–1% OsO4), plastic resin–embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they cease to function following heavy fixation, hindering advanced applications such as correlative light and electron microscopy. Here we report engineered variants of the photoconvertible Eos fluorescent protein that function normally in heavily fixed (0.5–1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

Talin determines the nanoscale architecture of focal adhesions

Significance Focal adhesions (FAs) mediate cell–extracellular matrix interactions and consist of integrin receptors linked to the actin cytoskeleton via multiprotein complexes organized into nanoscale strata. In this work, we sought to determine the molecular basis of FA nanostructure. Combining superresolution microscopy and protein engineering, we demonstrate the structural role of talin in regulating the nanoscale architecture of FAs. Talin specifies the dimension of the FA core, akin to a molecular ruler, in a remarkably modular manner. Our results define the molecular geometry of the integrin–talin–actin module that comprises the key mechanical linkage within FAs and elucidate how such interactions serve to integrate multiple cellular forces at adhesion sites. Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin–talin–actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.