Talin Autoinhibition Is Required for Morphogenesis

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Extracellular Norepinephrine Clearance by the Norepinephrine Transporter Is Required for Skeletal Homeostasis

Changes in bone remodeling induced by pharmacological and genetic manipulation of β-adrenergic receptor (βAR) signaling in osteoblasts support a role of sympathetic nerves in the regulation of bone remodeling. However, the contribution of endogenous sympathetic outflow and nerve-derived norepinephrine (NE) to bone remodeling under pathophysiological conditions remains unclear. We show here that differentiated osteoblasts, like neurons, express the norepinephrine transporter (NET), exhibit specific NE uptake activity via NET and can catabolize, but not generate, NE. Pharmacological blockade of NE transport by reboxetine induced bone loss in WT mice. Similarly, lack of NE reuptake in norepinephrine transporter (Net)-deficient mice led to reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and mechanical properties associated with low sympathetic outflow and high plasma NE levels. Last, daily sympathetic activation induced by mild chronic stress was unable to induce bone loss, unless NET activity was blocked. These findings indicate that the control of endogenous NE release and reuptake by presynaptic neurons and osteoblasts is an important component of the complex homeostatic machinery by which the sympathetic nervous system controls bone remodeling. These findings also suggest that drugs antagonizing NET activity, used for the treatment of hyperactivity disorders, may have deleterious effects on bone accrual.     

The Sec63p J-Domain Is Required for ERAD of Soluble Proteins in Yeast

How misfolded proteins are exported from the ER to the cytosol for degradation (ER-associated Degradation, ERAD) and which proteins are participating in this process is not understood. Several studies using a single, leaky mutant indicated that Sec63p might be involved in ERAD. More recently, Sec63p was also found strongly associated with proteasomes attached to the protein-conducting channel in the ER membrane which presumably form part of the export machinery. These observations prompted us to reinvestigate the role of Sec63p in ERAD by generating new mutants which were selected in a screen monitoring the intracellular accumulation of the ERAD substrate CPY*. We show that a mutation in the DnaJ-domain of Sec63p causes a defect in ERAD, whereas mutations in the Brl, acidic, and transmembrane domains only affect protein import into the ER. Unexpectedly, mutations in the acidic domain which mediates interaction of Sec63p with Sec62p also caused defects in cotranslational import. In contrast to mammalian cells where SEC63 expression levels affect steady-state levels of multi-spanning transmembrane proteins, the sec63 J-domain mutant was only defective in ERAD of soluble substrates.     

Itch Is Required for Lateral Line Development in Zebrafish

The zebrafish posterior lateral line is formed during early development by the deposition of neuromasts from a migrating primordium. The molecular mechanisms regulating the regional organization and migration of the primordium involve interactions between Fgf and Wnt/-catenin signaling and the establishment of specific cxcr4b and cxcr7b cytokine receptor expression domains. Itch has been identified as a regulator in several different signaling pathways, including Wnt and Cxcr4 signaling. We identified two homologous itch genes in zebrafish, itcha and itchb, with generalized expression patterns. By reducing itchb expression in particular upon morpholino knockdown, we demonstrated the importance of Itch in regulating lateral line development by perturbing the patterns of cxcr4b and cxcr7b expression. Itch knockdown results in a failure to down-regulate Wnt signaling and overexpression of cxcr4b in the primordium, slowing migration of the posterior lateral line primordium and resulting in abnormal development of the lateral line.     

The Epithelial Cell Adhesion Molecule EpCAM Is Required for Epithelial Morphogenesis and Integrity during Zebrafish Epiboly and Skin Development

The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell–cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell–cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers.     

Drosophila convoluted/dALS Is an Essential Gene Required for Tracheal Tube Morphogenesis and Apical Matrix Organization

Insulin-like growth factors (IGFs) control cell and organism growth through evolutionarily conserved signaling pathways. The mammalian acid-labile subunit (ALS) is a secreted protein that complexes with IGFs to modulate their activity. Recent work has shown that a Drosophila homolog of ALS, dALS, can also complex with and modulate the activity of a Drosophila IGF. Here we report the first mutations in the gene encoding dALS. Unexpectedly, we find that these mutations are allelic to a previously described mutation in convoluted (conv), a gene required for epithelial morphogenesis. In conv mutants, the tubes of the Drosophila tracheal system become abnormally elongated without altering tracheal cell number. conv null mutations cause larval lethality, but do not disrupt several processes required for tracheal tube size control, including septate junction formation, deposition of a lumenal/apical extracellular matrix, and lumenal secretion of Vermiform and Serpentine, two putative matrix-modifying proteins. Clearance of lumenal matrix and subcellular localization of clathrin also appear normal in conv mutants. However, we show that Conv/dALS is required for the dynamic organization of the transient lumenal matrix and normal structure of the cuticle that lines the tracheal lumen. These and other data suggest that the Conv/dALS-dependent tube size control mechanism is distinct from other known processes involved in tracheal tube size regulation. Moreover, we present evidence indicating that Conv/dALS has a novel, IGF-signaling independent function in tracheal morphogenesis.     

BAF200 Is Required for Heart Morphogenesis and Coronary Artery Development

ATP-dependent SWI/SNF chromatin remodeling complexes utilize ATP hydrolysis to non-covalently change nucleosome-DNA interactions and are essential in stem cell development, organogenesis, and tumorigenesis. Biochemical studies show that SWI/SNF in mammalian cells can be divided into two subcomplexes BAF and PBAF based on the subunit composition. ARID2 or BAF200 has been defined as an intrinsic subunit of PBAF complex. However, the function of BAF200 in vivo is not clear. To dissect the possible role of BAF200 in regulating embryogenesis and organ development, we generated BAF200 mutant mice and found they were embryonic lethal. BAF200 mutant embryos exhibited multiple cardiac defects including thin myocardium, ventricular septum defect, common atrioventricular valve, and double outlet right ventricle around E14.5. Moreover, we also detected reduced intramyocardial coronary arteries in BAF200 mutants, suggesting that BAF200 is required for proper migration and differentiation of subepicardial venous cells into arterial endothelial cells. Our work revealed that PBAF complex plays a critical role in heart morphogenesis and coronary artery angiogenesis.     

Binding of Extracellular Matrix Proteins by Enterococci

Forty-four enterococcal strains isolated from human clinical specimens were investigated for binding of ¹²⁵I-labeled fibronectin, vitronectin, thrombospondin, lactoferrin, and collagen type I and IV, and for cell surface hydrophobicity. Most strains expressed low binding of iodine-labeled human fibronectin, collagen I and IV, and higher binding of human vitronectin, human lactoferrin, and human thrombospondin. Bacteria grown in Todd-Hewitt broth exhibited increased binding to vitronectin and thrombospondin. In particle agglutination assays (PAA), Enterococcus faecalis strains reacted strongly with coated latex beads in contrast to E. faecium strains, which generally did not react. The ability of enterococci to bind ECM proteins was affected by heating and proteolytic digestion, suggesting that some protein-binding components become surface exposed after treatment with proteases. The binding of ¹²⁵I-labeled proteins to E. faecalis strain E70 was inhibited when cells were preincubated with unlabeled proteins. Preincubating cells with sulfated polymers such as dextran sulfate (M _r 5000 and 8000), pentosan sulfate and heparin decreased binding of vitronectin, lactoferrin, and thrombospondin. The binding of lactoferrin and thrombospondin was also decreased when bacteria were preincubated with galactose, fucose, and mannosamine, but not with mannose. All of 30 E. faecalis strains expressed pronounced surface hydrophobicity, but 10 of 14 E. faecium strains showed hydrophilic cell surface. Received: 22 April 1996 / Accepted: 29 June 1996     

Proper Activity of Histone H3 Lysine 4 (H3K4) Methyltransferase Is Required for Morphogenesis during Zebrafish Cardiogenesis

While increasing evidence indicates the important function of histone methylation during development, how this process influences cardiac development in vertebrates has not been explored. Here, we elucidate the functions of two histone H3 lysine 4 (H3K4) methylation enzymes, SMYD3 and SETD7, during zebrafish heart morphogenesis using gene expression profiling by whole mount in situ hybridization and antisense morpholino oligonucleotide (MO)-based gene knockdown. We find both smyd3 and setd7 are highly expressed within developing zebrafish heart and knock-down of these genes led to severe defects in cardiac morphogenesis without altering the expressions pattern of heart markers, including cmlc2, vmhc, and amhc. Furthermore, double knock-down by coinjection of smyd3 and setd7 MOs caused the synergistic defects in heart development. As similar to knock-down effect, overexpression of these genes also caused the heart morphogenesis defect in zebrafish. These results indicate that histone modifying enzymes, SMYD3 and SETD7, appear to function synergistically during heart development and their proper functioning is essential for normal heart morphogenesis during development.     

Nkx2.7 and Nkx2.5 Function Redundantly and Are Required for Cardiac Morphogenesis of Zebrafish Embryos

Background

Nkx2.7 is the tinman-related gene, as well as orthologs of Nkx2.5 and Nkx-2.3. Nkx2.7 and Nkx2.5 express in zebrafish heart fields of lateral plate mesoderm. The temporal and spatial expression patterns of Nkx2.7 are similar to those of Nkx2.5, but their functions during cardiogenesis remain unclear.

Methodology/Principal Findings

Here, Nkx2.7 is demonstrated to compensate for Nkx2.5 loss of function and play a predominant role in the lateral development of the heart, including normal cardiac looping and chamber formation. Knocking down Nkx2.5 showed that heart development was normal from 24 to 72 hpf. However, when knocking down either Nkx2.7 or Nkx2.5 together with Nkx2.7, it appeared that the heart failed to undergo looping and showed defective chambers, although embryos developed normally before the early heart tube stage. Decreased ventricular myocardium proliferation and defective myocardial differentiation appeared to result from late-stage up-regulation of bmp4, versican, tbx5 and tbx20, which were all expressed normally in hearts at an early stage. We also found that tbx5 and tbx20 were modulated by Nkx2.7 through the heart maturation stage because an inducible overexpression of Nkx2.7 in the heart caused down-regulation of tbx5 and tbx20. Although heart defects were induced by overexpression of an injection of 150-pg Nkx2.5 or 5-pg Nkx2.7 mRNA, either Nkx2.5 or Nkx2.7 mRNA rescued the defects induced by Nkx2.7-morpholino(MO) and Nkx2.5-MO with Nkx2.7-MO.

Conclusions and Significance

Therefore, we conclude that redundant activities of Nkx2.5 and Nkx2.7 are required for cardiac morphogenesis, but that Nkx2.7 plays a more critical function, specifically indicated by the gain-of-function and loss-of- function experiments where Nkx2.7 is observed to regulate the expressions of tbx5 and tbx20 through the maturation stage.     

Proteomics Analysis of Extracellular Matrix Remodeling During Zebrafish Heart Regeneration

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Highlights

• •We have developed a decellularization protocol for ECM protein enrichment.
• •We have characterized the proteome of adult zebrafish heart ECM.
• •We describe dynamic changes in heart ECM proteome during regeneration.
• •We describe changes in heart ECM stiffness during regeneration.

     

The Par-PrkC Polarity Complex Is Required for Cilia Growth in Zebrafish Photoreceptors

Specification and development of the apical membrane in epithelial cells requires the function of polarity proteins, including Pard3 and an atypical protein kinase C (PrkC). Many epithelial cells possess microtubule-based organelles, known as cilia, that project from their apical surface and the membrane surrounding the cilium is contiguous with the apical cell membrane. Although cilia formation in cultured cells required Pard3, the in vivo requirement for Pard3 in cilia development remains unknown. The vertebrate photoreceptor outer segment represents a highly specialized cilia structure in which to identify factors necessary for apical and ciliary membrane formation. Pard3 and PrkC localized to distinct domains within vertebrate photoreceptors. Using partial morpholino knockdown, photo-morpholinos, and pharmacological approaches, the function of Pard3 and PrkC were found to be required for the formation of both the apical and ciliary membrane of vertebrate photoreceptors. Inhibition of Pard3 or PrkC activity significantly reduced the size of photoreceptor outer segments and resulted in mislocalization of rhodopsin. Suppression of Pard3 or PrkC also led to a reduction in cilia size and cilia number in Kupffer’s Vesicle, which resulted in left-right asymmetry defects. Thus, the Par-PrkC complex functions in cilia formation in vivo and this likely reflects a general role in specifying non-ciliary and ciliary compartments of the apical domain.     

Cholesterol Is Required for Surface Transport of Influenza Virus Hemagglutinin

Transport from the TGN to the basolateral surface involves a rab/N-ethylmaleimide–sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)/SNAP receptor (SNARE) mechanism. Apical transport instead is thought to be mediated by detergent-insoluble sphingolipid–cholesterol rafts. By reducing the cholesterol level of living cells by 60–70% with lovastatin and methyl-β-cyclodextrin, we show that the TGN-to-surface transport of the apical marker protein influenza virus hemagglutinin was slowed down, whereas the transport of the basolateral marker vesicular stomatitis virus glycoprotein as well as the ER-to-Golgi transport of both membrane proteins was not affected. Reduction of transport of hemagglutinin was accompanied by increased solubility in the detergent Triton X-100 and by significant missorting of hemagglutinin to the basolateral membrane. In addition, depletion of cellular cholesterol by lovastatin and methyl-β-cyclodextrin led to missorting of the apical secretory glycoprotein gp-80, suggesting that gp-80 uses a raft-dependent mechanism for apical sorting. Our data provide for the first time direct evidence for the functional significance of cholesterol in the sorting of apical membrane proteins as well as of apically secreted glycoproteins.     

Sec3-containing Exocyst Complex Is Required for Desmosome Assembly in Mammalian Epithelial Cells

The Exocyst is a conserved multisubunit complex involved in the docking of post-Golgi transport vesicles to sites of membrane remodeling during cellular processes such as polarization, migration, and division. In mammalian epithelial cells, Exocyst complexes are recruited to nascent sites of cell–cell contact in response to E-cadherin–mediated adhesive interactions, and this event is an important early step in the assembly of intercellular junctions. Sec3 has been hypothesized to function as a spatial landmark for the development of polarity in budding yeast, but its role in epithelial cells has not been investigated. Here, we provide evidence in support of a function for a Sec3-containing Exocyst complex in the assembly or maintenance of desmosomes, adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. We show that Sec3 associates with a subset of Exocyst complexes that are enriched at desmosomes. Moreover, we found that membrane recruitment of Sec3 is dependent on cadherin-mediated adhesion but occurs later than that of the known Exocyst components Sec6 and Sec8 that are recruited to adherens junctions. RNA interference-mediated suppression of Sec3 expression led to specific impairment of both the morphology and function of desmosomes, without noticeable effect on adherens junctions. These results suggest that two different exocyst complexes may function in basal–lateral membrane trafficking and will enable us to better understand how exocytosis is spatially organized during development of epithelial plasma membrane domains.     

Extracellular DNA Is Required for Root Tip Resistance to Fungal Infection

Plant defense involves a complex array of biochemical interactions, many of which occur in the extracellular environment. The apical 1- to 2-mm root tip housing apical and root cap meristems is resistant to infection by most pathogens, so growth and gravity sensing often proceed normally even when other sites on the root are invaded. The mechanism of this resistance is unknown but appears to involve a mucilaginous matrix or “slime” composed of proteins, polysaccharides, and detached living cells called “border cells.” Here, we report that extracellular DNA (exDNA) is a component of root cap slime and that exDNA degradation during inoculation by a fungal pathogen results in loss of root tip resistance to infection. Most root tips (>95%) escape infection even when immersed in inoculum from the root-rotting pathogen Nectria haematococca. By contrast, 100% of inoculated root tips treated with DNase I developed necrosis. Treatment with BAL31, an exonuclease that digests DNA more slowly than DNase I, also resulted in increased root tip infection, but the onset of infection was delayed. Control root tips or fungal spores treated with nuclease alone exhibited normal morphology and growth. Pea (Pisum sativum) root tips incubated with [³²P]dCTP during a 1-h period when no cell death occurs yielded root cap slime containing ³²P-labeled exDNA. Our results suggest that exDNA is a previously unrecognized component of plant defense, an observation that is in accordance with the recent discovery that exDNA from white blood cells plays a key role in the vertebrate immune response against microbial pathogens.Root diseases caused by soil-borne plant pathogens are a perennial source of crop loss worldwide (Bruehl, 1986; Curl and Truelove, 1986). These diseases are of increasing concern, as pesticides like methyl bromide are removed from the market due to environmental concerns (Gilreath et al., 2005). One possible alternative means of crop protection is to exploit natural mechanisms of root disease resistance (Nelson, 1990; Goswami and Punja, 2008; Shittu et al., 2009). Direct observation of root systems under diverse conditions has revealed that root tips, in general, are resistant to infection even when lesions are initiated elsewhere on the same plant root (Foster et al., 1983; Bruehl, 1986; Curl and Truelove, 1986; Smith et al., 1992; Gunawardena et al., 2005; Wen et al., 2007). This form of disease resistance is important for crop production because root growth and its directional movement in response to gravity, water, and other signals can proceed normally as long as the root tip is not invaded. The 1- to 2-mm apical region of roots houses the root meristems required for root growth and cap development, and when infection does occur, root development ceases irreversibly within a few hours even in the absence of severe necrosis (Gunawardena and Hawes, 2002). Mechanisms underlying root tip resistance to infection are unclear, but the phenomenon appears to involve root cap “slime,” a mucilaginous matrix produced by the root cap (Morré et al., 1967; Rougier et al., 1979; Foster, 1982; Chaboud, 1983; Guinel and McCully, 1986; Moody et al., 1988; Knee et al., 2001; Barlow, 2003; Iijima et al., 2008). Within the root cap slime of cereals, legumes, and most other crop species are specialized populations of living cells called root “border cells” (Supplemental Fig. S1; Hawes et al., 2000). Border cell numbers increase in response to pathogens and toxins such as aluminum, and the cell populations maintain a high rate of metabolic activity even after detachment from the root cap periphery (Brigham et al., 1995; Miyasaka and Hawes, 2000).As border cells detach from roots of cereals and legumes, a complex of more than 100 proteins, termed the root cap secretome, is synthesized and exported from living cells into the matrix ensheathing the root tip (Brigham et al., 1995). The profile of secreted proteins changes in response to challenge with soil-borne bacteria (De-la-Peña et al., 2008). In pea (Pisum sativum), root tip resistance to infection is abolished in response to proteolytic degradation of the root cap secretome (Wen et al., 2007). In addition to an array of antimicrobial enzymes and other proteins known to be components of the extracellular matrix and apoplast of higher plants, the DNA-binding protein histone H4 unexpectedly was found to be present among the secreted proteins (Wen et al., 2007). One explanation for the presence of histone is global leakage of material from disrupted nuclei in dead cells, but no cell death occurs during delivery of the secretome (Brigham et al., 1995; Wen et al., 2007). An alternative explanation for the presence of a secreted DNA-binding protein is that extracellular DNA (exDNA) also is present in root cap slime.exDNA has long been known to be a component of slimy biological matrices ranging from purulent localized human infections to bacterial capsules, biofilms, and snail exudate (Sherry and Goeller, 1950; Leuchtenberger and Schrader, 1952; Braun and Whallon, 1954; Smithies and Gibbons, 1955; Catlin, 1956; Fahy et al., 1993; Allesen-Holm et al., 2006; Spoering and Gilmore, 2006; Qin et al., 2007; Izano et al., 2008). Specialized white blood cells in humans and other species including fish recently have been shown to deploy a complex neutrophil extracellular “trap” (NET), composed of DNA and a collection of enzymes, in response to infection (Brinkmann et al., 2004; Brinkmann and Zychlinsky, 2007; Palić et al., 2007; Wartha et al., 2007; Yousefi et al., 2008). NETs appear to kill bacterial, fungal, and protozoan pathogens by localizing them within a matrix of antimicrobial peptides and proteins (Urban et al., 2006; Wartha et al., 2007; Guimaraes-Costa et al., 2009). Several extracellular peptides and proteins implicated in neutrophil function, including histone, also are present within the pea root cap secretome (Wen et al., 2007). exDNA linked with extracellular histone is a structural component of NETs, and treatment with DNase destroys NET integrity and function (Wartha et al., 2007). Moreover, human pathogens including group A Streptococcus and Streptococcus pneumoniae release extracellular DNase (Sherry and Goeller, 1950). When these activities are eliminated by mutagenesis of the encoding genes, bacteria lose their normal ability to escape the NET and multiply at the site of infection (Sumby et al., 2005; Buchanan et al., 2006). Here, we report that, in addition to histone and other secretome proteins, exDNA also is a component of root cap slime. When this exDNA is digested enzymatically, root tip resistance to infection is abolished.     

Interaction with LC8 Is Required for Pak1 Nuclear Import and Is Indispensable for Zebrafish Development

Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.