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The linker of the nucleoskeleton and cytoskeleton (LINC) complex comprises SUN (Sad-1 and UNC-84) and KASH (Klarsicht, ANC-1, SYNE homology) domain proteins, whose conserved interactions provide a physical coupling between the cytoskeleton and the nucleoskeleton, thereby mediating the transfer of physical forces across the nuclear envelope. The LINC complex can perform distinct cellular functions by pairing various KASH domain proteins with the same SUN domain protein. Recent studies have suggested a higher-order assembly of SUN and KASH instead of a more widely accepted linear trimer model for the LINC complex. In the present study, we use molecular dynamics simulations to investigate the mechanism of force transfer across the two proposed models of LINC complex assembly, namely the 3:3 linear trimer model and the 6:6 higher-order model. Employing steered molecular dynamics simulations with various structures using forces at different rates and directions, we examine the structural stability of the two models under various biologically relevant conditions. Our results suggest that both models can withstand and transfer significant levels of force while retaining their structural integrity. However, the force response of various SUN KASH assemblies depended on the force direction and pulling rates. Slower pulling rates resulted in higher mean square fluctuations of the 3:3 assembly compared to the fast pulling. Interestingly, the 6:6 assembly tends to provide an additional range of motion flexibility and might be more suitable for describing the interaction between SUN and KASH under compressive and shear forces. These findings offer insights into how the SUN and KASH proteins maintain the structural integrity of the nuclear membrane.

Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used three-dimensional (3D) nanostructures such as nanopillars to imprint well-defined membrane curvatures (the "nano-bio interface"). In these studies, proteins and their interactions were probed by two-dimensional fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano-bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone-oil immersion objective and 3D double-helix point spread function microscopy. We carefully adjust the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/iR/i(the reciprocal of the radius) membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano-bio interface using 3D SR microscopy.

Both substrate stiffness and surface topography regulate cell behavior through mechanotransduction signaling pathways. Such intertwined effects suggest that engineered surface topographies might substitute or cancel the effects of substrate stiffness in biomedical applications. However, the mechanisms by which cells recognize topographical features are not fully understood. Here we demonstrate that the presence of nanotopography drastically alters cell behavior such that neurons and stem cells cultured on rigid glass substrates behave as if they were on soft hydrogels. We further show that rigid nanotopography resembles the effect of soft hydrogels in reducing cell stiffness and membrane tension as measured by atomic force microscopy. Finally, we demonstrate that nanotopography reduces focal adhesions and cell stiffness by enhancing the endocytosis and the subsequent removal of integrin receptors. This mechanistic understanding will support the rational design of nanotopography that directs cells on rigid materials to behave as if they were on soft ones.TOC graphic

Bioelectronics aims to design electronic devices which can be fully integrated within tissues to monitor or stimulate specific cell functions. The main challenge is the engineering of the cell–chip interface and diverse materials, and devices have been developed to recapitulate biological architectures and functionalities. In this Prospective article, the authors give an overview on how the bioelectronics community has exploited biomimetic approaches to emulate cell morphologies, interactions, and functions to design optimal electrical platforms to be coupled to living cells.

Drug-induced cardiotoxicity arises primarily when a compound alters the electrophysiological properties of cardiomyocytes. Features of intracellular action potentials (iAPs) are powerful biomarkers that predict proarrhythmic risks. However, the conventional patch clamp techniques for measuring iAPs are either laborious and low throughput or not suitable for measuring electrically connected cardiomyocytes. In the last decade, a number of vertical nanoelectrodes have been demonstrated to achieve parallel and minimally-invasive iAP recordings. Nanoelectrodes show great promise, but the large variability in success rate, signal strength, and the low throughput of device fabrication have hindered them from being broadly adopted for proarrhythmia drug assessment. In this work, we developed vertically-aligned and semi-hollow nanocrown electrodes that are mechanically robust and made through a scalable fabrication process. Nanocrown electrodes achieve >99% success rates in obtaining intracellular access through electroporation, allowing reliable and simultaneous iAP recordings from up to 57 human pluripotent stem-cell-derived cardiomyocytes (hPSC-CMs). The accuracy of nanocrown electrode recordings is validated by simultaneous patch clamp recording from the same cell. Nanocrown electrodes enable prolonged iAP recording for continual monitoring of the same cells upon the sequential addition of four to five incremental drug doses. In this way, the dose-response data is self-referencing, which avoids the cell-to-cell variations inherent to hPSC-CMs. We are hopeful that this technology development is a step towards establishing an iAP screening assay for preclinical evaluation of drug-induced arrhythmogenicity.

The linker of the nucleoskeleton and cytoskeleton (LINC) complex is formed by the conserved interactions between Sad-1 and UNC-84 (SUN) and Klarsicht, ANC-1, SYNE homology (KASH) domain proteins, providing a physical coupling between the nucleoskeleton and cytoskeleton that mediates the transfer of physical forces across the nuclear envelope. The LINC complex can perform distinct cellular functions by pairing various KASH domain proteins with the same SUN domain protein. For example, in Caenorhabditis elegans, SUN protein UNC-84 binds to two KASH proteins UNC-83 and ANC-1 to mediate nuclear migration and anchorage, respectively. In addition to distinct cytoplasmic domains, the luminal KASH domain also varies among KASH domain proteins of distinct functions. In this study, we combined in vivo C. elegans genetics and in silico molecular dynamics simulations to understand the relation between the length and amino acid composition of the luminal KASH domain, and the function of the SUN–KASH complex. We show that longer KASH domains can withstand and transfer higher forces and interact with the membrane through a conserved membrane proximal EEDY domain that is unique to longer KASH domains. In agreement with our models, our in vivo results show that swapping the KASH domains of ANC-1 and UNC-83, or shortening the KASH domain of ANC-1, both result in a nuclear anchorage defect in C. elegans.

Mechanical signals affect many aspects of biological processes. Physical forces from the extracellular microenvironment are ultimately transmitted to the nucleus and elicit a response that result in the deformation and remodeling of the nucleus. Recent studies have shown that nuclear deformation has several consequences such as reorganization of chromatin, changes in gene expression, and nuclear envelope rupture. It is widely believed that a direct coupling between the cytoskeleton and nucleoskeleton is required for nuclear deformation; however, some studies have proposed alternative mechanisms for nuclear deformation and the transmission of mechanical signals and stresses from the cytoskeleton to the nucleus. Herein, we review the processes, in which the cell nucleus experiences stresses and discuss the evidence of involvement of a direct link between the cytoskeleton and nucleoskeleton in nuclear deformation.

Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used 3D nanostructures such as nanopillars to imprint well-defined membrane curvatures (the “nano-bio interface”). In these studies, proteins and their interactions were probed by 2D fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano-bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone oil immersion objective and 3D double-helix point-spread function microscopy. We carefully optimize the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano-bio interface using 3D SR microscopy.

Large protein complexes assemble at the nuclear envelope to transmit mechanical signals between the cytoskeleton and nucleoskeleton. These protein complexes are known as the linkers of the nucleoskeleton and cytoskeleton complexes (LINC complexes) and are formed by the interaction of SUN and KASH domain proteins in the nuclear envelope. Ample evidence suggests that SUN–KASH complexes form higher-order assemblies to withstand and transfer forces across the nuclear envelope. Herein, we present a review of recent studies over the past few years that have shed light on the mechanisms of SUN–KASH interactions, their higher order assembly, and the molecular mechanisms of force transfer across these complexes.

Integrin αIIbβ3 is a predominant type of integrin abundantly expressed on the surface of platelets and its activation regulates the process of thrombosis. Talin and kindlin are cytoplasmic proteins that bind to integrin and modulate its affinity for extracellular ligands. While the molecular details of talin-mediated integrin activation are known, the mechanism of kindlin involvement in this process remains elusive. Here, we demonstrate that the interplay between talin and kindlin promotes integrin activation. Our all-atomic molecular dynamics simulations on complete transmembrane and cytoplasmic domains of integrin αIIbβ3, talin1 F2/F3 subdomains, and kindlin2 FERM domain in an explicit lipid-water environment over microsecond timescale, unraveled the role of kindlin as an enhancer of the talin interaction with the membrane proximal region of β–integrin. The cooperation of kindlin with talin results in a complete disruption of salt bridges between R995 on αIIb and D723/E726 on β3. Furthermore, kindlin modifies the molecular mechanisms of inside-out activation by decreasing the crossing angle between transmembrane helices of integrin αIIb-β3, which eventually results in parallelization of integrin dimer. In addition, our control simulation featuring integrin in complex with kindlin reveals that kindlin binding is not sufficient for unclasping the inner membrane and outer membrane interactions of integrin dimer, thus ruling out the possibility of solitary action of kindlin in integrin activation.Statement of SignificanceUsing the newly solved crystal structure of kindlin, we investigated, for the first time, the molecular mechanism of kindlin-mediated integrin activation through simultaneous binding of talin and kindlin. We demonstrate in atomist details how kindlin cooperates with talin to promote the activation of integrin αIIb-β3.

Linkers of the nucleoskeleton and cytoskeleton are key molecular complexes that span the nuclear envelope (NE) and provide a direct linkage between the nucleoskeleton and cytoskeleton. Two major components of these complexes are members of the SUN and KASH protein families that interact in the perinuclear space to allow the transmission of mechanochemical signals across the NE. Structural details of the mammalian SUN domain protein SUN2 have established that SUN2 must form a trimer to bind to KASH, and that this trimerization is mediated through two predicted coiled-coil regions of the protein, CC1 and CC2, which precede the SUN domain. Recent crystallographic data suggest that CC2-SUN formed an unexpected autoinhibited monomer unable to bind to KASH. These structural insights raise the question of how full-length SUN2 transitions from a monomer to a trimer inside the NE. In this study we used a computational approach to model a fragment of SUN2 containing CC1, CC2, and the SUN domain. We observed the dynamics of these modeled structures using ∼1 μs molecular dynamics simulations and showed that the interplay between CC1 and CC2 may be sufficient for the release of CC2-SUN2 from its autoinhibited state. Additionally, using our models and gel filtration analysis, we show the involvement of an E452 residue on CC1 in the monomer–­trimer transition of SUN2. Intriguingly, mutations in this residue have been seen in muscular dystrophy–associated SUN2 variants. Finally, we propose a Ca2+-dependent monomer–trimer transition of SUN2.

Bacteria have evolved as intelligent microorganisms that can colonize and form highly structured and cooperative multicellular communities with sophisticated singular and collective behaviors. The initial stages of colony formation and intercellular communication are particularly important to understand and depend highly on the spatial organization of cells. Controlling the distribution and growth of bacterial cells at the nanoscale is, therefore, of great interest in understanding the mechanisms of cell-cell communication at the initial stages of colony formation. Staphyloccocus aureus, a ubiquitous human pathogen, is of specific clinical importance due to the rise of antibiotic resistant strains of this species, which can cause life-threatening infections. Although several methods have attempted to pattern bacterial cells onto solid surfaces at single cell resolution, no study has truly controlled the 3D architectures of growing colonies. Herein, we present a simple, low-cost method to pattern S. aureus bacterial colonies and control the architecture of their growth. Using the wetting properties of micropatterened poly(dimethyl siloxane) platforms, with help from the physiological activities of the S. aureus cells, we fabricated connected networks of bacterial microcolonies of various sizes. Unlike conventional heterogeneous growth of biofilms on surfaces, the patterned S. aureus microcolonies in this work grow radially from nanostrings of a few bacterial cells, to form micrometer-thick rods when provided with a nutrient rich environment. This simple, efficient, and low-cost method can be used as a platform for studies of cell-cell communication phenomena, such as quorum sensing, horizontal gene transfer, and metabolic cross-feeding especially during initial stages of colony formation.

Size-dependent softening characteristics of electrodeposited solid core, hollow, C-shaped, and x-shaped nanocrystalline (nc) nickel pillars with outer diameters of ~ 520 nm were investigated by using uniaxial micro-compression techniques. Flow stress results show that only complex-shaped pillars with an average grain size of ~ 9.4 nm prepared with saccharine containing electrolytes soften with decreasing sample sizes and volume-to-surface area ratios. Interestingly, large grain (~ 18.2 nm) specimens prepared with saccharine-free electrolytes do not exhibit this softening behavior. Size-dependent softening may be due to the enhanced grain boundary-mediated deformation in the small grain pillars caused by segregation of the electrolyte additives at the grain boundaries.

Mechanical stresses directly regulate the function of several proteins of the integrin-mediated focal adhesion complex as they experience intra- and extracellular forces. Kindlin is a largely overlooked member of the focal adhesion complex whose roles in cellular mechanotransduction are only recently being identified. Recent crystallographic experiments have revealed that kindlins can form dimers that bind simultaneously to two integrins, providing a mechanistic explanation of how kindlins may promote integrin activation and clustering. In this study, using the newly identified molecular structure, we modeled the response of the kindlin2 dimer in complex with integrin β1 to mechanical cytoskeletal forces on integrins. Using molecular dynamics simulations, we show that forces on integrins are directly transmitted to the kindlin2 dimerization site, resulting in a shift in an R577-S550/E553 interaction network at this site. Under force, R577 on one protomer switches from interacting with S550 to forming new hydrogen bonds with E553 on the neighboring protomer, resulting in the strengthening of the kindlin2 dimer in complex with integrin β1. This force-induced strengthening is similar to the catch-bond mechanisms that have previously been observed in other adhesion molecules. Based on our results, we propose that the kindlin2 dimer is mechanosensitive and can strengthen integrin-mediated focal adhesions under force by shifting the interactions at its dimerization sites.

Author(s): Jahed, Z; Mofrad, MRK | Abstract: The transmission of mechanical signals across the nuclear envelope is primarily mediated by linkers of the nucleoskeleton and cytoskeleton (LINC complexes). These complexes bridge the inner and outer nuclear membrane and connect various elements of the cytoskeleton to the nucleoskeleton. Through their interaction with various cytoskeletal elements, LINC complexes repeatedly endure different types of mechanical loading. In this short review, we discuss the structural features of LINC complexes that allow them to withstand and transmit mechanical forces across the nuclear envelope, the types of mechanical forces on these complexes, and the consequences of these forces on the integrity of the nuclear envelope.

The LINC complex is found in a wide variety of organisms and is formed by the transluminal interaction between outer- and inner-nuclear-membrane KASH and SUN proteins, respectively. Most extensively studied are SUN1 and SUN2 proteins, which are widely expressed in mammals. Although SUN1 and SUN2 play functionally redundant roles in several cellular processes, more recent studies have revealed diverse and distinct functions for SUN1. While several recent in vitro structural studies have revealed the molecular details of various fragments of SUN2, no such structural information is available for SUN1. Herein, we conduct a systematic analysis of the molecular relationships between SUN1 and SUN2, highlighting key similarities and differences that could lead to clues into their distinct functions. We use a wide range of computational tools, including multiple sequence alignments, homology modeling, molecular docking, and molecular dynamic simulations, to predict structural differences between SUN1 and SUN2, with the goal of understanding the molecular mechanisms underlying SUN1 oligomerization in the nuclear envelope. Our simulations suggest that the structural model of SUN1 is stable in a trimeric state and that SUN1 trimers can associate through their SUN domains to form lateral complexes. We also ask whether SUN1 could adopt an inactive monomeric conformation as seen in SUN2. Our results imply that the KASH binding domain of SUN1 is also inhibited in monomeric SUN1 but through weaker interactions than in monomeric SUN2.

The effects of microstructure, sample dimensions, and cross-sectional geometry on the deformation characteristics of electroplated nanocrystalline copper sub-micron pillars are investigated. Nanocrystalline copper pillars were produced with four types of geometry—solid core, hollow, c-shaped, and x-shaped—with outer diameters of ~1000 or 220 nm and three different average grain sizes (between 5.1 and 49.3 nm). Flow stress results from uniaxial compression tests of 1000- and 220-nm-outer-diameter pillars, with average grain sizes in the range between ~32 and 50 nm, revealed there are no observable strength dependences with the pillar cross-sectional geometries. This suggests that they behave with bulk-like character: mechanical properties independent of size and sample geometry. All of the pillar specimens examined exhibit an increase in mechanical strength with reduction of grain sizes, but soften as the crystalline dimensions are smaller than 10 to 20 nm threshold limits. Interestingly, pillars with outer diameters of 220 nm are distinctively softer than the 1000-nm-diameter samples when their grain size is at and below this threshold limit. These results indicate a strength specimen size effect exists for such fine grain copper pillars.

The adhesion properties of Staphylococcus aureus on palladium–cobalt (Pd–Co) alloy nanostructures with various cross-sectional geometries have been characterised. They include solid core, hollow, c-shaped, and x-shaped pillars. These pillars have unique funnel-shaped geometric features on the top surfaces with average included angles between ∼142o and ∼149o. The success rates of cell attachment on these pillar tops were quantified by using field emission scanning electron microscopy techniques. Results show the Staphylococcus aureus attachment rates of Pd–Co solid core, hollow, and x-shaped pillars are statistically indistinguishable with success rate up to 82%. X-shaped pillars have the lowest attachment rate among the four geometries of 46 ± 5%.

A new methodology was developed to trap polystyrene or latex nanospheres in nanocrystalline nickel columnar nanostructures with various cross-sectional geometries. After filling the column interiors with polystyrene or latex nanospheres with diameters of ∼300 and 500 nm, respectively, the top openings of these small metallic nanopillars were capped with Staphylococcus aureus (S. aureus) bacterial cells. High resolution scanning electron microscopy inspection revealed that these bacterial cells were able to cover the top openings of C-shaped pillars within an hour of incubation at 37 °C. Results also demonstrated that capping S. aureus adhesive strengths to the nickel structures are greater than the cell wall cohesive strengths.

© 2017 by the authors. In this study we investigated the contact characteristics of human prostate cancer cells (PC3) on silicon micropillar arrays with complex shapes by using high-resolution confocal fluorescence microscopy techniques. These arrays consist of micropillars that are of various cross-sectional geometries which produce different deformation profiles in adherent cells. Fluorescence micrographs reveal that some DAPI (4′ ,6-diamidino-2-phenylindole)-stained nuclei from cells attached to the pillars develop nanometer scale slits and contain low concentrations of DNA. The lengths of these slits, and their frequency of occurrence, were characterized for various cross-sectional geometries. These DNA-depleted features are only observed in locations below the pillar's top surfaces. Results produced in this study indicate that surface topography can induce unique nanometer scale features in the PC3 cell.

A broad range of human diseases are associated with bacterial infections, often initiated by specific adhesion of a bacterium to the target environment. Despite the significant role of bacterial adhesion in human infectious diseases, details and mechanisms of bacterial adhesion have remained elusive. Herein, we study the physical interactions between Staphylococcus aureus, a type of micro-organism relevant to infections associated with medical implants, and nanocrystalline (nc) nickel nanostructures with various columnar features, including solid core, hollow, x-shaped and c-shaped pillars. Scanning electron microscopy results show the tendency of these bacterial cells to attach to the nickel nanostructures. Moreover, unique single bacterium attachment characteristics were observed on nickel nanostructures with dimensions comparable to the size of a single bacterium.

Sub-micron scale nanocrystalline rhodium pillars were fabricated by electron beam lithography and electroplating techniques. The fabricated specimens included solid core pillars and columnar structure with more complex cross-sectional geometries, including x-shaped and annulus shaped. Among these specimens, two groups of sub-micron scale annulus structures with sidewall thicknesses of 250 and 205 nm were fabricated. All of the structures have outer diameters of ~1 μm and consist of average grain size smaller than 22 nm. Uniaxial compression results reveal these rhodium pillars are very strong with true flow stresses exceeding 5 GPa and are not sensitive to the sample cross-sectional geometries.

Sub-micron scale columnar nickel structures with different cross-sectional geometries and grain sizes were successfully fabricated via electron beam lithography and electroplating methods. Uniaxial microcompression tests revealed the flow stresses of these structures increase with smaller grain sizes, in close agreement with the Hall-Petch relationship. Size dependent softening effects were only observed in nickel pillars with average grain sizes in the range of 9.4–13.2 nm. In contrast, pillars with grain size near ~22 nm do not exhibit any flow stress dependence on size, with the pillar strengths remaining relatively constant. In addition, for nickel pillars with the same microstructures, there is no observable dependence of flow stresses on the cross-sectional geometries.

Sub-micron scale nanocrystalline ruthenium columnar structures with average grain sizes of approximately 4 nm were fabricated by using electron beam lithography and electroplating techniques. Small scale ruthenium columns with several different cross-sectional geometries – solid core, keyhole, hollow, and x-shaped, were successfully manufactured. Uniaxial compressive plastic flow stress results of these ruthenium specimens revealed that they are not sensitive to the column diameters investigated, i.e. size independent. This suggests that the mechanical strength size dependence of nanocrystalline previously reported in literature may be suppressed or reduced if the grain size is significantly smaller than the external structure dimension. In addition, results indicated that the mechanical strengths of ruthenium nanocrystalline structures are not influenced by the cross-sectional geometry of the specimens.

Three-dimensional vertically aligned nano- and micropillars have emerged as promising tools for a variety of biological applications. Despite their increasing usage, the interaction mechanisms of cells with these rigid structures and their effect on single- and collective-cell behaviors are not well understood for different cell types. In the present study, we examine the response of glioma cells to micropillar arrays using a new microfabricated platform consisting of rigid silicon micropillar arrays of various shapes, sizes, and configurations fabricated on a single platform. We compare collective- and single-cell behaviors at micropillar array interfaces and show that glial cells under identical chemical conditions form distinct arrangements on arrays of different shapes and sizes. Tumor-like aggregation and branching of glial cells only occur on arrays with feature diameters greater than 2 μm, and distinct transitions are observed at interfaces between various arrays on the platform. Additionally, despite the same side-to-side spacing and gaps between micropillars, single glial cells interact with the flat silicon surface in the gap between small pillars but sit on top of larger micropillars. Furthermore, micropillars induced local changes in stress fibers and actin-rich filopodia protrusions as the cells conformed to the shape of spatial cues formed by these micropillars.

The genetic information of eukaryotic cells is enclosed within a double-layered nuclear envelope, which comprises an inner and outer nuclear membrane. Several transmembrane proteins locate to the nuclear envelope; however, only two integral protein complexes span the nuclear envelope and connect the inside of the nucleus to the cytoplasm. The nuclear pore complex (NPC) acts as a gateway for molecular exchange between the interior of the nucleus and the cytoplasm, whereas so-called LINC complexes physically link the nucleoskeleton and the cytoskeleton. In this Commentary, we will discuss recent studies that have established direct functional associations between these two complexes. The assembly of NPCs and their even distribution throughout the nuclear envelope is dependent on components of the LINC complex. Additionally, LINC complex formation is dependent on the successful localization of inner nuclear membrane components of LINC complexes and their transport through the NPC. Furthermore, the architecture of the nuclear envelope depends on both protein complexes. Finally, we will present recent evidence showing that LINC complexes can affect nucleo-cytoplasmic transport through the NPC, further highlighting the importance of understanding the associations of these essential complexes at the nuclear envelope.

© 2016, Biomedical Engineering Society. The nuclear pore complex (NPC) is primarily recognized for its function as the gateway for nucleocytoplasmic traffic, regulating biochemical exchange between the cytoplasm and the nucleoplasm. On the other hand, the LINC complex, comprised of SUN-domain and KASH-domain proteins, is typically credited as the main physical bridge across the nuclear envelope. However, recent evidence suggests that the NPC is also directly engaged with the cytoskeletal elements and the nucleoskeleton, and as such provides a direct physical association between the nucleus and the cytoskeleton. Moreover, by controlling the transport of inner nuclear membrane proteins, including components of the LINC complex, the NPC plays additional roles in physically connecting the cytoskeleton and the nucleus. This review examines the NPC’s direct and indirect contributions to nucleo-cytoskeletal coupling and mechanobiology.

Time-dependent nanoscale plasticity of nanocrystalline nickel at room temperature was critically explored through a series of micropillar creep and quasi-static compression experiments on rod and tube specimens fabricated by electron beam lithography and electroplating. Enhanced creep rates in tubes as compared to rods, establishes the facilitating role played by the free surface in time-dependent deformation. Creep stress exponent, n, and strain-rate sensitivity, m, were compared to examine connections between creep and the rate-dependent plasticity, if any. (C) 2015 Elsevier Ltd. All rights reserved.

We studied the properties of a fluoropolymer based on perfluoropolyether (PFPE) as the mold material for thermal nanoimprint lithography. The PFPE mold was duplicated from a hard master mold by casting and thermal-curing of the PFPE pre-polymer mixture. The PFPE mold was able to pattern 100nm half-pitch grating into a resist, with the very fine line edge roughness of the master mold faithfully duplicated into the resist. This implies PFPE is capable of a resolution far beyond 100nm, which is consistent with its high elastic modulus (two orders higher than PDMS) extracted from nanoindentation experiment. It was found that the elastic modulus is much higher near the film surface than deep inside the film. This is a very desirable property for an imprint mold, as the relatively stiff top surface offers high resolution, whereas the more flexible bulk is important for a conformal contact with the substrate and easy demolding.

Submicron-scale columnar cobalt structures with three different cross-sectional geometries – solid cylinders, x-shaped pillars and keyhole-shaped pillars – were characterized by uniaxial compression tests. All electroplated specimens possess a hybrid polycrystalline–nanocrystalline microstructure. Results of uniaxial compression revealed that the average mechanical strengths of submicron cobalt specimens are statistically indistinguishable, regardless of cross-sectional geometry. This surprising result is in contrast with previous studies of solid cylindrical pillars, where the mechanical strength shows a dependence on specimen size.

The mechanical properties exhibited by sub-micron scale columnar structures of cobalt, fabricated by electron beam lithography and electroplating techniques, were investigated through uniaxial compression. Transmission electron microscopy analyses show these specimens possess a microstructure with sub-micron grains which are elongated and aligned near to the pillar loading axis. In addition, small nanocrystalline cobalt crystals are also present within the columnar structure. These specimens display exceptional mechanical strength comparable with both bulk polycrystalline and nanocrystalline cobalt deposited by electroplating. Size-dependent softening with shrinking sample dimensions is also observed in this work. Additionally, the strength of these sub-micron structures appears to be strain rate sensitive and comparable with bulk nanocrystalline cobalt specimens.

The mechanical properties of submicron scale columnar zinc structures, with average diameters between 130 and 1060 nm, were characterized by uniaxial microcompression tests. The zinc pillars were fabricated by electron beam lithography and electroplating and were found to be generally single crystalline, with a preferred out-of-plane orientation close to the [0001] directions. Post deformation microstructural analysis suggests that the zinc pillars maintain their single-crystalline structure, but without twin boundary formation. Interestingly, the engineering flow stress results indicate that small-scale zinc structures are insensitive to both strain rate and size.

Sub-micron-scale columnar cadmium structures were tested in uniaxial compression. Transmission electron microscopy and electron diffraction analysis indicated that the cadmium pillars are virtually single crystalline. Compression results revealed that the mechanical strength of small-scale cadmium is strain rate sensitive and size dependent. Specimens with diameters near 0.5 and 1.1 μm are slightly stronger than bulk. However, as the cadmium structure size reduces to near 0.1 μm, the mechanical strengths exceed ∼1 GPa, approaching the theoretical strength of cadmium.

Novel fabrication techniques were developed to manufacture polycrystalline palladium, cobalt, and rhodium nanopillars with diameters of approximately 130 nm. These nanostructures have length-to-radius ratios in the range of 4 and 20. Using uniaxial nano-compression techniques, the buckling behaviors of the produced nanopillars were characterized and critical buckling loads were extracted. The tangent moduli, which were extracted from plots of critical buckling load as a function of effective specimen cross-sectional area, of palladium, cobalt, and rhodium nanopillars are 90 ± 6 GPa, 175 ± 9 GPa, and 375 ± 23 GPa, respectively. As expected, these calculated values are slightly smaller than the bulk polycrystalline elastic moduli for all three materials. Post compression scanning electron microscope images revealed ductile behavior for palladium nanostructures, while cobalt and rhodium specimens failed by fracture.