γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

Background

There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex.

Principal Findings

We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis.

Conclusions/Significance

These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein.     

RAD50 Is Required for Efficient Initiation of Resection and Recombinational Repair at Random, γ-Induced Double-Strand Break Ends

Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random “dirty-ended” DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this “PFGE-shift” to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after γ-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1–2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.     

Swing-Out of the β3 Hybrid Domain Is Required for αIIbβ3 Priming and Normal Cytoskeletal Reorganization,but Not Adhesion to Immobilized Fibrinogen

Structural and functional analyses of integrin αIIbβ3 has implicated swing-out motion of the β3 hybrid domain in αIIbβ3 activation and ligand binding. Using data from targeted molecular dynamics (TMD) simulations, we engineered two disulfide-bonded mutant receptors designed to limit swing-out (XS-O). XS-O mutants cannot bind the high Mr ligand fibrinogen in the presence of an activating mAb or after introducing mutations into the αIIb subunit designed to simulate inside-out signaling. They also have reduced capacity to be “primed” to bind fibrinogen by pretreatment with eptifibatide. They can, however, bind the small RGD venom protein kistrin. Despite their inability to bind soluble fibrinogen, the XS-O mutants can support adhesion to immobilized fibrinogen, although such adhesion does not initiate outside-in signaling leading to normal cytoskeletal reorganization. Collectively, our data further define the biologic role of β3 hybrid domain swing-out in both soluble and immobilized high Mr ligand binding, as well as in priming and outside-in signaling. We also infer that swing-out is likely to be a downstream effect of receptor extension.     

Actopaxin (α-Parvin) Phosphorylation Is Required for Matrix Degradation and Cancer Cell Invasion

Dysregulation of cell adhesion and motility is known to be an important factor in the development of tumor malignancy. Actopaxin (α-parvin) is a paxillin, integrin-linked kinase, and F-actin binding focal adhesion protein with several serine phosphorylation sites in the amino terminus that contribute to the regulation of cell spreading and migration. Here, phosphorylation of actopaxin is shown to contribute to the regulation of matrix degradation and cell invasion. Osteosarcoma cells stably expressing wild type (WT), nonphosphorylatable (Quint), and phosphomimetic (S4D/S8D) actopaxin demonstrate that actopaxin phosphorylation is necessary for efficient Src and matrix metalloproteinase-driven degradation of extracellular matrix. Rac1 was found to be required for actopaxin-induced matrix degradation whereas inhibition of myosin contractility promoted degradation in the phosphomutant-expressing Quint cells, indicating that a balance of Rho GTPase signaling and regulation of cellular tension are important for the process. Furthermore, actopaxin forms a complex with the Rac1/Cdc42 GEF β-PIX and Rac1/Cdc42 effector PAK1, to regulate actopaxin-dependent matrix degradation. Actopaxin phosphorylation is elevated in the invasive breast cancer cell line MDA-MB-231 compared with normal breast epithelial MCF10A cells. Expression of the nonphosphorylatable Quint actopaxin in MDA-MB-231 cells inhibits cell invasion whereas overexpression of WT actopaxin promotes invasion in MCF10A cells. Taken together, this study demonstrates a new role for actopaxin phosphorylation in matrix degradation and cell invasion via regulation of Rho GTPase signaling.     

Asic3?/? Female Mice with Hearing Deficit Affects Social Development of Pups

Background

Infant crying is an important cue for mothers to respond adequately. Inappropriate response to infant crying can hinder social development in infants. In rodents, the pup-mother interaction largely depends on pup''s calls. Mouse pups emit high frequency to ultrasonic vocalization (2–90 kHz) to communicate with their dam for maternal care. However, little is known about how the maternal response to infant crying or pup calls affects social development over the long term.

Methodology/Principal Findings

Here we used mice lacking acid-sensing ion channel 3 (Asic3^−/−) to create a hearing deficit to probe the effect of caregiver hearing on maternal care and adolescent social development. Female Asic3^−/− mice showed elevated hearing thresholds for low to ultrasonic frequency (4–32 kHz) on auditory brain stem response, which thus hindered their response to their pups'' wriggling calls and ultrasonic vocalization, as well as their retrieval of pups. In adolescence, pups reared by Asic3^−/− mice showed a social deficit in juvenile social behaviors as compared with those reared by wild-type or heterozygous dams. The social-deficit phenotype in juvenile mice reared by Asic3^−/− mice was associated with the reduced serotonin transmission of the brain. However, Asic3^−/− pups cross-fostered to wild-type dams showed rescued social deficit.

Conclusions/Significance

Inadequate response to pups'' calls as a result of ASIC3-dependent hearing loss confers maternal deficits in caregivers and social development deficits in their young.     

Saccharomyces cerevisiae–Based Platform for Rapid Production and Evaluation of Eukaryotic Nutrient Transporters and Transceptors for Biochemical Studies and Crystallography

To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins C-terminally-fused to a Tobacco Etch Virus (TEV) protease detachable Green Fluorescent Protein (GFP)-8His tag, which facilitates localization, quantification, quality control, and purification. Using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from S. cerevisiae that have not previously been overexpressed in S. cerevisiae and purified. Whole-cell GFP-fluorescence showed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15°C resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusions estimated by GFP-fluorescence were in the range of 0.4 mg to 1.7 mg transporter pr. liter cell culture. A detergent screen showed that n-dodecyl-ß-D-maltopyranoside (DDM) is acceptable for solubilization of the membrane-integrated fusions. Extracts of solubilized membranes were prepared with this detergent and used for purifications by Ni-NTA affinity chromatography, which yielded partially purified full-length fusions. Most of the fusions were readily cleaved at a TEV protease site between the membrane protein and the GFP-8His tag. Using the yeast oligopeptide transporter Ptr2 as an example, we further demonstrate that almost pure transporters, free of the GFP-8His tag, can be achieved by TEV protease cleavage followed by reverse immobilized metal-affinity chromatography. The quality of the GFP-fusions was analysed by fluorescence size-exclusion chromatography. Membranes solubilized in DDM resulted in preparations containing aggregated fusions. However, 9 of the fusions solubilized in DDM in presence of cholesteryl hemisuccinate and specific substrates, yielded monodisperse preparations with only minor amounts of aggregated membrane proteins. In conclusion, we developed a new effective S. cerevisiae expression system that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis.     

The Lewis X-related α1,3-Fucosyltransferase,Fut10, Is Required for the Maintenance of Stem Cell Populations

Lewis X (Le^X, Galβ1–4(Fucα1–3)GlcNAc) is a carbohydrate epitope that is present at the nonreducing terminus of sugar chains of glycoproteins and glycolipids, and is abundantly expressed in several stem cell populations. Le^X antigen can be used in conjunction with fluorescence-activated cell sorting to isolate neurosphere-forming neural stem cells (NSCs) from embryonic mouse brains. However, its function in the maintenance and differentiation of stem cells remains largely unknown. In this study, we examined mice deficient for fucosyltransferase 9 (Fut9), which is thought to synthesize most, if not all, of the Le^X moieties in the brain. We found that the number of NSCs was increased in the brain of Fut9^−/− embryos, suggesting that Fut9-synthesized Le^X is dispensable for the maintenance of NSCs. Another α1,3-fucosyltransferase gene, fucosyltransferase 10 (Fut10), is expressed in the ventricular zone of the embryonic brain. Overexpression of Fut10 enhanced the self-renewal of NSCs. Conversely, suppression of Fut10 expression induced the differentiation of NSCs and embryonic stem cells. In addition, knockdown of Fut10 expression in the cortical ventricular zone of the embryonic brain by in utero electroporation of Fut10-miRNAs impaired the radial migration of neural precursor cells. Our data suggest that Fut10 is involved in a unique α1,3-fucosyltransferase activity with stringent substrate specificity, and that this activity is required to maintain stem cells in an undifferentiated state.     

An Enhanced Immune Response of Mclk1 +/? Mutant Mice Is Associated with Partial Protection from Fibrosis,Cancer and the Development of Biomarkers of Aging

The immune response is essential for survival by destroying microorganisms and pre-cancerous cells. However, inflammation, one aspect of this response, can result in short- and long-term deleterious side-effects. Mclk1^+/− mutant mice can be long-lived despite displaying a hair-trigger inflammatory response and chronically activated macrophages as a result of high mitochondrial ROS generation. Here we ask whether this phenotype is beneficial or simply tolerated. We used models of infection by Salmonella serovars and found that Mclk1^+/− mutants mount a stronger immune response, control bacterial proliferation better, and are resistant to cell and tissue damage resulting from the response, including fibrosis and types of oxidative damage that are considered to be biomarkers of aging. Moreover, these same types of tissue damage were found to be low in untreated 23 months-old mutants. We also examined the initiation of tumour growth after transplantation of mouse LLC1 carcinoma cells into Mclk1^+/− mutants, as well as during spontaneous tumorigenesis in Mclk1^+/−Trp53^+/− double mutants. Tumour latency was increased by the Mclk1^+/− genotype in both models. Furthermore, we used the transplantation model to show that splenic CD8+ T lymphocytes from Mclk1^+/− graft recipients show enhanced cytotoxicity against LLC1 cells in vitro. Mclk1^+/− mutants thus display an association of an enhanced immune response with partial protection from age-dependent processes and from pathologies similar to those that are found with increased frequency during the aging process. This suggests that the immune phenotype of these mutants might contribute to their longevity. We discuss how these findings suggest a broader view of how the immune response might impact the aging process.     

RasGRP1, but Not RasGRP3, Is Required for Efficient Thymic β-Selection and ERK Activation Downstream of CXCR4

T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4⁻CD8⁻ ‘double negative’ (DN) thymocytes, pass through a checkpoint termed “β-selection” before maturing into CD4⁺CD8⁺ ‘double positive’ (DP) thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR) undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ⁺ DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.     

In Vitro and In Vivo Evaluation of Zinc-Modified Ca–Si-Based Ceramic Coating for Bone Implants

The host response to calcium silicate ceramic coatings is not always favorable because of their high dissolution rates, leading to high pH within the surrounding physiological environment. Recently, a zinc-incorporated calcium silicate-based ceramic Ca₂ZnSi₂O₇ coating, developed on a Ti-6Al-4V substrate using plasma-spray technology, was found to exhibit improved chemical stability and biocompatibility. This study aimed to investigate and compare the in vitro response of osteoblastic MC3T3-E1 cells cultured on Ca₂ZnSi₂O₇ coating, CaSiO₃ coating, and uncoated Ti-6Al-4V titanium control at cellular and molecular level. Our results showed Ca₂ZnSi₂O₇ coating enhanced MC3T3-E1 cell attachment, proliferation, and differentiation compared to CaSiO₃ coating and control. In addition, Ca₂ZnSi₂O₇ coating increased mRNA levels of osteoblast-related genes (alkaline phosphatase, procollagen α1(I), osteocalcin), insulin-like growth factor-I (IGF-I), and transforming growth factor-β1 (TGF-β1). The in vivo osteoconductive properties of Ca₂ZnSi₂O₇ coating, compared to CaSiO₃ coating and control, was investigated using a rabbit femur defect model. Histological and histomorphometrical analysis demonstrated new bone formation in direct contact with the Ca₂ZnSi₂O₇ coating surface in absence of fibrous tissue and higher bone-implant contact rate (BIC) in the Ca₂ZnSi₂O₇ coating group, indicating better biocompatibility and faster osseointegration than CaSiO₃ coated and control implants. These results indicate Ca₂ZnSi₂O₇ coated implants have applications in bone tissue regeneration, since they are biocompatible and able to osseointegrate with host bone.