Endothelial Cell Morphogenesis and Capillary-like Network Induced by Soluble and Bound VEGF in a Definite Biogel Composed of Collagen and Fibronectin

In vitro culture of endothelial cells to form capillary-like networks is essential in tissue engineering. Vascular endothelial growth factor (VEGF) is one of the primary signal proteins stimulating blood vessel formation. This growth factor can be soluble in the medium or protein-bound to the substrate. However, less attention has been paid to distinguishing the specific stimulations by soluble and bound VEGF. We conducted a series of experiments to explore the respective effects of these two VEGF forms. An in-house synthesized biogel comprising a definite concentration of collagen and fibronectin was designed to cultivate human umbilical vein endothelial cells to form the capillary-like network. Collagen served as the primary substrate for cell attachment. Fibronectin provided the surface to bind soluble VEGF in the culture medium to create the bound VEGF. The experiment of adding VEGF-blocking-peptide was conducted to prevent the formation of VEGF bound to the fibronectin domains, to distinguish the respective effects of the soluble and bound VEGF. With the in-house biogel of definite components, we were able to clarify the different roles of soluble and bound VEGF. The results indicated that the soluble VEGF promptly induced the cells to change from round to elongated shape, which contributed to forming network cords. Simultaneously, the bound VEGF provided long-term stimulation, causing the cells to migrate and differentiate into the final capillary-like network.

The Ephb2 Receptor Uses Homotypic, Head-to-Tail Interactions within Its Ectodomain as an Autoinhibitory Control Mechanism

The Eph receptor tyrosine kinases and their ephrin ligands direct axon pathfinding and neuronal cell migration, as well as mediate many other cell–cell communication events. Their dysfunctional signaling has been shown to lead to various diseases, including cancer. The Ephs and ephrins both localize to the plasma membrane and, upon cell–cell contact, form extensive signaling assemblies at the contact sites. The Ephs and the ephrins are divided into A and B subclasses based on their sequence conservation and affinities for each other. The molecular details of Eph–ephrin recognition have been previously revealed and it has been documented that ephrin binding induces higher-order Eph assemblies, which are essential for full biological activity, via multiple, distinct Eph–Eph interfaces. One Eph–Eph interface type is characterized by a homotypic, head-to-tail interaction between the ligand-binding and the fibronectin domains of two adjacent Eph molecules. While the previous Eph ectodomain structural studies were focused on A class receptors, we now report the crystal structure of the full ectodomain of EphB2, revealing distinct and unique head-to-tail receptor–receptor interactions. The EphB2 structure and structure-based mutagenesis document that EphB2 uses the head-to-tail interactions as a novel autoinhibitory control mechanism for regulating downstream signaling and that these interactions can be modulated by posttranslational modifications.

The cryo-EM structure of the endocytic receptor DEC-205

DEC-205 (CD205), a member of the macrophage mannose receptor protein family, is the prototypic endocytic receptor of dendritic cells, whose ligands include phosphorothioated cytosine-guanosine (CpG) oligonucleotides, a motif often seen in bacterial or viral DNA. However, despite growing biological and clinical significance, little is known about the structural arrangement of this receptor or any of its family members. Here we describe the 3.2 Å cryo-EM structure of human DEC-205, thereby illuminating the structure of the mannose receptor protein family. The DEC-205 monomer forms a compact structure comprising two intercalated rings of C-type lectin-like domains, where the N-terminal cysteine-rich and fibronectin domains reside at the central intersection. We establish a pH dependant oligomerisation pathway forming tetrameric DEC-205 using solution-based techniques and ultimately solved the 4.9 Å cryo-EM structure of the DEC-205 tetramer to identify the unfurling of the second lectin ring which enables tetramer formation. Furthermore, we suggest the relevance of this oligomerisation pathway within a cellular setting, whereby CpG binding appeared to disrupt this cell-surface oligomer. Accordingly, we provide insight into the structure and oligomeric assembly of the DEC-205 receptor.

RIM-binding protein couples synaptic vesicle recruitment to release sites

At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. We here show in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a “hinge” in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs’ conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites.

Fibronectin and Its Role in Human Infective Diseases

Fibronectin is a multidomain glycoprotein ubiquitously detected in extracellular fluids and matrices of a variety of animal and human tissues where it functions as a key link between matrices and cells. Fibronectin has also emerged as the target for a large number of microorganisms, particularly bacteria. There are clear indications that the binding of microorganism’ receptors to fibronectin promotes attachment to and infection of host cells. Each bacterium may use different receptors which recognize specific fibronectin domains, mostly the N-terminal domain and the central cell-binding domain. In many cases, fibronectin receptors have actions over and above that of simple adhesion: In fact, adhesion is often the prerequisite for invasion and internalization of microorganisms in the cells of colonized tissues. This review updates the current understanding of fibronectin receptors of several microorganisms with emphasis on their biochemical and structural properties and the role they can play in the onset and progression of host infection diseases. Furthermore, we describe the antigenic profile and discuss the possibility of designing adhesion inhibitors based on the structure of the fibronectin-binding site in the receptor or the receptor-binding site in fibronectin.

De Novo Isolation & Affinity Maturation of yeast-displayed Virion-binding human fibronectin domains by flow cytometric screening against Virions

Background The promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Identifying avenues for overcoming the challenges associated with using virions as screening reagents is paramount given the difficulties associated with obtaining high-purity virus-associated proteins that retain the conformation exhibited on the virion surface. Results Fluorescence activated cell sorting (FACS) of 1.5 × 107 clones taken from a naïve yeast surface-displayed human fibronectin domain (Fn3) against whole virions yielded two unique binders to Zika virions. Construction and FACS of site-directed binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zika-binding affinities. These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins in E. coli shake flask culture, with post-purification yields exceeding 10 mg/L. Conclusions FACS of a yeast-displayed binding domain library is an efficient method for de novo isolation of virion-binding domains. Affinities of isolated virion-binding clones are readily enhanced via FACS screening of mutant progeny libraries. Given that most binding domains are compatible with yeast display, the approach taken in this work may be broadly utilized for generating virion-binding domains against many different viruses for use in passive immunotherapy and the prevention of viral infection.

Twitchin kinase inhibits muscle activity

Muscle sarcomeres contain giant polypeptides composed of multiple immunoglobulin and fibronectin domains and one or two protein kinase domains. Although binding partners for a number of this family’s kinase domains have been identified, the catalytic necessity of these kinase domains remains unknown. In addition, various members of this kinase family are suspected pseudokinases with no or little activity. Here we address catalytic necessity for the first time, using the prototypic invertebrate representative twitchin (UNC-22) from Caenorhabditis elegans. In in vitro experiments, change of a conserved lysine (K) that is involved in ATP coordination to alanine (A) resulted in elimination of kinase activity without affecting the overall structure of the kinase domain. The same mutation, unc-22(sf21), was generated in the endogenous twitchin gene. The unc-22(sf21) worms have well-organized sarcomeres. However, unc-22(sf21) mutants move faster than wild-type worms and, by optogenetic experiments, contract more. Wild-type nematodes exhibited greater competitive fitness than unc-22(sf21) mutants. Thus the catalytic activity of twitchin kinase has a role in vivo, where it inhibits muscle activity and is likely maintained by selection.