Jesse K. Placone

The Extracellular Domain of Fibroblast Growth Factor Receptor 3 Inhibits Ligand-Independent Dimerization

The extracellular domain of a receptor tyrosine kinase has opposing effects on dimerization depending on whether ligand is present. Repulsive Potential Receptor tyrosine kinases (RTKs) consist of an extracellular region that binds to ligand, a single-pass transmembrane region, and an intracellular tyrosine kinase domain. Activation of RTKs requires receptor dimerization followed by ligand binding, which is thought to stabilize the dimer and alter its structure and activity. However, dimerization of RTKs can occur in the absence of ligand; indeed, some mutations in certain RTKs result in ligand-independent dimerization and activation. The contributions of the various extracellular domains of RTKs to ligand-independent receptor dimerization are unclear. Chen et al. used their quantitative imaging Förster resonance energy transfer technique to monitor ligand-independent dimerization of fluorescently tagged fibroblast growth factor receptor 3 (FGFR3) in plasma membrane–derived vesicles from transfected cells. They found that the extracellular domain, which is known to contribute to ligand-dependent dimerization, inhibited ligand-independent dimerization of the receptor. Thermodynamic analysis showed that the magnitude of this inhibitory effect was similar in extent, but opposite in effect, to that of activating mutations of FGFR3. Together, these data highlight the dual nature of the extracellular domain of FGFR3, which promotes RTK dimerization in the presence of ligand but inhibits dimerization when ligand is absent. Dysregulation of the ligand-independent dimerization of receptor tyrosine kinases (RTKs), which is the first step in the activation of RTKs, leads to various pathologies. A mechanistic understanding of the dimerization process is lacking, and this lack of basic knowledge is one bottleneck in the development of effective RTK-targeted therapies. For example, the roles and relative contributions of the different domains of RTKs to receptor dimerization are unknown. Here, we used quantitative imaging Förster resonance energy transfer (QI-FRET) to determine the contribution of the extracellular domain of fibroblast growth factor receptor 3 (FGFR3) to the dimerization of the receptor. We provide evidence that the contribution of the extracellular domain of FGFR3 to dimerization is repulsive in the absence of ligand and is on the order of ~1 kcal/mol. The repulsive contribution of the extracellular domain is similar in magnitude, but opposite in sign, to the contribution of pathogenic single–amino acid mutations to RTK signaling, and is therefore likely to be important for biological function. Together, these results highlight the fine balance in the domain interactions that regulate RTK dimerization and signaling.

Direct assessment of the effect of the Gly380Arg achondroplasia mutation on FGFR3 dimerization using quantitative imaging FRET.

The Gly380Arg mutation in FGFR3 is the genetic cause for achondroplasia (ACH), the most common form of human dwarfism. The mutation has been proposed to increase FGFR3 dimerization, but the dimerization propensities of wild-type and mutant FGFR3 have not been compared. Here we use quantitative imaging FRET to characterize the dimerization of wild-type FGFR3 and the ACH mutant in plasma membrane-derived vesicles from HEK293T cells. We demonstrate a small, but statistically significant increase in FGFR3 dimerization due to the ACH mutation. The data are consistent with the idea that the ACH mutation causes a structural change which affects both the stability and the activity of FGFR3 dimers in the absence of ligand.

Production of plasma membrane vesicles with chloride salts and their utility as a cell membrane mimetic for biophysical characterization of membrane protein interactions

Plasma membrane derived vesicles are used as a model system for the biochemical and biophysical investigations of membrane proteins and membrane organization. The most widely used vesiculation procedure relies on formaldehyde and dithiothreitol (DTT), but these active chemicals may introduce artifacts in the experimental results. Here we describe a procedure to vesiculate Chinese hamster ovary (CHO) cells, widely used for the expression of recombinant proteins, using a hypertonic vesiculation buffer containing chloride salts and no formaldehyde or DTT. We characterize the size distribution of the produced vesicles. We also show that these vesicles can be used for the biophysical characterization of interactions between membrane proteins.

Effect of Thanatophoric Dysplasia Type I Mutations on FGFR3 Dimerization

Thanatophoric dysplasia type I (TDI) is a lethal human skeletal growth disorder with a prevalence of 1 in 20,000 to 1 in 50,000 births. TDI is known to arise because of five different mutations, all involving the substitution of an amino acid with a cysteine in fibroblast growth factor receptor 3 (FGFR3). Cysteine mutations in receptor tyrosine kinases (RTKs) have been previously proposed to induce constitutive dimerization in the absence of ligand, leading to receptor overactivation. However, their effect on RTK dimer stability has never been measured experimentally. In this study, we characterize the effect of three TDI mutations, Arg248Cys, Ser249Cys, and Tyr373Cys, on FGFR3 dimerization in mammalian membranes, in the absence of ligand. We demonstrate that the mutations lead to surprisingly modest dimer stabilization and to structural perturbations of the dimers, challenging the current understanding of the molecular interactions that underlie TDI.

Extrusion-based 3D printing of poly(propylene fumarate) scaffolds with hydroxyapatite gradients

Abstract The primary focus of this work is to present the current challenges of printing scaffolds with concentration gradients of nanoparticles with an aim to improve the processing of these scaffolds. Furthermore, we address how print fidelity is related to material composition and emphasize the importance of considering this relationship when developing complex scaffolds for bone implants. The ability to create complex tissues is becoming increasingly relevant in the tissue engineering community. For bone tissue engineering applications, this work demonstrates the ability to use extrusion-based printing techniques to control the spatial deposition of hydroxyapatite (HA) nanoparticles in a 3D composite scaffold. In doing so, we combined the benefits of synthetic, degradable polymers, such as poly(propylene fumarate) (PPF), with osteoconductive HA nanoparticles that provide robust compressive mechanical properties. Furthermore, the final 3D printed scaffolds consisted of well-defined layers with interconnected pores, two critical features for a successful bone implant. To demonstrate a controlled gradient of HA, thermogravimetric analysis was carried out to quantify HA on a per-layer basis. Moreover, we non-destructively evaluated the tendency of HA particles to aggregate within PPF using micro-computed tomography (μCT). This work provides insight for proper fabrication and characterization of composite scaffolds containing particle gradients and has broad applicability for future efforts in fabricating complex scaffolds for tissue engineering applications.

Development and Characterization of a 3D Printed, Keratin-Based Hydrogel

Keratin, a naturally-derived polymer derived from human hair, is physiologically biodegradable, provides adequate cell support, and can self-assemble or be crosslinked to form hydrogels. Nevertheless, it has had limited use in tissue engineering and has been mainly used as casted scaffolds for drug or growth factor delivery applications. Here, we present and assess a novel method for the printed, sequential production of 3D keratin scaffolds. Using a riboflavin-SPS-hydroquinone (initiator–catalyst–inhibitor) photosensitive solution we produced 3D keratin constructs via UV crosslinking in a lithography-based 3D printer. The hydrogels obtained have adequate printing resolution and result in compressive and dynamic mechanical properties, uptake and swelling capacities, cytotoxicity, and microstructural characteristics that are comparable or superior to those of casted keratin scaffolds previously reported. The novel keratin-based printing resin and printing methodology presented have the potential to impact future research by providing an avenue to rapidly and reproducibly manufacture patient-specific hydrogels for tissue engineering and regenerative medicine applications.

Strong dimerization of wild-type ErbB2/Neu transmembrane domain and the oncogenic Val664Glu mutant in mammalian plasma membranes

Here, we study the homodimerization of the transmembrane domain of Neu, as well as an oncogenic mutant (V664E), in vesicles derived from the plasma membrane of mammalian cells. For the characterization, we use a Förster resonance energy transfer (FRET)-based method termed Quantitative Imaging-FRET (QI-FRET), which yields the donor and acceptor concentrations in addition to the FRET efficiencies in individual plasma membrane-derived vesicles. Our results demonstrate that both the wild-type and the mutant are 100% dimeric, suggesting that the Neu TM helix dimerizes more efficiently than other RTK TM domains in mammalian membranes. Furthermore, the data suggest that the V664E mutation causes a very small, but statistically significant change in dimer structure. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Increased expression of the integral membrane protein ErbB2 in Chinese hamster ovary cells expressing the anti-apoptotic gene Bcl-xL

Receptor tyrosine kinases (RTKs) are the second largest family of membrane receptors and play a key role in the regulation of vital cellular processes, such as control of cell growth, differentiation, metabolism, and migration. The production of whole-length RTKs in large quantities for biophysical or structural characterization, however, is a challenge. In this study, a cell engineering strategy using the anti-apoptotic Bcl-2 family protein, Bcl-x(L), was tested as a potential method for increasing stable expression levels of a recombinant RTK membrane protein in Chinese hamster ovary (CHO) cells. Wild-type and CHO cells stably overexpressing heterologous Bcl-x(L) were transformed with the gene for a model RTK membrane protein, ErbB2, on a plasmid also containing the Zeocin resistance gene. While CHO cells exhibited a gradual decrease in expression with passaging, CHO-bcl-x(L) cells offered an increased and sustained level of ErbB2 expression following continuous passaging over more than 33 days in culture. The increased ErbB2 expression in CHO-bcl-x(L) cells was evident both in stable transfected pools and in clonal isolates, and demonstrated both in Western blot analysis and flow cytometry. Furthermore, the sustained high-level protein expression in CHO-bcl-x(L) cells does not alter the correct membrane localization of the ErbB2 protein. Our results demonstrate that cellular engineering, specifically anti-apoptosis engineering, can provide increased and stable ErbB2 membrane protein expression in mammalian cells. This approach may also be useful for other membrane proteins in which large quantities are needed for biophysical and structural studies.

3D Printed Vascular Networks Enhance Viability in High-Volume Perfusion Bioreactor.

There is a significant clinical need for engineered bone graft substitutes that can quickly, effectively, and safely repair large segmental bone defects. One emerging field of interest involves the growth of engineered bone tissue in vitro within bioreactors, the most promising of which are perfusion bioreactors. Using bioreactor systems, tissue engineered bone constructs can be fabricated in vitro. However, these engineered constructs lack inherent vasculature and once implanted, quickly develop a necrotic core, where no nutrient exchange occurs. Here, we utilized COMSOL modeling to predict oxygen diffusion gradients throughout aggregated alginate constructs, which allowed for the computer-aided design of printable vascular networks, compatible with any large tissue engineered construct cultured in a perfusion bioreactor. We investigated the effect of 3D printed macroscale vascular networks with various porosities on the viability of human mesenchymal stem cells in vitro, using both gas-permeable, and non-gas permeable bioreactor growth chamber walls. Through the use of 3D printed vascular structures in conjunction with a tubular perfusion system bioreactor, cell viability was found to increase by as much as 50% in the core of these constructs, with in silico modeling predicting construct viability at steady state.

Understanding the extracellular forces that determine cell fate and maintenance

Stem cells interpret signals from their microenvironment while simultaneously modifying the niche through secreting factors and exerting mechanical forces. Many soluble stem cell cues have been determined over the past century, but in the past decade, our molecular understanding of mechanobiology has advanced to explain how passive and active forces induce similar signaling cascades that drive self-renewal, migration, differentiation or a combination of these outcomes. Improvements in stem cell culture methods, materials and biophysical tools that assess function have improved our understanding of these cascades. Here, we summarize these advances and offer perspective on ongoing challenges. Summary: This Review summarizes recent advances in stem cell culture methods, materials and biophysical tools, which reveal how various active and passive physical cues can influence cell behavior and regulate stem cell fate.