Maturation and Conformational Switching of a De Novo Designed Phase-Separating Polypeptide.

Cellular compartments formed by biomolecular condensation are widespread features of cell biology. These organelle-like assemblies compartmentalize macromolecules dynamically within the crowded intracellular environment. However, the intermolecular interactions that produce condensed droplets may also create arrested states and potentially pathological assemblies such as fibers, aggregates, and gels through droplet maturation. Protein liquid-liquid phase separation is a metastable process, so maturation may be an intrinsic property of phase-separating proteins, where nucleation of different phases or states arises in supersaturated condensates. Here, we describe the formation of both phase-separated droplets and proteinaceous fibers driven by a de novo designed polypeptide. We characterize the formation of supramolecular fibers in vitro and in bacterial cells. We show that client proteins can be targeted to the fibers in cells using a droplet-forming construct. Finally, we explore the interplay between phase separation and fiber formation of the de novo polypeptide, showing that the droplets mature with a post-translational switch to largely ß conformations, analogous to models of pathological phase separation.

Maintenance of complex I and its supercomplexes by NDUF-11 is essential for mitochondrial structure, function and health.

Mitochondrial supercomplexes form around a conserved core of monomeric complex I and dimeric complex III; wherein a subunit of the former, NDUFA11, is conspicuously situated at the interface. We identified nduf-11 (B0491.5) as encoding the Caenorhabditis elegans homologue of NDUFA11. Animals homozygous for a CRISPR-Cas9-generated knockout allele of nduf-11 arrested at the second larval (L2) development stage. Reducing (but not eliminating) expression using RNAi allowed development to adulthood, enabling characterisation of the consequences: destabilisation of complex I and its supercomplexes and perturbation of respiratory function. The loss of NADH dehydrogenase activity was compensated by enhanced complex II activity, with the potential for detrimental reactive oxygen species (ROS) production. Cryo-electron tomography highlighted aberrant morphology of cristae and widening of both cristae junctions and the intermembrane space. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development presumably arises due to its involvement in complex I and supercomplex maintenance. This highlights the importance of respiratory complex integrity for health and the potential for its perturbation to cause mitochondrial disease. This article has an associated First Person interview with Amber Knapp-Wilson, joint first author of the paper.

Endothelial glycocalyx is damaged in diabetic cardiomyopathy: angiopoietin 1 restores glycocalyx and improves diastolic function in mice.

AIMS/HYPOTHESIS: Diabetic cardiomyopathy (DCM) is a serious and under-recognised complication of diabetes. The first sign is diastolic dysfunction, which progresses to heart failure. The pathophysiology of DCM is incompletely understood but microcirculatory changes are important. Endothelial glycocalyx (eGlx) plays multiple vital roles in the microcirculation, including in the regulation of vascular permeability, and is compromised in diabetes but has not previously been studied in the coronary microcirculation in diabetes. We hypothesised that eGlx damage in the coronary microcirculation contributes to increased microvascular permeability and hence to cardiac dysfunction. METHODS: We investigated eGlx damage and cardiomyopathy in mouse models of type 1 (streptozotocin-induced) and type 2 (db/db) diabetes. Cardiac dysfunction was determined by echocardiography. We obtained eGlx depth and coverage by transmission electron microscopy (TEM) on mouse hearts perfusion-fixed with glutaraldehyde and Alcian Blue. Perivascular oedema was assessed from TEM images by measuring the perivascular space area. Lectin-based fluorescence was developed to study eGlx in paraformaldehyde-fixed mouse and human tissues. The eGlx of human conditionally immortalised coronary microvascular endothelial cells (CMVECs) in culture was removed with eGlx-degrading enzymes before measurement of protein passage across the cell monolayer. The mechanism of eGlx damage in the diabetic heart was investigated by quantitative reverse transcription-PCR array and matrix metalloproteinase (MMP) activity assay. To directly demonstrate that eGlx damage disturbs cardiac function, isolated rat hearts were treated with enzymes in a Langendorff preparation. Angiopoietin 1 (Ang1) is known to restore eGlx and so was used to investigate whether eGlx restoration reverses diastolic dysfunction in mice with type 1 diabetes. RESULTS: In a mouse model of type 1 diabetes, diastolic dysfunction (confirmed by echocardiography) was associated with loss of eGlx from CMVECs and the development of perivascular oedema, suggesting increased microvascular permeability. We confirmed in vitro that eGlx removal increases CMVEC monolayer permeability. We identified increased MMP activity as a potential mechanism of eGlx damage and we observed loss of syndecan 4 consistent with MMP activity. In a mouse model of type 2 diabetes we found a similar loss of eGlx preceding the development of diastolic dysfunction. We used isolated rat hearts to demonstrate that eGlx damage (induced by enzymes) is sufficient to disturb cardiac function. Ang1 restored eGlx and this was associated with reduced perivascular oedema and amelioration of the diastolic dysfunction seen in mice with type 1 diabetes. CONCLUSIONS/INTERPRETATION: The association of CMVEC glycocalyx damage with diastolic dysfunction in two diabetes models suggests that it may play a pathophysiological role and the enzyme studies confirm that eGlx damage is sufficient to impair cardiac function. Ang1 rapidly restores the CMVEC glycocalyx and improves diastolic function. Our work identifies CMVEC glycocalyx damage as a potential contributor to the development of DCM and therefore as a therapeutic target.

De Novo Designed Peptide and Protein Hairpins Self-Assemble into Sheets and Nanoparticles.

The design and assembly of peptide-based materials has advanced considerably, leading to a variety of fibrous, sheet, and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible to the same or similar peptide building blocks. Here a de novo peptide system is presented that forms nanoparticles or sheets depending on the strategic placement of a "disulfide pin" between two elements of secondary structure that drive self-assembly. Specifically, homodimerizing and homotrimerizing de novo coiled-coil α-helices are joined with a flexible linker to generate a series of linear peptides. The helices are pinned back-to-back, constraining them as hairpins by a disulfide bond placed either proximal or distal to the linker. Computational modeling indicates, and advanced microscopy shows, that the proximally pinned hairpins self-assemble into nanoparticles, whereas the distally pinned constructs form sheets. These peptides can be made synthetically or recombinantly to allow both chemical modifications and the introduction of whole protein cargoes as required.

ESCRT-III controls nuclear envelope reformation.

During telophase, the nuclear envelope (NE) reforms around daughter nuclei to ensure proper segregation of nuclear and cytoplasmic contents. NE reformation requires the coating of chromatin by membrane derived from the endoplasmic reticulum, and a subsequent annular fusion step to ensure that the formed envelope is sealed. How annular fusion is accomplished is unknown, but it is thought to involve the p97 AAA-ATPase complex and bears a topological equivalence to the membrane fusion event that occurs during the abscission phase of cytokinesis. Here we show that the endosomal sorting complex required for transport-III (ESCRT-III) machinery localizes to sites of annular fusion in the forming NE in human cells, and is necessary for proper post-mitotic nucleo-cytoplasmic compartmentalization. The ESCRT-III component charged multivesicular body protein 2A (CHMP2A) is directed to the forming NE through binding to CHMP4B, and provides an activity essential for NE reformation. Localization also requires the p97 complex member ubiquitin fusion and degradation 1 (UFD1). Our results describe a novel role for the ESCRT machinery in cell division and demonstrate a conservation of the machineries involved in topologically equivalent mitotic membrane remodelling events.

Interventions for improving self-direction in people with dementia: a systematic review.

BACKGROUND: Dementia is a progressive disease that affects people's everyday functioning, including the ability to express values, needs and wishes, which can be considered key elements of self-direction. For the purpose of this review, self-direction refers to the organization and/or coordination of your own life, including professional and other care, with the objective of having what you perceive to be a good life. The aim of this systematic review was to assess and describe interventions that aim to improve self-direction of people with dementia. METHODS: A systematic search was conducted in PubMed, Embase, CINAHL, PsycInfo and the Cochrane Library. Empirical studies up to April 2020 were included that used qualitative and/or quantitative methods and reported on interventions for people with dementia aimed at improving self-direction. Stepwise study selection and the assessment of methodological quality were conducted independently by two authors. Data on study and intervention characteristics, outcomes related to self-direction and well-being of people with dementia and factors influencing the feasibility were extracted systematically and described narratively. RESULTS: Ten studies were identified describing a total of nine interventions. Interventions varied in terms of goals, content, target population and duration. Overall, interventions consisted of multiple components focusing on identifying "Who am I?" (beliefs, strengths, values, goals), identifying "What is important to me?" (meaningful activities and goal setting) and/or communicating about preferences with professionals and/or caregivers. The review provides indications that people with dementia may benefit from the interventions included. Overall, positive effects were found in studies on outcomes related to self-direction and wellbeing. However, outcomes measured using quantitative methods showed inconsistent effects between the studies. CONCLUSIONS: Although the methodological quality of all the studies included was 'good' or at least 'fair', the evidence base of interventions aiming to improve self-direction is still limited due to the low number of studies, the low number of participants and the frequent use of and their authors' own non-standardized measures. Nevertheless, the review points towards positive effects on self-direction and well-being. Identifying individual beliefs, strengths, values, goals and meaningful activities can be essential components of these interventions, as well as communication about the desired care and support.

The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components.

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively.

Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm.

We have developed a system for producing a supramolecular scaffold that permeates the entire Escherichia coli cytoplasm. This cytoscaffold is constructed from a three-component system comprising a bacterial microcompartment shell protein and two complementary de novo coiled-coil peptides. We show that other proteins can be targeted to this intracellular filamentous arrangement. Specifically, the enzymes pyruvate decarboxylase and alcohol dehydrogenase have been directed to the filaments, leading to enhanced ethanol production in these engineered bacterial cells compared to those that do not produce the scaffold. This is consistent with improved metabolic efficiency through enzyme colocation. Finally, the shell-protein scaffold can be directed to the inner membrane of the cell, demonstrating how synthetic cellular organization can be coupled with spatial optimization through in-cell protein design. The cytoscaffold has potential in the development of next-generation cell factories, wherein it could be used to organize enzyme pathways and metabolite transporters to enhance metabolic flux.

Online Self-Management Support for Family Caregivers Dealing With Behavior Changes in Relatives With Dementia (Part 2): Randomized Controlled Trial.

BACKGROUND: Online contacts with a health professional have the potential to support family caregivers of people with dementia. OBJECTIVE: The goal of the research was to study the effects of an online self-management support intervention in helping family caregivers deal with behavior changes of a relative with dementia. The intervention-involving among others personal email contacts with a dementia nurse-was compared with online interventions without these email contacts. METHODS: A randomized controlled trial was conducted with 81 family caregivers of people with dementia who live at home. Participants were randomly assigned to a (1) major self-management support intervention consisting of personal email contacts with a specialist dementia nurse, online videos, and e-bulletins; (2) medium intervention consisting only of online videos and e-bulletins; or (3) minor intervention consisting of only the e-bulletins. The primary outcome was family caregivers' self-efficacy in dealing with behavior changes of the relative with dementia. Secondary outcomes were family caregivers' reports of behavior problems in the people with dementia and the quality of the relationship between the family caregiver and the person with dementia. Measurements were performed at the baseline and at 6 (T1) and 12 weeks (T2) after the baseline. A mixed-model analysis was conducted to compare the outcomes of the 3 intervention arms. RESULTS: Family caregivers participating in the major intervention involving email contacts showed no statistically significant differences in self-efficacy after the intervention compared with the minor intervention involving only e-bulletins (difference -0.02, P=.99). In the adjusted analysis, the medium intervention (involving videos and e-bulletins) showed a negative trend over time (difference -4.21, P=.09) and at T1 (difference -4.71, P=.07) compared with the minor intervention involving only e-bulletins. No statistical differences were found between the intervention arms in terms of the reported behavior problems and the quality of the relationship between the family caregiver and the person with dementia. CONCLUSIONS: The expectation that an online self-management support intervention involving email contacts would lead to positive effects and be more effective than online interventions without personal email contacts was not borne out. One explanation might be related to the fact that not all family caregivers who were assigned to that intervention actually made use of the opportunity for personal email contact. The online videos were also not always viewed. To obtain more definite conclusions, future research involving extra efforts to reach higher use rates is required. TRIAL REGISTRATION: Netherlands Trial Registry NTR6237; http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=6237 (Archived by WebCite at http://www.webcitation.org/6v0S4fxTC). INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/resprot.8365.

High-Contrast Imaging of Nanodiamonds in Cells by Energy Filtered and Correlative Light-Electron Microscopy: Toward a Quantitative Nanoparticle-Cell Analysis.

Fluorescent nanodiamonds (fNDs) represent an emerging class of nanomaterials offering great opportunities for ultrahigh resolution imaging, sensing and drug delivery applications. Their biocompatibility, exceptional chemical and consistent photostability renders them particularly attractive for correlative light-electron microscopy studies providing unique insights into nanoparticle-cell interactions. Herein, we demonstrate a stringent procedure to image and quantify fNDs with a high contrast down to the single particle level in cells. Individual fNDs were directly visualized by energy-filtered transmission electron microscopy, that is, inside newly forming, early endosomal vesicles during their cellular uptake processes as well as inside cellular organelles such as a mitochondrion. Furthermore, we demonstrate the unequivocal identification, localization, and quantification of individual fNDs in larger fND clusters inside intracellular vesicles. Our studies are of great relevance to obtain quantitative information on nanoparticle trafficking and their various interactions with cells, membranes, and organelles, which will be crucial to design-improved sensors, imaging probes, and nanotherapeutics based on quantitative data.

Lipid species affect morphology of endoplasmic reticulum: a sea urchin oocyte model of reversible manipulation.

The ER is a large multifunctional organelle of eukaryotic cells. Malfunction of the ER in various disease states, such as atherosclerosis, diabetes, cancer, Alzheimer's and Parkinson's and amyotrophic lateral sclerosis, often correlates with alterations in its morphology. The ER exhibits regionally variable membrane morphology that includes, at the extremes, large relatively flat surfaces and interconnected tubular structures highly curved in cross-section. ER morphology is controlled by shaping proteins that associate with membrane lipids. To investigate the role of these lipids, we developed a sea urchin oocyte model, a relatively quiescent cell in which the ER consists mostly of tubules. We altered levels of endogenous diacylglycerol (DAG), phosphatidylethanolamine (PtdEth), and phosphatidylcholine by microinjection of enzymes or lipid delivery by liposomes and evaluated shape changes with 2D and 3D confocal imaging and 3D electron microscopy. Decreases and increases in the levels of lipids such as DAG or PtdEth characterized by negative spontaneous curvature correlated with conversion to sheet structures or tubules, respectively. The effects of endogenous alterations of DAG were reversible upon exogenous delivery of lipids of negative spontaneous curvature. These data suggest that proteins require threshold amounts of such lipids and that localized deficiencies of the lipids could contribute to alterations of ER morphology. The oocyte modeling system should be beneficial to studies directed at understanding requirements of lipid species in interactions leading to alterations of organelle shaping.

Process Evaluation of Nurse-Led Online Self-Management Support for Family Caregivers to Deal With Behavior Changes of a Relative With Dementia (Part 1): Mixed Methods Study.

BACKGROUND: Coping with behavioral changes is a daily challenge for family caregivers in all phases of dementia, and assistance is needed for it. An online self-management support intervention was therefore developed and conducted involving the following elements: (1) email contact with a specialized dementia nurse, (2) online videos, and (3) e-bulletins containing information about behavior changes and how to manage them. OBJECTIVE: The aim of this study was to understand (1) family caregivers' actual use of various elements of the online self-management support, (2) family caregivers' evaluation and satisfaction with the various elements, and (3) nurses' usage and evaluations of the online support through the tailored email contacts. METHODS: A mixed methods design was used in this process evaluation, combining quantitative and qualitative methods including analyses of dementia nurses' registration forms, the number of clicks on online videos and e-bulletins, evaluation questions answered by family caregivers in a survey questionnaire, semistructured interviews with family caregivers and nurses, and analysis of the content of the email contacts. RESULTS: The actual use of various elements of the online self-management support by family caregivers varied: 78% (21/27) of family caregivers had an email contact with the specialist nurse, 80% (43/54) of family caregivers clicked on an online video, and 37% (30/81) clicked on an e-bulletin. Family caregivers showed positive evaluations and satisfaction. The tailor-made approach in the personal email contacts in particular was valued by the family caregivers. Nurses' evaluations about providing self-management support online were mixed as it was a relatively new task for them. CONCLUSIONS: An important insight is that not all participants made optimum use of the various elements of the intervention. Nurses also said that the email contacts were more often used to express feelings about coping with behavioral changes. More research is needed to investigate the reasons why people accept, adopt, and adhere to online interventions to reduce cases where they are not used and to back them up appropriately with tailored (online) information and advice for their personal situations.

Modifying Self-Assembled Peptide Cages To Control Internalization into Mammalian Cells.

Nanoparticles can be used to transport a variety of biological cargoes into eukaryotic cells. Polypeptides provide a versatile material for constructing such systems. Previously, we have assembled nanoscale peptide cages (SAGEs) from de novo designed coiled-coil modules. Here, we show that the modules can be extended with short charged peptides to alter endocytosis of the assembled SAGE particles by cultured human cells in a tunable fashion. First, we find that the peptide extensions affect coiled-coil stability predictably: N-terminal polylysine and C-terminal polyglutamate tags are destabilizing; whereas, the reversed arrangements have little impact. Second, the cationic assembled particles are internalized faster and to greater extents by cells than the parent SAGEs. By contrast, anionic decorations markedly inhibit both aspects of uptake. These studies highlight how the modular SAGE system facilitates rational peptide design to fine-tune the bioactivity of nanoparticles, which should allow engineering of tailored cell-delivery vehicles.

Acute depletion of diacylglycerol from the cis-Golgi affects localized nuclear envelope morphology during mitosis.

Dysregulation of nuclear envelope (NE) assembly results in various cancers; for example, renal and some lung carcinomas ensue due to NE malformation. The NE is a dynamic membrane compartment and its completion during mitosis is a highly regulated process, but the detailed mechanism still remains incompletely understood. Previous studies have found that isolated diacylglycerol (DAG)-containing vesicles are essential for completing the fusion of the NE in nonsomatic cells. We investigated the impact of DAG depletion from the cis-Golgi in mammalian cells on NE reassembly. Using advanced electron microscopy, we observed an enriched DAG population of vesicles at the vicinity of the NE gaps of telophase mammalian cells. We applied a mini singlet oxygen generator-C1-domain tag that localized DAG-enriched vesicles at the perinuclear region, which suggested the existence of NE fusogenic vesicles. We quantified the impact of Golgi-DAG depletion by measuring the in situ NE rim curvature of the reforming NE. The rim curvature in these cells was significantly reduced compared with controls, which indicated a localized defect in NE morphology. Our novel results demonstrate the significance of the role of DAG from the cis-Golgi for the regulation of NE assembly.

Species differences in the morphology of transverse tubule openings in cardiomyocytes.

AIMS: The ultrastructure of ventricular cardiomyocyte T-tubule connections with the outer cell surface ('mouth' regions) has been reported to differ between mice and rabbits. In mice, T-tubule mouths form convoluted narrow spaces filled with electron-dense matter that impedes diffusion between T-tubular lumen and bulk extracellular space. Here, we explore whether T-tubule mouths are also constricted in rat (another murine model used frequently for cardiac research) and whether pig and human T-tubule mouth configurations are structurally more similar to mice or rabbits. METHODS AND RESULTS: We used chemically-fixed tissue and high-pressure frozen isolated cardiomyocytes to compare T-tubule mouth architecture using transmission electron microscopy and three-dimensional electron tomography. We find that rat T-tubular mouth architecture is more similar to that of rabbits than mice, lacking the marked tortuosity and electron-dense ground substance that obstructs access to deeper portions of the T-tubular system in mice. Pilot observations in larger mammals (pig, human) suggest that mouse may be the least representative animal model of T-tubule connectivity with the outer cell surface in larger mammals. CONCLUSION: Rat T-tubular system architecture appears to be more similar in size and topology to larger mammals than mice. T-tubular mouth topology may contribute to differences in experimental model behaviour, underscoring the challenge of appropriate model selection for research into cell and tissue function.

The 2018 correlative microscopy techniques roadmap.

Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell-cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure-function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints.

The actin-driven spatiotemporal organization of T-cell signaling at the system scale.

T cells are activated through interaction with antigen-presenting cells (APCs). During activation, receptors and signaling intermediates accumulate in diverse spatiotemporal distributions. These distributions control the probability of signaling interactions and thus govern information flow through the signaling system. Spatiotemporally resolved system-scale investigation of signaling can extract the regulatory information thus encoded, allowing unique insight into the control of T-cell function. Substantial technical challenges exist, and these are briefly discussed herein. While much of the work assessing T-cell spatiotemporal organization uses planar APC substitutes, we focus here on B-cell APCs with often stark differences. Spatiotemporal signaling distributions are driven by cell biologically distinct structures, a large protein assembly at the interface center, a large invagination, the actin-supported interface periphery as extended by smaller individual lamella, and a newly discovered whole-interface actin-driven lamellum. The more than 60 elements of T-cell activation studied to date are dynamically distributed between these structures, generating a complex organization of the signaling system. Signal initiation and core signaling prefer the interface center, while signal amplification is localized in the transient lamellum. Actin dynamics control signaling distributions through regulation of the underlying structures and drive a highly undulating T-cell/APC interface that imposes substantial constraints on T-cell organization. We suggest that the regulation of actin dynamics, by controlling signaling distributions and membrane topology, is an important rheostat of T-cell signaling.