111 research outputs found
Loss of a Clueless-dGRASP complex results in ER stress and blocks Integrin exit from the perinuclear endoplasmic reticulum in Drosophila larval muscle
Citation: Wang, Z. H., Rabouille, C., & Geisbrecht, E. R. (2015). Loss of a Clueless-dGRASP complex results in ER stress and blocks Integrin exit from the perinuclear endoplasmic reticulum in Drosophila larval muscle. Biology Open, 4(5), 636-648. doi:10.1242/bio.201511551Drosophila Clueless (Clu) and its conserved orthologs are known for their role in the prevention of mitochondrial clustering. Here, we uncover a new role for Clu in the delivery of integrin subunits in muscle tissue. In clu mutants, alpha PS2 integrin, but not beta PS integrin, abnormally accumulates in a perinuclear endoplasmic reticulum (ER) subdomain, a site that mirrors the endogenous localization of Clu. Loss of components essential for mitochondrial distribution do not phenocopy the clu mutant alpha PS2 phenotype. Conversely, RNAi knockdown of the Drosophila Golgi reassembly and stacking protein GRASP55/65 (dGRASP) recapitulates clu defects, including the abnormal accumulation of alpha PS2 and larval locomotor activity. Both Clu and dGRASP proteins physically interact and loss of Clu displaces dGRASP from ER exit sites, suggesting that Clu cooperates with dGRASP for the exit of alpha PS2 from a perinuclear subdomain in the ER. We also found that Clu and dGRASP loss of function leads to ER stress and that the stability of the ER exit site protein Sec16 is severely compromised in the clu mutants, thus explaining the ER accumulation of alpha PS2. Remarkably, exposure of clu RNAi larvae to chemical chaperones restores both alpha PS2 delivery and functional ER exit sites. We propose that Clu together with dGRASP prevents ER stress and therefore maintains Sec16 stability essential for the functional organization of perinuclear early secretory pathway. This, in turn, is essential for integrin subunit alpha PS2 ER exit in Drosophila larval myofibers
Enterotoxigenic Escherichia coli Flagellin Inhibits TNF-Induced NF-κB Activation in Intestinal Epithelial Cells
Citation: Wang, G.; Geisbrecht, B.V.; Rueter, C.; Hardwidge, P.R. Enterotoxigenic Escherichia coli Flagellin Inhibits TNF-Induced NF-κB Activation in Intestinal Epithelial Cells. Pathogens 2017, 6, 18.Enterotoxigenic Escherichia coli (ETEC) causes childhood diarrhea in developing countries. ETEC strains produce the heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (ST) and encode a diverse set of colonization factors used for adherence to intestinal epithelial cells. We previously found that ETEC secretes a heat-stable protein we designated as ETEC Secreted Factor (ESF) that inhibits the extent of NF-κB activation normally induced by tumor necrosis factor alpha (TNF). Here we fractionated ETEC supernatants using fast protein liquid chromatography (FPLC) and determined that ETEC flagellin was necessary and sufficient to protect IκBα from degradation in response to TNF stimulation. These data suggest a potentially novel mechanism by which ETEC may evade the host innate immune response by down-regulating NF-κB-dependent host responses
Cell adhesion and cortex contractility determine cell patterning in the Drosophila retina
Hayashi and Carthew (Nature 431 [2004], 647) have shown that the packing of
cone cells in the Drosophila retina resembles soap bubble packing, and that
changing E- and N-cadherin expression can change this packing, as well as cell
shape.
The analogy with bubbles suggests that cell packing is driven by surface
minimization. We find that this assumption is insufficient to model the
experimentally observed shapes and packing of the cells based on their cadherin
expression. We then consider a model in which adhesion leads to a surface
increase, balanced by cell cortex contraction. Using the experimentally
observed distributions of E- and N-cadherin, we simulate the packing and cell
shapes in the wildtype eye. Furthermore, by changing only the corresponding
parameters, this model can describe the mutants with different numbers of
cells, or changes in cadherin expression.Comment: revised manuscript; 8 pages, 6 figures; supplementary information not
include
NlpC/P60 domain-containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan
A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain-containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine-155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain-containing proteins than simply hydrolases
Costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for Limb-girdle muscular dystrophy type 2H
Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of βPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and β-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration
The DOCK Protein Sponge Binds to ELMO and Functions in Drosophila Embryonic CNS Development
Cell morphogenesis, which requires rearrangement of the actin cytoskeleton, is
essential to coordinate the development of tissues such as the musculature and
nervous system during normal embryonic development. One class of signaling
proteins that regulate actin cytoskeletal rearrangement is the evolutionarily
conserved CDM (C. elegans
Ced-5, human DOCK180,
Drosophila
Myoblast city, or Mbc) family of proteins, which function
as unconventional guanine nucleotide exchange factors for the small GTPase Rac.
This CDM-Rac protein complex is sufficient for Rac activation, but is enhanced
upon the association of CDM proteins with the ELMO/Ced-12 family of proteins. We
identified and characterized the role of Drosophila Sponge
(Spg), the vertebrate DOCK3/DOCK4 counterpart as an ELMO-interacting protein.
Our analysis shows Spg mRNA and protein is expressed in the visceral musculature
and developing nervous system, suggesting a role for Spg in later embryogenesis.
As maternal null mutants of spg die early in development, we
utilized genetic interaction analysis to uncover the role of Spg in central
nervous system (CNS) development. Consistent with its role in ELMO-dependent
pathways, we found genetic interactions with spg and
elmo mutants exhibited aberrant axonal defects. In
addition, our data suggests Ncad may be responsible for recruiting Spg to the
membrane, possibly in CNS development. Our findings not only characterize the
role of a new DOCK family member, but help to further understand the role of
signaling downstream of N-cadherin in neuronal development
Identification of the Neogenin-Binding Site on the Repulsive Guidance Molecule A
Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the chick retinotectal system. RGMa, one of the 3 isoforms found in mammals, is involved in laminar patterning, cephalic neural tube closure, axon guidance, and inhibition of axonal regeneration. In addition to its roles in the nervous system, RGMa plays a role in enhancing helper T-cell activation. Binding of RGM to its receptor, neogenin, is considered necessary to transduce these signals; however, information on the binding of RGM to neogenin is limited. Using co-immunoprecipitation studies, we have identified that the RGMa region required for binding to neogenin contains amino acids (aa) 259–295. Synthesized peptide consisting of aa 284–293 directly binds to the extracellular domain (ECD) of recombinant neogenin, and addition of this peptide inhibits RGMa-induced growth cone collapse in mouse cortical neurons. Thus, we propose that this peptide is a promising lead in finding reagents capable of inhibiting RGMa signaling
Corpse Engulfment Generates a Molecular Memory that Primes the Macrophage Inflammatory Response
Macrophages are multifunctional cells that perform diverse roles in health and disease. Emerging evidence has suggested that these innate immune cells might also be capable of developing immunological memory, a trait previously associated with the adaptive system alone. While recent studies have focused on the dramatic macrophage reprogramming that follows infection and protects against secondary microbial attack, can macrophages also develop memory in response to other cues? Here, we show that apoptotic corpse engulfment by Drosophila macrophages is an essential primer for their inflammatory response to tissue damage and infection in vivo. Priming is triggered via calcium-induced JNK signaling, which leads to upregulation of the damage receptor Draper, thus providing a molecular memory that allows the cell to rapidly respond to subsequent injury or infection. This remarkable plasticity and capacity for memory places macrophages as key therapeutic targets for treatment of inflammatory disorders
Novel suppressors of α-synuclein toxicity identified using yeast
The mechanism by which the Parkinson’s disease-related protein α-synuclein (α-syn) causes neurodegeneration has not been elucidated. To determine the genes that protect cells from α-syn, we used a genetic screen to identify suppressors of the super sensitivity of the yeast Saccharomyces cerevisiae expressing α-syn to killing by hydrogen peroxide. Forty genes in ubiquitin-dependent protein catabolism, protein biosynthesis, vesicle trafficking and the response to stress were identified. Five of the forty genes—ENT3, IDP3, JEM1, ARG2 and HSP82—ranked highest in their ability to block α-syn-induced reactive oxygen species accumulation, and these five genes were characterized in more detail. The deletion of any of these five genes enhanced the toxicity of α-syn as judged by growth defects compared with wild-type cells expressing α-syn, which indicates that these genes protect cells from α-syn. Strikingly, four of the five genes are specific for α-syn in that they fail to protect cells from the toxicity of the two inherited mutants A30P or A53T. This finding suggests that α-syn causes toxicity to cells through a different pathway than these two inherited mutants. Lastly, overexpression of Ent3p, which is a clathrin adapter protein involved in protein transport between the Golgi and the vacuole, causes α-syn to redistribute from the plasma membrane into cytoplasmic vesicular structures. Our interpretation is that Ent3p mediates the transport of α-syn to the vacuole for proteolytic degradation. A similar clathrin adaptor protein, epsinR, exists in humans
Myosin VI in PC12 cells plays important roles in cell migration and proliferation but not in catecholamine secretion
Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments and is believed to play distinct role(s) than other myosins. We addressed a role of this unique motor in secretory PC12 cells, derived from rat adrenal medulla pheochromocytoma using cell lines with reduced MVI synthesis (produced by means of siRNA). Decrease of MVI expression caused severe changes in cell size and morphology, and profound defects in actin cytoskeleton organization and Golgi structure. Also, significant inhibition of cell migration as well as cell proliferation was observed. Flow cytometric analysis revealed that MVI-deficient cells were arrested in G0/G1 phase of the cell cycle but did not undergo increased senescence as compared with control cells. Also, neither polyploidy nor aneuploidy were detected. Surprisingly, no significant effect on noradrenaline secretion was observed. These data indicate that in PC12 cells MVI is involved in cell migration and proliferation but is not crucial for stimulation-dependent catecholamine release
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