Bottom-up assembly of biomedical relevant fully synthetic extracellular vesicles.

Extracellular vesicles (EVs) are fundamental for intercellular communication and influence nearly every process in cell physiology. However, because of their intricate molecular complexity, quantitative knowledge on their signaling mechanisms is missing, particularly impeding their therapeutic application. We used a complementary and quantitative engineering approach based on sequential synthetic bottom-up assembly of fully functional EVs with precisely controlled lipid, protein, and RNA composition. We show that the functionalities of synthetic EVs are analogous to natural EVs and demonstrate their programmable therapeutic administration for wound healing and neovascularization therapy. We apply transcriptome profiling to systematically decode synergistic effects between individual EV constituents, enabling analytical dissection and a fundamental understanding of EV signaling.

Pre-operative masseter muscle EMG activation during smile predicts synchronicity of smile development in facial palsy patients undergoing reanimation with the masseter nerve: A prospective cohort study✰.

BACKGROUND: Synchronicity of the oral commissure movement of a bilateral smile is a significant goal for reconstruction in facial reanimation and may only be guaranteed with use of the facial nerve as a donor nerve. Yet over the years several studies report some degree of spontaneity in certain patients when using a non-facial donor nerve, which indicates that synchronous initiation of the smile might be achievable with other donor nerves. We designed a prospective cohort study to evaluate whether pre-operative involuntary activation of the masseteric nerve during smile predicts development of a synchronous smile development when using the masseteric nerve for reanimation. METHODS: In a prospective cohort study unilateral long-standing facial palsy patients scheduled for dynamic smile reanimation with a free functional muscle transplant using the masseteric nerve as a donor nerve were preoperatively evaluated via EMG for involuntary activation of the masseter muscle upon smiling, which we called coactivation. Postoperatively, six months after noting the first muscle contraction smile synchronicity was evaluated. We analyzed the synchronicity of the bilateral smile development by analyzing slow-motion video sequences of the patients that were taken while the patients were watching funny video sequences. Results were then correlated with the pre-operative EMG. RESULTS: 30 patients were recruited for this prospective study and underwent facial reanimation surgery with a free gracilis transfer innervated by the masseteric nerve. 19 patients demonstrated involuntary coactivation of the masseter muscle upon smiling and 11 did not. Postoperatively all patients could demonstrate a voluntary smile. 94% of patients who had preoperative coactivation showed a synchronous movement of the oral commissure when smiling. In those patients, that did not show activation of the masseter muscle upon smiling 0% showed synchronicity. The preoperative coactivation of the masseter muscle is able to predict the outcome regarding synchronicity of the smile with a sensitivity of 99.7%, a specificity of 88.5% and 92.5% positive predictive value and 99.6% negative predictive value (p < 0.001 for all). CONCLUSIONS: The lack of masseter co-activation with smile predicts a lack of spontaneous involuntary smile after dynamic smile reconstruction using the masseteric nerve.

Characterization of spindle pole body duplication reveals a regulatory role for nuclear pore complexes.

The spindle pole body (SPB) of budding yeast duplicates once per cell cycle. In G1, the satellite, an SPB precursor, assembles next to the mother SPB (mSPB) on the cytoplasmic side of the nuclear envelope (NE). How the growing satellite subsequently inserts into the NE is an open question. To address this, we have uncoupled satellite growth from NE insertion. We show that the bridge structure that separates the mSPB from the satellite is a distance holder that prevents deleterious fusion of both structures. Binding of the γ-tubulin receptor Spc110 to the central plaque from within the nucleus is important for NE insertion of the new SPB. Moreover, we provide evidence that a nuclear pore complex associates with the duplicating SPB and helps to insert the SPB into the NE. After SPB insertion, membrane-associated proteins including the conserved Ndc1 encircle the SPB and retain it within the NE. Thus, uncoupling SPB growth from NE insertion unmasks functions of the duplication machinery.

IRSp53 mediates podosome formation via VASP in NIH-Src cells.

Podosomes are cellular "feet," characterized by F-actin-rich membrane protrusions, which drive cell migration and invasion into the extracellular matrix. Small GTPases that regulate the actin cytoskeleton, such as Cdc42 and Rac are central regulators of podosome formation. The adaptor protein IRSp53 contains an I-BAR domain that deforms membranes into protrusions and binds to Rac, a CRIB motif that interacts with Cdc42, an SH3 domain that binds to many actin cytoskeletal regulators with proline-rich peptides including VASP, and the C-terminal variable region by splicing. However, the role of IRSp53 and VASP in podosome formation had been unclear. Here we found that the knockdown of IRSp53 by RNAi attenuates podosome formation and migration in Src-transformed NIH3T3 (NIH-Src) cells. Importantly, the differences in the IRSp53 C-terminal splicing isoforms did not affect podosome formation. Overexpression of IRSp53 deletion mutants suggested the importance of linking small GTPases to SH3 binding partners. Interestingly, VASP physically interacted with IRSp53 in NIH-Src cells and was essential for podosome formation. These data highlight the role of IRSp53 as a linker of small GTPases to VASP for podosome formation.

How far in-silico computing meets real experiments. A study on the structure and dynamics of spin labeled vinculin tail protein by molecular dynamics simulations and EPR spectroscopy.

BACKGROUND: Investigation of conformational changes in a protein is a prerequisite to understand its biological function. To explore these conformational changes in proteins we developed a strategy with the combination of molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy. The major goal of this work is to investigate how far computer simulations can meet the experiments. METHODS: Vinculin tail protein is chosen as a model system as conformational changes within the vinculin protein are believed to be important for its biological function at the sites of cell adhesion. MD simulations were performed on vinculin tail protein both in water and in vacuo environments. EPR experimental data is compared with those of the simulated data for corresponding spin label positions. RESULTS: The calculated EPR spectra from MD simulations trajectories of selected spin labelled positions are comparable to experimental EPR spectra. The results show that the information contained in the spin label mobility provides a powerful means of mapping protein folds and their conformational changes. CONCLUSIONS: The results suggest the localization of dynamic and flexible regions of the vinculin tail protein. This study shows MD simulations can be used as a complementary tool to interpret experimental EPR data.

Metavinculin: New insights into functional properties of a muscle adhesion protein.

Metavinculin is a muscle-specific splice variant of the ubiquitously expressed cytoskeletal adaptor protein vinculin. Both proteins are thought to be co-expressed in all muscle types where they co-localize to microfilament-associated adhesion sites. It has been shown that a metavinculin-specific insertion of 68 amino acids alters the biochemical properties of the five-helix bundle in the tail domain. Here, we demonstrate that the metavinculin-specific helix H1' plays an important role for protein stability of the tail domain, since a point mutation in this helix, R975W, which is associated with the occurrence of dilated cardiomyopathy in man, further decreases thermal stability of the metavinculin tail domain. In striated muscle progenitor cells (myoblasts), both, metavinculin and the R975W mutant show significantly reduced, albeit distinctive residency and exchange rates in adhesion sites as compared to vinculin. In contrast to previous studies, we show that metavinculin is localized in a muscle fiber type-dependent fashion to the costameres of striated muscle, reflecting the individual metabolic and physiological status of a given muscle fiber. Metavinculin expression is highest in fast, glycolytic muscle fibers and virtually absent in M. diaphragmaticus, a skeletal muscle entirely lacking fast, glycolytic fibers. In summary, our data suggest that metavinculin enrichment in attachment sites of muscle cells leads to higher mechanical stability of adhesion complexes allowing for greater shear force resistance.

Orientation selective DEER measurements on vinculin tail at X-band frequencies reveal spin label orientations.

Double electron electron resonance (DEER) spectroscopy has been established as a valuable method to determine distances between spin labels bound to protein molecules. Caused by selective excitation of molecular orientations DEER primary data also depend on the mutual orientation of the spin labels. For a doubly spin labeled variant of the cytoskeletal protein vinculin tail strong orientation selection can be observed already at X-band frequencies, which allows us to reduce the problem to the relative orientation of two molecular axes and the spin-spin axis parameterized by three angles. A full grid search of parameter space reveals that the DEER experiment introduces parameter-space symmetry higher than the symmetry of the spin Hamiltonian. Thus, the number of equivalent parameter sets is twice as large as expected and the relative orientation of the two spin labels is ambiguous. Except for this inherent ambiguity the most probable relative orientation of the two spin labels can be determined with good confidence and moderate uncertainty by global fitting of a set of five DEER experiments at different offsets between pump and observer frequency. The experiment provides restraints on the angles between the z axis of the nitroxide molecular frame and the spin-spin vector and on the dihedral between the two z axes. When using the same type of label at both sites, assignment of the angle restraints is ambiguous and the sign of the dihedral restraint is also ambiguous.

Monomeric and dimeric conformation of the vinculin tail five-helix bundle in solution studied by EPR spectroscopy.

The cytoskeletal adaptor protein vinculin plays an important role in the control of cell adhesion and migration, linking the actin cytoskeleton to adhesion receptor complexes in cell adhesion sites. The conformation of the vinculin tail dimer, which is crucial for protein function, was analyzed using site-directed spin labeling in electron paramagnetic resonance spectroscopy. Interspin distances for a set of six singly and four doubly spin-labeled mutants of the tail domain of vinculin were determined and used as constraints for modeling of the vinculin tail dimer. A comparison of the results obtained by molecular dynamic simulations and a rotamer library approach reveals that the crystal structure of the vinculin tail monomer is essentially preserved in aqueous solution. The orientation of monomers within the dimer observed previously by x-ray crystallography agrees with the solution electron paramagnetic resonance data. Furthermore, the distance between positions 1033 is shown to increase by >3 nm upon interaction of the vinculin tail domain with F-actin.

Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels.

A prerequisite for successful tissue engineering is the existence of a functional microvascular network. We hypothesized that such networks can be created and quantified in an in vitro setting by co-culturing endothelial cells (ECs) with tissue-specific 'bystander cells' in 3-D gel matrices. To test this hypothesis we adapted a previously described in vitro microcarrier-based angiogenesis assay (V. Nehls and D. Drenckhahn, 1995, Microvasc Res 50: 311-322). On optimizing this assay, we noted that the initial EC-microcarrier coverage depended on EC type and seeding technique employed to coat the microcarrier beads with the ECs. A confluent EC monolayer on the microcarrier surfaces formed only when bovine aortic endothelial cells (BAECs) were admixed to the beads under gentle agitation on an orbital shaker. After embedding BAEC-covered microcarrier beads into a sandwich-like arrangement of collagen or fibrin gels, we assessed cellular outgrowth at different serum concentrations in terms of migration distance and sprout formation. Quantifiable sprout formation was highest at 1% fetal bovine serum (FBS) in collagen matrices and at 0.1% FBS in fibrin matrices. At higher serum concentration, excess cell migration and formation of clusters prevented quantitative analysis of sprouting. Following the fine-tuning of this angiogenesis assay, we co-cultured BAECs with adipose tissue-derived fibroblasts (FBs) and vascular smooth muscle cells (SMCs). While FBs were able to increase the average migration distance of BAECs in both matrices, SMCs enhanced BAEC migration in fibrin, but not in collagen gels. By contrast, the number of newly formed sprouts in fibrin gels was increased by both cell types. We conclude that in this model bystander cells enhance EC network formation in a matrix-dependent manner. Additionally, these results stress the importance of carefully selecting the experimental parameters of a given in vitro angiogenesis model.

Topographic guidance of endothelial cells on silicone surfaces with micro- to nanogrooves: orientation of actin filaments and focal adhesions.

To mimic the uniformly elongated endothelium in natural linear vessels, bovine aortic endothelial cells (BAECs) are cultured on micro- to nanogrooved, model poly(dimethylsiloxane) (PDMS) substrates preadsorbed with about 300 ng/cm(2) of fibronectin. BAEC alignment, elongation, and projected area were investigated for channel depths of 200 nm, 500 nm, 1 microm, and 5 microm, as well as smooth surfaces. Except for the 5 microm case, the ridge and channel widths were held nearly constant about 3.5 microm. With increasing channel depth, the percentage of aligned BAECs increased by factors of 2, 2, 1.8, and 1.7 for 1, 4, 24, and 48 h. Maximum alignment, about 90%, was observed for 1 microm deep channels at 1 h. The alignment of BAECs on grooved PDMS was maintained at least until cells reached near confluence. F-actin and vinculin at focal adhesions also aligned with channel direction. Analysis of confocal microscopy images showed that focal adhesions localized at corners and along the sidewalls of 1-microm deep channels. In contrast, focal adhesions could not form on the bottom of the 5-microm deep channels. Cell proliferation was similar on grooved and smooth substrates. In summary, PDMS substrates engraved with micro- and nanochannels provide a powerful method for investigating the interplay between topography and cell/cytoskeletal alignment.