scholarly journals Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1631
Author(s):  
Cole P. Frisbie ◽  
Alexander Y. Lushnikov ◽  
Alexey V. Krasnoslobodtsev ◽  
Jean-Jack M. Riethoven ◽  
Jennifer L. Clarke ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Brefeldin A ◽  
Direct Interaction ◽  
Complete Recovery ◽  
Proximity Ligation Assay ◽  
Conformational Structure ◽  
Force Microscopy ◽  
Proximity Ligation ◽  
Golgi Membranes ◽  
Response To Stress

Background: The Golgi apparatus undergoes disorganization in response to stress, but it is able to restore compact and perinuclear structure under recovery. This self-organization mechanism is significant for cellular homeostasis, but remains mostly elusive, as does the role of giantin, the largest Golgi matrix dimeric protein. Methods: In HeLa and different prostate cancer cells, we used the model of cellular stress induced by Brefeldin A (BFA). The conformational structure of giantin was assessed by proximity ligation assay and atomic force microscopy. The post-BFA distribution of Golgi resident enzymes was examined by 3D SIM high-resolution microscopy. Results: We detected that giantin is rather flexible than an extended coiled-coil dimer and BFA-induced Golgi disassembly was associated with giantin monomerization. A fusion of the nascent Golgi membranes after BFA washout is forced by giantin re-dimerization via disulfide bond in its luminal domain and assisted by Rab6a GTPase. GM130-GRASP65-dependent enzymes are able to reach the nascent Golgi membranes, while giantin-sensitive enzymes appeared at the Golgi after its complete recovery via direct interaction of their cytoplasmic tail with N-terminus of giantin. Conclusion: Post-stress recovery of Golgi is conducted by giantin dimer and Golgi proteins refill membranes according to their docking affiliation rather than their intra-Golgi location.

scholarly journals PGRMC1 localization and putative function in the nucleolus of bovine granulosa cells and oocytes

Reproduction ◽  
2018 ◽  
Vol 155 (3) ◽  
pp. 273-282 ◽  
Author(s):  
Laura Terzaghi ◽  
Alberto Maria Luciano ◽  
Priscila C Dall’Acqua ◽  
Silvia C Modina ◽  
John J Peluso ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Granulosa Cells ◽  
Direct Interaction ◽  
Proximity Ligation Assay ◽  
Multifunctional Protein ◽  
Proximity Ligation ◽  
Response To Stress ◽  
Interfering Rna ◽  
Cellular Stressors

Progesterone receptor membrane component-1 (PGRMC1) is a highly conserved multifunctional protein that is found in numerous systems, including reproductive system. Interestingly, PGRMC1 is expressed at several intracellular locations, including the nucleolus. The aim of this study is to investigate the functional relationship between PGRMC1 and nucleolus. Immunofluorescence experiments confirmed PGRMC1’s nucleolar localization in cultured bovine granulosa cells (bGC) and oocytes. Additional experiments conducted on bGC revealed that PGRMC1 co-localizes with nucleolin (NCL), a major nucleolar protein. Furthermore, small interfering RNA (RNAi)-mediated gene silencing experiments showed that when PGRMC1 expression was depleted, NCL translocated from the nucleolus to the nucleoplasm. Similarly, oxidative stress induced by hydrogen peroxide (H2O2) treatment, reduced PGRMC1 immunofluorescent signal in the nucleolus and increased NCL nucleoplasmic signal, when compared to non-treated cells. Although PGRMC1 influenced NCL localization, a direct interaction between these two proteins was not detected using in situ proximity ligation assay. This suggests the involvement of additional molecules in mediating the co-localization of PGRMC1 and nucleolin. Since nucleolin translocates into the nucleoplasm in response to various cellular stressors, PGRMC1’s ability to regulate its localization within the nucleolus is likely an important component of mechanism by which cells response to stress. This concept is consistent with PGRMC1’s well-described ability to promote ovarian cell survival and provides a rationale for future studies on PGRMC1, NCL and the molecular mechanism by which these two proteins protect against the adverse effect of cellular stressors, including oxidative stress.

Identification and characterization of a novel human plant pathogenesis-related protein that localizes to lipid-enriched microdomains in the Golgi complex

2002 ◽  
Vol 115 (4) ◽  
pp. 827-838 ◽  
Author(s):  
Heike B. Eberle ◽  
Ramon L. Serrano ◽  
Joachim Füllekrug ◽  
Andreas Schlosser ◽  
Wolf D. Lehmann ◽  
...  
Keyword(s):  
Immune System ◽  
Mammalian Cells ◽  
Electrostatic Interactions ◽  
Brefeldin A ◽  
Direct Interaction ◽  
Insoluble Fraction ◽  
Golgi Membranes ◽  
Pathogenesis Related Protein ◽  
Pathogenesis Related ◽  
Plant Pathogenesis

Group 1 of plant pathogenesis-related proteins (PR-1) and a variety of related mammalian proteins constitute a superfamily of proteins that share structural similarities. Little is known about their function, but all the family members identified to date are co-translationally translocated to the lumen of the endoplasmic reticulum and are secreted as soluble proteins or are targeted to vacuoles. Here we report the identification of a novel family member that localizes to the cytosolic site of the endomembrane system in mammalian cells. After detergent solubilization of isolated Golgi membranes, a 17 kDa protein was found associated with a low-density detergent-insoluble fraction. The amino-acid sequence, determined by microsequencing and molecular cloning, revealed a significant homology with the superfamily of PR-1 proteins. Golgi-associated PR-1 protein (GAPR-1) showed a brefeldin-A-sensitive Golgi localization in immunofluorescence. Interestingly,the protein remained associated with the microdomain fraction in the presence of Brefeldin A. By mass spectrometry, GAPR-1 was shown to be myristoylated. Immunoprecipitation of GAPR- 1 from Golgi membranes resulted in the coimmunoprecipitation of caveolin-1, indicating a direct interaction between these two proteins. Myristoylation, together with protein-protein or electrostatic interactions at physiological pH owing to the highly basic pI of GAPR-1 (pI 9.4) could explain the strong membrane association of GAPR-1. Tissue screening revealed that GAPR-1 is not detectably expressed in liver,heart or adrenal glands. High expression was found in monocytes, leukocytes,lung, spleen and embryonic tissue. Consistent with the involvement of PR-1 proteins in the plant immune system, these data could indicate that GAPR-1 is involved in the immune system.

scholarly journals OR12-02 When the Glucocorticoid Receptor Meets the Mineralocorticoid Receptor in the Nucleus of Human Cells

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Maria G Petrillo ◽  
Christine M Jewell ◽  
Robert H Oakley ◽  
John A Cidlowski
Keyword(s):  
Glucocorticoid Receptor ◽  
Cell Lines ◽  
Mineralocorticoid Receptor ◽  
Nuclear Translocation ◽  
Transcriptional Response ◽  
Proximity Ligation Assay ◽  
Osteosarcoma Cell ◽  
Proximity Ligation ◽  
Gene Response ◽  
Response To Stress

Abstract Adrenal corticosteroids, such as glucocorticoids and mineralocorticoids, are indispensable for mediating response to stress, development, limiting inflammation, and maintaining energy and fluid homeostasis. These hormones exert their actions via binding to two closely related nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). The GR has low affinity for corticosteroids, but is expressed in nearly every cell. In contrast, the MR shows a higher affinity for corticosteroids and its expression is largely confined to those tissues where electrolyte exchange and fluid balance are required. GR and MR act as ligand-activated transcription factors which, following interaction with co-regulators and DNA responsive elements, either promote or repress gene transcription. The affinity for the same ligands, structural homology, and binding to the same DNA regions suggest GR and MR can compensate for each other’s actions. Yet, there are specific glucocorticoid and mineralocorticoid-mediated responses indicating GR-MR functional diversity. To investigate this interplay, we developed U-2 OS (human osteosarcoma) cell lines stably expressing GR, MR, and both GR and MR (GRMR). Immunofluorescence analysis showed that treatment of these cell lines with 1 nM of the synthetic glucocorticoid dexamethasone (Dex) induced nuclear traslocation of GR and MR. Conversely, treatment with 1 nM of aldosterone (Aldo) promoted nuclear translocation of the MR only. Moreover, Proximity Ligation Assay revealed that, in the absence of ligand, GR associated with MR in the cytoplasm and, upon 1 nM Dex exposure, GR-MR dimers were detected in the nucleus of GRMR cells. Surprisingly, nuclear GR-MR dimers were also detected in the presence of Aldo, suggesting that it is necessary to activate at least one receptor to induce nuclear traslocation of the heterocomplex. To decipher the functional contribution of GR-MR dimers in the transcriptional response of GR to Dex and MR to Aldo, we performed RNA-seq in GR, MR, and GRMR cells treated with 1 nM of Dex or Aldo. Transcriptome analysis revealed that Dex-activated GR regulated the transcription of 6180 genes. Co-expression of MR resulted in a blunted Dex-mediated gene response which affected only 1608 genes, suggesting a functional antagonism of MR. Aldo-activated MR regulated the transcription of 1660 genes. However, co-expression of GR expanded the Aldo-mediated gene response to 3150 genes. Strikingly, 74% of these genes were also regulated by Dex via GR, suggesting that GR-MR dimers in the presence of aldosterone are able to mimic the glucocorticod transcriptional response. Our data suggest that the role of distinct GR and MR homo- and hetero-dimers is relevant for regulating gene expression. Dissecting the mechanism and investigating the cross-talk between GR and MR may be useful to understanding these two receptors in heath and disease.

scholarly journals The Association between α-Synuclein and α-Tubulin in Brain Synapses

2021 ◽  
Vol 22 (17) ◽  
pp. 9153
Author(s):  
Alida Amadeo ◽  
Sara Pizzi ◽  
Alessandro Comincini ◽  
Debora Modena ◽  
Alessandra Maria Calogero ◽  
...  
Keyword(s):  
Potential Role ◽  
Physiological Role ◽  
Direct Interaction ◽  
Substantia Nigra Pars Compacta ◽  
Proximity Ligation Assay ◽  
Proximity Ligation ◽  
Cytoskeletal Component ◽  
Presynaptic Boutons

α-synuclein is a small protein that is mainly expressed in the synaptic terminals of nervous tissue. Although its implication in neurodegeneration is well established, the physiological role of α-synuclein remains elusive. Given its involvement in the modulation of synaptic transmission and the emerging role of microtubules at the synapse, the current study aimed at investigating whether α-synuclein becomes involved with this cytoskeletal component at the presynapse. We first analyzed the expression of α-synuclein and its colocalization with α-tubulin in murine brain. Differences were found between cortical and striatal/midbrain areas, with substantia nigra pars compacta and corpus striatum showing the lowest levels of colocalization. Using a proximity ligation assay, we revealed the direct interaction of α-synuclein with α-tubulin in murine and in human brain. Finally, the previously unexplored interaction of the two proteins in vivo at the synapse was disclosed in murine striatal presynaptic boutons through multiple approaches, from confocal spinning disk to electron microscopy. Collectively, our data strongly suggest that the association with tubulin/microtubules might actually be an important physiological function for α-synuclein in the synapse, thus suggesting its potential role in a neuropathological context.

scholarly journals Protein flexibility is required for vesicle tethering at the Golgi

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Pak-yan Patricia Cheung ◽  
Charles Limouse ◽  
Hideo Mabuchi ◽  
Suzanne R Pfeffer
Keyword(s):  
Coiled Coil ◽  
Rab Gtpases ◽  
Force Microscopy ◽  
Vesicle Tethering ◽  
Proximity Ligation ◽  
Atomic Force ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Specific Sequences

The Golgi is decorated with coiled-coil proteins that may extend long distances to help vesicles find their targets. GCC185 is a trans Golgi-associated protein that captures vesicles inbound from late endosomes. Although predicted to be relatively rigid and highly extended, we show that flexibility in a central region is required for GCC185’s ability to function in a vesicle tethering cycle. Proximity ligation experiments show that that GCC185’s N-and C-termini are within <40 nm of each other on the Golgi. In physiological buffers without fixatives, atomic force microscopy reveals that GCC185 is shorter than predicted, and its flexibility is due to a central bubble that represents local unwinding of specific sequences. Moreover, 85% of the N-termini are splayed, and the splayed N-terminus can capture transport vesicles in vitro. These unexpected features support a model in which GCC185 collapses onto the Golgi surface, perhaps by binding to Rab GTPases, to mediate vesicle tethering.

Significance of the IL-3 Receptor Beta Chain (IL-3Rbc) for FLT3-ITD Dependent Oncogeneic Signaling in AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3660-3660
Author(s):  
Christoph Rummelt ◽  
Sivahari Prasad Gorantla ◽  
Philip Keye ◽  
Kathrin Klaesener ◽  
Venkatesh Rao ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Direct Interaction ◽  
Proximity Ligation Assay ◽  
Secondary Resistance ◽  
Knock Down ◽  
Proximity Ligation ◽  
Interaction Studies ◽  
Flt3 Mutations ◽  
Tki Resistance

Abstract Introduction Activating FLT3 mutations are found in 30% of AML patients. Internal tandem duplication (ITD) mutations are most common, and are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKI) were shown to be effective in clinical trials. However, complete remissions are rare, responses are short-lived, and the majority of patients display primary or secondary resistance to FLT3 inhibition. In FLT3 kinase inhibitor resistant, FLT3-ITD positive cell lines, we identified a direct interaction between FLT3-ITD and IL-3Rβc. We therefore sought to characterize this interaction and determine its' role in FLT3-ITD oncogenic signaling. Methods Drug sensitive BA/F3 FLT3-ITD cells and sublines resistant to FLT3 TKIs were subjected to analysis of activated IL-3Rβc - JAK1/2 - STAT signaling by PCR, Western-Blot (WB) and Immunoprecipitation (IP). Interaction studies were performed in vitro using IVT FLT3-ITD and GST IL-3Rβc, and in cell lines using γ2A cells transfected with human FLT3-ITD and human IL-3Rβc, human MOLM13 and MV4-11 AML cell lines, and primary AML patient samples by WB, IP and proximity ligation assay (PLA). Mapping experiments were performed in mouse embryonic fibroblasts (MEF) transduced with FLT3-ITD and flag-IL-3Rβc full-length or cytoplasmic Y to F mutant constructs. Knockdown-experiments were performed in BA/F3 cells with stably transfected FLT3-ITD and inducable IL-3Rβc shRNA. Results In TKI resistant FLT3-ITD positive cell-lines that did not harbor secondary FLT3 mutations inducing TKI resistance, we observed phosphorylation of IL-3Rβc in 5/16 resistant lines (30%) in the presence of FLT3 TKI. In these cells, IL-3Rβc phosphorylation was mediated by an activating JAK1 V658F mutation that bypasses FLT3-ITD dependent IL-3Rβc phosphorylation. Thus, IL-3Rβc in FLT3-ITD expressing cells mediates JAK1/2-dependent TKI resistance. Of note in inhibitor sensitive cells, IL-3Rβc interacted with and was phosphorylated by FLT3-ITD in a JAK1/2 independent manner, suggesting that IL-3Rβc participates in FLT3-ITD dependent oncogeneic signaling. Indeed, in IL-3Rβc and JAK2 deficient γ2A cells, expression of human FLT3-ITD was sufficient to induce interaction with and phosphorylation of human IL-3Rβc. In human FLT3-ITD-positive AML cell lines MOLM13 and MV4-11, IL-3Rβc phosphorylation occurred in a FLT3-ITD dependent fashion and IL-3Rβc interacted with FLT3-ITD. Proximity ligation assay (PLA) experiments detected FTL3-ITD and IL-3Rβc in close proximity suggesting a direct interaction of both proteins in MOLM13 and MV4-11 cells, as well as in primary cells from FLT3-ITD positive AML patients. Interaction studies revealed that binding to FLT3-ITD occurred independent of IL-3Rβc cytoplasmic tyrosines, and phosphorylation of IL-3Rβc by FLT3-ITD did not require the presence of IL-3Rαc. Preliminary results from knock down experiments in FLT3-ITD expressing BA/F3 cells indicated that knock-down of IL-3Rβc renders cells more susceptible to FLT3 TKI inhibition. Conclusion These data suggest an entirely novel model of FLT3-ITD "physiologically" employing IL-3Rβc as signaling intermediate, and IL-3Rβc serving as a signaling module mediating JAK1/2-dependent TKI resistance. These findings point toward the significance of IL-3Rβc for FLT3-ITD dependent transformation and treatment resistance, supporting the relevance of IL-3Rβc as a possible treatment target in FLT3-ITD positive AML. Disclosures von Bubnoff: Novartis: Research Funding; BMS: Speakers Bureau.

scholarly journals Proximity Ligation Assay Detection of Protein–DNA Interactions—Is There a Link between Heme Oxygenase-1 and G-quadruplexes?

Antioxidants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 94
Author(s):  
Wojciech Krzeptowski ◽  
Patryk Chudy ◽  
Grzegorz Sokołowski ◽  
Monika Żukowska ◽  
Anna Kusienicka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Heme Oxygenase ◽  
Direct Interaction ◽  
Proximity Ligation Assay ◽  
Limiting Factor ◽  
Heme Oxygenase 1 ◽  
Hematopoietic Stem ◽  
Proximity Ligation ◽  
Rare Cells ◽  
Induced Pluripotent

G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1−/− mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4–protein colocalizations can be quantitatively analyzed using PLA, even in rare cells.

Proximity Ligation Assay (PLA) Protocol Using Duolink® for T Cells

BIO-PROTOCOL ◽  
2016 ◽  
Vol 6 (10) ◽  
Author(s):  
Valentin Derangère ◽  
Mélanie Bruchard ◽  
Frédérique Végran ◽  
François Ghiringhelli
Keyword(s):  
T Cells ◽  
Proximity Ligation Assay ◽  
Proximity Ligation
Sign in / Sign up

Export Citation Format

Share Document