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Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75-80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains., Results: Contrary to lactate dehydrogenase, deleting phosphotransacetylase ( pta , acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A , ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively., Conclusions: Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB . Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings., Competing Interests: Competing interestsLRL is a shareholder in a start-up company focusing on cellulosic biofuel production and conversion. There are no other competing interests., (© The Author(s) 2020.)

The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum , we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I-B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus , is active in C. thermocellum . We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF . For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo / beta homologs, isolated from Acidithiobacillus caldus , are functional in C. thermocellum . For the Type I-B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lee Lynd is a co-founder of the Enchi Corporation, which has a financial interest in C. thermocellum. Work within was filed as a provisional patent, Provisional Patent Application No. 62/896,555 titled Novel Recombineering Machinery to Increase Homology Directed Genome Editing in Thermophilic Microbes.

Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that has been engineered to produce high amounts of ethanol, reaching ~90% theoretical yield at a titer of 70 g/L. Here we report the physiological changes that occur upon deleting the redox-sensing transcriptional regulator Rex in wild type T. saccharolyticum: a single deletion of rex resulted in a two-fold increase in ethanol yield (from 40% to 91% theoretical yield), but the resulting strains grew only about a third as fast as the wild type strain. Deletion of the rex gene also had the effect of increasing expression of alcohol dehydrogenase genes, adhE and adhA. After several serial transfers, the ethanol yield decreased from an average of 91% to 55%, and the growth rates had increased. We performed whole-genome resequencing to identify secondary mutations in the Δrex strains adapted for faster growth. In several cases, secondary mutations had appeared in the adhE gene. Furthermore, in these strains the NADH-linked alcohol dehydrogenase activity was greatly reduced. Complementation studies were done to reintroduce rex into the Δrex strains: reintroducing rex decreased ethanol yield to below wild type levels in the Δrex strain without adhE mutations, but did not change the ethanol yield in the Δrex strain where an adhE mutation occurred.

Background: Clostridium thermocellum is a promising microorganism for conversion of cellulosic biomass to biofuel, without added enzymes; however, the low ethanol titer produced by strains developed thus far is an obstacle to industrial application., Results: Here, we analyzed changes in the relative concentration of intracellular metabolites in response to gradual addition of ethanol to growing cultures. For C. thermocellum , we observed that ethanol tolerance, in experiments with gradual ethanol addition, was twofold higher than previously observed in response to a stepwise increase in the ethanol concentration, and appears to be due to a mechanism other than mutation. As ethanol concentrations increased, we found accumulation of metabolites upstream of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction and depletion of metabolites downstream of that reaction. This pattern was not observed in the more ethanol-tolerant organism Thermoanaerobacterium saccharolyticum . We hypothesize that the Gapdh enzyme may have different properties in the two organisms. Our hypothesis is supported by enzyme assays showing greater sensitivity of the C. thermocellum enzyme to high levels of NADH, and by the increase in ethanol tolerance and production when the T. saccharolyticum gapdh was expressed in C. thermocellum ., Conclusions: We have demonstrated that a metabolic bottleneck occurs at the GAPDH reaction when the growth of C. thermocellum is inhibited by high levels of ethanol. We then showed that this bottleneck could be relieved by expression of the gapdh gene from T. saccharolyticum . This enzyme is a promising target for future metabolic engineering work.

Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields and titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. This suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE . To explore the effects of overexpressing wild-type, mutant, and exogenous adhE s, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum . As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.

Background: Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30-70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities., Results: It was found that pyruvate ferredoxin oxidoreductase enzyme (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield., Conclusion: PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.

In mice and humans, loss of myosin VI (Myo6) function results in deafness, and certain Myo6 mutations also result in cardiomyopathies in humans. The current studies have utilized the Snell's waltzer (sv) mouse (a functional null mutation for Myo6) to determine if this mouse also exhibits cardiac defects and thus used to determine the cellular and molecular basis for Myo6-associated heart disease. Myo6 is expressed in mouse heart where it is predominantly expressed in vascular endothelial cells (VECs) based on co-localization with the VEC cell marker CD31. Sv/sv heart mass is significantly greater than that of sv/+ littermates, a result of left ventricle hypertrophy. The left ventricle of the sv/sv exhibits extensive fibrosis, both interstitial and perivascular, based on histologic staining, and immunolocalization of several markers for fibrosis including fibronectin, collagen IV, and the fibroblast marker vimentin. Myo6 is also expressed in lung VECs but not in VECs of intestine, kidney, or liver. Sv/sv lungs exhibit increased periaveolar fibrosis and enlarged air sacs. Electron microscopy of sv/sv cardiac and lung VECs revealed abnormal ultrastructure, including luminal protrusions and increased numbers of cytoplasmic vesicles. Previous studies have shown that loss of function of either Myo6 or its adaptor binding partner synectin/GIPC results in impaired arterial development due to defects in VEGF signaling. However, examination of synectin/GIPC-/- heart revealed no fibrosis or significantly altered VEC ultrastructure, suggesting that the cardiac and lung defects observed in the sv/sv mouse are not due to Myo6 function in arterial development., (© 2015 Wiley Periodicals, Inc.)

A key tool for metabolic engineering is the ability to express heterologous genes. One obstacle to gene expression in non-model organisms, and especially in relatively uncharacterized bacteria, is the lack of well-characterized promoters. Here we test 17 promoter regions for their ability to drive expression of the reporter genes β-galactosidase ( lacZ ) and NADPH-alcohol dehydrogenase ( adhB ) in Clostridium thermocellum , an important bacterium for the production of cellulosic biofuels. Only three promoters have been commonly used for gene expression in C. thermocellum , gapDH, cbp and eno. Of the new promoters tested, 2638, 2926, 966 and 815 showed reliable expression. The 2638 promoter showed relatively higher activity when driving adhB (compared to lacZ ), and the 815 promoter showed relatively higher activity when driving lacZ (compared to adhB )., (© 2015 The Authors.)

Background: Regulation of vascular endothelial growth factor receptor-2 (VEGFR2) signaling is a control point that determines the extent of vascular tree formation. Recent studies demonstrated an important role played by VEGFR2 endothelial trafficking in control of its activity and suggested the involvement of a phosphotyrosine phosphatase 1b (PTP1b) in this process. This study was designed to define the role of PTP1b in endothelial VEGFR2 signaling and its role in regulation of angiogenesis and arteriogenesis., Methods and Results: We generated mice carrying an endothelial-specific deletion of PTP1b and examined the effect of this knockout on VEGF signaling, angiogenesis, and arteriogenesis in vitro and in vivo. PTP1b knockout endothelial cells had increased VEGF-dependent activation of extracellular signal-regulated kinase signaling, sprouting, migration, and proliferation compared with controls. Endothelial PTP1b null mice had increased retinal and Matrigel implant angiogenesis and accelerated wound healing, pointing to enhanced angiogenesis. Increased arteriogenesis was demonstrated by observations of faster recovery of arterial blood flow and large numbers of newly formed arterioles in the hindlimb ischemia mouse model. PTP1b endothelial knockout also rescued impaired blood flow recovery after common femoral artery ligation in synectin null mice., Conclusions: PTP1b is a key regulator of endothelial VEGFR2 signaling and plays an important role in regulation of the extent of vascular tree formation., (© 2014 American Heart Association, Inc.)

Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor that is involved in the regulation of vascular integrity, quiescence, and inflammation. The mode of secretion of Ang2 has never been established, however. Here, we provide evidence that Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway, whereas it is positively regulated by the syndecan-4/syntenin pathway. Vascular defects in Akt1 null mice arise, in part, because of excessive Ang2 secretion and can be rescued by the syndecan-4 knock-out that reduces extracellular Ang2 levels. This novel mechanism connects three critical signaling pathways: angiopoietin/Tie2, PI3K/Akt/eNOS, and syndecan/syntenin, which play important roles in vascular growth and stabilization.

Rationale: Arteriogenesis is the process of formation of arterial conduits. Its promotion is an attractive therapeutic strategy in occlusive atherosclerotic diseases. Despite the functional and clinical importance of arteriogenesis, the biology of the process is poorly understood. Synectin, a gene previously implicated in the regulation of vascular endothelial cell growth factor signaling, offers a unique opportunity to determine relative contributions of various cell types to arteriogenesis., Objective: We investigated the cell-autonomous effects of a synectin knockout in arterial morphogenesis., Methods and Results: A floxed synectin knockin mouse line was crossbred with endothelial-specific (Tie2, Cdh5, Pdgfb) and smooth muscle myosin heavy chain-specific Cre driver mouse lines to produce cell type-specific deletions. Ablation of synectin expression in endothelial, but not smooth muscle cells resulted in the presence of developmental arterial morphogenetic defects (smaller size of the arterial tree, reduced number of arterial branches and collaterals) and impaired arteriogenesis in adult mice., Conclusions: Synectin modulates developmental and adult arteriogenesis in an endothelial cell-autonomous fashion. These findings show for the first time that endothelial cells are central to both developmental and adult arteriogenesis and provide a model for future studies of factors involved in this process.

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The fibroblast growth factor (FGF) system plays a critical role in the maintenance of vascular integrity via enhancing the stability of VE-cadherin at adherens junctions. However, the precise molecular mechanism is not well understood. In the present study, we aimed to investigate the detailed mechanism of FGF regulation of VE-cadherin function that leads to endothelial junction stabilization., Methods and Findings: In vitro studies demonstrated that the loss of FGF signaling disrupts the VE-cadherin-catenin complex at adherens junctions by increasing tyrosine phosphorylation levels of VE-cadherin. Among protein tyrosine phosphatases (PTPs) known to be involved in the maintenance of the VE-cadherin complex, suppression of FGF signaling reduces SHP2 expression levels and SHP2/VE-cadherin interaction due to accelerated SHP2 protein degradation. Increased endothelial permeability caused by FGF signaling inhibition was rescued by SHP2 overexpression, indicating the critical role of SHP2 in the maintenance of endothelial junction integrity., Conclusions: These results identify FGF-dependent maintenance of SHP2 as an important new mechanism controlling the extent of VE-cadherin tyrosine phosphorylation, thereby regulating its presence in adherens junctions and endothelial permeability.

GIPC1 is a cytoplasmic scaffold protein that interacts with numerous receptor signaling complexes, and emerging evidence suggests that it plays a role in tumorigenesis. GIPC1 is highly expressed in a number of human malignancies, including breast, ovarian, gastric, and pancreatic cancers. Suppression of GIPC1 in human pancreatic cancer cells inhibits in vivo tumor growth in immunodeficient mice. To better understand GIPC1 function, we suppressed its expression in human breast and colorectal cancer cell lines and human mammary epithelial cells (HMECs) and assayed both gene expression and cellular phenotype. Suppression of GIPC1 promotes apoptosis in MCF-7, MDA-MD231, SKBR-3, SW480, and SW620 cells and impairs anchorage-independent colony formation of HMECs. These observations indicate GIPC1 plays an essential role in oncogenic transformation, and its expression is necessary for the survival of human breast and colorectal cancer cells. Additionally, a GIPC1 knock-down gene signature was used to interrogate publically available breast and ovarian cancer microarray datasets. This GIPC1 signature statistically correlates with a number of breast and ovarian cancer phenotypes and clinical outcomes, including patient survival. Taken together, these data indicate that GIPC1 inhibition may represent a new target for therapeutic development for the treatment of human cancers.

VEGF is the key growth factor regulating arterial morphogenesis. However, molecular events involved in this process have not been elucidated. Synectin null mice demonstrate impaired VEGF signaling and a marked reduction in arterial morphogenesis. Here, we show that this occurs due to delayed trafficking of VEGFR2-containing endosomes that exposes internalized VEGFR2 to selective dephosphorylation by PTP1b on Y(1175) site. Synectin involvement in VEGFR2 intracellular trafficking requires myosin-VI, and myosin-VI knockout in mice or knockdown in zebrafish phenocopy the synectin null phenotype. Silencing of PTP1b restores VEGFR2 activation and significantly recovers arterial morphogenesis in myosin-VI(-/-) knockdown zebrafish and synectin(-/-) mice. We conclude that activation of the VEGF-mediated arterial morphogenesis cascade requires phosphorylation of the VEGFR2 Y(1175) site that is dependent on trafficking of internalized VEGFR2 away from the plasma membrane via a synectin-myosin-VI complex. This key event in VEGF signaling occurs at an intracellular site and is regulated by a novel endosomal trafficking-dependent process., (Copyright 2010 Elsevier Inc. All rights reserved.)

Arterial morphogenesis is an important and poorly understood process. In particular, the signaling events controlling arterial formation have not been established. We evaluated whether alterations in the balance between ERK1/2 and PI3K signaling pathways could stimulate arterial formation in the setting of defective arterial morphogenesis in mice and zebrafish. Increased ERK1/2 activity in mouse ECs with reduced VEGF responsiveness was achieved in vitro and in vivo by downregulating PI3K activity, suppressing Akt1 but not Akt2 expression, or introducing a constitutively active ERK1/2 construct. Such restoration of ERK1/2 activation was sufficient to restore impaired arterial development and branching morphogenesis in synectin-deficient mice and synectin-knockdown zebrafish. The same approach effectively stimulated arterial growth in adult mice, restoring arteriogenesis in mice lacking synectin and in atherosclerotic mice lacking both LDL-R and ApoB48. We therefore conclude that PI3K-ERK1/2 crosstalk plays a key role in the regulation of arterial growth and that the augmentation of ERK signaling via suppression of the PI3K signaling pathway can effectively stimulate arteriogenesis.

A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2(+/-) mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.

Synectin, a ubiquitously expressed PDZ scaffold protein, has been shown to be a key regulator in the formation of arterial vasculature. Examination of the retinal vasculature in synectin(-/-) mice demonstrated poor mural cell coverage of and attachment to the forming arterial tree, a defect reminiscent of retinal abnormalities observed in platelet derived growth factor (PDGF) -B(-/-) mice. Primary cultures of synectin(-/-) smooth muscle cells had normal expression of PDGFR-beta and migrated normally in response to PDGF-BB. However, expression of PDGF-BB protein, but not mRNA, was reduced in lysates from arterial, but not venous, primary synectin(-/-) endothelial cells (EC), that was restored by inhibition of proteosomal degradation. Transduction of synectin(-/-) and (+/+) EC with a bicistronic Pdgfb/gfp construct, resulted in comparable expression of green fluorescent protein in both EC populations while PDGF-BB expression was severely reduced in synectin(-/-) EC. Finally, synectin expression in synectin(-/-) arterial EC restored PDGF-BB protein levels. These results suggest that synectin deficiency results in increased degradation of PDGF-BB protein in arterial EC and, consequently, reduced recruitment of mural cells to newly forming arteries. This observation may explain the selective reduction in arterial morphogenesis observed in synectin knockout mice., ((c) 2009 Wiley-Liss, Inc.)

The neuropilin (Nrp)1 receptor is essential for both nervous and vascular system development. Nrp1 is unusually versatile, because it transmits both chemoattractive and repulsive signals in response to vascular endothelial growth factor (VEGF)-A and class 3 semaphorins, respectively. Both Nrp1 and VEGF receptor 2 undergo ligand-dependent endocytosis. We sought to establish the endocytic pathway of Nrp1 and to determine whether uptake is required for its signaling. Whereas Nrp1 underwent clathrin-dependent endocytosis in response to VEGFA(165) treatment, semaphorin 3C (sema3C) induced lipid raft-dependent endocytosis. The myosin VI PDZ (postsynaptic density 95, Disk large, Zona occludens-1) adaptor protein synectin was essential for Nrp1 trafficking. Sema3C failed to inhibit migration of synectin(-/-) endothelial cells, mirroring the lower migratory response of these cells to VEGFA(165). These results show that the endocytic pathway of Nrp1 is determined by its ligand and that the trafficking of Nrp1 is essential for its signaling.

Vascular endothelial growth factor (VEGF) acts as a hierarchically high switch of the angiogenic cascade by interacting with its high affinity VEGF receptors and with neuropilin co-receptors. VEGF(165) binds to both Neuropilin-1 (NP-1) and VEGFR-2, and it is believed that ligand binding forms an extracellular bridge between both molecules. This leads to complex formation, thereby enhancing VEGFR-2 phosphorylation and subsequent signaling. We found that inhibition of VEGF receptor (VEGFR) phosphorylation reduced complex formation between NP-1 and VEGFR-2, suggesting a functional role of the cytoplasmic domain of VEGFR-2 for complex formation. Correspondingly, deleting the PDZ-binding domain of NP-1 decreased complex formation, indicating that extracellular VEGF(165) binding is not sufficient for VEGFR-2-NP-1 interaction. Synectin is an NP-1 PDZ-binding domain-interacting molecule. Experiments in Synectin-deficient endothelial cells revealed reduced VEGFR-2-NP-1 complex formation, suggesting a role for Synectin in VEGFR-2-NP-1 signaling. Taken together, the experiments have identified a novel mechanism of NP-1 interaction with VEGFR-2, which involves the cytoplasmic domain of NP-1.

Synectin (GIPC1), a receptor scaffold protein, has been isolated by our laboratory as a syndecan-4 cytoplasmic domain binding partner that regulates important aspects of cell motility (Gao Y, Li M, Chen W, Simons M. J Cell Physiol 184: 373-379, 2000; Tkachenko E, Elfenbein A, Tirziu D, Simons M. Circ Res 98: 1398-1404, 2006). Moreover, synectin plays a major role in arterial morphogenesis and in growth factor signaling in arterial endothelial cells by regulating Rac1 activity (Chittenden TW, Claes F, Lanahan AA, Autiero M, Palac RT, Tkachenko EV, Elfenbein A, Ruiz de Almodovar C, Dedkov E, Tomanek R, Li W, Westmore M, Singh J, Horowitz A, Mulligan-Kehoe MJ, Moodie KL, Zhuang ZW, Carmeliet P, Simons M. Dev Cell 10: 783-795, 2006). The present study was carried out to characterize changes in synectin-dependent gene expression induced by homozygous disruption of the gene in endothelial cells. Using a combination of suppression subtraction hybridization and high throughput microarray technology, we have identified aberrant biological processes of transcriptional regulation in synectin(-/-) primary endothelial cells including abnormal basal regulation of genes associated with development, cell organization and biogenesis, intracellular tracking, and cell adhesion. Analysis of gene expression following FGF2 treatment demonstrated significant abnormalities in transcription, cytoskeletal organization and biogenesis, and protein modification and transport in synectin(-/-) compared with synectin(+/+) endothelial cells. These results confirm synectin involvement in FGF2-dependent signal transduction and provide insights into synectin-dependent gene expression in the endothelium.

Branching morphogenesis is a key process in the formation of vascular networks. To date, little is known regarding the molecular events regulating this process. We investigated the involvement of synectin in this process. In zebrafish embryos, synectin knockdown resulted in a hypoplastic dorsal aorta and hypobranched, stunted, and thin intersomitic vessels due to impaired migration and proliferation of angioblasts and arterial endothelial cells while not affecting venous development. Synectin(-/-) mice demonstrated decreased body and organ size, reduced numbers of arteries, and an altered pattern of arterial branching in multiple vascular beds while the venous system remained normal. Murine synectin(-/-) primary arterial, but not venous, endothelial cells showed decreased in vitro tube formation, migration, and proliferation and impaired polarization due to abnormal localization of activated Rac1. We conclude that synectin is involved in selective regulation of arterial, but not venous, growth and branching morphogenesis and that Rac1 plays an important role in this process.

Vascular endothelium forms a continuous, semipermeable barrier that regulates the transvascular movement of hormones, macromolecules, and other solutes. Here, we describe a novel immediate early gene that is expressed selectively in vascular endothelial cells, verge (vascular early response gene). Verge protein includes an N-terminal region of approximately 70 amino acids with modest homology (approximately 30% identity) to Apolipoprotein L but is otherwise unique. Verge mRNA and protein are induced selectively in the endothelium of adult vasculature by electrical or chemical seizures. Verge expression appears to be responsive to local tissue conditions, because it is induced in the hemisphere ipsilateral to transient focal cerebral ischemia. In contrast to the transient expression in adult, Verge mRNA and protein are constitutively expressed at high levels in the endothelium of developing tissues (particularly heart) in association with angiogenesis. Verge mRNA is induced in cultured endothelial cells by defined growth factors and hypoxia. Verge protein is dramatically increased by cysteine proteinase inhibitors, suggesting rapid turnover, and is localized to focal regions near the periphery of the cells. Endothelial cell lines that stably express Verge form monolayers that show enhanced permeability in response to activation of protein kinase C by phorbol esters. This response is accompanied by reorganization of the actin cytoskeleton and the formation of paracellular gaps. These studies suggest that Verge functions as a dynamic regulator of endothelial cell signaling and vascular function.

Neuronal activity is a requirement for the plasticity and normal development of the central nervous system. We have used differential cloning techniques to identify an immediate-early gene (IEG) that is rapidly induced in neurons by activity in both adult and developmental models of plasticity. Here we describe the key regulatory enzyme of polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT), as a neuronal IEG. In the rat brain, kainate-induced seizures result in a 5.5-fold increase in the amount of SSAT mRNA above basal levels and the enzymatic activity is increased twofold. Expression of SSAT mRNA is rapidly and transiently upregulated in the cerebral cortex and hippocampus by seizure-induced neuronal activation. In hippocampal neurons, SSAT expression is dynamically responsive to synaptic activity in the long-term potentiation (LTP) paradigm. In developing brain, region-specific expression of SSAT mRNA is first detected at postnatal day 9 (P9) and subsequently increases through days P15, P20, before reaching maximal level in adult animals. This dynamic transcriptional and translational control suggests that SSAT may play a role in activity-dependent neuronal plasticity and development.

Shank is a recently described family of postsynaptic proteins that function as part of the NMDA receptor-associated PSD-95 complex (Naisbitt et al., 1999 [this issue of Neuron]). Here, we report that Shank proteins also bind to Homer. Homer proteins form multivalent complexes that bind proline-rich motifs in group 1 metabotropic glutamate receptors and inositol trisphosphate receptors, thereby coupling these receptors in a signaling complex. A single Homer-binding site is identified in Shank, and Shank and Homer coimmunoprecipitate from brain and colocalize at postsynaptic densities. Moreover, Shank clusters mGluR5 in heterologous cells in the presence of Homer and mediates the coclustering of Homer with PSD-95/GKAP. Thus, Shank may cross-link Homer and PSD-95 complexes in the PSD and play a role in the signaling mechanisms of both mGluRs and NMDA receptors.

Parkinson disease (PD) is a neurodegenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. Post-mortem examination shows loss of neurons and Lewy bodies, which are cytoplasmic eosinophilic inclusions, in the substantia nigra and other brain regions. A few families have PD caused by mutations (A53T or A30P) in the gene SNCA (encoding alpha-synuclein). Alpha-synuclein is present in Lewy bodies of patients with sporadic PD, suggesting that alpha-synuclein may be involved in the pathogenesis of PD. It is unknown how alpha-synuclein contributes to the cellular and biochemical mechanisms of PD, and its normal functions and biochemical properties are poorly understood. To determine the protein-interaction partners of alpha-synuclein, we performed a yeast two-hybrid screen. We identified a novel interacting protein, which we term synphilin-1 (encoded by the gene SNCAIP). We found that alpha-synuclein interacts in vivo with synphilin-1 in neurons. Co-transfection of both proteins (but not control proteins) in HEK 293 cells yields cytoplasmic eosinophilic inclusions.

Synaptic activity induces a rapid transcriptional response that is essential for the establishment of long-term neuronal plasticity. Using a differential cloning technique, we have identified a gene induced by seizure activity in the brain as RB3. RB3 is a recently cloned gene belonging to the stathmin family of phosphoproteins. Like SCG10, RB3 is brain-specific, although in situ hybridization results show that the expression of RB3 is more ubiquitous than is that of SCG10. Using genomic DNA sequencing, we show that the 27 amino acid sequence unique to the RB3" transcript is encoded by an alternatively spliced exon, exon 2'. Using a peptide antibody raised against exon 2' to detect RB3" and an anti-Flag antibody to detect an epitope-tagged version of RB3, we show that both proteins are localized to the Golgi apparatus of transfected COS7 cells. Of particular interest, RB3 mRNA, but not SCG10 mRNA, is rapidly induced in the dentate gyrus granule layer of the hippocampus after electrically induced seizure activity as well as stimuli leading to long-term potentiation (LTP). In addition, RB3 mRNA is induced in pheochromocytoma (PC12) cells treated with 250 ng/ml NGF. These results suggest that RB3 may play a role in activity-induced neuronal plasticity and neuronal differentiation.

Homer is a neuronal immediate early gene (IEG) that is enriched at excitatory synapses and binds group 1 metabotropic glutamate receptors (mGluRs). Here, we characterize a family of Homer-related proteins derived from three distinct genes. Like Homer IEG (now termed Homer 1a), all new members bind group 1 mGluRs. In contrast to Homer 1a, new members are constitutively expressed and encode a C-terminal coiled-coil (CC) domain that mediates self-multimerization. CC-Homers form natural complexes that cross-link mGluRs and are enriched at the postsynaptic density. Homer 1a does not multimerize and blocks the association of mGluRs with CC-Homer complexes. These observations support a model in which the dynamic expression of Homer 1a competes with constitutively expressed CC-Homers to modify synaptic mGluR properties.

Group I metabotropic glutamate receptors (mGluRs) activate PI turnover and thereby trigger intracellular calcium release. Previously, we demonstrated that mGluRs form natural complexes with members of a family of Homer-related synaptic proteins. Here, we present evidence that Homer proteins form a physical tether linking mGluRs with the inositol trisphosphate receptors (IP3R). A novel proline-rich "Homer ligand" (PPXXFr) is identified in group 1 mGluRs and IP3R, and these receptors coimmunoprecipitate as a complex with Homer from brain. Expression of the IEG form of Homer, which lacks the ability to cross-link, modulates mGluR-induced intracellular calcium release. These studies identify a novel mechanism in calcium signaling and provide evidence that an IEG, whose expression is driven by synaptic activity, can directly modify a specific synaptic function.

Long-term neuronal plasticity is known to be dependent on rapid de novo synthesis of mRNA and protein, and recent studies provide insight into the molecules involved in this response. Here, we demonstrate that mRNA encoding a member of the regulator of G-protein signaling (RGS) family, RGS2, is rapidly induced in neurons of the hippocampus, cortex, and striatum in response to stimuli that evoke plasticity. Although several members of the RGS family are expressed in brain with discrete neuronal localizations, RGS2 appears unique in that its expression is dynamically responsive to neuronal activity. In biochemical assays, RGS2 stimulates the GTPase activity of the alpha subunit of Gq and Gi1. The effect on Gi1 was observed only after reconstitution of the protein in phospholipid vesicles containing M2 muscarinic acetylcholine receptors. RGS2 also inhibits both Gq- and Gi-dependent responses in transfected cells. These studies suggest a novel mechanism linking neuronal activity and signal transduction.

AMPA glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system and play a role in the synaptic plasticity underlying learning and memory. AMPA receptors are heteromeric complexes of four homologous subunits (GluR1-4) that differentially combine to form a variety of AMPA receptor subtypes. These subunits are thought to have a large extracellular amino-terminal domain, three transmembrane domains and an intracellular carboxy-terminal domain. AMPA receptors are localized at excitatory synapses and are not found on adjacent inhibitory synapses enriched in GABA(A) receptors. The targeting of neurotransmitter receptors, such as AMPA receptors, and ion channels to synapses is essential for efficient transmission. A protein motif called a PDZ domain is important in the targeting of a variety of membrane proteins to cell-cell junctions including synapses. Here we identify a synaptic PDZ domain-containing protein GRIP (glutamate receptor interacting protein) that specifically interacts with the C termini of AMPA receptors. GRIP is a new member of the PDZ domain-containing protein family which has seven PDZ domains and no catalytic domain. GRIP appears to serve as an adapter protein that links AMPA receptors to other proteins and may be critical for the clustering of AMPA receptors at excitatory synapses in the brain.

Spatial localization and clustering of membrane proteins is critical to neuronal development and synaptic plasticity. Recent studies have identified a family of proteins, the PDZ proteins, that contain modular PDZ domains and interact with synaptic ionotropic glutamate receptors and ion channels. PDZ proteins are thought to have a role in defining the cellular distribution of the proteins that interact with them. Here we report a novel dendritic protein, Homer, that contains a single, PDZ-like domain and binds specifically to the carboxy terminus of phosphoinositide-linked metabotropic glutamate receptors. Homer is highly divergent from known PDZ proteins and seems to represent a novel family. The Homer gene is also distinct from members of the PDZ family in that its expression is regulated as an immediate early gene and is dynamically responsive to physiological synaptic activity, particularly during cortical development. This dynamic transcriptional control suggests that Homer mediates a novel cellular mechanism that regulates metabotropic glutamate signalling.

Murine fibroblasts respond to mitogens by sequential gene expression in which immediate-early, or primary response, gene transcription factors direct expression of secondary transcripts encoded by delayed-early response (DER) genes. DER gene products include growth progression factors, but the products of several novel cDNAs are unknown. Murine and human cDNAs derived from one novel DER gene (DER12) were characterized to identify its product and to probe its role in the growth response. Both sequences encoded a hydrophobic 36 kD protein (HNP36) that was related to the yeast protein, FUN26. Anti-murine HNP36 antibodies were prepared and shown to immunoprecipitate both mammalian in vitro translation products. Immunocytochemical staining indicated localization of HNP36 to the nucleolus where its concentration increased after mitogen stimulation. Although HNP36 protein function is unknown, its identification as a nucleolar gene transcriptionally activated by growth factors implicates it as participating in the proliferative response.

Neuronal activity is an essential stimulus for induction of plasticity and normal development of the CNS. We have used differential cloning techniques to identify a novel immediate-early gene (IEG) cDNA that is rapidly induced in neurons by activity in models of adult and developmental plasticity. Both the mRNA and the encoded protein are enriched in neuronal dendrites. Analysis of the deduced amino acid sequence indicates a region of homology with alpha-spectrin, and the full-length protein, prepared by in vitro transcription/translation, coprecipitates with F-actin. Confocal microscopy of the native protein in hippocampal neurons demonstrates that the IEG-encoded protein is enriched in the subplasmalemmal cortex of the cell body and dendrites and thus colocalizes with the actin cytoskeletal matrix. Accordingly, we have termed the gene and encoded protein Arc (activity-regulated cytoskeleton-associated protein). Our observations suggest that Arc may play a role in activity-dependent plasticity of dendrites.

Disruption of the 75-kD low-affinity nerve growth factor (NGF) receptor (p75) has been shown to result in sensory and sympathetic nervous system deficits (Lee et al., 1992a,b). In order to establish precisely which subsets of neurons are capable of responding to neurotrophins (NTs) through the low-affinity NGF receptor, p75 was localized in the primate autonomic and somatic sensory nervous systems. In the autonomic system, cell bodies of some parasympathetic and enteric neurons expressed detectable levels of p75, whereas all sympathetic neurons expressed the protein. In the sensory system, some, but not all, cell bodies were labeled in cranial and spinal sensory ganglia and in the mesencephalic nucleus. Some peripheral and central projections of the sensory neurons were also labeled. Centrally, most of the labeled processes were found in regions containing primarily small unmyelinated fibers, including lamina II of Rexed and areas of the solitary tract and nucleus. Peripherally, labeled processes were associated with unmyelinated nerves and specialized structures such as taste buds and Meissner corpuscles, but not with myelinated processes. This study indicates that the subset of neurons in the autonomic nervous system likely to be capable of responding to neurotrophins is broader than generally thought, and that p75-expressing neurons tend to be clustered. Moreover, in the sensory nervous system p75 is expressed by most cell bodies, but expression in their projections is restricted both peripherally and centrally to unmyelinated processes and nerve terminals.

Primary human urinary cells support growth and plaque formation by papovavirus BK, permitting plaque purification of the virus. After plaquing, the virus forms uniform plaques and its DNA has a homogeneous restriction enzyme fragment profile. Support of BK replication as well as morphological and cultural characteristics suggest an epithelial origin for the cells.

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

Alleles for the single human nerve growth factor receptor gene (NGFR) on chromosome 17q can be distinguished by two polymorphic restriction sites for XmnI and one for HincII. The combined information content for haplotypes is quite high, making the NGFR locus an excellent genetic marker. Two of these polymorphisms were used to follow the inheritance of NGFR alleles in families with two or more members affected with familial dysautonomia. This rare disease is inherited in an autosomal recessive mode in the Ashkenazic Jewish population. Affected individuals show a severe depletion of NGF-dependent nerve populations from birth. Linkage analysis excluded a role for NGFR in this disease with odds of greater than 10(6):1 against the dysautonomia gene being within 1 centiMorgan of the mutation. In a previous study the gene for the beta subunit of NGF (NGFB) was also excluded in this disease. A possible role for other genes involved in NGF action or those coding for other developmentally determining neuronal factors is indicated.