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It is increasingly appreciated that the cell nucleus is not only a home for DNA but also a complex material that resists physical deformations and dynamically responds to external mechanical cues. The molecules that confer mechanical properties to nuclei certainly contribute to laminopathies and possibly contribute to cellular mechanotransduction and physical processes in cancer such as metastasis. Studying nuclear mechanics and the downstream biochemical consequences or their modulation requires a suite of complex assays for applying, measuring, and visualizing mechanical forces across diverse length, time, and force scales. Here, we review the current methods in nuclear mechanics and mechanobiology, placing specific emphasis on each of their unique advantages and limitations. Furthermore, we explore important considerations in selecting a new methodology as are demonstrated by recent examples from the literature. We conclude by providing an outlook on the development of new methods and the judicious use of the current techniques for continued exploration into the role of nuclear mechanobiology.

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.

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.

We developed VIEW-MOD (Versatile Illumination Engine with a Modular Optical Design): a compact, multi-modality microscope, which accommodates multiple illumination schemes including variable angle total internal reflection, point scanning and vertical/horizontal light sheet. This system allows combining and flexibly switching between different illuminations and imaging modes by employing three electrically tunable lenses and two fast-steering mirrors. This versatile optics design provides control of 6 degrees of freedom of the illumination source (3 translation, 2 tilt, and beam shape) plus the axial position of the imaging plane. We also developed standalone software with an easy-to-use GUI to calibrate and control the microscope. We demonstrate the applications of this system and software in biosensor imaging, optogenetics and fast 3D volume imaging. This system is ready to fit into complex imaging circumstances requiring precise control of illumination and detection paths, and has a broad scope of usability for a myriad of biological applications.

Here we describe development of a microfluidic viscometer based on arrays of magnetically actuated micro-posts. Quantitative viscosities over a range of three orders of magnitude were determined for samples of less than 20 μL. This represents the first demonstration of quantitative viscometry using driven flexible micropost arrays. Critical to the success of our system is a comprehensive analytical model that includes the mechanical and magnetic properties of the actuating posts, the optical readout, and fluid-structure interactions. We found that alterations of the actuator beat shape as parameterized by the dimensionless “sperm number” must be taken into account to determine the fluid properties from the measured actuator dynamics. Beyond our particular system, the model described here can provide dynamics predictions for a broad class of flexible microactuator designs. We also show how the model can guide the design of new arrays that expand the accessible range of measurements.

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. Yan et al. identify the major microtubule acetylase in Drosophila and show that the enzyme and microtubule acetylation broadly control mechanosensation, but not other sensory modalities. Acetylation is required for mechanosensation by the TRP channel NOMPC, and possibly other channels, by virtue of its effects on microtubule mechanical stability and/or dynamics.

We quantify nanorod magnetophoresis through the extracellular matrix (ECM) via single particle observations in vitro. We show that smaller nanorods experience bimodal stick-slip motion through ECM, as well as larger deviations in their orientation angle with respect to the magnetic field, as compared to larger nanorods.

The mechanical properties of blood clots are of central importance to hemostasis, thrombosis and embolism. Fibrin fiber networks are the major structural constituent of clots, and numerous studies dating back several decades have characterized their macroscopic viscoelastic properties. The fiber-level and molecular details giving rise to these properties have not been established, however. The correlation between mechanical properties and amino acid sequence is critical for a predictive understanding of the role of genetic defects in clot pathologies. To address this issue, we have developed a nanomanipulation technique for evaluating individual fibrin fibers. It consists of a combination fluorescence/atomic force microscope system that permits viewing of fiber deformation simultaneous with quantitative strain data. Recently we and our colleagues reported on the high extensibility of individual human fibrin fibers, with extensibility (or strain at breaking) of some fibers exceeding 300%, and elastic recovery with strains of up to 180%. This places human fibrin among the most extensible protein polymers, exceeding elastin and resilin in extensibility. Here we test the hypothesis that the majority of the strain is taken up by the tandem repeat segment of the flexible αC region of fibrin. Our study focused on this portion of the protein by mechanically evaluating fibrins with varying lengths of the tandem repeat segment. Using our integrated nanomanipulation system, we stretched individual fibrin fibers made of human, mouse and chicken fibrinogen which have long, intermediate and zero length tandem repeat segments respectively. We found that extensibility correlated with the lengths of the tandem repeat segments.

Fibrin polymerizes into the fibrous network that is the major structural component of blood clots and thrombi. We demonstrate that fibrin from three different species can also spontaneously polymerize into extensive, molecularly thin, 2D sheets. Sheet assembly occurs in physiologic buffers on both hydrophobic and hydrophilic surfaces, but is routinely observed only when polymerized using very low concentrations of fibrinogen and thrombin. Sheets may have been missed in previous studies because they may be very short-lived at higher concentrations of fibrinogen and thrombin, and their thinness makes them very difficult to detect. We were able to distinguish fluorescently labeled fibrin sheets by polymerizing fibrin onto micro-patterned structured surfaces that suspended polymers 10 μm above and parallel to the cover-glass surface. We used a combined fluorescence/atomic force microscope system to determine that sheets were ≈5 nm thick, flat, elastic and mechanically continuous. Video microscopy of assembling sheets showed that they could polymerize across 25-μm channels at hundreds of μm2/sec (≈1013 subunits/s·M), an apparent rate constant many times greater than those of other protein polymers. Structural transitions from sheets to fibers were observed by fluorescence, transmission, and scanning electron microscopy. Sheets appeared to fold and roll up into larger fibers, and also to develop oval holes to form fiber networks that were “pre-attached” to the substrate and other fibers. We propose a model of fiber formation from sheets and compare it with current models of end-wise polymerization from protofibrils. Sheets could be an unanticipated factor in clot formation and adhesion in vivo, and are a unique material in their own right.

The authors have developed steerable locomotive devices as small as 30 μm using the inertial impact drive as the thrust method. The devices consist of three-legged, thin-metal-film bimorphs designed to rest on three sharp tips with the device body curved up off the surface. Rapid, thermally induced curvature of one leg leads to stepwise translation on a low friction surface. A focused laser was used to supply energy and its parameters controlled the velocity and direction of motion of the device.

A vexing problem in cystic fibrosis (CF) pathogenesis has been to explain the high prevalence of Pseudomonas aeruginosa biofilms in CF airways. We speculated that airway surface liquid (ASL) hyperabsorption generates a concentrated airway mucus that interacts with P. aeruginosa to promote biofilms. To model CF vs. normal airway infections, normal (2.5% solids) and CF-like concentrated (8% solids) mucus were prepared, placed in flat chambers, and infected with an ≈5 × 103 strain PAO1 P. aeruginosa. Although bacteria grew to 1010 cfu/ml in both mucus concentrations, macrocolony formation was detected only in the CF-like (8% solids) mucus. Biophysical and functional measurements revealed that concentrated mucus exhibited properties that restrict bacterial motility and small molecule diffusion, resulting in high local bacterial densities with high autoinducer concentrations. These properties also rendered secondary forms of antimicrobial defense, e.g., lactoferrin, ineffective in preventing biofilm formation in a CF-like mucus environment. These data link airway surface liquid hyperabsorption to the high incidence of P. aeruginosa biofilms in CF via changes in the hydration-dependent physical–chemical properties of mucus and suggest that the thickened mucus gel model will be useful to develop therapies of P. aeruginosa biofilms in CF airways.

We report on the characterization of nanometer-scale resonators. Each device incorporates one multiwalled carbon nanotube (MWNT) as a torsional spring. The devices are actuated electrostatically, and their deflections, both low frequency and on resonance, are detected optically. These are some of the smallest electromechanical devices ever created and are a demonstration of practical integrated MWNT-based oscillators. The results also show surprising intershell mechanical coupling behavior in the MWNTs.

Adenovirus (Ad) has been extensively studied as a eukaryotic viral vector. As these vectors have evolved from first-generation vectors to vectors that contain either very few or no viral genes (“gutless” Ad), significant reductions in the host innate immune response upon infection have been observed. Regardless of these vector improvements an unknown amount of toxicity has been associated with the virion structural proteins. Here we demonstrate the ability to generate high particle numbers (1011 to 1012) of Ad empty virions based on a modification of Cre/lox gutless Ad vectors. Using a battery of analyses (electron microscopy, atomic force microscopy, confocal images, and competition assays) we characterized this reagent and determined that it (i) makes intact virion particles, (ii) competes for receptor binding with wild-type Ad, and (iii) enters the cell proficiently, demonstrating an ability to carry out essential steps of viral entry. To further study the biological impact of these Ad empty virions on infected cells, we carried out DNA microarray analysis. Compared to that for recombinant Ad, the number of mRNAs modulated upon infection was significantly reduced but the expression signatures were similar. This reagent provides a valuable tool for studies of Ad in that researchers can examine the effect of infection in the presence of the virion capsid alone.

We present a quartz tuning-fork-based atomic force microscopy(AFM) setup that is capable of mapping the surfacecontact resistance while scanning topography. The tuning-fork setup allows us to use etched Pt/Ir tips, which have higher durability and better conductivity than probes used in earlier AFM conductance measurements. The performance of the method is demonstrated with contact resistance measurements of gold lines on silicon dioxide and carbon nanotubes on graphite.

We report, through experimental observations and computer simulations, that atomic lattice interlocking can determine whether an object rolls or slides on a surface. We have quantitatively manipulated carbon nanotubes (CNTs) on a variety of substrates with an atomic force microscope (AFM) and observe rolling to occur only on graphite. We measure the forces when the CNT is in-registry with the graphite lattice, and observe rolling only in this lock-in state. Atomistic computer simulations identify the energy barriers for sliding and rolling, elucidate atomic-scale features of slip-roll motion, and explain the details of the lateral force data in terms of the intrinsic faceting of multiwall CNTs., Comment: 7 pages, 4 figures

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