scholarly journals Evidence that Golgi structure depends on a p115 activity that is independent of the vesicle tether components giantin and GM130

2001 ◽  
Vol 155 (2) ◽  
pp. 227-238 ◽  
Author(s):  
Manojkumar A. Puthenveedu ◽  
Adam D. Linstedt
Keyword(s):  
Brefeldin A ◽  
Detectable Effect ◽  
Vesicle Tethering ◽  
Culture Cells ◽  
Binding Partners ◽  
Golgi Fragmentation ◽  
Copi Vesicle ◽  
Golgi Structure

Inhibition of the putative coatomer protein I (COPI) vesicle tethering complex, giantin–p115–GM130, may contribute to mitotic Golgi breakdown. However, neither this, nor the role of the giantin–p115–GM130 complex in the maintenance of Golgi structure has been demonstrated in vivo. Therefore, we generated antibodies directed against the mapped binding sites in each protein of the complex and injected these into mammalian tissue culture cells. Surprisingly, the injected anti-p115 and antigiantin antibodies caused proteasome-mediated degradation of the corresponding antigens. Reduction of p115 levels below detection led to COPI-dependent Golgi fragmentation and apparent accumulation of Golgi-derived vesicles. In contrast, neither reduction of giantin below detectable levels, nor inhibition of p115 binding to GM130, had any detectable effect on Golgi structure or Golgi reassembly after cell division or brefeldin A washout. These observations indicate that inhibition of p115 can induce a mitotic-like Golgi disassembly, but its essential role in Golgi structure is independent of its Golgi-localized binding partners giantin and GM130.

ANGPTL4 modulates vascular junction integrity by integrin signaling and disruption of intercellular VE-cadherin and claudin-5 clusters

Blood ◽  
2011 ◽  
Vol 118 (14) ◽  
pp. 3990-4002 ◽  
Author(s):  
Royston-Luke Huang ◽  
Ziqiang Teo ◽  
Han Chung Chong ◽  
Pengcheng Zhu ◽  
Ming Jie Tan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Integrin Signaling ◽  
Wild Type ◽  
Integrin Α5β1 ◽  
Vascular Integrity ◽  
Vascular Disruption ◽  
Binding Partners ◽  
Adhesive Proteins

Abstract Vascular disruption induced by interactions between tumor-secreted permeability factors and adhesive proteins on endothelial cells facilitates metastasis. The role of tumor-secreted C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4) in vascular leakiness and metastasis is controversial because of the lack of understanding of how cANGPTL4 modulates vascular integrity. Here, we show that cANGPTL4 instigated the disruption of endothelial continuity by directly interacting with 3 novel binding partners, integrin α5β1, VE-cadherin, and claudin-5, in a temporally sequential manner, thus facilitating metastasis. We showed that cANGPTL4 binds and activates integrin α5β1-mediated Rac1/PAK signaling to weaken cell–cell contacts. cANGPTL4 subsequently associated with and declustered VE-cadherin and claudin-5, leading to endothelial disruption. Interfering with the formation of these cANGPTL4 complexes delayed vascular disruption. In vivo vascular permeability and metastatic assays performed using ANGPTL4-knockout and wild-type mice injected with either control or ANGPTL4-knockdown tumors confirmed that cANGPTL4 induced vascular leakiness and facilitated lung metastasis in mice. Thus, our findings elucidate how cANGPTL4 induces endothelial disruption. Our findings have direct implications for targeting cANGPTL4 to treat cancer and other vascular pathologies.

scholarly journals DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

2017 ◽  
Vol 114 (28) ◽  
pp. 7408-7413 ◽  
Author(s):  
Yan Jin ◽  
Yaohui Chen ◽  
Shimin Zhao ◽  
Kun-Liang Guan ◽  
Yuan Zhuang ◽  
...  
Keyword(s):  
Complex Formation ◽  
Germ Line ◽  
Mechanistic Explanation ◽  
Mass Spectrometry Analysis ◽  
Physical Interaction ◽  
Spectrometry Analysis ◽  
Culture Cells ◽  
Underlying Mechanisms

The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. Modified piggyBac (PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of PB among different cell lines have restricted its application. We discovered that the DNA–PK complex facilitates PB transposition by binding to PB transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA–PK components Ku70, Ku80, and DNA-PKcs. Overexpression or knockdown of DNA–PK components enhances or suppresses PB transposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency of PB is significantly reduced in Ku80 heterozygous mutant mice, confirming the role of DNA–PK in facilitating PB transposition in vivo. Fused dimer PBase can efficiently promote transposition. FRET experiments with tagged dimer PBase molecules indicated that DNA–PK promotes the paired-end complex formation of the PB transposon. These data provide a mechanistic explanation for the role of DNA–PK in facilitating PB transposition and suggest a transposition-promoting manipulation by enhancing the interaction of the PB ends. Consistent with this, deletions shortening the distance between the two PB ends, such as PB vectors with closer ends (PB-CE vectors), have a profound effect on transposition efficiency. Taken together, our study indicates that in addition to regulating DNA repair fidelity during transposition, DNA–PK also affects transposition efficiency by promoting paired-end complex formation. The approach of CE vectors provides a simple practical solution for designing efficient transposon vectors.

scholarly journals A comprehensive protein–protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1

2014 ◽  
Vol 25 (14) ◽  
pp. 2199-2215 ◽  
Author(s):  
Desiree DeMille ◽  
Benjamin T. Bikman ◽  
Andrew D. Mathis ◽  
John T. Prince ◽  
Jordan T. Mackay ◽  
...  
Keyword(s):  
Glucose Homeostasis ◽  
Protein Modification ◽  
Molecular Mechanisms ◽  
Binding Partners ◽  
Pas Kinase ◽  
Protein Interactome ◽  
Deficient Mice

Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein–protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase–deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis.

scholarly journals The tomosyn homologue, Sro7, is a direct effector of the Rab GTPase, Sec4, in post-Golgi vesicle tethering

2018 ◽  
Vol 29 (12) ◽  
pp. 1476-1486 ◽  
Author(s):  
Guendalina Rossi ◽  
Kelly Watson ◽  
Wade Kennedy ◽  
Patrick Brennwald
Keyword(s):  
Plasma Membrane ◽  
Simple Model ◽  
Genetic Analysis ◽  
Rab Gtpase ◽  
Vesicle Tethering ◽  
Effector Pathways ◽  
Rab Effector

The tomosyn/Sro7 family is thought to play an important role in cell surface trafficking both as an effector of Rab family GTPases and as a regulator of plasma-membrane SNARE function. Recent work has determined the binding site of GTP-bound Sec4 on Sro7. Here we examine the effect of mutations in Sro7 that block Sec4 binding in determining the role of this interaction in Sro7 function. Using an in vitro vesicle:vesicle tethering assay, we find that most of Sro7’s ability to tether vesicles is blocked by mutations that disrupt binding to Sec4-GTP. Similarly, genetic analysis demonstrates that the interaction with Sec4 is important for most of Sro7’s functions in vivo. The interaction of Sro7 with Sec4 appears to be particularly important when exocyst function is compromised. This provides strong evidence that Sro7 and the exocyst act as dual effector pathways downstream of Sec4. We also demonstrate that Sro7 tethering requires the presence of Sec4 on both opposing membranes and that homo-oligomerization of Sro7 occurs during vesicle tethering. This suggests a simple model for Sro7 function as a Rab effector in tethering post-Golgi vesicles to the plasma membrane in a pathway parallel to that of the exocyst complex.

scholarly journals The LINC00961 transcript and its encoded micropeptide, small regulatory polypeptide of amino acid response, regulate endothelial cell function

2020 ◽  
Vol 116 (12) ◽  
pp. 1981-1994 ◽  
Author(s):  
Helen L Spencer ◽  
Rachel Sanders ◽  
Mounia Boulberdaa ◽  
Marco Meloni ◽  
Amy Cochrane ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Amino Acid ◽  
Full Length ◽  
Binding Partners ◽  
Full Length Transcript ◽  
Opposing Effects ◽  
Amino Acid Response

Abstract Aims Long non-coding RNAs (lncRNAs) play functional roles in physiology and disease, yet understanding of their contribution to endothelial cell (EC) function is incomplete. We identified lncRNAs regulated during EC differentiation and investigated the role of LINC00961 and its encoded micropeptide, small regulatory polypeptide of amino acid response (SPAAR), in EC function. Methods and results Deep sequencing of human embryonic stem cell differentiation to ECs was combined with Encyclopedia of DNA Elements (ENCODE) RNA-seq data from vascular cells, identifying 278 endothelial enriched genes, including 6 lncRNAs. Expression of LINC00961, first annotated as an lncRNA but reassigned as a protein-coding gene for the SPAAR micropeptide, was increased during the differentiation and was EC enriched. LINC00961 transcript depletion significantly reduced EC adhesion, tube formation, migration, proliferation, and barrier integrity in primary ECs. Overexpression of the SPAAR open reading frame increased tubule formation; however, overexpression of the full-length transcript did not, despite production of SPAAR. Furthermore, overexpression of an ATG mutant of the full-length transcript reduced network formation, suggesting a bona fide non-coding RNA function of the transcript with opposing effects to SPAAR. As the LINC00961 locus is conserved in mouse, we generated an LINC00961 locus knockout (KO) mouse that underwent hind limb ischaemia (HLI) to investigate the angiogenic role of this locus in vivo. In agreement with in vitro data, KO animals had a reduced capillary density in the ischaemic adductor muscle after 7 days. Finally, to characterize LINC00961 and SPAAR independent functions in ECs, we performed pull-downs of both molecules and identified protein-binding partners. LINC00961 RNA binds the G-actin sequestering protein thymosin beta-4x (Tβ4) and Tβ4 depletion phenocopied the overexpression of the ATG mutant. SPAAR binding partners included the actin-binding protein, SYNE1. Conclusion The LINC00961 locus regulates EC function in vitro and in vivo. The gene produces two molecules with opposing effects on angiogenesis: SPAAR and LINC00961.

scholarly journals Coupling of vesicle tethering and Rab binding is required for in vivo functionality of the golgin GMAP-210

2015 ◽  
Vol 26 (3) ◽  
pp. 537-553 ◽  
Author(s):  
Keisuke Sato ◽  
Peristera Roboti ◽  
Alexander A. Mironov ◽  
Martin Lowe
Keyword(s):  
Golgi Apparatus ◽  
Functional Significance ◽  
Coiled Coil ◽  
Rab Gtpases ◽  
Vesicle Tethering ◽  
Amino Terminal ◽  
Membrane Tethering ◽  
Per Se ◽  
Golgi Structure

Golgins are extended coiled-coil proteins believed to participate in membrane-tethering events at the Golgi apparatus. However, the importance of golgin-mediated tethering remains poorly defined, and alternative functions for golgins have been proposed. Moreover, although golgins bind to Rab GTPases, the functional significance of Rab binding has yet to be determined. In this study, we show that depletion of the golgin GMAP-210 causes a loss of Golgi cisternae and accumulation of numerous vesicles. GMAP-210 function in vivo is dependent upon its ability to tether membranes, which is mediated exclusively by the amino-terminal ALPS motif. Binding to Rab2 is also important for GMAP-210 function, although it is dispensable for tethering per se. GMAP-210 length is also functionally important in vivo. Together our results indicate a key role for GMAP-210–mediated membrane tethering in maintaining Golgi structure and support a role for Rab2 binding in linking tethering with downstream docking and fusion events at the Golgi apparatus.

scholarly journals Resistance of neoplasms to immunological destruction: role of a macrophage chemotaxis inhibitor.

1978 ◽  
Vol 148 (1) ◽  
pp. 93-102 ◽  
Author(s):  
G R Pasternack ◽  
R Snyderman ◽  
M C Pike ◽  
R J Johnson ◽  
H S Shin
Keyword(s):  
Growth Rate ◽  
Tissue Culture ◽  
Tumor Cells ◽  
Culture Cells ◽  
Macrophage Chemotaxis ◽  
Immunological Destruction ◽  
Increased Susceptibility ◽  
Thymectomized Mice

Several tissue culture lines of 6C3HED, a murine lymphoma, were more susceptible to immunologic destruction in vivo than the highly virulent 6C3HED line maintained by serial intramuscular transplantation. The attenuated tissue culture cells were rejected by normal syngeneic recipients, but thymectomized mice were unable to reject attenuated cells. In such mice, the growth rate of attenuated cells was equivalent to the growth rate of virulent cells in normal syngeneic mice. The increased susceptibility of attenuated cells to destruction by syngeneic hosts was shown to correlate with decreased production by the tumor cells of a macrophage chemotaxis inhibitor, and not with altered antigen density. In addition, when inhibitor isolated from virulent cells was administered to mice challenged with attenuated cells, the latter cells became virulent in vivo. When attenuated and virulent cells were administered simultaneously in the same host, the attenuated cells were able to develop into progressively growing tumors. The data suggest that the successful growth of neoplastic cells in normal may require tumor cells to produce factors which subvert the ability of the host to mobilize macrophages rapidly at the tumor site.

scholarly journals The inflammasome components NLRP3 and ASC act in concert with IRGM to rearrange the Golgi during HCV infection.

2020 ◽  
Author(s):  
Coralie F. Daussy ◽  
Sarah C. Monard ◽  
Coralie Guy ◽  
Sara Muñoz-González ◽  
Maxime Chazal ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hcv Infection ◽  
Pyrin Domain ◽  
Inflammasome Component ◽  
Infected Cells ◽  
Golgi Fragmentation ◽  
Golgi Protein ◽  
Golgi Structure

Hepatitis C virus (HCV) infection triggers Golgi fragmentation through the Golgi-resident protein immunity-related GTPase M (IRGM). Here, we report the role of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) and ASC (Apoptosis-associated speck-like protein containing a CARD), two inflammasome components, in the initial events leading to this fragmentation. We show that ASC resides at the Golgi with IRGM at homeostasis. Upon infection, ASC dissociates from both IRGM and Golgi and associates with HCV-induced NLRP3. NLRP3 silencing inhibits Golgi fragmentation. ASC silencing disrupts the Golgi structure in both control and infected cells and reduces the localization of IRGM at the Golgi. IRGM-depletion in the ASC silenced cells cannot totally restore the Golgi structure. These data highlight a role for ASC, upstream of the formation of the inflammasome, in regulating IRGM through its control on the Golgi. A similar mechanism occurs in response to Nigericin treatment, but not in cells infected with another member of the Flaviviridae family, Zika virus (ZIKV). We propose a model for a newly ascribed function of the inflammasome components in Golgi structural remodeling during certain stimuli. IMPORTANCE Numerous pathogens can affect cellular homeostasis and organelle dynamics. Hepatitis C virus (HCV) triggers Golgi fragmentation through the immunity-related GTPase M (IRGM), a resident Golgi protein to enhance its lipid supply for replication. Here, we reveal the role of the inflammasome components NLRP3 and ASC in this process, thus uncovering a new interplay between effectors of inflammation and viral infection or stress. We show that the inflammasome component ASC resides at the Golgi under homeostasis and associates with IRGM. Upon HCV infection, ASC is recruited to NLRP3 and dissociates from IRGM causing Golgi fragmentation. Our results uncover that aside from their known function in the inflammation response, this host defense regulators also ensure the maintenance of intact intracellular structure in homeostasis status, while their activation relieves factors leading to Golgi remodeling.

scholarly journals Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure

1997 ◽  
Vol 139 (5) ◽  
pp. 1109-1118 ◽  
Author(s):  
Alexander Mironov ◽  
Antonino Colanzi ◽  
Maria Giuseppina Silletta ◽  
Giusy Fiucci ◽  
Silvio Flati ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Golgi Complex ◽  
Brefeldin A ◽  
Coat Proteins ◽  
Cytosolic Proteins ◽  
Permeabilized Cells ◽  
Adp Ribosylation ◽  
Golgi Membranes ◽  
Golgi Structure

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD+ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD+-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD+ triggered Golgi disassembly by BFA. This effect of NAD+ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.

scholarly journals GCC185 plays independent roles in Golgi structure maintenance and AP-1–mediated vesicle tethering

2011 ◽  
Vol 194 (5) ◽  
pp. 779-787 ◽  
Author(s):  
Frank C. Brown ◽  
Carmel H. Schindelhaim ◽  
Suzanne R. Pfeffer
Keyword(s):  
Coiled Coil ◽  
Retrograde Transport ◽  
Vesicle Tethering ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Transport Vesicle ◽  
Golgi Structure ◽  
Clathrin Adaptor ◽  
Golgi Network

GCC185 is a long coiled-coil protein localized to the trans-Golgi network (TGN) that functions in maintaining Golgi structure and tethering mannose 6-phosphate receptor (MPR)–containing transport vesicles en route to the Golgi. We report the identification of two distinct domains of GCC185 needed either for Golgi structure maintenance or transport vesicle tethering, demonstrating the independence of these two functions. The domain needed for vesicle tethering binds to the clathrin adaptor AP-1, and cells depleted of GCC185 accumulate MPRs in transport vesicles that are AP-1 decorated. This study supports a previously proposed role of AP-1 in retrograde transport of MPRs from late endosomes to the Golgi and indicates that docking may involve the interaction of vesicle-associated AP-1 protein with the TGN-associated tethering protein GCC185.

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