Biology subjects

Medalia, O.

Publications and source records attributed to Medalia, O..

28 records · Page 2Linked to original sources

bioRxiv2021

Vimentin Intermediate Filaments and Filamentous Actin Form Unexpected Interpenetrating Networks That Redefine the Cell Cortex

Vimentin intermediate filaments (VIFs) and F-actin are filamentous cytoskeletal proteins generally thought to form completely independent networks that have vastly different properties and functions. Here, we show that, unexpectedly, there exist both extensive structural and functional interactions between VIFs and F-actin. We show that VIFs and F-actin form an interpenetrating network (IPN) within the cell cortex and interact synergistically at multiple length scales. This IPN structure has important functional consequences in cells: The IPN results in enhanced contractile forces in the cell. In addition, VIFs influence the diffusive behavior of actin monomers, suggesting specific associations between actin and vimentin proteins in the cytoplasm; this facilitates formation of the IPN and has downstream effects on other actin-driven processes. The results suggest that contributions of VIFs and F-actin are strongly correlated. Such interactions counter generally accepted behavior and are broadly significant given the wide range of processes currently attributed to F-actin alone.

cell biology↗

bioRxiv2021

The molecular architecture of vimentin filaments

Intermediate filaments are integral components of the cytoskeleton in metazoan cells. Due to their specific viscoelastic properties they are principal contributors to flexibility and tear strength of cells and tissues. Vimentin, an intermediate filament protein expressed in fibroblasts and endothelial cells, assembles into ~11 nm thick biopolymers, that are involved in a wide variety of cellular functions in health and disease. Here, we reveal the structure of in-situ polymerized vimentin filaments to a subnanometer resolution by applying cryo-electron tomography to mouse embryonic fibroblasts grown on electron microscopy grids. We show that vimentin filaments are tube-like assemblies with a well-defined helical symmetry. Their structure is comprised of five octameric, spring-like protofibrils harboring 40 vimentin polypeptide chains in cross-section. The protofibrils are connected by the intrinsically disordered head and helix 1A domains of vimentin. Individual filaments display two polymerization states characterized by either the presence or absence of a luminal density along the helical axis. The structure of vimentin filaments unveils the generic building plan of the intermediate filament superfamily in molecular details.

cell biology↗

bioRxiv2021

Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy

Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery. Keratin filaments provide cellular resistance to mechanical stresses, ensure cell and tissue integrity in addition to regulatory functions. Mutations in keratin genes are related to a variety of epithelial tissue diseases that mostly affect skin and hair. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Interestingly, two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Reconstitution of filaments of substantial length from keratin segments uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex architecture of keratin filaments, which demonstrate a remarkable degree of heterogeneity, suggesting that they are highly flexible, dynamic cytoskeletal structures.

cell biology↗

bioRxiv2021

Nanoscale resolution of microbial fiber degradation in action

Deconstruction of plant cell walls is imperative to global carbon cycling and sustainability efforts. Selected microbes degrade plant fibers using extremely efficient multi-enzymatic cellulosomes assemblies. Organization of cellulosomes on the bacterial cell surface and their ecological regulation remain elusive. By combining structural methodologies with molecular and biochemical approaches on the canonical Clostridium thermocellum system, we provide an unprecedented view into the in-situ structure and distribution of cellulosomal enzymes while interacting with their cellulosic substrate during fiber degradation. Structural exploration of growing cultures revealed isogenic phenotypic heterogeneity of cellulosome organization on single cells across the bacterial population, suggesting a division-of labor strategy driven by product-dependent dynamics. This study demonstrates how structural biology under near-physiological conditions can be employed to develop ecological hypotheses to understand microbial plant-fiber degradation at the single-cell nanoscale level. One Sentence SummaryThis study contributes critical insights into the in-situ organization of cellulosomes and their cellulosic substrates and provides evidence for phenotypic heterogeneity, with dynamic, growth phase-dependent organization of the fiber-degrading machinery.

microbiology↗

bioRxiv2020

A lamin A/C variant causing striated muscle disease provides insights into filament organization

The LMNA gene encodes the A-type lamins that polymerize into ~3.5 nm thick filaments, and together with B-type lamins and lamin binding proteins form the nuclear lamina. Mutations in LMNA are associated with a wide variety of pathologies. In this study, we analyzed the nuclear lamina of embryonic fibroblasts from LmnaH222P/H222P mice, which develop cardiomyopathy and muscular dystrophy. Although the organization of the lamina appeared unaltered, there were changes in chromatin and B-type lamin expression. An increase in nuclear size and consequently a relative reduction in heterochromatin near the lamina allowed for a higher resolution structural analysis of lamin filaments using cryo-electron tomography. This was most apparent when visualizing lamin filaments in situ, and using a nuclear extraction protocol. Averaging of individual segments of filaments in LmnaH222P/H222P mouse fibroblasts resolved two-polymers that constitute the mature filaments. Our findings provide better views of the organization of lamin filaments and the effect of a striated muscle disease-causing mutation on nuclear structure.

cell biology↗

bioRxiv2020

Nuclear Lamins A/C and B1 Provide a Structural Framework That Organizes and Anchors Nuclear Pore Complexes

Nuclear lamin isoforms form fibrous meshworks associated with nuclear pore complexes (NPCs). Using data sets prepared from sub-pixel and segmentation analyses of 3D-Structured Illumination Microscopy images of WT and lamin isoform knockout mouse embryo fibroblasts, we determined with high precision the spatial association of NPCs with specific lamin isoform fibers. These relationships are retained in the enlarged lamin meshworks of Lmna-/- and Lmnb1-/- fibroblast nuclei. Cryo-ET observations reveal that the lamin filaments composing the fibers contact the nucleoplasmic ring of NPCs. Knockdown of the ring-associated nucleoporin ELYS induces NPC clusters that exclude lamin A/C fibers, but include LB1 and LB2 fibers. Knockdown of the nucleoporins TPR or NUP153 alter the arrangement of lamin fibers and NPCs. Evidence that the number of NPCs is regulated by specific lamin isoforms is presented. Overall the results demonstrate that lamin isoforms and nucleoporins act together to maintain the normal organization of lamin meshworks and NPCs within the nuclear envelope.

cell biology↗

bioRxiv2020

Revealing the polarity of actin filaments in intact cells

The actin cytoskeleton plays a fundamental role in numerous cellular processes, such as cell motility, cytokinesis, and adhesion to the extracellular matrix. Revealing the polarity of individual actin filaments in cells, would foster an unprecedented understanding of cytoskeletal processes and their associated mechanical forces. Cryo-electron tomography provides the means for high-resolution structural imaging of cells. However, the low signal-to-noise ratio of cryo-tomograms obscures the high frequencies and therefore the polarity of actin filaments cannot be directly measured. Here, we developed an approach that enables to determine the polarity of actin filaments in cellular cryo-tomograms. We applied it to reveal the actin polarity distribution in focal adhesions, and show a linear relation between actin polarity and distance from the apical boundary of the adhesion site.

cell biology↗

bioRxiv2019

Nonlinear mechanics of lamin filaments and the meshwork topology build an emergent nuclear lamina

The nuclear lamina - a meshwork of intermediate filaments termed lamins - functions as a mechanotransduction interface between the extracellular matrix and the nucleus via the cytoskeleton. Although lamins are primarily responsible for the mechanical stability of the nucleus in multicellular organisms, in situ characterization of lamin filaments under tension has remained elusive. Here, we apply an integrative approach combining atomic force microscopy, cryo-electron tomography, network analysis, and molecular dynamics simulations to directly measure the mechanical response of single lamin filaments in its three-dimensional meshwork. Endogenous lamin filaments portray non-Hookean behavior - they deform reversibly under a force of a few hundred picoNewtons and stiffen at nanoNewton forces. The filaments are extensible, strong and tough, similar to natural silk and superior to the synthetic polymer Kevlar(R). Graph theory analysis shows that the lamin meshwork is not a random arrangement of filaments but the meshwork topology follows small world properties. Our results suggest that the lamin filaments arrange to form a robust, emergent meshwork that dictates the mechanical properties of individual lamin filaments. The combined approach provides quantitative insights into the structure-function organization of lamins in situ, and implies a role of meshwork topology in laminopathies.

biophysics↗

bioRxiv2018

Morphometric analysis of spread platelets identifies integrin αIIbβ3-specific contractile phenotype

Haemostatic platelet function is intimately linked to cellular mechanics and cytoskeletal morphology. How cytoskeletal reorganizations give rise to a highly contractile phenotype that is necessary for clot contraction remains poorly understood. To elucidate this process in vitro, we developed a morphometric screen to quantify the spatial organization of actin fibres and vinculin adhesion sites in single spread platelets. Platelets from healthy donors predominantly adopted a bipolar morphology on fibrinogen and fibronectin, whereas distinguishable, more isotropic phenotypes on collagen type I or laminin. Specific integrin IIb{beta}3 inhibitors induced an isotropic cytoskeletal organization in a dose-dependent manner. The same trend was observed with decreasing matrix stiffness. Circular F-actin arrangements in platelets from a patient with type II Glanzmann thrombasthenia (GT) were consistent with the residual activity of a small number of IIb{beta}3 integrins. Cytoskeletal morphologies in vitro can thus inform about platelet adhesion receptor identity and functionality, and integrin IIb{beta}3 mechanotransduction fundamentally determines the adoption of a highly contractile bipolar phenotype. Super-resolution microscopy and electron microscopies further confirmed the stress fibre-like contractile actin architecture. For the first time, our assay allows the unbiased and quantitative assessment of platelet morphologies and could help to identify defective platelet contractility contributing to elusive bleeding phenotypes.

cell biology↗

bioRxiv2017

Conformational states during vinculin unlocking differentially regulate focal adhesion properties

Focal adhesions (FAs) are multi-protein complexes that connect the actin cytoskeleton to the extracellular matrix, via integrin receptors. The growth, stability and adhesive functionality of these structures are tightly regulated by mechanical stress, yet, despite the extensive characterization of the integrin adhesome, the mechanisms underlying FA mechanosensitivity are still poorly understood. One of the key candidates for regulating FA-associated mechanosensing is vinculin, a prominent FA component, which was proposed to possess either closed (\"auto-inhibited\") or open (active) conformations. However, a direct demonstration of the nature of conformational transition between the two states is still absent. In this study we combined multiple structural and biological approaches to probe the transition from auto-inhibited to active conformation, and determine its effects on FA structure and dynamics. We further show here that the closed to open transition requires two sequential steps that can differentially regulate FA growth and stability.

biochemistry↗