Temperature-Sensitive Mutations That Cause Stage-Specific Defects in Zebrafish Fin Regeneration

When amputated, the fins of adult zebrafish rapidly regenerate the missing tissue. Fin regeneration proceeds through several stages, including wound healing, establishment of the wound epithelium, recruitment of the blastema from mesenchymal cells underlying the wound epithelium, and differentiation and outgrowth of the regenerate. We screened for temperature-sensitive mutations that affect the regeneration of the fin. Seven mutations were identified, including five that fail to regenerate their fins, one that causes slow growth during regeneration, and one that causes dysmorphic bumps or tumors to develop in the regenerating fin. reg5 mutants fail to regenerate their caudal fins, whereas reg6 mutants develop dysmorphic bumps in their regenerates at the restrictive temperature. Temperature-shift experiments indicate that reg5 and reg6 affect different stages of regeneration. The critical period for reg5 occurs during the early stages of regeneration before or during establishment of the blastema, resulting in defects in subsequent growth of the blastema and failure to differentiate bone-forming cells. The critical period for reg6 occurs after the onset of bone differentiation and during early stages of regenerative outgrowth. Both reg5 and reg6 also show temperature-sensitive defects in embryonic development or in ontogenetic outgrowth of the juvenile fin.     

PIK3R1 Mutations Cause Syndromic Insulin Resistance with Lipoatrophy

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.     

The Endolysin-Binding Domain Encompasses the N-Terminal Region of the Mycobacteriophage Ms6 Gp1 Chaperone

The intermolecular interactions of the mycobacteriophage Ms6 secretion chaperone with endolysin were characterized. The 384-amino-acid lysin (lysin₃₈₄)-binding domain was found to encompass the N-terminal region of Gp1, which is also essential for a lysis phenotype in Escherichia coli. In addition, a GXXXG-like motif involved in Gp1 homo-oligomerization was identified within the C-terminal region.     

Sec3 Mutations Are Synthetically Lethal with Profilin Mutations and Cause Defects in Diploid-Specific Bud-Site Selection

Replacement of the wild-type yeast profilin gene (PFY1) with a mutated form (pfy1-111) that has codon 72 changed to encode glutamate rather than arginine results in defects similar to, but less severe than, those that result from complete deletion of the profilin gene. We have used a colony color-sectoring assay to identify mutations that cause pfy1-111, but not wild-type, cells to be inviable. These profilin synthetic lethal (psl) mutations result in various degrees of abnormal growth, morphology, and temperature sensitivity in PFY1 cells. We have examined psl1 strains in the most detail. Interestingly, these strains display a diploid-specific defect in bud-site selection; haploid strains bud normally, while homozygous diploid strains show a dramatic increase in random budding. We discovered that PSL1 is the late secretory gene, SEC3, and have found that mutations in several other late secretory genes are also synthetically lethal with pfy1-111. Our results are likely to reflect an interdependence between the actin cytoskeleton and secretory processes in directing cell polarity and growth. Moreover, they indicate that the secretory pathway is especially crucial for maintaining budding polarity in diploids.     

Vesicular Stomatitis Virus Matrix Protein Mutations That Affect Association with Host Membranes and Viral Nucleocapsids

Viral matrix (M) proteins bind the nucleoprotein core (nucleocapsid) to host membranes during the process of virus assembly by budding. Previous studies using truncated M proteins had implicated the N-terminal 50 amino acids of the vesicular stomatitis virus M protein in binding both membranes and nucleocapsids and a sequence from amino acids 75-106 as an additional membrane binding region. Structure-based mutations were introduced into these two regions, and their effects on membrane association and incorporation into nucleocapsid-M protein complexes were determined using quantitative assays. The results confirmed that the N terminus of M protein is involved in association with plasma membranes as well as nucleocapsids, although these two activities were differentially affected by individual mutations. Mutations in the 75-106 region affected incorporation into nucleocapsid-M complexes but had only minor effects on association with membranes. The ability of site-specific mutant M proteins to complement growth of temperature-sensitive M mutant virus did not correlate well with the ability to associate with membranes or nucleocapsids, suggesting that complementation involves an additional activity of M protein. Mutants with similar abilities to associate with membranes and nucleocapsids but differing in complementation activity were incorporated into infectious cDNA clones. Infectious virus was repeatedly recovered containing mutant M proteins capable of complementation but was never recovered with mutant M proteins that lacked complementation activity, providing further evidence for a separate activity of M protein that is essential for virus replication.Most viruses that have a membrane or envelope as part of their structure acquire their envelopes by budding from the plasma membrane of the host cell. For budding to occur, the nucleoprotein core of the virus (nucleocapsid) must interact with the cytoplasmic surface of the host membrane. For many viruses this interaction is mediated by a matrix (M)² protein that binds to both the nucleocapsid and the host membrane (1, 2). Despite the similarity in the functions of viral M proteins, there is little structural or sequence similarity among the M proteins of different virus families (3). Thus, understanding the relationship of structure to function must be undertaken for individual M proteins before the general principles involved in virus budding can be understood. The goal of the experiments described here was to determine sequences in the M protein of vesicular stomatitis virus (VSV) involved in binding to membranes and nucleocapsids.VSV is the prototype member of the Rhabdoviridae family and has been widely studied to determine mechanisms involved in virus budding (2). The core of the virus contains an ∼11-kilobase negative-stranded RNA genome covered by 1300 copies of a single nucleocapsid protein (4). The nucleocapsid also contains lesser amounts of two proteins, P and L, which constitute the viral RNA-dependent RNA polymerase. The envelope contains a single species of transmembrane glycoprotein (G protein) that mediates virus attachment and entry into host cells. The virion contains ∼2000 copies of the M protein (4), which binds the nucleocapsid to the envelope and condenses the nucleocapsid into a tightly coiled helical nucleocapsid-M protein (NCM) complex that gives the virion its bullet-like shape (5-8). In cells infected with VSV and in transfected cells that express M protein in the absence of other VSV components, M protein is present both in a soluble form and bound to the cytoplasmic surface of the host plasma membrane (9-18). Mutagenesis studies, affinity labeling, and membrane reconstitution experiments have suggested that a combination of hydrophobic and ionic interactions mediate M protein binding to membranes by binding acidic phospholipids on the inner surface of the host plasma membrane (for review, see Ref. 19). Binding of M protein to nucleocapsids is less well understood than its binding to membranes. Most of the M protein in isolated NCM complexes is bound in a rapidly reversible equilibrium (20). However, M protein does not bind to nucleocapsids from which all of the M protein has been dissociated or to intracellular nucleocapsids that have never been assembled with M protein (11, 20). This suggests that binding of M protein to nucleocapsids in infected cells must be initiated in a separate step, after which most of the M protein is recruited into the NCM complex through the reversible binding step.M protein does not have separately folded domains that mediate binding to membranes versus nucleocapsids. The 229-amino acid (aa) M protein contains a positively charged N terminus (aa 1-50) that is highly exposed to proteolysis. The remainder of M protein (aa 51-229) is compactly folded to form a protease-resistant core (16, 21-23). The ability to obtain crystals of M protein required proteolytic removal of both the N-terminal sequence (aa 1-47) and a hydrophobic sequence (aa 121-124) to prevent M protein self-association (21, 22); however, the resulting structure showed a single-domain fold for the crystallized portion of M. In the present study we focused on two regions of the M protein structure that had been suggested to be involved in binding to either membranes or nucleocapsids; 1) previous data had implicated the N-terminal sequence in binding to both nucleocapsids and membranes (9, 10, 16, 22-25) and 2) deletion mutagenesis studies had implicated an additional region from aa 75-106 in membrane binding (16).In the experiments described here, M protein sequence substitutions were made using a scanning approach in the N-terminal sequence, and substitutions were based on the crystal structure in the 75-106-aa region. These mutants were used to determine the specific amino acids involved in these interactions. The results confirm that the N terminus of M protein is involved in association with plasma membranes as well as nucleocapsids, although these two activities are differentially affected by individual mutations. Mutations in the 75-106-aa region affected incorporation into NCM complexes but had only minor effects on association with membranes. Furthermore, the ability of mutant M proteins to function in the context of virus infection suggested that a new activity of M protein that is separate from its ability to associate with membranes or NCM complexes is critical for virus assembly.     

Mutations in the Endothelin Receptor Type A Cause Mandibulofacial Dysostosis with Alopecia

The endothelin receptor type A (EDNRA) signaling pathway is essential for the establishment of mandibular identity during development of the first pharyngeal arch. We report four unrelated individuals with the syndrome mandibulofacial dysostosis with alopecia (MFDA) who have de novo missense variants in EDNRA. Three of the four individuals have the same substitution, p.Tyr129Phe. Tyr129 is known to determine the selective affinity of EDNRA for endothelin 1 (EDN1), its major physiological ligand, and the p.Tyr129Phe variant increases the affinity of the receptor for EDN3, its non-preferred ligand, by two orders of magnitude. The fourth individual has a somatic mosaic substitution, p.Glu303Lys, and was previously described as having Johnson-McMillin syndrome. The zygomatic arch of individuals with MFDA resembles that of mice in which EDNRA is ectopically activated in the maxillary prominence, resulting in a maxillary to mandibular transformation, suggesting that the p.Tyr129Phe variant causes an EDNRA gain of function in the developing upper jaw. Our in vitro and in vivo assays suggested complex, context-dependent effects of the EDNRA variants on downstream signaling. Our findings highlight the importance of finely tuned regulation of EDNRA signaling during human craniofacial development and suggest that modification of endothelin receptor-ligand specificity was a key step in the evolution of vertebrate jaws.     

Opposite Effects of Insulin on Focal Adhesion Proteins in 3T3-L1 Adipocytes and in Cells Overexpressing the Insulin Receptor

Insulin can regulate the abundance and organization of filamentous actin within cells in culture. Early studies using cell lines that overexpress the insulin receptor demonstrated that insulin caused a rapid reversible disassembly of actin filaments that coincided with the rapid tyrosine dephosphorylation of focal adhesion kinase. We have extended these studies by demonstrating that paxillin, another focal adhesion protein, and Src undergo tyrosine dephosphorylation in response to insulin in Chinese hamster ovary (CHO) and rat hepatoma (HTC) cells that overexpress the insulin receptor. This contrasted with the effect of insulin in parental CHO and HTC cells in which focal adhesion proteins were not dephosphorylated in response to the hormone. In addition, insulin caused a dispersion of focal adhesion proteins and disruption of actin filament bundles only in cells that overexpressed the insulin receptor. Moreover, in 3T3-L1 adipocytes, which are considered prototypic insulin-responsive cells, actin filament assembly was stimulated, and focal adhesion protein tyrosine phosphorylation was not altered. 3T3-L1 cells have more insulin receptors than either parental CHO or HTC cells but have fivefold less insulin receptors than the overexpressing cell lines. We hypothesize that a threshold may exist in which the overexpression of insulin receptors determines how insulin signaling pathways regulate the actin cytoskeleton.     

Structural Determinants of the Insulin Receptor-related Receptor Activation by Alkali

IRR is a member of the insulin receptor (IR) family that does not have any known agonist of a peptide nature but can be activated by mildly alkaline medium and was thus proposed to function as an extracellular pH sensor. IRR activation by alkali is defined by its N-terminal extracellular region. To reveal key structural elements involved in alkali sensing, we developed an in vitro method to quantify activity of IRR and its mutants. Replacing the IRR L1C domains (residues 1–333) or L2 domain (residues 334–462) or both with the homologous fragments of IR reduced the receptor activity to 35, 64, and 7% percent, respectively. Within L1C domains, five amino acid residues (Leu-135, Gly-188, Arg-244, and vicinal His-318 and Lys-319) were identified as IRR-specific by species conservation analysis of the IR family. These residues are exposed and located in junctions between secondary structure folds. The quintuple mutation of these residues to alanine had the same negative effect as the entire L1C domain replacement, whereas none of the single mutations was as effective. Separate mutations of these five residues and of L2 produced partial negative effects that were additive. The pH dependence of cell-expressed mutants (L1C and L2 swap, L2 plus triple LGR mutation, and L2 plus quintuple LGRHK mutation) was shifted toward alkalinity and, in contrast with IRR, did not show significant positive cooperativity. Our data suggest that IRR activation is not based on a single residue deprotonation in the IRR ectodomain but rather involves synergistic conformational changes at multiple points.     

20-Hydroxyeicosatetraenoic Acid Impairs Endothelial Insulin Signaling by Inducing Phosphorylation of the Insulin Receptor Substrate-1 at Ser616

20-hydroxyeicosatetraenoic acid (20-HETE) induces endothelial dysfunction and is correlated with diabetes. This study was designed to investigate the effects of 20-HETE on endothelial insulin signaling.Human umbilical vein endothelial cells (HUVECs) or C57BL/6J mice were treated with 20-HETE in the presence or absence of insulin, and p-ERK1/2, p-JNK, IRS-1/PI3K/AKT/eNOS pathway, were examined in endothelial cells and aortas by immunoblotting. eNOS activity and nitric oxide production were measured. 20-HETE increased ERK1/2 phosphorylation and IRS-1 phosphorylation at Ser⁶¹⁶; these effects were reversed by ERK1/2 inhibition. We further observed that 20-HETE treatment resulted in impaired insulin-stimulated IRS-1 phosphorylation at Tyr⁶³² and subsequent PI3-kinase/Akt activation. Furthermore, 20-HETE treatment blocked insulin-stimulated phosphorylation of eNOS at the stimulatory Ser¹¹⁷⁷ site, eNOS activation and NO production; these effects were reversed by inhibiting ERK1/2. Treatment of C57BL/6J mice with 20-HETE resulted in ERK1/2 activation and impaired insulin-dependent activation of the IRS-1/PI3K/Akt/eNOS pathway in the aorta. Our data suggest that the 20-HETE activation of IRS-1 phosphorylation at Ser⁶¹⁶ is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway.     

Differential Effects of Human L1CAM Mutations on Complementing Guidance and Synaptic Defects in Drosophila melanogaster

A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin–moesin–radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis.