Deformation of caveolae impacts global transcription and translation processes through relocalization of cavin-1

Caveolae are invaginated membrane domains that provide mechanical strength to cells in addition to being focal points for the localization of signaling molecules. Caveolae are formed through the aggregation of caveolin-1 or -3 (Cav1/3), membrane proteins that assemble into multifunctional complexes with the help of caveola-associated protein cavin-1. In addition to its role in the formation of caveolae, cavin-1, also called polymerase I and transcript release factor, is further known to promote ribosomal RNA transcription in the nucleus. However, the mechanistic link between these functions is not clear. Here, we found that deforming caveolae by subjecting cells to mild osmotic stress (150–300 mOsm) changes levels of GAPDH, Hsp90, and Ras only when Cav1/cavin-1 levels are reduced, suggesting a link between caveola deformation and global protein expression. We show that this link may be due to relocalization of cavin-1 to the nucleus upon caveola deformation. Cavin-1 relocalization is also seen when Cav1-Gαq contacts change upon stimulation. Furthermore, Cav1 and cavin-1 levels have been shown to have profound effects on cytosolic RNA levels, which in turn impact the ability of cells to form stress granules and RNA-processing bodies (p-bodies) which sequester and degrade mRNAs, respectively. Our studies here using a cavin-1-knockout cell line indicate adaptive changes in cytosolic RNA levels but a reduced ability to form stress granules. Taken together, our findings suggest that caveolae, through release of cavin-1, communicate extracellular cues to the cell interior to impact transcriptional and translational. Caveolae are invaginated membrane domains that provide mechanical strength to cells in addition to being focal points for the localization of signaling molecules. Caveolae are formed through the aggregation of caveolin-1 or -3 (Cav1/3), membrane proteins that assemble into multifunctional complexes with the help of caveola-associated protein cavin-1. In addition to its role in the formation of caveolae, cavin-1, also called polymerase I and transcript release factor, is further known to promote ribosomal RNA transcription in the nucleus. However, the mechanistic link between these functions is not clear. Here, we found that deforming caveolae by subjecting cells to mild osmotic stress (150–300 mOsm) changes levels of GAPDH, Hsp90, and Ras only when Cav1/cavin-1 levels are reduced, suggesting a link between caveola deformation and global protein expression. We show that this link may be due to relocalization of cavin-1 to the nucleus upon caveola deformation. Cavin-1 relocalization is also seen when Cav1-Gαq contacts change upon stimulation. Furthermore, Cav1 and cavin-1 levels have been shown to have profound effects on cytosolic RNA levels, which in turn impact the ability of cells to form stress granules and RNA-processing bodies (p-bodies) which sequester and degrade mRNAs, respectively. Our studies here using a cavin-1-knockout cell line indicate adaptive changes in cytosolic RNA levels but a reduced ability to form stress granules. Taken together, our findings suggest that caveolae, through release of cavin-1, communicate extracellular cues to the cell interior to impact transcriptional and translational. Caveolae are flask-shaped membrane invaginations that can flatten to provide more membrane area and are implicated in mechanosensation, electric sensation, endocytosis, and vasodilation through modulating the NO pathway (1Parton R.G. Caveolae: structure, function, and relationship to disease.Annu. Rev. Cell Dev. Biol. 2018; 34: 111-136Crossref PubMed Scopus (123) Google Scholar, 2Bernatchez P. Sharma A. Bauer P.M. Marin E. Sessa W.C. A noninhibitory mutant of the caveolin-1 scaffolding domain enhances eNOS-derived NO synthesis and vasodilation in mice.J. Clin. Invest. 2011; 121: 3747-3755Crossref PubMed Scopus (95) Google Scholar, 3Parton R.G. Tillu V.A. Collins B.M. Caveolae.Curr. Biol. 2018; 28: R402-R405Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 4Buwa N. Mazumdar D. Balasubramanian N. Caveolin1 tyrosine-14 phosphorylation: role in cellular responsiveness to mechanical cues.J. Membr. Biol. 2020; 253: 509-534Crossref PubMed Scopus (7) Google Scholar). In previous studies, our lab found that populations of Gαq and their receptors reside in caveola domains and this localization is assisted by interactions between Gαq and caveolin molecules (5Sengupta P. Philip F. Scarlata S. Caveolin-1 alters Ca2+ signal duration through specific interaction with the G{alpha}q family of G proteins.J. Cell Sci. 2008; 121: 1363-1372Crossref PubMed Scopus (34) Google Scholar, 6Calizo R.C. Scarlata S. A role for G-proteins in directing G-protein-coupled receptor–caveolae localization.Biochemistry. 2012; 51: 9513-9523Crossref PubMed Scopus (25) Google Scholar). Activation of Gαq by hormones or neurotransmitters strengthens these interactions resulting in enhancement of calcium signals. Deformation of caveolae by mild osmotic stress disrupts this stabilization and returns calcium signals to levels observed in the absence of caveolae (7Guo Y. Golebiewska U. Scarlata S. Modulation of Ca2+ activity in cardiomyocytes through caveolae-G[alpha]q interactions.Biophys. J. 2011; 100: 1599-1607Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 8Guo Y. Yang L. Haught K. Scarlata S. Osmotic stress reduces Ca2+ signals through deformation of caveolae.J. Biol. Chem. 2015; 290: 16698-16707Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 9Yang L. Scarlata S. Super-resolution visualization of caveola deformation in response to osmotic stress.J. Biol. Chem. 2017; 292: 3779-3788Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Additionally, when cells are subjected to either bidirectional static or oscillating mechanical stretch, calcium release through activation of Gαq/PLCβ is intact, but contacts between Gαq and caveolin are disrupted (10Aisiku O. Dowal L. Scarlata S. Protein kinase C phosphorylation of PLC[beta]1 regulates its cellular localization.Arch. Biochem. Biophys. 2011; 509: 186-190Crossref PubMed Scopus (24) Google Scholar, 11Qifti A. Garwain O. Scarlata S. Mechanical stretch redefines membrane galphaq-calcium signaling complexes.J. Membr. Biol. 2019; 252: 307-315Crossref PubMed Scopus (7) Google Scholar). In separate series of studies, our lab found that activation of the Gαq/PLCβ triggers a novel calcium-independent pathway that is linked to regulation of GAPDH protein production but not Hsp90 (12Philip F. Guo Y. Aisiku O. Scarlata S. Phospholipase Cβ1 is linked to RNA interference of specific genes through translin-associated factor X.FASEB J. 2012; 26: 4903-4913Crossref PubMed Scopus (22) Google Scholar). Cavins are a family of four proteins that regulate the curvature of the caveola membrane by anchoring caveolins to the cytoskeleton (for reviews, refer to the studies by Briand et al (13Briand N. Dugail I. Le Lay S. Cavin proteins: new players in the caveolae field.Biochimie. 2011; 93: 71-77Crossref PubMed Scopus (79) Google Scholar), Kovtun et al (14Kovtun O. Tillu V.A. Ariotti N. Parton R.G. Collins B.M. Cavin family proteins and the assembly of caveolae.J. Cell Sci. 2015; 128: 1269-1278Crossref PubMed Scopus (137) Google Scholar), and Williams and Palmer (15Williams J.J. Palmer T.M. Cavin-1: caveolae-dependent signalling and cardiovascular disease.Biochem. Soc. Trans. 2014; 42: 284-288Crossref PubMed Scopus (19) Google Scholar)). The most abundantly expressed is cavin-1, also known as polymerase 1 and transcript release factor or cav-p60. Since its discovery in 1998, cavin-1 has been found to be a necessary component of caveola formation by mediating the sequestration of caveolin molecules into immobile caveola domains (refer to the study by Briand et al (13Briand N. Dugail I. Le Lay S. Cavin proteins: new players in the caveolae field.Biochimie. 2011; 93: 71-77Crossref PubMed Scopus (79) Google Scholar)). Several studies, including those here, strongly suggest that expression of cavin-1 and caveolin are interdependent; Cav1-knockout (KO) mice have nearly no cavin-1 expression, and cavin-1 KO mice have diminished Cav1 expression (15Williams J.J. Palmer T.M. Cavin-1: caveolae-dependent signalling and cardiovascular disease.Biochem. Soc. Trans. 2014; 42: 284-288Crossref PubMed Scopus (19) Google Scholar, 16Liu L. Pilch P.F. A critical role of cavin (polymerase I and transcript release factor) in caveolae formation and organization.J. Biol. Chem. 2008; 283: 4314-4322Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). When fibroblasts are swelled by a 10-fold decrease in osmotic strength, cavin-1 is released from the plasma membrane as caveolae disassemble to provide more membrane area (17Sinha B. Köster D. Ruez R. Gonnord P. Bastiani M. Abankwa D. et al.Cells respond to mechanical stress by rapid disassembly of caveolae.Cell. 2011; 144: 402-413Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). Before cavin-1 was identified as a structural adapter for caveolae, it was recognized for its role in modulating cellular transcriptional activity (18Jansa P. Mason S.W. Hoffmann-Rohrer U. Grummt I. Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary mortality complexes.EMBO J. 1998; 17: 2855-2864Crossref PubMed Scopus (120) Google Scholar). Cavin-1/polymerase 1 and transcript release factor promotes ribosomal DNA transcription by binding to the 3′ pre-RNA, allowing the release of pre-RNA and Polymerase I from the transcription complex (19Liu L. Pilch P.F. PTRF/Cavin-1 promotes efficient ribosomal RNA transcription in response to metabolic challenges.Elife. 2016; 5e17508Crossref Google Scholar). Cavin-1 not only plays a role in transcript release but also increases the overall rate of transcription in a concentration-dependent manner. In adipocytes, insulin stimulation causes phosphorylation of cavin-1 promoting its translocation from caveolae to the nucleus (19Liu L. Pilch P.F. PTRF/Cavin-1 promotes efficient ribosomal RNA transcription in response to metabolic challenges.Elife. 2016; 5e17508Crossref Google Scholar) suggesting a role in signal transduction. The importance of cavin-1 expression is seen in KO mice that show diverse abnormalities consistent with impaired ribosome biogenesis including abnormal growth failure, loss in fat, resistance to obesity, impaired exercise ability, muscle hypertrophy, altered cardiac, and lung function (20Liu L. Lessons from cavin-1 deficiency.Biochem. Soc. Trans. 2020; 48: 147-154Crossref PubMed Scopus (11) Google Scholar). Pertinent for this study are reports suggesting that increased cavin-1 expression promotes cellular stress responses to toxic agents which may be traced to binding to p53 in the cytosol (21Bai L. Deng X. Li J. Wang M. Li Q. An W. et al.Regulation of cellular senescence by the essential caveolar component PTRF/Cavin-1.Cell Res. 2011; 21: 1088-1101Crossref PubMed Scopus (51) Google Scholar). The connection between cavin-1’s ability to regulate caveola structure and promote ribosomal RNA production suggests that any mechanism that destabilizes cavin-1’s interactions with caveolin including caveola deformation would impact transcription and transitional processes through cavin-1. Here, we show that changes in environmental conditions, such as mild osmotic stress, addition of neurotransmitter, or exposure toxins, drive cavin-1 from the plasma membrane to impact RNA transcription and two processes that regulate protein translation (i.e., stress granule and p-body formation). Our results suggest that cavin-1 molecules of caveolae act as sensors to inform the cell interior of environmental stress. We have previously found that the binding of cytosolic PLCβ to C3PO, the promoter of the RNA-induced silencing complex (RISC), inhibits its activity to regulate the silencing of select genes and that this effect is reversed upon Gαq activation (12Philip F. Guo Y. Aisiku O. Scarlata S. Phospholipase Cβ1 is linked to RNA interference of specific genes through translin-associated factor X.FASEB J. 2012; 26: 4903-4913Crossref PubMed Scopus (22) Google Scholar, 22Philip F. Sahu S. Golebiewska U. Scarlata S. RNA-induced silencing attenuates G protein-mediated calcium signals.FASEB J. 2016; 30: 1958-1967Crossref PubMed Scopus (11) Google Scholar, 23Sahu S. Philip F. Scarlata S. Hydrolysis rates of different small interfering RNAs (siRNAs) by the RNA silencing promoter complex, C3PO, determines their regulation by phospholipase cβ.J. Biol. Chem. 2014; 289: 5134-5144Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 24Sahu S. Williams L. Perez A. Philip F. Caso G. Zurawsky W. et al.Regulation of the activity of the promoter of RNA-induced silencing, C3PO.Protein Sci. 2017; 26: 1807-1818Crossref PubMed Scopus (8) Google Scholar). Specifically, we found that activation of Gαq drives cytosolic PLCβ to the plasma membrane releasing inhibition of the promoter of RISC and reversing silencing of GAPDH by siRNA, but not Hsp90. We tested the idea that because the caveola enhances activation of Gαq (5Sengupta P. Philip F. Scarlata S. Caveolin-1 alters Ca2+ signal duration through specific interaction with the G{alpha}q family of G proteins.J. Cell Sci. 2008; 121: 1363-1372Crossref PubMed Scopus (34) Google Scholar), which shifts the cytosolic population of PLCβ to the plasma membrane, then caveola expression could indirectly regulate GAPDH production. Using rat aortic smooth muscle (A10) cells, we quantified the production of GAPDH, along with Hsp90 and Ras for comparison, when Cav1, Gαq, and PLCβ were downregulated. We first found that reducing Cav1 changes the level of actin and reduces the level of other cellular proteins (discussed in the later part of the article), making direct impact of Cav1 levels difficult to assess. We therefore took a more indirect approach. Because caveola provides mechanical strength to cells, we subjected cells to mild osmotic stress that will deform caveola and eliminate its stabilization of the Gαq/PLCβ pathways (8Guo Y. Yang L. Haught K. Scarlata S. Osmotic stress reduces Ca2+ signals through deformation of caveolae.J. Biol. Chem. 2015; 290: 16698-16707Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). We reduced the osmolarity of the media from 300 to 150 mOsm in control cells and cells treated with siRNA(Cav1) and quantified changes in GAPDH, Hsp90, and Ras levels (Fig. 1, where samples blots are shown in Fig. S1). Differences between the two cell groups were not immediate but appeared ∼12 h after continuous osmotic stress, suggesting that Cav1 impacts slower transcription and/or translation processes rather than more rapid degradation or other downregulation mechanisms. In contrast to Cav1, downregulating PLCβ1 or Gαq had little or no effect, arguing that the Gαq/PLCβ pathway is not the primary factor underlying changes in protein production. These data suggest that Cav1 levels impact the ability of cells to produce proteins under hypo-osmotic stress conditions. Expression of Cav1 and cavin-1 are interdependent ((16Liu L. Pilch P.F. A critical role of cavin (polymerase I and transcript release factor) in caveolae formation and organization.J. Biol. Chem. 2008; 283: 4314-4322Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) and Fig. S2A), and so we tested whether the protein changes observed in Figure 1 might be due to cavin-1 levels since cavin-1 promotes transcription of ribosomal RNA (18Jansa P. Mason S.W. Hoffmann-Rohrer U. Grummt I. Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary mortality complexes.EMBO J. 1998; 17: 2855-2864Crossref PubMed Scopus (120) Google Scholar). While previous studies found that a tenfold change in osmotic stress disassembles caveolae releasing cavin-1 from the plasma membrane (17Sinha B. Köster D. Ruez R. Gonnord P. Bastiani M. Abankwa D. et al.Cells respond to mechanical stress by rapid disassembly of caveolae.Cell. 2011; 144: 402-413Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar), it is unclear whether release will occur at the mild, physiological stress conditions used here which deforms but not disassembles caveolae (9Yang L. Scarlata S. Super-resolution visualization of caveola deformation in response to osmotic stress.J. Biol. Chem. 2017; 292: 3779-3788Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Cavin-1 relocalization studies were carried out in intact Wistar Kyoto smooth muscle cells (WKO-3M22) which are flat and easily imaged. Cells were transfected with eGFP-cavin-1, and shifts in the cellular distribution of fluorescence intensity with hypo-osmotic stress by confocal were measured. Under basal conditions, we find that eGFP-cavin-1 localizes on the plasma membrane with small amounts in the cytoplasm and nucleus (Fig. 2A). Because the additional, transfected protein would increase total cellular cavin-1 and may effect localization, we repeated these studies by first downregulating cavin-1 by ∼95% before transfecting with eGFP-cavin-1 (see Fig. S2, A and B). We then quantified the eGFP-cavin-1 fluorescence intensity on or close to the plasma membrane, the cytosol, and the nucleus (see Experimental procedures). In this case, the observations were similar with the intensity being slightly lower on the plasma membrane than on the nucleus, and no intensity could be detected in the cytosol, suggesting that endogenous cavin-1 is mainly distributed between the plasma membrane and nuclear compartments (see Fig. S2B).Figure 2Cavin-1 relocalization in WKO-3M22 cells. A, sample fluorescence images of fixed WKO-3M22 cells transfected with eGFP-Cavin-1 (green) and stained with CellMask Deep Red Plasma Membrane stain (red) and DAPI (blue) under control conditions as obtained at 60X magnification and focusing on the bottom of the cell. Scale bars are 20 μm long. B and C, cell localization of eGFP-cavin-1 was assessed in cells subjected to hypo-osmotic (orange), arsenite (purple), Gαq stimulation with carbachol (pink), bradykinin stimulation (green), and isoproterenol treatment (yellow). eGFP-cavin-1 localization was determined by measuring the pixel intensities to coordinates to the plasma membrane, cytosolic, and nucleus compartments based on the location of DAPI and the CellMask plasma membrane stain (see Experimental procedures). Changes in membrane (B) and nuclear localization (C) in wildtype cells. Intensities were normalized to the plasma membrane control. The asterisks correspond to p values, with one asterisk being 0.1