Curved crease origami and topological singularities enable hyperextensibility of L. olor.

Fundamental limits of cellular deformations, such as hyperextension of a living cell, remain poorly understood. Here, we describe how the single-celled protist Lacrymaria olor, a 40-micrometer cell, is capable of reversibly and repeatably extending its necklike protrusion up to 1200 micrometers in 30 seconds. We discovered a layered cortical cytoskeleton and membrane architecture that enables hyperextensions through the folding and unfolding of cellular-scale origami. Physical models of this curved crease origami display topological singularities, including traveling developable cones and cytoskeletal twisted domain walls, which provide geometric control of hyperextension. Our work unravels how cell geometry encodes behavior in single cells and provides inspiration for geometric control in microrobotics and deployable architectures.

ß-Cell keratin 8 maintains islet mechanical integrity, mitochondrial ultrastructure, and ß-cell glucose transporter 2 plasma membrane targeting.

Islet ß-cell dysfunction is an underlying factor for type I diabetes (T1D) development. Insulin sensing and secretion are tightly regulated in ß-cells at multiple subcellular levels. The epithelial intermediate filament (IF) protein keratin (K) 8 is the main ß-cell keratin, constituting the filament network with K18. To identify the cell-autonomous functions of K8 in ß-cells, mice with targeted deletion of ß-cell K8 (K8flox/flox; Ins-Cre) were analyzed for islet morphology, ultrastructure, and integrity, as well as blood glucose regulation and streptozotocin (STZ)-induced diabetes development. Glucose transporter 2 (GLUT2) localization was studied in ß-cells in vivo and in MIN6 cells with intact or disrupted K8/K18 filaments. Loss of ß-cell K8 leads to a major reduction in K18. Islets without ß-cell K8 are more fragile, and these ß-cells display disjointed plasma membrane organization with less membranous E-cadherin and smaller mitochondria with diffuse cristae. Lack of ß-cell K8 also leads to a reduced glucose-stimulated insulin secretion (GSIS) response in vivo, despite undisturbed systemic blood glucose regulation. K8flox/flox, Ins-Cre mice have a decreased sensitivity to STZ compared with K8 wild-type mice, which is in line with decreased membranous GLUT2 expression observed in vivo, as GLUT2 is required for STZ uptake in ß-cells. In vitro, MIN6 cell plasma membrane GLUT2 is rescued in cells overexpressing K8/K18 filaments but mistargeted in cells with disrupted K8/K18 filaments. ß-Cell K8 is required for islet and ß-cell structural integrity, normal mitochondrial morphology, and GLUT2 plasma membrane targeting, and has implications on STZ sensitivity as well as systemic insulin responses.NEW & NOTEWORTHY Keratin 8 is the main cytoskeletal protein in the cytoplasmic intermediate filament network in ß-cells. Here for the first time, we assessed the ß-cell autonomous mechanical and nonmechanical roles of keratin 8 in ß-cell function. We demonstrated the importance of keratin 8 in islet and ß-cell structural integrity, maintaining mitochondrial morphology and GLUT2 plasma membrane targeting.

A tiny, long-distance hunter.

Lacrymaria olor cytoskeleton and membrane "origami" enables rapid cell hyperextension.

Novel Transmission Electron Microscopic Method to Quantify Protein Clustering on the Cell Surface.

The cell surface distribution patterns (clustering) of membrane proteins have been widely investigated in cell biology. Here we describe a novel transmission electron microscopic (TEM) protocol designed to improve the quality of information obtained about the protein distribution patterns detected. This novel method makes it possible to study the clustering of all transmembrane proteins on one half of the cytoplasmic membrane of a whole cell. To achieve better imaging, we combine various methods, including critical-point drying, fixation of gold beads with a carbon layer, and a newly developed chemical thinning method. In addition, in our image-processing algorithm, we implemented pair correlation and pair cross-correlation functions, providing more details and better quantitative accuracy in characterizing the size and numbers of possible protein clusters. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and transmission electron micrography Alternate Protocol: Direct cell labeling for transmission electron micrography Basic Protocol 2: Analysis of TEM images to detect immunogold-labeled proteins.

Cutinsomes of Malus Mill. (Rosaceae) leaf and pericarp: genesis, localization, and transport.

New data were obtained on specific bionanostructures, cutinsomes, which are involved in the formation of cuticles on the surface of leaf blades and pericarp of Malus domestica Borkh (Malus Mill., Rosaceae)introduced to the mountains at the altitudes of 1200 and 1700 m above sea level. Cutinsomes, which are electron-dense structures of spherical shape, have been identified by transmission electron microscopy. It was demonstrated that plastids can be involved in the synthesis of their constituent nanocomponents. The greatest number of nanoparticles was observed in the granal thylakoid lumen of the chloroplasts in palisade mesophyll cells and pericarp hypodermal cells. The transmembrane transport of cutinsomes into the cell wall cuticle proper by exocytosis has been visualized for the first time. The plasma membrane is directly involved in the excretion of nanostructures from the cell. Nanoparticles of cutinsomes in the form of necklace-like formations line up in a chain near cell walls, merge into larger conglomerates and are loaded into plasmalemma invaginations, and then, in membrane packing, they move into the cuticle, which covers both outer and inner cell walls of external tissues. The original materials obtained by us supplement the ideas about the non-enzymatic synthesis of cuticle components available in the literature and expand the cell compartment geography involved in this process.

Lifecycle of a predatory bacterium vampirizing its prey through the cell envelope and S-layer.

Predatory bacteria feed upon other bacteria in various environments. Bdellovibrio exovorus is an obligate epibiotic predator that attaches on the prey cell surface, where it grows and proliferates. Although the mechanisms allowing feeding through the prey cell envelope are unknown, it has been proposed that the prey's proteinaceous S-layer may act as a defensive structure against predation. Here, we use time-lapse and cryo-electron microscopy to image the lifecycle of B. exovorus feeding on Caulobacter crescentus. We show that B. exovorus proliferates by non-binary division, primarily generating three daughter cells. Moreover, the predator feeds on C. crescentus regardless of the presence of an S-layer, challenging its assumed protective role against predators. Finally, we show that apparently secure junctions are established between prey and predator outer membranes.

Molecular insights into capsular polysaccharide secretion.

Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.

Chlamydia psittaci infected cell studies by 4Pi Raman and atomic force microscopy.

Chlamydia psittaci is an avian bacterial pathogen that can cause atypical pneumonia in humans via zoonotic transmission. It is a Gram-negative intracellular bacterium that proliferates inside membrane bound inclusions in the cytoplasm of living eukaryotic cells. The study of such cells with C. psittaci inside without destroying them poses a significant challenge. We demonstrated in this work the utility of a combined multitool approach to analyze such complex samples. Atomic force microscopy was applied to obtain high-resolution images of the surface of infected cells upon entrance of bacteria. Atomic force microscopy scans revealed the morphological changes of the cell membrane of Chlamydia infected cells such as changes in roughness of cell membrane and the presence of micro vesicles. 4Pi Raman microscopy was used to image and probe the molecular composition of intracellular bacteria inside intact cells. Information about the structure of the inclusion produced by C. psittaci was obtained and it was found to have a similar molecular fingerprint as that of an intracellular lipid droplet but with less proteins and unsaturated lipids. The presented approach demonstrates complementarity of various microscopy-based approaches and might be useful for characterization of intracellular bacteria.

ColabSeg: An interactive tool for editing, processing, and visualizing membrane segmentations from cryo-ET data.

Cellular cryo-electron tomography (cryo-ET) has emerged as a key method to unravel the spatial and structural complexity of cells in their near-native state at unprecedented molecular resolution. To enable quantitative analysis of the complex shapes and morphologies of lipid membranes, the noisy three-dimensional (3D) volumes must be segmented. Despite recent advances, this task often requires considerable user intervention to curate the resulting segmentations. Here, we present ColabSeg, a Python-based tool for processing, visualizing, editing, and fitting membrane segmentations from cryo-ET data for downstream analysis. ColabSeg makes many well-established algorithms for point-cloud processing easily available to the broad community of structural biologists for applications in cryo-ET through its graphical user interface (GUI). We demonstrate the usefulness of the tool with a range of use cases and biological examples. Finally, for a large Mycoplasma pneumoniae dataset of 50 tomograms, we show how ColabSeg enables high-throughput membrane segmentation, which can be used as valuable training data for fully automated convolutional neural network (CNN)-based segmentation.

HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes.

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD41-4. Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes5-13. Here we used cryo-electron tomography to monitor Env-CD4 interactions at the membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design.

Membrane structure and internalization dynamics of human Flower isoforms hFWE3 and hFWE4 indicate a conserved endocytic role for hFWE4.

Human Flower (hFWE) isoforms hFWE1-4 are putative transmembrane (TM) proteins that reportedly mediate fitness comparisons during cell competition through extracellular display of their C-terminal tails. Isoform topology, subcellular localization, and duration of plasma membrane presentation are essential to this function. However, disagreement persists regarding the structure of orthologous fly and mouse FWEs, and experimental evidence for hFWE isoform subcellular localization or membrane structure is lacking. Here, we used AlphaFold2 and subsequent molecular dynamics-based structural predictions to construct epitope-tagged hFWE3 and hFWE4, the most abundant human isoforms, for experimental determination of their structure and internalization dynamics. We demonstrate that hFWE3 resides in the membrane of the endoplasmic reticulum (ER), while hFWE4 partially colocalizes with Rab4-, Rab5-, and Rab11-positive vesicles as well as with the plasma membrane. An array of imaging techniques revealed that hFWE4 positions both N- and C-terminal tails and a loop between second and third TM segments within the cytosol, while small (4-12aa) loops between the first and second and the third and fourth TM segments are either exposed to the extracellular space or within the lumen of cytoplasmic vesicles. Similarly, we found hFWE3 positions both N- and C-terminal tails in the cytosol, while a short loop between TM domains extends into the ER lumen. Finally, we demonstrate that hFWE4 exists only transiently at the cell surface and is rapidly internalized in an AP-2- and dynamin-1-dependent manner. Collectively, these data are consistent with a conserved role for hFWE4 in endocytic processes.

Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Structural basis of NINJ1-mediated plasma membrane rupture in cell death.

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1-7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.

Cryo-tomography reveals rigid-body motion and organization of apicomplexan invasion machinery.

The apical complex is a specialized collection of cytoskeletal and secretory machinery in apicomplexan parasites, which include the pathogens that cause malaria and toxoplasmosis. Its structure and mechanism of motion are poorly understood. We used cryo-FIB-milling and cryo-electron tomography to visualize the 3D-structure of the apical complex in its protruded and retracted states. Averages of conoid-fibers revealed their polarity and unusual nine-protofilament arrangement with associated proteins connecting and likely stabilizing the fibers. Neither the structure of the conoid-fibers nor the architecture of the spiral-shaped conoid complex change during protrusion or retraction. Thus, the conoid moves as a rigid body, and is not spring-like and compressible, as previously suggested. Instead, the apical-polar-rings (APR), previously considered rigid, dilate during conoid protrusion. We identified actin-like filaments connecting the conoid and APR during protrusion, suggesting a role during conoid movements. Furthermore, our data capture the parasites in the act of secretion during conoid protrusion.

The trajectory patterns of single HIV-1 virus-like particle in live CD4 cells: A real time three-dimensional multi-resolution microscopy study using encapsulated nonblinking giant quantum dot.

BACKGROUND: The exploration of virology knowledge was limited by the optical technology for the observation of virus. Previously, a three-dimensional multi-resolution real-time microscope system (3D-MRM) was developed to observe the uptake of HIV-1-tat peptide-modified nanoparticles in cell membrane. In this study, we labeled HIV-1 virus-like particles (VLPs) with passivated giant quantum dots (gQDs) and recorded their interactive trajectories with human Jurkat CD4 cells through 3D-MRM. METHODS: The labeled of gQDs of the HIV-1 VLPs in sucrose-gradient purified viral lysates was first confirmed by Cryo-electronic microscopy and Western blot assay. After the infection with CD4 cells, the gQD-labeled VLPs were visualized and their extracellular and intracellular trajectories were recorded by 3D-MRM. RESULTS: A total of 208 prime trajectories was identified and classified into three distinct patterns: cell-free random diffusion pattern, directional movement pattern and cell-associated movement pattern, with distributions and mean durations were 72.6%/87.6 s, 9.1%/402.7 s and 18.3%/68.7 s, respectively. Further analysis of the spatial-temporal relationship between VLP trajectories and CD4 cells revealed the three stages of interactions: (1) cell-associated (extracellular) diffusion stage, (2) cell membrane surfing stage and (3) intracellular directional movement stage. CONCLUSION: A complete trajectory of HIV-1 VLP interacting with CD4 cells was presented in animation. This encapsulating method could increase the accuracy for the observation of HIV-1-CD4 cell interaction in real time and three dimensions.

Structure of a cholinergic cell membrane.

Cell membranes are complex assemblies of proteins and lipids making transient or long-term associations that have yet to be characterized at a molecular level. Here, cryo-electron microscopy is applied to determine how phospholipids and cholesterol arrange between neighboring proteins (nicotinic acetylcholine receptors) of Torpedo cholinergic membrane. The lipids exhibit distinct properties in the two leaflets of the bilayer, influenced by the protein surfaces and by differences in cholesterol concentration. In the outer leaflet, the lipids show no consistent motif away from the protein surfaces, in keeping with their assumed fluidity. In the inner leaflet, where the cholesterol concentration is higher, the lipids organize into extensive close-packed linear arrays. These arrays are built from the sterol groups of cholesterol and the initial saturated portions of the phospholipid hydrocarbon chains. Together, they create an ordered ∼7 Å-thick "skin" within the hydrophobic core of the bilayer. The packing of lipids in the arrays appears to bear a close relationship to the linear cholesterol arrays that form crystalline monolayers at the air-water interface.

Total synthesis of himastatin.

The natural product himastatin has an unusual homodimeric structure that presents a substantial synthetic challenge. We report the concise total synthesis of himastatin from readily accessible precursors, incorporating a final-stage dimerization strategy that was inspired by a detailed consideration of the compound's biogenesis. Combining this approach with a modular synthesis enabled expedient access to more than a dozen designed derivatives of himastatin, including synthetic probes that provide insight into its antibiotic activity.

Topological Relationships Cytoskeleton-Membrane Nanosurface-Morphology as a Basic Mechanism of Total Disorders of RBC Structures.

The state of red blood cells (RBCs) and their functional possibilities depend on the structural organization of the membranes. Cell morphology and membrane nanostructure are compositionally and functionally related to the cytoskeleton network. In this work, the influence of agents (hemin, endogenous oxidation during storage of packed RBCs, ultraviolet (UV) radiation, temperature, and potential of hydrogen (pH) changes) on the relationships between cytoskeleton destruction, membrane nanostructure, and RBC morphology was observed by atomic force microscope. It was shown that the influence of factors of a physical and biochemical nature causes structural rearrangements in RBCs at all levels of organization, forming a unified mechanism of disturbances in relationships "cytoskeleton-membrane nanosurface-cell morphology". Filament ruptures and, consequently, large cytoskeleton pores appeared. The pores caused membrane topological defects in the form of separate grain domains. Increasing loading doses led to an increase in the number of large cytoskeleton pores and defects and their fusion at the membrane nanosurfaces. This caused the changes in RBC morphology. Our results can be used in molecular cell biology, membrane biophysics, and in fundamental and practical medicine.

High-speed atomic force microscopy reveals a three-state elevator mechanism in the citrate transporter CitS.

The secondary active transporter CitS shuttles citrate across the cytoplasmic membrane of gram-negative bacteria by coupling substrate translocation to the transport of two Na+ ions. Static crystal structures suggest an elevator type of transport mechanism with two states: up and down. However, no dynamic measurements have been performed to substantiate this assumption. Here, we use high-speed atomic force microscopy for real-time visualization of the transport cycle at the level of single transporters. Unexpectedly, instead of a bimodal height distribution for the up and down states, the experiments reveal movements between three distinguishable states, with protrusions of ∼0.5 nm, ∼1.0 nm, and ∼1.6 nm above the membrane, respectively. Furthermore, the real-time measurements show that the individual protomers of the CitS dimer move up and down independently. A three-state elevator model of independently operating protomers resembles the mechanism proposed for the aspartate transporter GltPh Since CitS and GltPh are structurally unrelated, we conclude that the three-state elevators have evolved independently.