Computational modeling of TGF-ß2:TßRI:TßRII receptor complex assembly as mediated by the TGF-ß coreceptor betaglycan.

Transforming growth factor-ß1, -ß2, and -ß3 (TGF-ß1, -ß2, and -ß3) are secreted signaling ligands that play essential roles in tissue development, tissue maintenance, immune response, and wound healing. TGF-ß ligands form homodimers and signal by assembling a heterotetrameric receptor complex comprised of two type I receptor (TßRI):type II receptor (TßRII) pairs. TGF-ß1 and TGF-ß3 ligands signal with high potency due to their high affinity for TßRII, which engenders high-affinity binding of TßRI through a composite TGF-ß:TßRII binding interface. However, TGF-ß2 binds TßRII 200-500 more weakly than TGF-ß1 and TGF-ß3 and signals with lower potency compared with these ligands. Remarkably, the presence of an additional membrane-bound coreceptor, known as betaglycan, increases TGF-ß2 signaling potency to levels similar to TGF-ß1 and -ß3. The mediating effect of betaglycan occurs even though it is displaced from and not present in the heterotetrameric receptor complex through which TGF-ß2 signals. Published biophysics studies have experimentally established the kinetic rates of the individual ligand-receptor and receptor-receptor interactions that initiate heterotetrameric receptor complex assembly and signaling in the TGF-ß system; however, current experimental approaches are not able to directly measure kinetic rates for the intermediate and latter steps of assembly. To characterize these steps in the TGF-ß system and determine the mechanism of betaglycan in the potentiation of TGF-ß2 signaling, we developed deterministic computational models with different modes of betaglycan binding and varying cooperativity between receptor subtypes. The models identified conditions for selective enhancement of TGF-ß2 signaling. The models provide support for additional receptor binding cooperativity that has been hypothesized but not evaluated in the literature. The models further showed that betaglycan binding to the TGF-ß2 ligand through two domains provides an effective mechanism for transfer to the signaling receptors that has been tuned to efficiently promote assembly of the TGF-ß2(TßRII)2(TßRI)2 signaling complex.

"I Think I Am Getting There" Understanding the Computational Identity of Engineering Students Participating in a Computationally Intensive Thermodynamics Course.

In response to the growing computational intensity of the healthcare industry, biomedical engineering (BME) undergraduate education is placing increased emphasis on computation. The presence of substantial gender disparities in many computationally intensive disciplines suggests that the adoption of computational instruction approaches that lack intentionality may exacerbate gender disparities. Educational research suggests that the development of an engineering and computational identity is one factor that can support students' decisions to enter and persist in an engineering major. Discipline-based identity research is used as a lens to understand retention and persistence of students in engineering. Our specific purpose is to apply discipline-based identity research to define and explore the computational identities of undergraduate engineering students who engage in computational environments. This work will inform future studies regarding retention and persistence of students who engage in computational courses. Twenty-eight undergraduate engineering students (20 women, 8 men) from three engineering majors (biomedical engineering, agricultural engineering, and biological engineering) participated in semi-structured interviews. The students discussed their experiences in a computationally-intensive thermodynamics course offered jointly by the Biomedical Engineering and Agricultural & Biological Engineering departments. The transcribed interviews were analyzed through thematic coding. The gender stereotypes associated with computer programming also come part and parcel with computer programming, possibly threatening a student's sense of belonging in engineering. The majority of the participants reported that their computational identity was "in the making." Students' responses also suggested that their engineering identity and their computational identity were in congruence, while some incongruence is found between their engineering identity and a creative identity as well as between computational identity and perceived feminine norms. Responses also indicate that students associate specific skills with having a computational identity. This study's findings present an emergent thematic definition of a computational person constructed from student perceptions and experiences. Instructors can support students' nascent computational identities through intentional mitigation of the gender stereotypes and biases, and by framing assignments to focus on developing specific skills associated with the computational modeling processes.

Supporting Computational Apprenticeship Through Educational and Software Infrastructure: A Case Study in a Mathematical Oncology Research Lab.

There is growing awareness of the need for mathematics and computing to quantitatively understand the complex dynamics and feedbacks in the life sciences. Although several institutions and research groups are conducting pioneering multidisciplinary research, communication and education across fields remain a bottleneck. The opportunity is ripe for using education research-supported mechanisms of cross-disciplinary training at the intersection of mathematics, computation, and biology. This case study uses the computational apprenticeship theoretical framework to describe the efforts of a computational biology lab to rapidly prototype, test, and refine a mentorship infrastructure for undergraduate research experiences. We describe the challenges, benefits, and lessons learned, as well as the utility of the computational apprenticeship framework in supporting computational/math students learning and contributing to biology, and biologists in learning computational methods. We also explore implications for undergraduate classroom instruction and cross-disciplinary scientific communication.

Heterodimer-heterotetramer formation mediates enhanced sensor activity in a biophysical model for BMP signaling.

Numerous stages of organismal development rely on the cellular interpretation of gradients of secreted morphogens including members of the Bone Morphogenetic Protein (BMP) family through transmembrane receptors. Early gradients of BMPs drive dorsal/ventral patterning throughout the animal kingdom in both vertebrates and invertebrates. Growing evidence in Drosophila, zebrafish, murine and other systems suggests that BMP ligand heterodimers are the primary BMP signaling ligand, even in systems in which mixtures of BMP homodimers and heterodimers are present. Signaling by heterodimers occurs through a hetero-tetrameric receptor complex comprising of two distinct type one BMP receptors and two type II receptors. To understand the system dynamics and determine whether kinetic assembly of heterodimer-heterotetramer BMP complexes is favored, as compared to other plausible BMP ligand-receptor configurations, we developed a kinetic model for BMP tetramer formation based on current measurements for binding rates and affinities. We find that contrary to a common hypothesis, heterodimer-heterotetramer formation is not kinetically favored over the formation of homodimer-tetramer complexes under physiological conditions of receptor and ligand concentrations and therefore other mechanisms, potentially including differential kinase activities of the formed heterotetramer complexes, must be the cause of heterodimer-heterotetramer signaling primacy. Further, although BMP complex assembly favors homodimer and homomeric complex formation over a wide range of parameters, ignoring these signals and instead relying on the heterodimer improves the range of morphogen interpretation in a broad set of conditions, suggesting a performance advantage for heterodimer signaling in patterning multiple cell types in a gradient.

Diversity and robustness of bone morphogenetic protein pattern formation.

Pattern formation by bone morphogenetic proteins (BMPs) demonstrates remarkable plasticity and utility in several contexts, such as early embryonic development, tissue patterning and the maintenance of stem cell niches. BMPs pattern tissues over many temporal and spatial scales: BMP gradients as short as 1-2 cell diameters maintain the stem cell niche of the Drosophila germarium over a 24-h cycle, and BMP gradients of several hundred microns establish dorsal-ventral tissue specification in Drosophila, zebrafish and Xenopus embryos in timescales between 30 min and several hours. The mechanisms that shape BMP signaling gradients are also incredibly diverse. Although ligand diffusion plays a dominant role in forming the gradient, a cast of diffusible and non-diffusible regulators modulate gradient formation and confer robustness, including scale invariance and adaptability to perturbations in gene expression and growth. In this Review, we document the diverse ways that BMP gradients are formed and refined, and we identify the core principles that they share to achieve reliable performance.

α2ß1 Integrin.

The α2ß1 integrin, also known as VLA-2, GPIa-IIa, CD49b, was first identified as an extracellular matrix receptor for collagens and/or laminins [55, 56]. It is now recognized that the α2ß1 integrin serves as a receptor for many matrix and nonmatrix molecules [35, 79, 128]. Extensive analyses have clearly elucidated the α2 I domain structural motifs required for ligand binding, and also defined distinct conformations that lead to inactive, partially active or highly active ligand binding [3, 37, 66, 123, 136, 137, 140]. The mechanisms by which the α2ß1 integrin plays a critical role in platelet function and homeostasis have been carefully defined via in vitro and in vivo experiments [76, 104, 117, 125]. Genetic and epidemiologic studies have confirmed human physiology and disease states mediated by this receptor in immunity, cancer, and development [6, 20, 21, 32, 43, 90]. The role of the α2ß1 integrin in these multiple complex biologic processes will be discussed in the chapter.

Mitigation of oxygen-induced retinopathy in α2ß1 integrin-deficient mice.

PURPOSE: The α2ß1 integrin plays an important but complex role in angiogenesis and vasculopathies. Published GWAS studies established a correlation between genetic polymorphisms of the α2ß1 integrin gene and incidence of diabetic retinopathy. Recent studies indicated that α2-null mice demonstrate superior vascularization in both the wound and diabetic microenvironments. The goal of this study was to determine whether the vasculoprotective effects of α2-integrin deficiency extended to the retina, using the oxygen-induced retinopathy (OIR) model for retinopathy of prematurity (ROP). METHODS: In the OIR model, wild-type (WT) and α2-null mice were exposed to 75% oxygen for 5 days (postnatal day [P] 7 to P12) and subsequently returned to room air for 6 days (P12-P18). Retinas were collected at postnatal day 7, day 13, and day 18 and examined via hematoxylin and eosin and Lectin staining. Retinas were analyzed for retinal vascular area, neovascularization, VEGF expression, and Müller cell activation. Primary Müller cell cultures from WT and α2-null mice were isolated and analyzed for hypoxia-induced VEGF-A expression. RESULTS: In the retina, the α2ß1 integrin was minimally expressed in endothelial cells and strongly expressed in activated Müller cells. Isolated α2-null primary Müller cells demonstrated decreased hypoxia-induced VEGF-A expression. In the OIR model, α2-null mice displayed reduced hyperoxia-induced vaso-attenuation, reduced pathological retinal neovascularization, and decreased VEGF expression as compared to WT counterparts. CONCLUSIONS: Our data suggest that the α2ß1 integrin contributes to the pathogenesis of retinopathy. We describe a newly identified role for α2ß1 integrin in mediating hypoxia-induced Müller cell VEGF-A production.

Flipping the switch: integrin switching provides metastatic competence.

Integrin switching plays a critical role in the progression to metastatic disease, but the mechanism by which it contributes remains poorly understood. In the 11 February 2014 issue of Science Signaling, Truong et al. identified a transforming growth factor-ß-mediated, prometastatic switch that is activated by ß1 integrin inhibition in triple-negative breast cancers (TNBCs). Their work provides insight into the complex signaling changes that arise from integrin switching. Further characterization of ß-integrin switching will require elucidation of the distribution of specific α-ß integrin heterodimers and the role of ligand binding. Identifying the nature of the molecular interactions and the influence of a specific oncogenic context, including the status of driver mutations such as those in Myc and p53, will define the next phase in integrin cancer biology.

The α2ß1 integrin is a metastasis suppressor in mouse models and human cancer.

Integrins regulate cell-cell and cell-matrix adhesion and thereby play critical roles in tumor progression and metastasis. Although work in preclinical models suggests that ß1 integrins may stimulate metastasis of a number of cancers, expression of the ß1 subunit alone has not been shown to be a useful prognostic indicator in human cancer patients. Here we have demonstrated that the α2ß1 integrin suppresses metastasis in a clinically relevant spontaneous mouse model of breast cancer. These data are consistent with previous studies indicating high expression of α2ß1 integrin in normal breast epithelium and loss of α2ß1 in poorly differentiated breast cancer. They are also consistent with our systematic analysis of microarray databases of human breast and prostate cancer, which revealed that decreased expression of the gene encoding α2 integrin, but not genes encoding α1, α3, or ß1 integrin, was predictive of metastatic dissemination and decreased survival. The predictive value of α2 expression persisted within both good-risk and poor-risk cohorts defined by estrogen receptor and lymph node status. Thus, the α2ß1 integrin functionally inhibits breast tumor metastasis, and α2 expression may serve as an important biomarker of metastatic potential and patient survival.

Reduced spontaneous relaxation in immature guinea pig airway smooth muscle is associated with increased prostanoid release.

Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-1 antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane B2 was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age.

Protein kinase activity of phosphoinositide 3-kinase regulates beta-adrenergic receptor endocytosis.

Phosphoinositide 3-kinase (PI(3)K) is a unique enzyme characterized by both lipid and protein kinase activities. Here, we demonstrate a requirement for the protein kinase activity of PI(3)K in agonist-dependent beta-adrenergic receptor (betaAR) internalization. Using PI(3)K mutants with either protein or lipid phosphorylation activity, we identify the cytoskeletal protein non-muscle tropomyosin as a substrate of PI(3)K, which is phosphorylated in a wortmannin-sensitive manner on residue Ser 61. A constitutively dephosphorylated (S61A) tropomyosin mutant blocks agonist-dependent betaAR internalization, whereas a tropomyosin mutant that mimics constitutive phosphorylation (S61D) complements the PI(3)K mutant, with only lipid phosphorylation activity reversing the defective betaAR internalization. Notably, knocking down endogenous tropomyosin expression using siRNAs that target different regions if tropomyosin resulted in complete inhibition of betaAR endocytosis, showing that non-muscle tropomyosin is essential for agonist-mediated receptor internalization. These studies demonstrate a previously unknown role for the protein phosphorylation activity of PI(3)K in betaAR internalization and identify non-muscle tropomyosin as a cellular substrate for protein kinase activity of PI(3)K.