Extracellular vesicles from retinal pigment epithelial cells expressing R345W-Fibulin-3 induce epithelial-mesenchymal transition in recipient cells.

We have shown previously that expression of R345W-Fibulin-3 induces epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W-Fibulin-3 mutation are sufficient to induce EMT in recipient cells. ARPE-19 cells were infected with luciferase-tagged wild-type (WT)- Fibulin-3 or luciferase-tagged R345W-Fibulin-3 (R345W) using lentiviruses.

Cryoelectron tomography reveals the multiplex anatomy of condensed native chromatin and its unfolding by histone citrullination.

Nucleosome chains fold and self-associate to form higher-order structures whose internal organization is unknown. Here, cryoelectron tomography (cryo-ET) of native human chromatin reveals intrinsic folding motifs such as (1) non-uniform nucleosome stacking, (2) intermittent parallel and perpendicular orientations of adjacent nucleosome planes, and (3) a regressive nucleosome chain path, which deviates from the direct zigzag topology seen in reconstituted nucleosomal arrays. By examining the self-associated structures, we observed prominent nucleosome stacking in cis and anti-parallel nucleosome interactions, which are consistent with partial nucleosome interdigitation in trans.

Rapid Synthesis of Cryo-ET Data for Training Deep Learning Models.

Deep learning excels at cryo-tomographic image restoration and segmentation tasks but is hindered by a lack of training data. Here we introduce cryo-TomoSim (CTS), a MATLAB-based software package that builds coarse-grained models of macromolecular complexes embedded in vitreous ice and then simulates transmitted electron tilt series for tomographic reconstruction. We then demonstrate the effectiveness of these simulated datasets in training different deep learning models for use on real cryotomographic reconstructions.

Visualizing Cofilin-Actin Filaments by Immunofluorescence and CryoEM: Essential Steps for Observing Cofilactin in Cells.

Fluorescence microscopy of cytoskeletal proteins in situ using immunolabeling, fluorescent reagents, or expression of tagged proteins has been a common practice for decades but often with too little regard for what might not be visualized. This is especially true for assembled filamentous actin (F-actin), for which binding of fluorescently labeled phalloidin is taken as the gold standard for its quantification even though it is well known that F-actin saturated with cofilin (cofilactin) binds neither fluorescently labeled phalloidin nor genetically encoded F-actin reporters, such as LifeAct. Here, using expressed fluorescent cofilactin reporters, we show that cofilactin is the major component of some actin-containing structures in both normal and stressed neurons and present various fixation, permeabilization, and cryo-preservation methods for optimizing its observation.

Monosialotetrahexosylganglioside Promotes Early Aβ42 Oligomer Formation and Maintenance.

The aggregation of the amyloid beta (Aβ) peptide is associated with Alzheimer's disease (AD) pathogenesis. Cell membrane composition, especially monosialotetrahexosylganglioside (GM1), is known to promote the formation of Aβ fibrils, yet little is known about the roles of GM1 in the early steps of Aβ oligomer formation. Here, by using GM1-contained liposomes as a mimic of the neuronal cell membrane, we demonstrate that GM1 is a critical trigger of Aβ oligomerization and aggregation.

Cofilactin filaments regulate filopodial structure and dynamics in neuronal growth cones.

Cofilin is best known for its ability to sever actin filaments and facilitate cytoskeletal recycling inside of cells, but at higher concentrations in vitro, cofilin stabilizes a more flexible, hyper-twisted state of actin known as "cofilactin". While this filament state is well studied, a structural role for cofilactin in dynamic cellular processes has not been observed. With a combination of cryo-electron tomography and fluorescence imaging in neuronal growth cones, we observe that filopodial actin filaments switch between a fascin-linked and a cofilin-decorated state, and that cofilactin is associated with a variety of dynamic events within filopodia.

Structure of -Synthesized Cellulose Fibrils Viewed by Cryo-Electron Tomography and C Natural-Abundance Dynamic Nuclear Polarization Solid-State NMR.

Cellulose, the most abundant biopolymer, is a central source for renewable energy and functionalized materials. synthesis of cellulose microfibrils (CMFs) has become possible using purified cellulose synthase (CESA) isoforms from and hybrid aspen. The exact nature of these fibrils remains unknown.

Challenges and triumphs in cryo-electron tomography.

Cryo-electron tomography has stepped fully into the spotlight. Enthusiasm is high. Fortunately for us, this is an exciting time to be a cryotomographer, but there is still a way to go before declaring victory.

Cryo-Electron Tomography and Automatic Segmentation of Cultured Hippocampal Neurons.

Cryo-electron tomography is fast becoming a preferred method for studying intracellular environments at the molecular scale. Increases in data collection throughput means that large numbers of tomograms can be generated at rates too fast for humans to easily explore quantitatively. Currently, there is a large effort to make data collection and segmentation tools more automated.

Assessing constituent volumes and morphology of bovine casein micelles using cryo-electron tomography.

We investigated the applicability of cryo-electron tomography as a method to quantify changes in the major constituents of casein micelles (i.e., casein proteins, putative colloidal calcium phosphate nanoclusters, and serum-filled voids and channels) in response to their environment.

Coarse-grained simulations of actomyosin rings point to a nodeless model involving both unipolar and bipolar myosins.

Cytokinesis in many eukaryotic cells is orchestrated by a contractile actomyosin ring. While many of the proteins involved are known, the mechanism of constriction remains unclear. Informed by the existing literature and new three-dimensional (3D) molecular details from electron cryotomography, here we develop 3D coarse-grained models of actin filaments, unipolar and bipolar myosins, actin cross-linkers, and membranes and simulate their interactions.

Structure of the fission yeast actomyosin ring during constriction.

Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane, remains unclear. Here we use cryosectioning and cryofocused ion beam milling to gain access to cryopreserved actomyosin rings in for direct 3D imaging by electron cryotomography.

The helical MreB cytoskeleton in Escherichia coli MC1000/pLE7 is an artifact of the N-Terminal yellow fluorescent protein tag.

Based on fluorescence microscopy, the actin homolog MreB has been thought to form extended helices surrounding the cytoplasm of rod-shaped bacterial cells. The presence of these and other putative helices has come to dominate models of bacterial cell shape regulation, chromosome segregation, polarity, and motility. Here we use electron cryotomography to show that MreB does in fact form extended helices and filaments in Escherichia coli when yellow fluorescent protein (YFP) is fused to its N terminus but native (untagged) MreB expressed to the same levels does not.

Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria.

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (> 80 nm) helical filaments exist near or along either surface of the inner membrane.

Structure and composition of the postsynaptic density during development.

In this study, we used electron tomography as well as immunogold labeling to analyze the morphology and distribution of proteins within postsynaptic densities (PSDs) isolated from rats before birth (embryonic day 19) and at postnatal days 2, 21, and 60. Our data provide direct evidence of distinct morphological and compositional differences in PSDs throughout development. Not all PSD components are present at the early stages of development, with a near lack of the scaffolding molecule PSD-95 at E19 and P2.

{beta}CaMKII regulates actin assembly and structure.

Ca(2+)-Calmodulin-dependent protein kinase II (CaMKII) is an abundant synaptic protein that was recently shown to regulate the organization of actin filaments leading to structural modifications of synapses. CaMKII is a dodecameric complex with a special architecture that provides it with unique potential for organizing the actin cytoskeleton. We report using biochemical assays that the beta isoform of CaMKII binds to and bundles actin filaments, and the disposition of betaCaMKII within the actin bundles was revealed by cryoelectron tomography.

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive.

Human endoplasmic reticulum mannosidase I is subject to regulated proteolysis.

In the early secretory pathway, opportunistic cleavage of asparagine-linked oligosaccharides by endoplasmic reticulum (ER) mannosidase I targets misfolded glycoproteins for dislocation into the cytosol and destruction by 26 S proteasomes. The low basal concentration of the glycosidase is believed to coordinate the glycan cleavage with prolonged conformation-based ER retention, ensuring that terminally misfolded glycoproteins are preferentially targeted for destruction. Herein the intracellular fate of human ER mannosidase I was monitored to determine whether a post-translational process might contribute to the regulation of its intracellular concentration.

Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation.

The exocytic pathway provides a physical route through which newly synthesized secretory and membrane proteins are deployed to the eukaryote cell surface. For newly synthesized alpha1-antitrypsin (AAT), the modification of its asparagine-linked oligosaccharides by a slow-acting mannosidase partitions the misfolded monomer into the proteasomal degradation pathway. Herein, we asked whether, and how, modification by endoplasmic reticulum mannosidase I (ERManI) contributes to the preferential selection of the misfolded AAT monomer for proteasomal degradation.