Utility of protein-protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display.

Over the past 3 decades, a diverse collection of small protein domains have been used as scaffolds to generate general purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from reversion-inducing Cysteine-rich Protein with Kazal Motifs and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and next generation sequencing, we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low µM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were inserted into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general purpose protein-binding reagents.

Structural insights into plasmalemma vesicle-associated protein (PLVAP): Implications for vascular endothelial diaphragms and fenestrae.

In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low-molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration-presumably a coiled coil-held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP-i.e., the long dimension of the pore-and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.

The WNT7A/WNT7B/GPR124/RECK signaling module plays an essential role in mammalian limb development.

In central nervous system vascular endothelial cells, signaling via the partially redundant ligands WNT7A and WNT7B requires two co-activator proteins, GPR124 and RECK. WNT7A and RECK have been shown previously to play a role in limb development, but the mechanism of RECK action in this context is unknown. The roles of WNT7B and GPR124 in limb development have not been investigated. Using combinations of conventional and/or conditional loss-of-function alleles for mouse Wnt7a, Wnt7b, Gpr124 and Reck, including a Reck allele that codes for a protein that is specifically defective in WNT7A/WNT7B signaling, we show that reductions in ligand and/or co-activator function synergize to cause reduced and dysmorphic limb bone growth. Two additional limb phenotypes - loss of distal Lmx1b expression and ectopic growth of nail-like structures - occur with reduced Wnt7a/Wnt7b gene copy number and, respectively, with Reck mutations and with combined Reck and Gpr124 mutations. A third limb phenotype - bleeding into a digit - occurs with the most severe combinations of Wnt7a/Wnt7b, Reck and Gpr124 mutations. These data imply that the WNT7A/WNT7B-FRIZZLED-LRP5/LRP6-GPR124-RECK signaling system functions as an integral unit in limb development.

Structure of the RECK CC domain, an evolutionary anomaly.

Five small protein domains, the CC-domains, at the N terminus of the RECK protein, play essential roles in signaling by WNT7A and WNT7B in the context of central nervous system angiogenesis and blood-brain barrier formation and maintenance. We have determined the structure of CC domain 4 (CC4) at 1.65-Å resolution and find that it folds into a compact four-helix bundle with three disulfide bonds. The CC4 structure, together with homology modeling of CC1, reveals the surface locations of critical residues that were shown in previous mutagenesis studies to mediate GPR124 binding and WNT7A/WNT7B recognition and signaling. Surprisingly, sequence and structural homology searches reveal no other cell-surface or secreted domains in vertebrates that resemble the CC domain, a pattern that is in striking contrast to other ancient and similarly sized domains, such as Epidermal Growth Factor, Fibronectin Type 3, Immunoglobulin, and Thrombospondin type 1 domains, which are collectively present in hundreds of proteins.

A mouse model for kinesin family member 11 (Kif11)-associated familial exudative vitreoretinopathy.

During mitosis, Kif11, a kinesin motor protein, promotes bipolar spindle formation and chromosome movement, and during interphase, Kif11 mediates diverse trafficking processes in the cytoplasm. In humans, inactivating mutations in KIF11 are associated with (1) retinal hypovascularization with or without microcephaly and (2) multi-organ syndromes characterized by variable combinations of lymphedema, chorioretinal dysplasia, microcephaly and/or mental retardation. To explore the pathogenic basis of KIF11-associated retinal vascular disease, we generated a Kif11 conditional knockout (CKO) mouse and investigated the consequences of early postnatal inactivation of Kif11 in vascular endothelial cells (ECs). The principal finding is that postnatal EC-specific loss of Kif11 leads to severely stunted growth of the retinal vasculature, mildly stunted growth of the cerebellar vasculature and little or no effect on the vasculature elsewhere in the central nervous system (CNS). Thus, in mice, Kif11 function in early postnatal CNS ECs is most significant in the two CNS regions-the retina and cerebellum-that exhibit the most rapid rate of postnatal growth, which may sensitize ECs to impaired mitotic spindle function. Several lines of evidence indicate that these phenotypes are not caused by reduced beta-catenin signaling in ECs, despite the close resemblance of the Kif11 CKO phenotype to that caused by EC-specific reductions in beta-catenin signaling. Based on prior work, defective beta-catenin signaling had been the only known mechanism responsible for monogenic human disorders of retinal hypovascularization. The present study implies that retinal hypovascularization can arise from a second and mechanistically distinct cause.

Defining the binding interface of Amyloid Precursor Protein (APP) and Contactin3 (CNTN3) by site-directed mutagenesis.

The Amyloid Precursor Protein (APP) and Contactin (CNTN) families of cell-surface proteins have been intensively studied in the context of neural development and neuropsychiatric diseases. Earlier studies demonstrated both genetic and biochemical interactions between the extracellular domains of APP and CNTN3, but their precise binding interfaces were not defined. In the present study, we have used binding assays between APP-alkaline phosphatase (AP) fusion proteins and CNTN-Fc fusion proteins, together with alanine substitution mutagenesis, to show that: (i) the second Fibronectin domain (Fn(2)) in CNTN3 mediates APP binding; (ii) the copper binding domain (CuBD) in APP mediates CNTN3 binding; and (iii) the most important amino acids for APP-CNTN3 binding reside on one face of CNTN3-Fn(2) and on one face of APP-CuBD. These experiments define the regions of direct contact that mediate the binding interaction between APP and CNTN3.

Molecular determinants in Frizzled, Reck, and Wnt7a for ligand-specific signaling in neurovascular development.

The molecular basis of Wnt-Frizzled specificity is a central question in developmental biology. Reck, a multi-domain and multi-functional glycosylphosphatidylinositol-anchored protein, specifically enhances beta-catenin signaling by Wnt7a and Wnt7b in cooperation with the 7-transmembrane protein Gpr124. Among amino acids that distinguish Wnt7a and Wnt7b from other Wnts, two clusters are essential for signaling in a Reck- and Gpr124-dependent manner. Both clusters are far from the site of Frizzled binding: one resides at the amino terminus and the second resides in a protruding loop. Within Reck, the fourth of five tandem repeats of an unusual domain with six-cysteines (the CC domain) is essential for Wnt7a stimulation: substitutions P256A and W261A in CC4 eliminate this activity without changing protein abundance or surface localization. Mouse embryos carrying ReckP256A,W261A have severe defects in forebrain angiogenesis, providing the strongest evidence to date that Reck promotes CNS angiogenesis by specifically stimulating Wnt7a and Wnt7b signaling.

Dlg1 activates beta-catenin signaling to regulate retinal angiogenesis and the blood-retina and blood-brain barriers.

Beta-catenin (i.e., canonical Wnt) signaling controls CNS angiogenesis and the blood-brain and blood-retina barriers. To explore the role of the Discs large/membrane-associated guanylate kinase (Dlg/MAGUK) family of scaffolding proteins in beta-catenin signaling, we studied vascular endothelial cell (EC)-specific knockout of Dlg1/SAP97. EC-specific loss of Dlg1 produces a retinal vascular phenotype that closely matches the phenotype associated with reduced beta-catenin signaling, synergizes with genetically-directed reductions in beta-catenin signaling components, and can be rescued by stabilizing beta-catenin in ECs. In reporter cells with CRISPR/Cas9-mediated inactivation of Dlg1, transfection of Dlg1 enhances beta-catenin signaling ~4 fold. Surprisingly, Frizzled4, which contains a C-terminal PDZ-binding motif that can bind to Dlg1 PDZ domains, appears to function independently of Dlg1 in vivo. These data expand the repertoire of Dlg/MAGUK family functions to include a role in beta-catenin signaling, and they suggest that proteins other than Frizzled receptors interact with Dlg1 to enhance beta-catenin signaling.

Interplay of the Norrin and Wnt7a/Wnt7b signaling systems in blood-brain barrier and blood-retina barrier development and maintenance.

ß-Catenin signaling controls the development and maintenance of the blood-brain barrier (BBB) and the blood-retina barrier (BRB), but the division of labor and degree of redundancy between the two principal ligand-receptor systems-the Norrin and Wnt7a/Wnt7b systems-are incompletely defined. Here, we present a loss-of-function genetic analysis of postnatal BBB and BRB maintenance in mice that shows striking threshold and partial redundancy effects. In particular, the combined loss of Wnt7a and Norrin or Wnt7a and Frizzled4 (Fz4) leads to anatomically localized BBB defects that are far more severe than observed with loss of Wnt7a, Norrin, or Fz4 alone. In the cerebellum, selective loss of Wnt7a in glia combined with ubiquitous loss of Norrin recapitulates the phenotype observed with ubiquitous loss of both Wnt7a and Norrin, implying that glia are the source of Wnt7a in the cerebellum. Tspan12, a coactivator of Norrin signaling in the retina, is also active in BBB maintenance but is less potent than Norrin, consistent with a model in which Tspan12 enhances the amplitude of the Norrin signal in vascular endothelial cells. Finally, in the context of a partially impaired Norrin system, the retina reveals a small contribution to BRB development from the Wnt7a/Wnt7b system. Taken together, these experiments define the extent of CNS region-specific cooperation for several components of the Norrin and Wnt7a/Wnt7b systems, and they reveal substantial regional heterogeneity in the extent to which partially redundant ligands, receptors, and coactivators maintain the BBB and BRB.

Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions.

Defining protein-protein interactions (PPIs) is central to the biological sciences. Here, we present a novel platform - Affinity Capture of Polyribosomes followed by RNA sequencing (ACAPseq) - for identifying PPIs. ACAPseq harnesses the power of massively parallel RNA sequencing (RNAseq) to quantify the enrichment of polyribosomes based on the affinity of their associated nascent polypeptides for an immobilized protein 'bait'. This method was developed and tested using neonatal mouse brain polyribosomes and a variety of extracellular domains as baits. Of 92 baits tested, 25 identified one or more binding partners that appear to be biologically relevant; additional candidate partners remain to be validated. ACAPseq can detect binding to targets that are present at less than 1 part in 100,000 in the starting polyribosome preparation. One of the observed PPIs was analyzed in detail, revealing the mode of homophilic binding for Protocadherin-9 (PCDH9), a non-clustered Protocadherin family member.

Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation.

Reck, a GPI-anchored membrane protein, and Gpr124, an orphan GPCR, have been implicated in Wnt7a/Wnt7b signaling in the CNS vasculature. We show here that vascular endothelial cell (EC)-specific reduction in Reck impairs CNS angiogenesis and that EC-specific postnatal loss of Reck, combined with loss of Norrin, impairs blood-brain barrier (BBB) maintenance. The most N-terminal domain of Reck binds to the leucine-rich repeat (LRR) and immunoglobulin (Ig) domains of Gpr124, and weakening this interaction by targeted mutagenesis reduces Reck/Gpr124 stimulation of Wnt7a signaling in cell culture and impairs CNS angiogenesis. Finally, a soluble Gpr124(LRR-Ig) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Reck, and a soluble Reck(CC1-5) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Gpr124. These experiments indicate that Reck and Gpr124 are part of the cell surface protein complex that transduces Wnt7a- and Wnt7b-specific signals in mammalian CNS ECs to promote angiogenesis and regulate the BBB.

Intramembrane Proteolysis of Astrotactins.

Astrotactins are vertebrate-specific membrane proteins implicated in neuron-glia interactions during central nervous system development and in hair follicle polarity during skin development. By studying epitope-tagged derivatives of mouse astrotactin-2 (Astn2) produced in transfected cells, we determined that the amino and carboxyl termini reside in the extracellular space and are initially linked by two transmembrane segments and a single cytoplasmic domain. We further show that Astn2 undergoes proteolytic cleavage in the second transmembrane domain (TM2) and that a disulfide bond holds the resulting two fragments together. Recombinant Astn1 also undergoes TM2 cleavage, as does Astn2 isolated from mouse cerebellum. Astn2 intramembrane proteolysis is insensitive to replacement of TM2 by the transmembrane domain of CD74 or by 21 alanines. However, replacement of TM2 by the transmembrane domain of CD4, the asialoglycoprotein receptor, or the transferrin receptor eliminates intramembrane proteolysis, as does leucine substitution of residues that overlap or are immediately upstream of the cleavage site. Replacement of the transmembrane domain of CD74 or the asialoglycoprotein receptor with Astn2 TM2 leads to the appearance of a carboxyl-terminal fragment consistent with intramembrane proteolysis. These experiments define a highly unusual transmembrane topology for the astrotactins, reveal intramembrane proteolysis as a feature of astrotactin maturation, and constrain the substrate sequences that are permissive for cleavage of one type 2 transmembrane segment.

The spatio-temporal domains of Frizzled6 action in planar polarity control of hair follicle orientation.

In mammals, hair follicles cover most of the body surface and exhibit precise and stereotyped orientations relative to the body axes. Follicle orientation is controlled by the planar cell polarity (PCP; or, more generally, tissue polarity) system, as determined by the follicle mis-orientation phenotypes observed in mice with PCP gene mutations. The present study uses conditional knockout alleles of the PCP genes Frizzled6 (Fz6), Vangl1, and Vangl2, together with a series of Cre drivers to interrogate the spatio-temporal domains of PCP gene action in the developing mouse epidermis required for follicle orientation. Fz6 is required starting between embryonic day (E)11.5 and E12.5. Eliminating Fz6 in either the anterior or the posterior halves of the embryo or in either the feet or the torso leads to follicle mis-orientation phenotypes that are limited to the territories associated with Fz6 loss, implying either that PCP signaling is required for communicating polarity information on a local but not a global scale, or that there are multiple independent sources of global polarity information. Eliminating Fz6 in most hair follicle cells or in the inter-follicular epidermis at E15.5 suggests that PCP signaling in developing follicles is not required to maintain their orientation. The asymmetric arrangement of Merkel cells around the base of each guard hair follicle dependents on Fz6 expression in the epidermis but not in differentiating Merkel cells. These experiments constrain current models of PCP signaling and the flow of polarity information in mammalian skin.

Sox7, Sox17, and Sox18 Cooperatively Regulate Vascular Development in the Mouse Retina.

Vascular development and maintenance are controlled by a complex transcriptional program, which integrates both extracellular and intracellular signals in endothelial cells. Here we study the roles of three closely related SoxF family transcription factors-Sox7, Sox17, and Sox18 -in the developing and mature mouse vasculature using targeted gene deletion on a mixed C57/129/CD1 genetic background. In the retinal vasculature, each SoxF gene exhibits a distinctive pattern of expression in different classes of blood vessels. On a mixed genetic background, vascular endothelial-specific deletion of individual SoxF genes has little or no effect on vascular architecture or differentiation, a result that can be explained by overlapping function and by reciprocal regulation of gene expression between Sox7 and Sox17. By contrast, combined deletion of Sox7, Sox17, and Sox18 at the onset of retinal angiogenesis leads to a dense capillary plexus with a nearly complete loss of radial arteries and veins, whereas the presence of a single Sox17 allele largely restores arterial identity, as determined by vascular smooth muscle cell coverage. In the developing retina, expression of all three SoxF genes is reduced in the absence of Norrin/Frizzled4-mediated canonical Wnt signaling, but SoxF gene expression is unaffected by reduced VEGF signaling in response to deletion of Neuropilin1 (Npn1). In adulthood, Sox7, Sox17, and Sox18 act in a largely redundant manner to maintain blood vessel function, as adult onset vascular endothelial-specific deletion of all three SoxF genes leads to massive edema despite nearly normal vascular architecture. These data reveal critical and partially redundant roles for Sox7, Sox17 and Sox18 in vascular growth, differentiation, and maintenance.

Identification of Astrotactin2 as a Genetic Modifier That Regulates the Global Orientation of Mammalian Hair Follicles.

Planar cell polarity (PCP) signaling controls the global orientation of surface structures, such as hairs and bristles, in both vertebrates and invertebrates. In Frizzled6(-/-) (Fz6(-/-)) mice, hair follicle orientations on the head and back are nearly random at birth, but reorient during early postnatal development to eventually generate a nearly parallel anterior-to-posterior array. We report the identification of a naturally occurring exon 5 deletion in Astrotactin2 (Astn2) that acts as a recessive genetic modifier of the Fz6(-/-) hair patterning phenotype. A genetically engineered Astn2 exon 5 deletion recapitulates the modifier phenotype. In Fz6(-/-);Astn2(ex5del/del) mice, hair orientation on the back is subtly biased from posterior-to-anterior, leading to a 180-degree orientation reversal in mature mice. These experiments suggest that Astn2, an endosomal membrane protein, modulates PCP signaling.

Partial interchangeability of Fz3 and Fz6 in tissue polarity signaling for epithelial orientation and axon growth and guidance.

In mammals, a set of anatomically diverse polarity processes - including axon growth and guidance, hair follicle orientation, and stereociliary bundle orientation in inner ear sensory hair cells - appear to be mechanistically related, as judged by their dependence on vertebrate homologues of core tissue polarity/planar cell polarity (PCP) genes in Drosophila. To explore more deeply the mechanistic similarities between different polarity processes, we have determined the extent to which frizzled 3 (Fz3) can rescue the hair follicle and Merkel cell polarity defects in frizzled 6-null (Fz6(-/-)) mice, and, reciprocally, the extent to which Fz6 can rescue the axon growth and guidance defects in Fz3(-/-) mice. These experiments reveal full rescue of the Fz6(-/-) phenotype by Fz3 and partial rescue of the Fz3(-/-) phenotype by Fz6, implying that these two proteins are likely to act in a conserved manner in these two contexts. Stimulated by these observations, we searched for additional anatomical structures that exhibit macroscopic polarity and that might plausibly use Fz3 and/or Fz6 signaling. This search has revealed a hitherto unappreciated pattern of papillae on the dorsal surface of the tongue that depends, at least in part, on redundant signaling by Fz3 and Fz6. Taken together, these experiments provide compelling evidence for a close mechanistic relationship between multiple anatomically diverse polarity processes.

Canonical WNT signaling components in vascular development and barrier formation.

Canonical WNT signaling is required for proper vascularization of the CNS during embryonic development. Here, we used mice with targeted mutations in genes encoding canonical WNT pathway members to evaluate the exact contribution of these components in CNS vascular development and in specification of the blood-brain barrier (BBB) and blood-retina barrier (BRB). We determined that vasculature in various CNS regions is differentially sensitive to perturbations in canonical WNT signaling. The closely related WNT signaling coreceptors LDL receptor-related protein 5 (LRP5) and LRP6 had redundant functions in brain vascular development and barrier maintenance; however, loss of LRP5 alone dramatically altered development of the retinal vasculature. The BBB in the cerebellum and pons/interpeduncular nuclei was highly sensitive to decrements in canonical WNT signaling, and WNT signaling was required to maintain plasticity of barrier properties in mature CNS vasculature. Brain and retinal vascular defects resulting from ablation of Norrin/Frizzled4 signaling were ameliorated by stabilizing ß-catenin, while inhibition of ß-catenin-dependent transcription recapitulated the vascular development and barrier defects associated with loss of receptor, coreceptor, or ligand, indicating that Norrin/Frizzled4 signaling acts predominantly through ß-catenin-dependent transcriptional regulation. Together, these data strongly support a model in which identical or nearly identical canonical WNT signaling mechanisms mediate neural tube and retinal vascularization and maintain the BBB and BRB.

Cellular resolution maps of X chromosome inactivation: implications for neural development, function, and disease.

Female eutherian mammals use X chromosome inactivation (XCI) to epigenetically regulate gene expression from ∼4% of the genome. To quantitatively map the topography of XCI for defined cell types at single cell resolution, we have generated female mice that carry X-linked, Cre-activated, and nuclear-localized fluorescent reporters--GFP on one X chromosome and tdTomato on the other. Using these reporters in combination with different Cre drivers, we have defined the topographies of XCI mosaicism for multiple CNS cell types and of retinal vascular dysfunction in a model of Norrie disease. Depending on cell type, fluctuations in the XCI mosaic are observed over a wide range of spatial scales, from neighboring cells to left versus right sides of the body. These data imply a major role for XCI in generating female-specific, genetically directed, stochastic diversity in eutherian mammals on spatial scales that would be predicted to affect CNS function within and between individuals.

Frizzled3 controls axonal development in distinct populations of cranial and spinal motor neurons.

Disruption of the Frizzled3 (Fz3) gene leads to defects in axonal growth in the VII(th) and XII(th) cranial motor nerves, the phrenic nerve, and the dorsal motor nerve in fore- and hindlimbs. In Fz3(-/-) limbs, dorsal axons stall at a precise location in the nerve plexus, and, in contrast to the phenotypes of several other axon path-finding mutants, Fz3(-/-) dorsal axons do not reroute to other trajectories. Affected motor neurons undergo cell death 2 days prior to the normal wave of developmental cell death that coincides with innervation of muscle targets, providing in vivo evidence for the idea that developing neurons with long-range axons are programmed to die unless their axons arrive at intermediate targets on schedule. These experiments implicate planar cell polarity (PCP) signaling in motor axon growth and they highlight the question of how PCP proteins, which form cell-cell complexes in epithelia, function in the dynamic context of axonal growth. DOI: http://dx.doi.org/10.7554/eLife.01482.001.