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[
Dev Biol,
2017]
Actin is an integral component of epithelial apical junctions, yet the interactions of branched actin regulators with apical junction components are still not clear. Biochemical data have shown that -catenin inhibits Arp2/3-dependent branched actin. These results suggested that branched actin is only needed at earliest stages of apical junction development. We use live imaging in developing C. elegans embryos to test models for how WAVE-induced branched actin collaborates with other apical junction proteins during the essential process of junction formation and maturation. We uncover both early and late essential roles for WAVE in apical junction formation. Early, as the C. elegans intestinal epithelium becomes polarized, we find that WAVE components become enriched concurrently with the Cadherin components and before the DLG-1 apical accumulation. Live imaging of F-actin accumulation in polarizing intestine supports that the Cadherin complex components and branched actin regulators work together for apical actin enrichment. Later in junction development, the apical accumulation of WAVE and Cadherin components is shown to be interdependent: Cadherin complex loss alters WAVE accumulation, and WAVE complex loss increases Cadherin accumulation. To determine why Cadherin levels rise when WVE-1 is depleted, we use FRAP to analyze Cadherin dynamics and find that loss of WAVE as well as of the trafficking protein EHD-1/RME-1 increases Cadherin dynamics. EM studies in adults depleted of branched actin regulators support that WVE-1 maintains established junctions, presumably through its trafficking effect on Cadherin. Thus we propose a developmental model for junction formation where branched actin regulators are tightly interconnected with Cadherin junctions through their previously unappreciated role in Cadherin transport.
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[
International Worm Meeting,
2017]
Our lab studies embryonic morphogenesis in C. elegans. Our earlier work showed that branched actin, powered by the WAVE/SCAR and Arp2/3 complexes, promotes polarized events during epithelial tissue development. These events include epidermal cell migration during ventral enclosure, and the polarized formation of the embryonic intestine. Actin is an integral component of the epithelial apical junction, yet the interactions of branched actin regulators with apical junction components are still not clear. We used live imaging of C. elegans embryos and newly available CRISPR-tagged strains (from our lab and the Goldstein lab) to define the developmental time course of accumulation of apical junction components relative to the branched actin regulators WVE-1 and ARP-2/ARX-2 as the embryonic intestinal epithelium becomes polarized. Live imaging of apical F-actin accumulation in the developing intestine showed that the two main apical junction complexes, namely Cadherin Catenin Complex (CCC) and DLG-1 AJM-1 complex (DAC), branched actin regulators, and non-muscle myosin were essential for apical actin enrichment. Biochemical data have shown that alpha catenin (a CCC component) inhibits Arp2/3-dependent branched actin, which suggests branched actin is only needed at the earliest stages of apical junction development. We found that loss of WVE-1 led to increased levels of CCC components after junction formation. The CCC components, in turn, regulated the levels of WAVE. Intriguingly, loss of two components of the CCC, E-Cdh/HMR-1 and beta -catenin/HMP-2, reduced levels of WVE-1 at apical junctions whereas loss of a-catenin/HMP-1 led to elevated apical WVE-1. Due to the interdependency in the localization of CCC and WVE-1 at the apical junction (AJ) and the temporal analysis of CCC and WAVE complex accumulation, we conclude that the CCC accumulates at AJ first and aids in recruitment of WAVE complex. Later CCC and DAC help in the maintenance of WAVE complex levels at AJ. WVE-1 in turn regulates HMR-1 turnover and shows inversely complemental levels with respect to a-catenin at the AJ. Therefore branched actin regulators are continually recruited to junctions to set up dynamic junction formation and maintenance of the AJ. Our on-going studies are investigating the mechanism for the interdependency of WAVE and CCC components at the apical junction.W
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[
International Worm Meeting,
2019]
Cadherin levels and localization are continuously adjusted throughout embryonic development to promote the movements of morphogenesis. In adult tissues apically enriched Cadherin supports the apical junction. We became intrigued with how branched actin helps regulate Cadherin/HMR-1 levels and localization when we discovered elevated levels of Cadherin complex components (Cadherin/HMR-1::GFP, beta catenin/HMP-2::GFP and alpha catenin/HMP-1::GFP) in embryos depleted of WAVE/Scar components. This seemed paradoxical, since loss of either Cadherin or WAVE/Scar components reduces apical actin accumulation in the embryonic and adult intestine. We are testing the model that branched actin promotes endosomal transport of Cadherin that is required for healthy apical junctions. Our analysis of Cadherin transport through multiple endosomal compartments shows that at least two populations of Cadherin are strongly altered in the absence of WAVE-dependent branched actin, including increased Cadherin at late endosomes. We will share our latest attempts to image the dynamics of vesicles at distinct compartments, and how these dynamics depend on branched actin. These studies could provide a mechanistic understanding to explain why Cadherin levels and distribution are altered in cancers where WAVE/Scar components are mutated.
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[
J Biol Chem,
2007]
The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.
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Olendrowitz C, Sasidharan N, Hegermann J, Rizzoli SO, Liewald JF, Hannemann M, Grant BD, Eimer S, Gottschalk A, Sumakovic M, Koenig S
[
Proc Natl Acad Sci U S A,
2012]
Neurons secrete neuropeptides from dense core vesicles (DCVs) to modulate neuronal activity. Little is known about how neurons manage to differentially regulate the release of synaptic vesicles (SVs) and DCVs. To analyze this, we screened all Caenorhabditis elegans Rab GTPases and Tre2/Bub2/Cdc16 (TBC) domain containing GTPase-activating proteins (GAPs) for defects in DCV release from C. elegans motoneurons.
rab-5 and
rab-10 mutants show severe defects in DCV secretion, whereas SV exocytosis is unaffected. We identified TBC-2 and TBC-4 as putative GAPs for RAB-5 and RAB-10, respectively. Multiple Rabs and RabGAPs are typically organized in cascades that confer directionality to membrane-trafficking processes. We show here that the formation of release-competent DCVs requires a reciprocal exclusion cascade coupling RAB-5 and RAB-10, in which each of the two Rabs recruits the other's GAP molecule. This contributes to a separation of RAB-5 and RAB-10 domains at the Golgi-endosomal interface, which is lost when either of the two GAPs is inactivated. Taken together, our data suggest that RAB-5 and RAB-10 cooperate to locally exclude each other at an essential stage during DCV sorting.
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Lou Y, Haque A, Freyzon Y, Farese RV, Terry-Kantor E, Hofbauer HF, Termine D, Welte MA, Barrasa MI, Imberdis T, Noble T, Lindquist S, Clish CB, Jaenisch R, Pincus D, Nuber S, Sandoe J, Kohlwein SD, Kim TE, Ho GPH, Ramalingam N, Walther TC, Baru V, Selkoe D, Srinivasan S, Landgraf D, Soldner F, Dettmer U, Fanning S, Becuwe M, Newby G
[
Mol Cell,
2018]
In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.
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[
PLoS One,
2017]
In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.
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[
Pathog Dis,
2014]
Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.
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[
International Worm Meeting,
2017]
During embryonic morphogenesis organs form and migrate to their final positions. This process requires continuous assembly and remodeling of adherens junctions (AJ). Our studies have identified Arp2/3 (branched actin nucleator) and its nucleation promoting factor, WAVE/Scar, as essential regulators of epithelial junctions. E-Cadherin/HMR-1 is a transmembrane receptor and an essential component of the AJ. Recent studies have shown that the turnover of Cadherin at the AJ is much more dynamic than previously thought. Adherens junctions rely heavily on the architecture and dynamics of the actin cytoskeleton. However, determining the function of the branched actin network at the junctions has proven elusive. Biochemical data have shown that AJ component a-catenin/HMP-1 inhibits Arp2/3-dependent branched actin, which suggested that branched actin is only needed at the earliest stages of apical junction development. We have addressed the role of branched actin at epithelial junctions using Transmission Electron Microscopy and live imaging. We found that branched actin is continuously recruited during both the dynamic process of junction formation and the later process of junction maintenance. Our results demonstrated an essential role for branched actin regulators in apical junction maintenance, and interdependence between the branched actin regulators and the Cadherin-Catenin complex (CCC). Therefore, we propose that there is a feedback mechanism present in which AJ-to-WAVE crosstalk dictates how the branched actin network is functioning. To our surprise, the three main components of the CCC complex, including Cadherin/HMR-1, accumulated to higher levels in embryos depleted of WAVE. We turned to FRAP imaging technique and biochemical approaches on C. elegans embryos to address this observation. We predicted that the increased levels of HMR-1::GFP in WAVE mutants resulted from slower turnover of HMR-1 at the membrane. Instead, we observed higher HMR-1::GFP mobility in the intestinal and epidermal junctions of WAVE-defective embryos. Several competitive models could be used to explain these results. Our goal is to determine which of the proposed mechanisms or Cadherin turnover, lateral-to-apical diffusion or vesicular trafficking, is significantly disrupted in WAVE mutants. We will interpret the fluorescence recovery traces by kinetic modeling. In addition we will quantify HMR-1 levels and mobility in embryos that are devoid of trafficking proteins that regulate distinct trafficking steps. These studies will advance understanding of the branched actin role at the forming and established adherens junctions. We will elucidate how branched actin affects Cadherin turnover at the apical cell-cell contacts in an in vivo system.
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Haass C, Hegermann J, Giese A, Eimer S, Kamp F, Lutz AK, Nuscher B, Wender N, Brunner B, Winklhofer KF, Exner N, Beyer K, Bartels T
[
EMBO J,
2010]
Aggregation of -synuclein (S) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of S is largely unknown. We demonstrate with in vitro vesicle fusion experiments that S has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, S binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous S. In contrast, siRNA-mediated downregulation of S results in elongated mitochondria in cell culture. S can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, S prevents fusion of differently labelled mitochondrial populations. Thus, S inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of S is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin 1-79 or by DJ-1 C106A.