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.

scholarly journals Progesterone Regulation of Progesterone Receptor Membrane Component 1 (PGRMC1) Sumoylation and Transcriptional Activity in Spontaneously Immortalized Granulosa Cells

Endocrinology ◽  
2012 ◽  
Vol 153 (8) ◽  
pp. 3929-3939 ◽  
Author(s):  
John J. Peluso ◽  
Valentina Lodde ◽  
Xiufang Liu
Keyword(s):  
Molecular Mass ◽  
Granulosa Cells ◽  
Cell Function ◽  
Direct Interaction ◽  
Membrane Component ◽  
T Cell Factor ◽  
Proximity Ligation ◽  
Receptor Membrane ◽  
Interfering Rna

Progesterone (P4) receptor membrane component (PGRMC)1 is detected as a 22-kDa band as well as higher molecular mass bands (>50 kDa) in spontaneously immortalized granulosa cells. That these higher molecular mass bands represent PGRMC1 is supported by the findings that they are not detected when either the primary antibody is omitted or the PGRMC1 antibody is preabsorbed with recombinant PGRMC1. Some but not most of the higher molecular mass bands are due to oligomerization. At least one of the higher molecular mass bands is sumoylated, because PGRMC1 coimmunoprecipitates with small ubiquitin-like modifier protein-1. Moreover, in situ proximity ligation assays reveal a direct interaction between PGRMC1 and small ubiquitin-like modifier protein-1. This interaction is increased by P4. Finally, the higher molecular mass forms of PGRMC1 localize to the nucleus. An analysis of transcription factor activity demonstrates that P4 suppresses T-cell factor/lymphoid enhancer factor (Tcf/Lef) activity through a PGRMC1-dependent mechanism, because treatment with PGRMC1 small interfering RNA depletes PGRMC1 levels and attenuates P4's effects on Tcf/Lef activity. In addition, transfection of a PGRMC1-Flag fusion protein enhances basal Tcf/Lef activity, which is suppressed by P4 treatment. Conversely, transfection of a PGRMC1-Flag protein in which all the sumoylation sites are mutated increases basal Tcf/Lef activity but attenuates P4's ability to suppress Tcf/Lef activity. Therefore, the ability to suppress Tcf/Lef activity is likely an essential part of the mechanism through which P4 activation of PGRMC1 regulates the gene cascades that control granulosa cell function with this action being dependent in part on the sumoylation status of PGRMC1.

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.

Selecting the appropriate in situ proximity ligation assay protocol

2017 ◽  
pp. 119-132
Author(s):  
Doroteya Raykova ◽  
Linda Arngården ◽  
Axel Klaesson ◽  
Johan Heldin ◽  
Ola Söderberg
Keyword(s):  
Proximity Ligation Assay ◽  
Proximity Ligation ◽  
Assay Protocol

scholarly journals Direct intracellular visualization of Ebola virus-receptor interaction by in situ proximity ligation

2020 ◽  
Author(s):  
Eva Mittler ◽  
Tanwee P Alkutkar ◽  
Rohit K Jangra ◽  
Kartik Chandran
Keyword(s):  
Receptor Binding ◽  
Ebola Virus ◽  
Host Cells ◽  
Proximity Ligation Assay ◽  
Viral Glycoprotein ◽  
Virus Receptor ◽  
Proximity Ligation ◽  
Infected Cells ◽  
Viral Surface

ABSTRACTEbola virus (EBOV) entry into host cells comprises stepwise and extensive interactions of the sole viral surface glycoprotein GP with multiple host factors. During the intricate process, following virus uptake and trafficking to late endosomal/lysosomal compartments, GP is proteolytically processed to GPCL by the endosomal proteases cathepsin B and L unmasking GP’s receptor-binding site. Engagement of GPCL with the universal filoviral intracellular receptor Niemann-Pick C1 (NPC1) eventually culminates in fusion between viral and cellular membranes, cytoplasmic escape of the viral nucleocapsid and subsequent infection. Mechanistic delineation of the indispensable GPCL:NPC1 binding step has been severely hampered by the unavailability of a robust cell-based assay assessing interaction of GPCL with full-length endosomal NPC1.Here, we describe a novel in situ assay to monitor GPCL:NPC1 engagement in intact, infected cells. Visualization of the subcellular localization of binding complexes is based on the principle of DNA-assisted, antibody-mediated proximity ligation. Virus-receptor binding monitored by proximity ligation was contingent on GP’s proteolytic cleavage, and was sensitive to perturbations in the GPCL:NPC1 interface. Our assay also specifically decoupled detection of virus-receptor binding from steps post-receptor binding, such as membrane fusion and infection. Testing of multiple FDA-approved small molecule inhibitors revealed that drug treatments inhibited virus entry and GPCL:NPC1 recognition by distinctive mechanisms. Together, here we present a newly established proximity ligation assay, which will allow us to dissect cellular and viral requirements for filovirus-receptor binding, and to delineate the mechanisms of action of inhibitors on filovirus entry in a cell-based system.IMPORTANCEEbola virus causes episodic but increasingly frequent outbreaks of severe disease in Middle Africa, as shown by a currently ongoing outbreak in the Democratic Republic of Congo. Despite considerable effort, FDA-approved anti-filoviral therapeutics or targeted interventions are not available yet. Virus host-cell invasion represents an attractive target for antivirals; however our understanding of the inhibitory mechanisms of novel therapeutics is often hampered by fragmented knowledge of the filovirus-host molecular interactions required for viral infection. To help close this critical knowledge gap, here, we report an in situ assay to monitor binding of the EBOV glycoprotein to its receptor NPC1 in intact, infected cells. We demonstrate that our in situ assay based on proximity ligation represents a powerful tool to delineate receptor-viral glycoprotein interactions. Similar assays can be utilized to examine receptor interactions of diverse viral surface proteins whose studies have been hampered until now by the lack of robust in situ assays.

scholarly journals Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction

2021 ◽  
Vol 9 ◽  
Author(s):  
Sara Benhammouda ◽  
Anjali Vishwakarma ◽  
Priya Gatti ◽  
Marc Germain
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescence Probes ◽  
Proximity Ligation Assay ◽  
Proximity Ligation ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Endogenous Proteins ◽  
Underlying Mechanisms

Organelles cooperate with each other to regulate vital cellular homoeostatic functions. This occurs through the formation of close connections through membrane contact sites. Mitochondria-Endoplasmic-Reticulum (ER) contact sites (MERCS) are one of such contact sites that regulate numerous biological processes by controlling calcium and metabolic homeostasis. However, the extent to which contact sites shape cellular biology and the underlying mechanisms remain to be fully elucidated. A number of biochemical and imaging approaches have been established to address these questions, resulting in the identification of a number of molecular tethers between mitochondria and the ER. Among these techniques, fluorescence-based imaging is widely used, including analysing signal overlap between two organelles and more selective techniques such as in-situ proximity ligation assay (PLA). While these two techniques allow the detection of endogenous proteins, preventing some problems associated with techniques relying on overexpression (FRET, split fluorescence probes), they come with their own issues. In addition, proper image analysis is required to minimise potential artefacts associated with these methods. In this review, we discuss the protocols and outline the limitations of fluorescence-based approaches used to assess MERCs using endogenous proteins.

scholarly journals Metformin Reverses the Enhanced Myocardial SR/ER–Mitochondria Interaction and Impaired Complex I-Driven Respiration in Dystrophin-Deficient Mice

2021 ◽  
Vol 8 ◽  
Author(s):  
Claire Angebault ◽  
Mathieu Panel ◽  
Mathilde Lacôte ◽  
Jennifer Rieusset ◽  
Alain Lacampagne ◽  
...  
Keyword(s):  
Complex I ◽  
Early Stage ◽  
Intrinsic Activity ◽  
Proximity Ligation Assay ◽  
Proximity Ligation ◽  
Skeletal Muscle Dysfunction ◽  
Species Production ◽  
Therapeutic Perspective ◽  
Deficient Mice

Besides skeletal muscle dysfunction, Duchenne muscular dystrophy (DMD) exhibits a progressive cardiomyopathy characterized by an impaired calcium (Ca2+) homeostasis and a mitochondrial dysfunction. Here we aimed to determine whether sarco-endoplasmic reticulum (SR/ER)–mitochondria interactions and mitochondrial function were impaired in dystrophic heart at the early stage of the pathology. For this purpose, ventricular cardiomyocytes and mitochondria were isolated from 3-month-old dystrophin-deficient mice (mdx mice). The number of contacts points between the SR/ER Ca2+ release channels (IP3R1) and the porine of the outer membrane of the mitochondria, VDAC1, measured using in situ proximity ligation assay, was greater in mdx cardiomyocytes. Expression levels of IP3R1 as well as the mitochondrial Ca2+ uniporter (MCU) and its regulated subunit, MICU1, were also increased in mdx heart. MICU2 expression was however unchanged. Furthermore, the mitochondrial Ca2+ uptake kinetics and the mitochondrial Ca2+ content were significantly increased. Meanwhile, the Ca2+-dependent pyruvate dehydrogenase phosphorylation was reduced, and its activity significantly increased. In Ca2+-free conditions, pyruvate-driven complex I respiration was decreased whereas in the presence of Ca2+, complex I-mediated respiration was boosted. Further, impaired complex I-mediated respiration was independent of its intrinsic activity or expression, which remains unchanged but is accompanied by an increase in mitochondrial reactive oxygen species production. Finally, mdx mice were treated with the complex I modulator metformin for 1 month. Metformin normalized the SR/ER-mitochondria interaction, decreased MICU1 expression and mitochondrial Ca2+ content, and enhanced complex I-driven respiration. In summary, before any sign of dilated cardiomyopathy, the DMD heart displays an aberrant SR/ER-mitochondria coupling with an increase mitochondrial Ca2+ homeostasis and a complex I dysfunction. Such remodeling could be reversed by metformin providing a novel therapeutic perspective in DMD.

Sign in / Sign up

Export Citation Format

Share Document