Subcellular Length Scales Research Articles

Concave/Convex Curvature of Anisotropic Grooves Differentially Alters Cellular Morphology, Adhesion, and Proliferation.

Curvature is an integral part of the complex in vivo tissue architecture across various length scales. Therefore, several in vitro models with a patterned curvature in different length scales have been developed to understand the role of this in cellular behavior. At the subcellular scale, wavy patterns have been reported wherein concave and convex grooves are adjacently present. However, the independent effect of continuous subcellular concave and convex shapes has not been reported, mainly owing to the limitations in fabricating such patterns. In this study, we developed continuous concave and convex grooves on polydimethylsiloxane (PDMS) using a Dracaena sanderiana (bamboo) leaf as a template. The first (negative) replica from the abaxial side of the bamboo leaf, which imparted concave grooves on PDMS, was subsequently used as a template to fabricate a positive replica of the leaf, resulting in convex grooves of the same size and arrangement as the concave grooves. We examined the influence of the groove curvature on the morphology of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) and skeletal muscle cells (C2C12). BM-hMSCs and C2C12 cells aligned on both concave and convex grooves as compared to the random orientation on a flat substrate. The significant difference was observed in the morphology of both cells, in terms of area, aspect ratio, number, and length of protrusions on concave and convex patterns. We found that the number of protrusions was also dependent on the ratio of cell to pattern length scale for convex-shaped grooves but independent of length scale for concave-shaped grooves. The proliferation of BM-hMSCs was also found to be different on concave and convex shapes. Therefore, this study shows the importance of (1) convex and concave curvatures of the subcellular length scale in cellular response, (2) dependence on the ratio of cell and curvature length scale, and (3) use of natural templates for overcoming fabrication challenges.

Chondroitin sulfate, dermatan sulfate, and hyaluronic acid differentially modify the biophysical properties of collagen-based hydrogels

Fibrillar collagens and glycosaminoglycans (GAGs) are structural biomolecules that are natively abundant to the extracellular matrix (ECM). Prior studies have quantified the effects of GAGs on the bulk mechanical properties of the ECM. However, there remains a lack of experimental studies on how GAGs alter other biophysical properties of the ECM, including ones that operate at the length scales of individual cells such as mass transport efficiency and matrix microstructure. This study focuses on the GAG molecules chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronic acid (HA). CS and DS are stereoisomers while HA is the only non-sulfated GAG. We characterized and decoupled the effects of these GAG molecules on the stiffness, transport, and matrix microarchitecture properties of type I collagen hydrogels using mechanical indentation testing, microfluidics, and confocal reflectance imaging, respectively. We complement these biophysical measurements with turbidity assays to profile collagen aggregate formation. Surprisingly, only HA enhanced the ECM indentation modulus, while all three GAGs had no effect on hydraulic permeability. Strikingly, we show that CS, DS, and HA differentially regulate the matrix microarchitecture of hydrogels due to their alterations to the kinetics of collagen self-assembly. In addition to providing information on how GAGs define key physical properties of the ECM, this work shows new ways in which stiffness measurements, microfluidics, microscopy, and turbidity kinetics can be used complementarily to reveal details of collagen self-assembly and structure. Statement of SignificanceCollagen and glycosaminoglycans (GAGs) are integral to the structure, function, and bioactivity of the extracellular matrix (ECM). Despite widespread interest in collagen-GAG composite hydrogels, there is a lack of quantitative understanding of how different GAGs alter the biophysical properties of the ECM across tissue, cellular, and subcellular length scales. Here we show using mechanical, microfluidic, microscopy, and analytical methods and measurements that the GAG molecules chondroitin sulfate, dermatan sulfate, and hyaluronic acid differentially regulate the mechanical, transport, and microstructural properties of hydrogels due to their alterations to the kinetics of collagen self-assembly. As such, these results will inform improved design and utilization of collagen-based scaffolds of tailored composition, mechanical properties, molecular availability due to mass transport, and microarchitecture.

Correlated cryo-SEM and CryoNanoSIMS imaging of biological tissue

BackgroundThe development of nanoscale secondary ion mass spectrometry (NanoSIMS) has revolutionized the study of biological tissues by enabling, e.g., the visualization and quantification of metabolic processes at subcellular length scales. However, the associated sample preparation methods all result in some degree of tissue morphology distortion and loss of soluble compounds. To overcome these limitations an entirely cryogenic sample preparation and imaging workflow is required.ResultsHere, we report the development of a CryoNanoSIMS instrument that can perform isotope imaging of both positive and negative secondary ions from flat block-face surfaces of vitrified biological tissues with a mass- and image resolution comparable to that of a conventional NanoSIMS. This capability is illustrated with nitrogen isotope as well as trace element mapping of freshwater hydrozoan Green Hydra tissue following uptake of 15N-enriched ammonium.ConclusionWith a cryo-workflow that includes vitrification by high pressure freezing, cryo-planing of the sample surface, and cryo-SEM imaging, the CryoNanoSIMS enables correlative ultrastructure and isotopic or elemental imaging of biological tissues in their most pristine post-mortem state. This opens new horizons in the study of fundamental processes at the tissue- and (sub)cellular level.TeaserCryoNanoSIMS: subcellular mapping of chemical and isotopic compositions of biological tissues in their most pristine post-mortem state.

OptoRheo: Simultaneous in situ micro-mechanical sensing and imaging of live 3D biological systems

Biomechanical cues from the extracellular matrix (ECM) are essential for directing many cellular processes, from normal development and repair, to disease progression. To better understand cell-matrix interactions, we have developed a new instrument named ‘OptoRheo’ that combines light sheet fluorescence microscopy with particle tracking microrheology. OptoRheo lets us image cells in 3D as they proliferate over several days while simultaneously sensing the mechanical properties of the surrounding extracellular and pericellular matrix at a sub-cellular length scale. OptoRheo can be used in two operational modalities (with and without an optical trap) to extend the dynamic range of microrheology measurements. We corroborated this by characterising the ECM surrounding live breast cancer cells in two distinct culture systems, cell clusters in 3D hydrogels and spheroids in suspension culture. This cutting-edge instrument will transform the exploration of drug transport through complex cell culture matrices and optimise the design of the next-generation of disease models.

Controlled Glioma Cell Migration and Confinement Using Biomimetic‐Patterned Hydrogels

Cancer cell migration is significantly influenced by the physical properties (e.g., mechanics, topography) of the local extracellular matrix (ECM). However, how cells guide their movement in a complex, heterogeneous landscape while interpreting various physical factors, especially those with sizes on the subcellular length scale, is not completely understood. Herein, a photodegradable hydrogel is fabricated with control over the spatial distribution of features with different physical characteristics. On the surface of this hydrogel, patterns of features with sizes and spacings on the subcellular to cellular length scale are generated using a digital micromirror device, and the influence of the organization of these patterns on cellular migratory behavior is investigated. The size and spatial arrangement of hard (or stiff) and soft domains, more so than their topography, is found to have a significant impact on the migration speed. Using this approach, the maximum migration speed is observed on hydrogels with 10 μm pattern sizes spaced 10 μm apart. In this regime, the cells are confined and interact with both hard and soft regions at their periphery. Together, this study of cell–ECM interactions at the interface of hard and soft domains provides insights into how cancer cells interact with their environment.

The precision of macroscale mechanical measurements is limited by the inherent structural heterogeneity of human stratum corneum

Biological tissues are structurally heterogenous mosaics at cellular and sub-cellular length scales. Some tissues, like the outermost layer of human skin, or stratum corneum (SC), also exhibit a rich topography of microchannels at larger mesoscopic length scales. Although this is well understood, modern studies continue to characterize the mechanical properties of biological tissues, including the SC, using macroscale techniques that assume these materials are homogenous in structure, thickness, and composition. Macroscale failure testing of SC is commonly associated with large sample to sample variability. We anticipate that microscale heterogeneities play an important role in defining the global mechanical response of the tissue. To evaluate the validity of the prevailing paradigm that macroscopic testing techniques can provide meaningful information about failure in soft heterogenous tissues, the macroscale work of fracture in isolated human SC samples is measured using conventional macroscale testing techniques and compared with the energy cost of creating new crack interfaces at the microscale, measured using a modified traction force microscopy technique. Results show that measured micro- and macroscale energy costs per unit crack path length are highly consistent. However, crack propagation is found to be guided by microscale topographical features in the tissue. This correlation reveals that macroscale mechanical sample to sample variability is caused by notable differences in crack propagation pathways. Statement of significanceAlthough designed to test homogeneous materials, macroscopic uniaxial tensometry is currently the gold standard for measuring the mechanical properties of biological tissues. All tissues, including human stratum corneum are structurally heterogeneous at the microscale and mechanical measurements are commonly highly variable, even for specimens from the same source. This study explores the fundamental causes of this disparity and evaluates the prevailing paradigm that macroscopic testing techniques can provide meaningful information about failure in soft heterogeneous tissues. Results conclude that the cause of large variability in mechanical work of fracture is due to inherent structural heterogeneities governing crack propagation pathways and altering the total crack length. Structural heterogeneities in tissue therefore limits the precision of macroscale biomechanical testing.

Mechanical heterogeneity along single cell-cell junctions is driven by lateral clustering of cadherins during vertebrate axis elongation.

Morphogenesis is governed by the interplay of molecular signals and mechanical forces across multiple length scales. The last decade has seen tremendous advances in our understanding of the dynamics of protein localization and turnover at subcellular length scales, and at the other end of the spectrum, of mechanics at tissue-level length scales. Integrating the two remains a challenge, however, because we lack a detailed understanding of the subcellular patterns of mechanical properties of cells within tissues. Here, in the context of the elongating body axis of Xenopus embryos, we combine tools from cell biology and physics to demonstrate that individual cell-cell junctions display finely-patterned local mechanical heterogeneity along their length. We show that such local mechanical patterning is essential for the cell movements of convergent extension and is imparted by locally patterned clustering of a classical cadherin. Finally, the patterning of cadherins and thus local mechanics along cell-cell junctions are controlled by Planar Cell Polarity signaling, a key genetic module for CE that is mutated in diverse human birth defects.

In Situ Studies of Fibrillar Polymorphs in Alzheimer's Disease

Aggregates of Aβ peptides and tau protein are defining features of Alzheimer's disease (AD) but the contribution of these structures to the etiology of disease remains uncertain. High resolution structures of fibrillar polymorphs have provided a basis for studies of disease, but fall short of defining the molecular mechanisms underlying disease progression. Evidence is accumulating that the peptide conformation within fibrils correlates with disease subtype and suggests that protofilament conformation represents the physical embodiment of a ‘strain’ of AD. Within a single strain the fold of peptides may be invariant. But the fibrils are capable of polymorphism that includes variation in the packing of protofilaments into fibrils; the pitch of the resultant fibrils and the higher order organization of plaques into which they aggregate. These intra-strain polymorphisms are divided by low-energy barriers allowing multiple configurations to coexist within a single preparation or tissue. Clinical presentation of different strains may be determined by variation in the way different protofilament structures generate relevant toxic species, be they monomers, oligomers or higher order structures. To better understand their role in disease, in situ studies of fibril structure have been carried out on histological thin sections of human brain tissue. Scanning x-ray microdiffraction is providing information on the variation of fibrillar polymorphism at sub-cellular length scales and makes possible mapping the distribution of polymorphs within tissue. Scattering in the wide-angle regime produces information on fibrillar twist. Scattering in the small-angle regime can be used to generate the cross-sectional shape of fibrils. Mapping the variation of these structural parameters across and among plaques and tangles in different regions of the brain provides insight into the development and evolution of neurodegenerative lesions.

Nanoscale x-ray holotomography of human brain tissue with phase retrieval based on multienergy recordings.

.X-ray cone-beam holotomography of unstained tissue from the human central nervous system reveals details down to subcellular length scales. This visualization of variations in the electron density of the sample is based on phase-contrast techniques using intensities formed by self-interference of the beam between object and detector. Phase retrieval inverts diffraction and overcomes the phase problem by constraints such as several measurements at different Fresnel numbers for a single projection. Therefore, the object-to-detector distance (defocus) can be varied. However, for cone-beam geometry, changing defocus changes magnification, which can be problematic in view of image processing and resolution. Alternatively, the photon energy can be altered (multi-E). Far from absorption edges, multi-E data yield the wavelength-independent electron density. We present the multi-E holotomography at the Göttingen Instrument for Nano-Imaging with X-Rays (GINIX) setup of the P10 beamline at Deutsches Elektronen-Synchrotron. The instrument is based on a combined optics of elliptical mirrors and an x-ray waveguide positioned in the focal plane for further coherence, spatial filtering, and high numerical aperture. Previous results showed the suitability of this instrument for nanoscale tomography of unstained brain tissue. We demonstrate that upon energy variation, the focal spot is stable enough for imaging. To this end, a double-crystal monochromator and automated alignment routines are required. Three tomograms of human brain tissue were recorded and jointly analyzed using phase retrieval based on the contrast transfer function formalism generalized to multiple photon energies. Variations of the electron density of the sample are successfully reconstructed.

Heterogeneities in Biological Tissues Limit the Precision of Macroscale Mechanical Tests

Biological tissues are structurally heterogenous mosaics at cellular and sub-cellular length scales. Some tissues, like the outermost layer of human skin, or stratum corneum (SC), also exhibit a rich topography at larger mesoscopic length scales. Although this is well understood, modern studies continue to characterize the mechanical properties of biological tissues, including the SC, using macroscale techniques that assume these materials are homogenous in structure, thickness, and composition. Macroscale failure testing of biological tissues is commonly associated with large sample to sample variability. We anticipate that microscale heterogeneities play an important role in defining the global mechanical response of tissues. To evaluate the validity of the prevailing paradigm that macroscopic testing techniques can provide meaningful information about failure in soft heterogenous tissues, the macroscale work of fracture in isolated human SC samples is measured using conventional macroscale testing techniques and compared with the energy cost of creating new crack interfaces at the microscale, measured using a modified traction force microscopy technique. Results show that measured micro- and macroscale energy costs per unit crack length are highly consistent. However, microscale crack propagation is found to be guided by topographical features in the tissue. This correlation reveals that macroscale mechanical sample to sample variability is primarily caused by notable differences in crack propagation pathways.

Spectral image scanning microscopy.

For decades, the confocal microscope has represented one of the dominant imaging systems in biomedical imaging at sub-cellular lengthscales. Recently, however, it has increasingly been replaced by a related, but more powerful successor technique termed image scanning microscopy (ISM). In this article, we present ISM capable of measuring spectroscopic information such as that contained in fluorescence or Raman images. Compared to established confocal spectroscopic imaging systems, our implementation offers similar spectral resolution, but higher spatial resolution and detection efficiency. Color sensitivity is achieved by a grating placed in the detection path in conjunction with a camera collecting both spatial and spectral information. The multidimensional data is processed using multi-view maximum likelihood image reconstruction. Our findings are supported by numerical simulations and experiments on micro beads and double-stained HeLa cells.

Cell Printing in Complex Hydrogel Scaffolds.

Advancements in the microfabrication of soft materials have enabled the creation of increasingly sophisticated functional synthetic tissue structures for a myriad of tissue engineering applications. A challenge facing the field is mimicking the complex microarchitecture necessary to recapitulate proper morphology and function of many endogenous tissue constructs. This paper describes the creation of PEGDA hydrogel microenvironments (microgels) that maintain a high level of viability at single cell patterning scales and can be integrated into composite scaffolds with tunable modulus. PEGDA was stereolithographically patterned using a digital micromirror device to print single cell microgels at progressively decreasing length scales. The effect of feature size on cell viability was assessed and inert gas purging was introduced to preserve viability. A composite PEGDA scaffold created by this technique was mechanically tested and found to enable dynamic adjustability of the modulus. Together this approach advances the ability to microfabricate tissues that better mimic native constructs on cellular and subcellular length scales.

Protein concentration gradients and switching diffusions.

Morphogen gradients play a vital role in developmental biology by enabling embryonic cells to infer their spatial location and determine their developmental fate accordingly. The standard mechanism for generating a morphogen gradient involves a morphogen being produced from a localized source and subsequently degrading. While this mechanism is effective over the length and time scales of tissue development, it fails over typical subcellular length scales due to the rapid dissipation of spatial asymmetries. In a recent theoretical work, we found an alternative mechanism for generating concentration gradients of diffusing molecules, in which the molecules switch between spatially constant diffusivities at switching rates that depend on the spatial location of a molecule. Independently, an experimental and computational study later found that Caenorhabditis elegans zygotes rely on this mechanism for cell polarization. In this paper, we extend our analysis of switching diffusivities to determine its role in protein concentration gradient formation. In particular, we determine how switching diffusivities modifies the standard theory and show how space-dependent switching diffusivities can yield a gradient in the absence of a localized source. Our mathematical analysis yields explicit formulas for the intracellular concentration gradient which closely match the results of previous experiments and numerical simulations.

Quantum versus classical Monte Carlo simulation of low-energy electron transport in condensed amorphous media

PurposeClassical trajectory Monte Carlo (MC) simulations modelling radiation-induced damage on subcellular length scales ignore quantum effects that may be non-negligible as electron energy decreases below 1 keV. This work investigates quantum mechanical (QM) treatments of low-energy electron transport in condensed media, comparing with classical MC. MethodsQM calculations involve a simplified model of electron transport in water with a plane wave incident on a cylinder (“droplet”) consisting of a cluster of point scatterers (positioned randomly but constrained by a minimum separation, dmin). The system of coupled equations for the electron wavefield incident on each scatterer is solved numerically and results are averaged over many clusters with different point scatterer positions. Average QM cluster cross sections and scattering event densities are compared with analogues computed within the corresponding classical MC model, and relative errors on MC results are calculated. ResultsDifferences between QM and MC results for both cluster cross section and scattering event density are sensitive to electron energy (wavelength), structure (dmin), and single-scatterer elastic/inelastic cross sections. Relative errors on cluster cross sections generally differ from errors on scattering event densities. The introduction of inelastic scatter generally increases relative errors (compared to calculations with the same single-scatterer elastic cross section) with some exceptions. Accounting for structure (dmin≠0) enhances differences between QM and MC results. ConclusionsThe quantum wave nature of electrons is non-negligible for simulations of low-energy electron transport within small-scale biological targets. The development of more realistic models of electron transport in condensed media is motivated for future work.

Texturing Silicon Nanowires for Highly Localized Optical Modulation of Cellular Dynamics.

Engineered silicon-based materials can display photoelectric and photothermal responses under light illumination, which may lead to further innovations at the silicon-biology interfaces. Silicon nanowires have small radial dimensions, promising as highly localized cellular modulators, however the single crystalline form typically has limited photothermal efficacy due to the poor light absorption and fast heat dissipation. In this work, we report strategies to improve the photothermal response from silicon nanowires by introducing nanoscale textures on the surface and in the bulk. We next demonstrate high-resolution extracellular modulation of calcium dynamics in a number of mammalian cells including glial cells, neurons, and cancer cells. The new materials may be broadly used in probing and modulating electrical and chemical signals at the subcellular length scale, which is currently a challenge in the field of electrophysiology or cellular engineering.

Probing nanostructured materials using X‐ray fluorescence analysis

Dispersion of metal nanoparticles on flat surfaces has attracted considerable interest in many technological applications. Numerous advantages of functionalized patterned nanostructures especially in medical physics, chemistry, biology, and semiconductor quantum dot applications have rapidly driven the growth of this field in the last two decades. In particular, for applications involving biomedical imaging, the nanoparticles have attracted perhaps the greatest attention as these materials provide a direct interface at the subcellular length scale. Reliable characterization methods are thus often needed to infer their detailed structural properties such as average particle size, particle shape, internal structure, surface morphology, distribution of particles, and their chemical composition. As a nondestructive probe, X‐ray fluorescence measurements at grazing incidence provide sensitive information about the physical and chemical nature of the nanoparticles over the large surface area of a substrate. Herein, we demonstrate the potential capabilities of the X‐ray fluorescence technique for reliable and precise determination of surface morphology of metal nanoparticles, dispersed on a flat surface. Our results show that X‐ray standing wave assisted fluorescence measurements are markedly sensitive to the nature of dispersion of nanoparticles on a substrate surface. We have analyzed different types of nanostructured materials; e.g. a W thin film structure and Au nanoparticles deposited on a Si surface. The average size of the Au nanoparticles estimated, using X‐ray standing wave fluorescence analysis, was found to closely agree with the atomic force microscopy measurements. The inherent features of the X‐ray fluorescence technique permit us to analyze distribution of all kinds of metal and metalloid nanoparticles on a flat surface. Copyright © 2017 John Wiley & Sons, Ltd.

Guiding cell migration with microscale stiffness patterns and undulated surfaces

By placing stiff structures under soft materials, prior studies have demonstrated that cells sense and prefer to position themselves over the stiff structures. However, an understanding of how cells migrate on such surfaces has not been established. Many studies have also shown that cells readily align to surface topography. Here we investigate the influence of these two aspects in directing cell migration on surfaces with 5 and 10μm line stiffness patterns (a cellular to subcellular length scale). A simple approach to create flat, stiffness-patterned surfaces by suspending a thin, low modulus polydimethylsiloxane (PDMS) film over a high modulus PDMS structure is presented, as well as a route to add undulations. We confirm that cells are able to sense through the thin film by observation of focal adhesions being positioned on stiff regions. We examine migration by introducing migration efficiency, a quantitative parameter to determine how strongly cells migrate in a certain direction. We found that cells have a preference to align and migrate along stiffness patterns while the addition of undulations boosts this effect, significantly increasing migration efficiency in either case. Interestingly, we found speed to play little role in the migration efficiency and to be mainly influenced by the top layer modulus. Our results demonstrate that both stiffness patterns and surface undulations are important considerations when investigating the interactions of cells with biomaterial surfaces. Statement of SignificanceTwo common physical considerations for cell-surface interactions include patterned stiffness and patterned topography. However, their relative influences on cell migration behavior have not been established, particularly on cellular to subcellular scale patterns. For stiffness patterning, it has been recently shown that cells tend to position themselves over a stiff structure that is placed under a thin soft layer. By quantifying the directional migration efficiency on such surfaces with and without undulations, we show that migration can be manipulated by flat stiffness patterns, although surface undulations also play a strong role. Our results offer insight on the effect of cellular scale stiffness and topographical patterns on cell migration, which is critical for the development of fundamental cell studies and engineered implants.

Noninvasive quantification of alveolar morphometry in elderly never- and ex-smokers.

Diffusion-weighted magnetic resonance imaging (MRI) provides a way to generate in vivo lung images with contrast sensitive to the molecular displacement of inhaled gas at subcellular length scales. Here, we aimed to evaluate hyperpolarized 3He MRI estimates of the alveolar dimensions in 38 healthy elderly never-smokers (73 ± 6 years, 15 males) and 21 elderly ex-smokers (70 ± 10 years, 14 males) with (n = 8, 77 ± 6 years) and without emphysema (n = 13, 65 ± 10 years). The ex-smoker and never-smoker subgroups were significantly different for FEV1/FVC (P = 0.0001) and DLCO (P = 0.009); while ex-smokers with emphysema reported significantly diminished FEV1/FVC (P = 0.02) and a trend toward lower DLCO (P = 0.05) than ex-smokers without emphysema. MRI apparent diffusion coefficients (ADC) and CT measurements of emphysema (relative area–CT density histogram, RA950) were significantly different (P = 0.001 and P = 0.007) for never-smoker and ex-smoker subgroups. In never-smokers, the MRI estimate of mean linear intercept (260 ± 27 μm) was significantly elevated as compared to the results previously reported in younger never-smokers (210 ± 30 μm), and trended smaller than in the age-matched ex-smokers (320 ± 72 μm, P = 0.06) evaluated here. Never-smokers also reported significantly smaller internal (220 ± 24 μm, P = 0.01) acinar radius but greater alveolar sheath thickness (120 ± 4 μm, P < 0.0001) than ex-smokers. Never-smokers were also significantly different than ex-smokers without emphysema for alveolar sheath thickness but not ADC, while ex-smokers with emphysema reported significantly different ADC but not alveolar sheath thickness compared to ex-smokers without CT evidence of emphysema. Differences in alveolar measurements in never- and ex-smokers demonstrate the sensitivity of MRI measurements to the different effects of smoking and aging on acinar morphometry.

Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth

Patterning of neuronal outgrowth in vitro is important in tissue engineering as well as for the development of neuronal interfaces with desirable characteristics. To date, this has been achieved with the aid of micro- and nanofabrication techniques giving rise to various anisotropic topographies, either in the form of continuous or discontinuous structures. In this study we propose a currently unexplored geometry of a 3D culture substrate for neuronal cell growth comprising discontinuous subcellular microstructures with anisotropic geometrical cross-section. Specifically, using laser precision 3D micro/nano fabrication techniques, silicon substrates comprising arrays of parallel oriented elliptical microcones (MCs) were fabricated to investigate whether a discontinuous geometry comprising anisotropic features at the subcellular level could influence the alignment of peripheral nervous system cell populations. It was shown that both Schwann cells and axons of sympathetic neurons were parallel oriented onto the MCs of elliptical shape, while they exhibited a random orientation onto the MCs of arbitrary shape. Notably, this topography-induced guidance effect was also observed in more complex cell culture systems, such as the organotypic culture whole dorsal root ganglia (DRG) explants. Our results suggest that a discontinuous topographical pattern could promote Schwann cell and axonal alignment, provided that it hosts anisotropic geometrical features, even though the sizes of those range at the subcellular lengthscale. The laser-patterned arrays of MCs presented here could potentially be a useful platform for patterning neurons into artificial networks, allowing the study of neuronal cells interactions under 3D ex-vivo conditions.

Localized light-induced protein dimerization in living cells using a photocaged dimerizer.

Regulated protein localization is critical for many cellular processes. Several techniques have been developed for experimental control over protein localization, including chemically induced and light-induced dimerization, which both provide temporal control. Light-induced dimerization offers the distinct advantage of spatial precision within subcellular length scales. A number of elegant systems have been reported that utilize natural light-sensitive proteins to induce dimerization via direct protein-protein binding interactions, but the application of these systems at cellular locations beyond the plasma membrane has been limited. Here we present a new technique to rapidly and reversibly control protein localization in living cells with subcellular spatial resolution using a cell-permeable, photoactivatable chemical inducer of dimerization. We demonstrate light-induced recruitment of a cytosolic protein to individual centromeres, kinetochores, mitochondria and centrosomes in human cells, indicating that our system is widely applicable to many cellular locations.