Search

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.

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.

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.

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.

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.

Many nuclear positioning events involve linker of nucleoskeleton and cytoskeleton (LINC) complexes, which transmit forces generated by the cytoskeleton across the nuclear envelope. LINC complexes are formed by trans-luminal interactions between inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins, but how these interactions are regulated is poorly understood. We combine in vivo C. elegans genetics, in vitro wounded fibroblast polarization, and in silico molecular dynamics simulations to elucidate mechanisms of LINC complexes. The extension of the KASH domain by a single alanine residue or the mutation of the conserved tyrosine at -7 completely blocked the nuclear migration function of C. elegans UNC-83. Analogous mutations at -7 of mouse nesprin-2 disrupted rearward nuclear movements in NIH 3T3 cells, but did not disrupt ANC-1 in nuclear anchorage. Furthermore, conserved cysteines predicted to form a disulfide bond between SUN and KASH proteins are important for the function of certain LINC complexes, and might promote a developmental switch between nuclear migration and nuclear anchorage. Mutations of conserved cysteines in SUN or KASH disrupted ANC-1-dependent nuclear anchorage in C. elegans and Nesprin-2G-dependent nuclear movements in polarizing fibroblasts. However, the SUN cysteine mutation did not disrupt nuclear migration. Moreover, molecular dynamics simulations showed that a disulfide bond is necessary for the maximal transmission of cytoskeleton-generated forces by LINC complexes in silico. Thus, we have demonstrated functions for SUN-KASH binding interfaces, including a predicted intermolecular disulfide bond, as mechanistic determinants of nuclear positioning that may represent targets for regulation.

Many nuclear positioning events involve linker of nucleoskeleton and cytoskeleton (LINC) complexes, which transmit forces generated by the cytoskeleton across the nuclear envelope. LINC complexes are formed by trans-luminal interactions between inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins, but how these interactions are regulated is poorly understood. We combine in vivo C. elegans genetics, in vitro wounded fibroblast polarization, and in silico molecular dynamics simulations to elucidate mechanisms of LINC complexes. The extension of the KASH domain by a single alanine residue or the mutation of the conserved tyrosine at -7 completely blocked the nuclear migration function of C. elegans UNC-83. Analogous mutations at -7 of mouse nesprin-2 disrupted rearward nuclear movements in NIH 3T3 cells, but did not disrupt ANC-1 in nuclear anchorage. Furthermore, conserved cysteines predicted to form a disulfide bond between SUN and KASH proteins are important for the function of certain LINC complexes, and might promote a developmental switch between nuclear migration and nuclear anchorage. Mutations of conserved cysteines in SUN or KASH disrupted ANC-1-dependent nuclear anchorage in C. elegans and Nesprin-2G-dependent nuclear movements in polarizing fibroblasts. However, the SUN cysteine mutation did not disrupt nuclear migration. Moreover, molecular dynamics simulations showed that a disulfide bond is necessary for the maximal transmission of cytoskeleton-generated forces by LINC complexes in silico. Thus, we have demonstrated functions for SUN-KASH binding interfaces, including a predicted intermolecular disulfide bond, as mechanistic determinants of nuclear positioning that may represent targets for regulation.

Numerous biological functions of a cell, including polarization, differentiation, division, and migration, rely on its ability to endure mechanical forces generated by the cytoskeleton on the nucleus. Coupling of the cytoskeleton and nucleoskeleton is ultimately mediated by LINC complexes that are formed via a strong interaction between SUN- and KASH-domain-containing proteins in the nuclear envelope. These complexes are mechanosensitive and essential for the transmission of forces between the cytoskeleton and nucleoskeleton, and the progression of cellular mechanotransduction. Herein, using molecular dynamics, we examine the effect of tension on the human SUN2-KASH2 complex and show that it is remarkably stable under physiologically relevant tensile forces and large strains. However, a covalent disulfide bond between two highly conserved cysteine residues of SUN2 and KASH2 is crucial for the stability of this interaction and the transmission of forces through the complex.

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.

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. © 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. © 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.

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. © 2017 by the authors.

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. © 2016 American Chemical Society.

Cells contain several mechanosensing components that transduce mechanical signals into biochemical cascades. During cell–ECM adhesion, a complex network of molecules mechanically couples the extracellular matrix (ECM), cytoskeleton, and nucleoskeleton. The network comprises transmembrane receptor proteins and focal adhesions, which link the ECM and cytoskeleton. Additionally, recently identified protein complexes extend this linkage to the nucleus by linking the cytoskeleton and the nucleoskeleton. Despite numerous studies in this field, due to the complexity of this network, our knowledge of the mechanisms of cell–ECM adhesion at the molecular level remains remarkably incomplete. Herein, we present a review of the structures of key molecules involved in cell-ECM adhesion, along with an evaluation of their predicted roles in mechanical sensing. Additionally, specific binding events prompted by force-induced conformational changes of each molecule are discussed. Finally, we propose a model for the biomechanical events prominent in cell–ECM adhesion.

Cells contain several mechanosensing components that transduce mechanical signals into biochemical cascades. During cell–ECM adhesion, a complex network of molecules mechanically couples the extracellular matrix (ECM), cytoskeleton, and nucleoskeleton. The network comprises transmembrane receptor proteins and focal adhesions, which link the ECM and cytoskeleton. Additionally, recently identified protein complexes extend this linkage to the nucleus by linking the cytoskeleton and the nucleoskeleton. Despite numerous studies in this field, due to the complexity of this network, our knowledge of the mechanisms of cell–ECM adhesion at the molecular level remains remarkably incomplete. Herein, we present a review of the structures of key molecules involved in cell-ECM adhesion, along with an evaluation of their predicted roles in mechanical sensing. Additionally, specific binding events prompted by force-induced conformational changes of each molecule are discussed. Finally, we propose a model for the biomechanical events prominent in cell–ECM adhesion.