Combined Selective Plane Illumination Microscopy and FRAP Maps Intranuclear Diffusion of NLS-GFP.

Since its initial development in 1976, fluorescence recovery after photobleaching (FRAP) has been one of the most popular tools for studying diffusion and protein dynamics in living cells. Its popularity is derived from the widespread availability of confocal microscopes and the relative ease of the experiment and analysis. FRAP, however, is limited in its ability to resolve spatial heterogeneity. Here, we combine selective plane illumination microscopy (SPIM) and FRAP to create SPIM-FRAP, wherein we use a sheet of light to bleach a two-dimensional (2D) plane and subsequently image the recovery of the same image plane. This provides simultaneous quantification of diffusion or protein recovery for every pixel in a given 2D slice, thus moving FRAP measurements beyond these previous limitations. We demonstrate this technique by mapping both intranuclear diffusion of NLS-GFP and recovery of 53BP1-mCherry, a marker for DNA damage, in live MDA-MB-231 cells. SPIM-FRAP proves to be an order of magnitude faster than fluorescence-correlation-spectroscopy-based techniques for such measurements. We observe large length-scale (>∼500 nm) heterogeneity in the recovery times of NLS-GFP, which is validated against simulated data sets. 2D maps of NLS-GFP recovery times showed no pixel-by-pixel correlation with histone density, although slower diffusion was observed in nucleoli. Additionally, recovery of 53BP1-mCherry was observed to be slowed at sites of DNA damage. We finally developed a diffusion simulation for our SPIM-FRAP experiments to compare across techniques. Our measured diffusion coefficients are on the order of previously reported results, thus validating the quantitative accuracy of SPIM-FRAP relative to well-established methods. With the recent rise of accessibility of SPIM systems, SPIM-FRAP is set to provide a straightforward means of quantifying the spatial distribution of protein recovery or diffusion in living cells.

Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics.

Nuclei are often under external stress, be it during migration through tight constrictions or compressive pressure by the actin cap, and the mechanical properties of nuclei govern their subsequent deformations. Both altered mechanical properties of nuclei and abnormal nuclear morphologies are hallmarks of a variety of disease states. Little work, however, has been done to link specific changes in nuclear shape to external forces. Here, we utilize a combined atomic force microscope and light sheet microscope to show SKOV3 nuclei exhibit a two-regime force response that correlates with changes in nuclear volume and surface area, allowing us to develop an empirical model of nuclear deformation. Our technique further decouples the roles of chromatin and lamin A/C in compression, showing they separately resist changes in nuclear volume and surface area, respectively; this insight was not previously accessible by Hertzian analysis. A two-material finite element model supports our conclusions. We also observed that chromatin decompaction leads to lower nuclear curvature under compression, which is important for maintaining nuclear compartmentalization and function. The demonstrated link between specific types of nuclear morphological change and applied force will allow researchers to better understand the stress on nuclei throughout various biological processes.

Vinculin and metavinculin exhibit distinct effects on focal adhesion properties, cell migration, and mechanotransduction.

Vinculin (Vcn) is a ubiquitously expressed cytoskeletal protein that links transmembrane receptors to actin filaments, and plays a key role in regulating cell adhesion, motility, and force transmission. Metavinculin (MVcn) is a Vcn splice isoform that contains an additional exon encoding a 68-residue insert within the actin binding tail domain. MVcn is selectively expressed at sub-stoichiometic amounts relative to Vcn in smooth and cardiac muscle cells. Mutations in the MVcn insert are linked to various cardiomyopathies. In vitro analysis has previously shown that while both proteins can engage filamentous (F)-actin, only Vcn can promote F-actin bundling. Moreover, we and others have shown that MVcn can negatively regulate Vcn-mediated F-actin bundling in vitro. To investigate functional differences between MVcn and Vcn, we stably expressed either Vcn or MVcn in Vcn-null mouse embryonic fibroblasts. While both MVcn and Vcn were observed at FAs, MVcn-expressing cells had larger but fewer focal adhesions per cell compared to Vcn-expressing cells. MVcn-expressing cells migrated faster and exhibited greater persistence compared to Vcn-expressing cells, even though Vcn-containing FAs assembled and disassembled faster. Magnetic tweezer measurements on Vcn-expressing cells show a typical cell stiffening phenotype in response to externally applied force; however, this was absent in Vcn-null and MVcn-expressing cells. Our findings that MVcn expression leads to larger but fewer FAs per cell, in conjunction with the inability of MVcn to bundle F-actin in vitro and rescue the cell stiffening response, are consistent with our previous findings of actin bundling deficient Vcn variants, suggesting that deficient actin-bundling may account for some of the differences between Vcn and MVcn.

Microtubule Acetylation Is Required for Mechanosensation in Drosophila.

At the cellular level, α-tubulin acetylation alters the structure of microtubules to render them mechanically resistant to compressive forces. How this biochemical property of microtubule acetylation relates to mechanosensation remains unknown, although prior studies have shown that microtubule acetylation influences touch perception. Here, we identify the major Drosophila α-tubulin acetylase (dTAT) and show that it plays key roles in several forms of mechanosensation. dTAT is highly expressed in the larval peripheral nervous system (PNS), but it is largely dispensable for neuronal morphogenesis. Mutation of the acetylase gene or the K40 acetylation site in α-tubulin impairs mechanical sensitivity in sensory neurons and behavioral responses to gentle touch, harsh touch, gravity, and vibration stimuli, but not noxious thermal stimulus. Finally, we show that dTAT is required for mechanically induced activation of NOMPC, a microtubule-associated transient receptor potential channel, and functions to maintain integrity of the microtubule cytoskeleton in response to mechanical stimulation.

Site-specific glycosylation regulates the form and function of the intermediate filament cytoskeleton.

Intermediate filaments (IF) are a major component of the metazoan cytoskeleton and are essential for normal cell morphology, motility, and signal transduction. Dysregulation of IFs causes a wide range of human diseases, including skin disorders, cardiomyopathies, lipodystrophy, and neuropathy. Despite this pathophysiological significance, how cells regulate IF structure, dynamics, and function remains poorly understood. Here, we show that site-specific modification of the prototypical IF protein vimentin with O-linked ß-N-acetylglucosamine (O-GlcNAc) mediates its homotypic protein-protein interactions and is required in human cells for IF morphology and cell migration. In addition, we show that the intracellular pathogen Chlamydia trachomatis, which remodels the host IF cytoskeleton during infection, requires specific vimentin glycosylation sites and O-GlcNAc transferase activity to maintain its replicative niche. Our results provide new insight into the biochemical and cell biological functions of vimentin O-GlcNAcylation, and may have broad implications for our understanding of the regulation of IF proteins in general.

Analysis-aware microscopy video compression.

This article introduces an analysis-aware microscopy video compression method designed for microscopy videos that are consumed by analysis algorithms rather than by the human visual system. We define the quality of a microscopy video based on the level of preservation of analysis results. We evaluated our method with a bead tracking analysis program. For the same error level in the analysis result, our method can achieve 1,000× compression on certain test microscopy videos. Compared with a previous technique that yields exactly the exact same results by analysis algorithms, our method gives more flexibility for a user to control the quality. A modification to the new method also provides faster compression speed.

Vertical Light Sheet Enhanced Side-View Imaging for AFM Cell Mechanics Studies.

The ability to measure dynamic structural changes within a cell under applied load is essential for developing more accurate models of cell mechanics and mechanotransduction. Atomic force microscopy is a powerful tool for evaluating cell mechanics, but the dominant applied forces and sample strains are in the vertical direction, perpendicular to the imaging plane of standard fluorescence imaging. Here we report on a combined sideways imaging and vertical light sheet illumination system integrated with AFM. Our system enables high frame rate, low background imaging of subcellular structural dynamics in the vertical plane synchronized with AFM force data. Using our system for cell compression measurements, we correlated stiffening features in the force indentation data with onset of nuclear deformation revealed in the imaging data. In adhesion studies we were able to correlate detailed features in the force data during adhesive release events with strain at the membrane and within the nucleus.

An Automated High-throughput Array Microscope for Cancer Cell Mechanics.

Changes in cellular mechanical properties correlate with the progression of metastatic cancer along the epithelial-to-mesenchymal transition (EMT). Few high-throughput methodologies exist that measure cell compliance, which can be used to understand the impact of genetic alterations or to screen the efficacy of chemotherapeutic agents. We have developed a novel array high-throughput microscope (AHTM) system that combines the convenience of the standard 96-well plate with the ability to image cultured cells and membrane-bound microbeads in twelve independently-focusing channels simultaneously, visiting all wells in eight steps. We use the AHTM and passive bead rheology techniques to determine the relative compliance of human pancreatic ductal epithelial (HPDE) cells, h-TERT transformed HPDE cells (HPNE), and four gain-of-function constructs related to EMT. The AHTM found HPNE, H-ras, Myr-AKT, and Bcl2 transfected cells more compliant relative to controls, consistent with parallel tests using atomic force microscopy and invasion assays, proving the AHTM capable of screening for changes in mechanical phenotype.

Analysis-preserving video microscopy compression via correlation and mathematical morphology.

The large amount video data produced by multi-channel, high-resolution microscopy system drives the need for a new high-performance domain-specific video compression technique. We describe a novel compression method for video microscopy data. The method is based on Pearson's correlation and mathematical morphology. The method makes use of the point-spread function (PSF) in the microscopy video acquisition phase. We compare our method to other lossless compression methods and to lossy JPEG, JPEG2000, and H.264 compression for various kinds of video microscopy data including fluorescence video and brightfield video. We find that for certain data sets, the new method compresses much better than lossless compression with no impact on analysis results. It achieved a best compressed size of 0.77% of the original size, 25× smaller than the best lossless technique (which yields 20% for the same video). The compressed size scales with the video's scientific data content. Further testing showed that existing lossy algorithms greatly impacted data analysis at similar compression sizes.

The RhoA guanine nucleotide exchange factor, LARG, mediates ICAM-1-dependent mechanotransduction in endothelial cells to stimulate transendothelial migration.

RhoA-mediated cytoskeletal rearrangements in endothelial cells (ECs) play an active role in leukocyte transendothelial cell migration (TEM), a normal physiological process in which leukocytes cross the endothelium to enter the underlying tissue. Although much has been learned about RhoA signaling pathways downstream from ICAM-1 in ECs, little is known about the consequences of the tractional forces that leukocytes generate on ECs as they migrate over the surface before TEM. We have found that after applying mechanical forces to ICAM-1 clusters, there is an increase in cellular stiffening and enhanced RhoA signaling compared with ICAM-1 clustering alone. We have identified that leukemia-associated Rho guanine nucleotide exchange factor (LARG), also known as Rho GEF 12 (ARHGEF12) acts downstream of clustered ICAM-1 to increase RhoA activity, and that this pathway is further enhanced by mechanical force on ICAM-1. Depletion of LARG decreases leukocyte crawling and inhibits TEM. To our knowledge, this is the first report of endothelial LARG regulating leukocyte behavior and EC stiffening in response to tractional forces generated by leukocytes.

Differential expression of genes in cells cultured from juxtacanalicular trabecular meshwork and Schlemm's canal.

PURPOSE: The purpose of this study was to distinguish differences in gene expression between cells cultured from the juxtacanalicular trabecular meshwork (JCTM) and those from Schlemm's canal (SC), to gain clues to differences between those cell types, and to add to our baseline knowledge of gene expression differences in these cell types for later comparison between cells from nonprimary open-angle glaucoma (POAG) and POAG outflow tissues. METHODS: A set of JCTM and SC cells was cultured from each of 2 donor eyes by an explant method, grown to passage 3, and frozen in liquid nitrogen. The cells were thawed, total RNA was extracted, and the probes made from total RNAs were hybridized to MICROMAX human cDNA microarray slides in 2 separate trials. Differentially expressed genes were analyzed using PubMed, Prosite, and IPA software, and the expression of several of the genes including intercellular adhesion molecule-1 (ICAM-1), tenascin, and ß-spectrin was assessed by immunofluorescence. RESULTS: Schlemm's canal cells differentially expressed ICAM-1, spectrin, complement, fibulin-1, and several genes consistent with an endothelial origin in both arrays, while the JCTM cells more often overexpressed genes consistent with contractile, matrix function, and neural character. At the same time, many genes highly expressed in the first array were not highly overexpressed in the second. One highly overexpressed gene in the JCTM in both arrays, that for heparan sulfate 3-O-sulfotransferase-1 precursor, is thought to be somewhat unique, and could affect the glycosaminoglycan functionality in the extracellular matrix (ECM). CONCLUSIONS: We found generally good agreement between the 2 array trials, but some contradictions as well. Many of the genes overexpressed in each cell type had been described in earlier work, but several were new. Tables of genes, grouped by cellular function, and the complete datasets are provided for the development of new hypotheses.

Submillisecond elastic recoil reveals molecular origins of fibrin fiber mechanics.

Fibrin fibers form the structural scaffold of blood clots. Thus, their mechanical properties are of central importance to understanding hemostasis and thrombotic disease. Recent studies have revealed that fibrin fibers are elastomeric despite their high degree of molecular ordering. These results have inspired a variety of molecular models for fibrin's elasticity, ranging from reversible protein unfolding to rubber-like elasticity. An important property that has not been explored is the timescale of elastic recoil, a parameter that is critical for fibrin's mechanical function and places a temporal constraint on molecular models of fiber elasticity. Using high-frame-rate imaging and atomic force microscopy-based nanomanipulation, we measured the recoil dynamics of individual fibrin fibers and found that the recoil was orders of magnitude faster than anticipated from models involving protein refolding. We also performed steered discrete molecular-dynamics simulations to investigate the molecular origins of the observed recoil. Our results point to the unstructured αC regions of the otherwise structured fibrin molecule as being responsible for the elastic recoil of the fibers.

Localized tensional forces on PECAM-1 elicit a global mechanotransduction response via the integrin-RhoA pathway.

BACKGROUND: Mechanical forces regulate cell behavior and function during development, differentiation, and tissue morphogenesis. In the vascular system, forces produced by blood flow are critical determinants not only of morphogenesis and function, but also of pathological states such as atherosclerosis. Endothelial cells (ECs) have numerous mechanotransducers, including platelet endothelial cell adhesion molecule-1 (PECAM-1) at cell-cell junctions and integrins at cell-matrix adhesions. However, the processes by which forces are transduced to biochemical signals and subsequently translated into downstream effects are poorly understood. RESULTS: Here, we examine mechanochemical signaling in response to direct force application on PECAM-1. We demonstrate that localized tensional forces on PECAM-1 result in, surprisingly, global signaling responses. Specifically, force-dependent activation of phosphatidylinositol 3-kinase (PI3K) downstream of PECAM-1 promotes cell-wide activation of integrins and the small GTPase RhoA. These signaling events facilitate changes in cytoskeletal architecture, including growth of focal adhesions and adaptive cytoskeletal stiffening. CONCLUSIONS: Taken together, our work provides the first evidence of a global signaling event in response to a localized mechanical stress. In addition, these data provide a possible mechanism for the differential stiffness of vessels exposed to distinct hemodynamic force patterns in vivo.

The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins.

How individual cells respond to mechanical forces is of considerable interest to biologists as force affects many aspects of cell behaviour. The application of force on integrins triggers cytoskeletal rearrangements and growth of the associated adhesion complex, resulting in increased cellular stiffness, also known as reinforcement. Although RhoA has been shown to play a role during reinforcement, the molecular mechanisms that regulate its activity are unknown. By combining biochemical and biophysical approaches, we identified two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, as key molecules that regulate the cellular adaptation to force. We show that stimulation of integrins with tensional force triggers activation of these two GEFs and their recruitment to adhesion complexes. Surprisingly, activation of LARG and GEF-H1 involves distinct signalling pathways. Our results reveal that LARG is activated by the Src family tyrosine kinase Fyn, whereas GEF-H1 catalytic activity is enhanced by ERK downstream of a signalling cascade that includes FAK and Ras.

Evidence that αC region is origin of low modulus, high extensibility, and strain stiffening in fibrin fibers.

Fibrin fibers form the structural scaffold of blood clots and perform the mechanical task of stemming blood flow. Several decades of investigation of fibrin fiber networks using macroscopic techniques have revealed remarkable mechanical properties. More recently, the microscopic origins of fibrin's mechanics have been probed through direct measurements on single fibrin fibers and individual fibrinogen molecules. Using a nanomanipulation system, we investigated the mechanical properties of individual fibrin fibers. The fibers were stretched with the atomic force microscope, and stress-versus-strain data was collected for fibers formed with and without ligation by the activated transglutaminase factor XIII (FXIIIa). We observed that ligation with FXIIIa nearly doubled the stiffness of the fibers. The stress-versus-strain behavior indicates that fibrin fibers exhibit properties similar to other elastomeric biopolymers. We propose a mechanical model that fits our observed force extension data, is consistent with the results of the ligation data, and suggests that the large observed extensibility in fibrin fibers is mediated by the natively unfolded regions of the molecule. Although some models attribute fibrin's force-versus-extension behavior to unfolding of structured regions within the monomer, our analysis argues that these models are inconsistent with the measured extensibility and elastic modulus.

Stiffening of individual fibrin fibers equitably distributes strain and strengthens networks.

As the structural backbone of blood clots, fibrin networks carry out the mechanical task of stemming blood flow at sites of vascular injury. These networks exhibit a rich set of remarkable mechanical properties, but a detailed picture relating the microscopic mechanics of the individual fibers to the overall network properties has not been fully developed. In particular, how the high strain and failure characteristics of single fibers affect the overall strength of the network is not known. Using a combined fluorescence/atomic force microscope nanomanipulation system, we stretched 2-D fibrin networks to the point of failure, while recording the strain of individual fibers. Our results were compared to a pair of model networks: one composed of linearly responding elements and a second of nonlinear, strain-stiffening elements. We find that strain-stiffening of the individual fibers is necessary to explain the pattern of strain propagation throughout the network that we observe in our experiments. Fiber strain-stiffening acts to distribute strain more equitably within the network, reduce strain maxima, and increase network strength. Along with its physiological implications, a detailed understanding of this strengthening mechanism may lead to new design strategies for engineered polymeric materials.

Force generation and dynamics of individual cilia under external loading.

Motile cilia are unique multimotor systems that display coordination and periodicity while imparting forces to biological fluids. They play important roles in normal physiology, and ciliopathies are implicated in a growing number of human diseases. In this work we measure the response of individual human airway cilia to calibrated forces transmitted via spot-labeled magnetic microbeads. Cilia respond to applied forces by 1), a reduction in beat amplitude (up to an 85% reduction by 160-170 pN of force); 2), a decreased tip velocity proportionate to applied force; and 3), no significant change in beat frequency. Tip velocity reduction occurred in each beat direction, independently of the direction of applied force, indicating that the cilium is "driven" in both directions at all times. By applying a quasistatic force model, we deduce that axoneme stiffness is dominated by the rigidity of the microtubules, and that cilia can exert 62 +/- 18 pN of force at the tip via the generation of 5.6 +/- 1.6 pN/dynein head.

High throughput system for magnetic manipulation of cells, polymers, and biomaterials.

In the past decade, high throughput screening (HTS) has changed the way biochemical assays are performed, but manipulation and mechanical measurement of micro- and nanoscale systems have not benefited from this trend. Techniques using microbeads (particles approximately 0.1-10 mum) show promise for enabling high throughput mechanical measurements of microscopic systems. We demonstrate instrumentation to magnetically drive microbeads in a biocompatible, multiwell magnetic force system. It is based on commercial HTS standards and is scalable to 96 wells. Cells can be cultured in this magnetic high throughput system (MHTS). The MHTS can apply independently controlled forces to 16 specimen wells. Force calibrations demonstrate forces in excess of 1 nN, predicted force saturation as a function of pole material, and powerlaw dependence of F approximately r(-2.7+/-0.1). We employ this system to measure the stiffness of SR2+ Drosophila cells. MHTS technology is a key step toward a high throughput screening system for micro- and nanoscale biophysical experiments.

Agnostic particle tracking for three-dimensional motion of cellular granules and membrane-tethered bead dynamics.

The ability to detect biological events at the single-molecule level provides unique biophysical insights. Back-focal-plane laser interferometry is a promising technique for nanoscale three-dimensional position measurements at rates far beyond the capability of standard video. We report an in situ calibration technique for back-focal-plane, low-power (nontrapping) laser interferometry. The technique does not rely on any a priori model or calibration knowledge, hence the name "agnostic". We apply the technique to track long-range (up to 100 microm) motion of a variety of particles, including magnetic beads, in three-dimensions with high spatiotemporal resolution ( approximately 2 nm, 100 micros). Our tracking of individual unlabeled vesicles revealed a previously unreported grouping of mean-squared displacement curves at short timescales (<10 ms). Also, tracking functionalized magnetic beads attached to a live cell membrane revealed an anchorage-dependent nonlinear response of the membrane. The software-based technique involves injecting small perturbations into the probe position by driving a precalibrated specimen-mounting stage while recording the quadrant photodetector signals. The perturbations and corresponding quadrant photodetector signals are analyzed to extract the calibration parameters. The technique is sufficiently fast and noninvasive that the calibration can be performed on-the-fly without interrupting or compromising high-bandwidth, long-range tracking of a particle.

Thin-foil magnetic force system for high-numerical-aperture microscopy.

Forces play a key role in a wide range of biological phenomena from single-protein conformational dynamics to transcription and cell division, to name a few. The majority of existing microbiological force application methods can be divided into two categories: those that can apply relatively high forces through the use of a physical connection to a probe and those that apply smaller forces with a detached probe. Existing magnetic manipulators utilizing high fields and high field gradients have been able to reduce this gap in maximum applicable force, but the size of such devices has limited their use in applications where high force and high-numerical-aperture (NA) microscopy must be combined. We have developed a magnetic manipulation system that is capable of applying forces in excess of 700 pN on a 1 mum paramagnetic particle and 13 nN on a 4.5 mum paramagnetic particle, forces over the full 4pi sr, and a bandwidth in excess of 3 kHz while remaining compatible with a commercially available high-NA microscope objective. Our system design separates the pole tips from the flux coils so that the magnetic-field geometry at the sample is determined by removable thin-foil pole plates, allowing easy change from experiment to experiment. In addition, we have combined the magnetic manipulator with a feedback-enhanced, high-resolution (2.4 nm), high-bandwidth (10 kHz), long-range (100 mum xyz range) laser tracking system. We demonstrate the usefulness of this system in a study of the role of forces in higher-order chromosome structure and function.