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Automated Detection and Quantification of Granular Cell Compartments

Published online by Cambridge University Press:  03 April 2013

Hicham Mahboubi ,
Mohamed Kodiha  and
Ursula Stochaj
Hicham Mahboubi
Affiliation:
Department of Physiology, McGill University, Montreal H3G 1Y6, Quebec, Canada
Mohamed Kodiha
Affiliation:
Department of Physiology, McGill University, Montreal H3G 1Y6, Quebec, Canada
Ursula Stochaj*
Affiliation:
Department of Physiology, McGill University, Montreal H3G 1Y6, Quebec, Canada
*
*Corresponding author. E-mail: [email protected]
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Abstract

Many cellular processes are organized in a compartmentalized and dynamic fashion to ensure effective adaptation to physiological changes. Thus, in response to stress and disease, cells initiate protective mechanisms to restore homeostasis. Among these mechanisms are the arrest of translation and remodeling of ribonucleoprotein complexes into granular compartments in the cytoplasm, known as stress granules (SGs). To date, the analysis of SGs has relied on the manual demarcation and measurement of the compartment, making quantitative studies time-consuming, while preventing the efficient use of high-throughput technology. We developed the first fully automated, computer-based procedures that measure the association of fluorescent molecules with granular compartments. Our methods quantify automatically multiple granule parameters and generate data at the level of single cells or individual SGs. These techniques detect simultaneously in an automated fashion proteins and RNAs located in SGs. The effectiveness of our protocols is demonstrated by studies that reveal several of the unique biological and structural characteristics of SGs. In particular, we show that the type of stress determines granule size and composition, as illustrated by the concentration of poly(A)-RNA and a specific SG marker protein. Furthermore, we took advantage of the computer-based and automated methods to design assays suitable for high-throughput screening.

Keywords

automated compartment detectionfluorescence quantificationhigh-throughput screening assaystress granulepoly(A)-RNA
Type
Equipment and Techniques Development: Biological
Information
Microscopy and Microanalysis , Volume 19 , Issue 3 , June 2013 , pp. 617 - 628
Copyright
Copyright © Microscopy Society of America 2013 

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Footnotes

†

Both authors contributed equally to the article.

References

Anderson, P. & Kedersha, N. (2002). Stressful initiations. J Cell Sci 115(Pt 16), 3227–3234.CrossRefGoogle ScholarPubMed
Anderson, P. & Kedersha, N. (2009). RNA granules: Post-transcriptional and epigenetic modulators of gene expression. Nat Rev Mol Cell Biol 10(6), 430–436.Google Scholar
Baguet, A., Degot, S., Cougot, N., Bertrand, E., Chenard, M.P., Wendling, C., Kessler, P., Le Hir, H., Rio, M.C. & Tomasetto, C. (2007). The exon-junction-complex-component metastatic lymph node 51 functions in stress-granule assembly. J Cell Sci 120(Pt 16), 2774–2784.CrossRefGoogle ScholarPubMed
Balagopal, V. & Parker, R. (2009). Polysomes, P bodies and stress granules: States and fates of eukaryotic mRNAs. Curr Opin Cell Biol 21(3), 403–408.Google Scholar
Buchan, J.R., Nissan, T. & Parker, R. (2010). Analyzing P-bodies and stress granules in Saccharomyces cerevisiae . Methods Enzymol 470, 619–640.CrossRefGoogle ScholarPubMed
Buchan, J.R. & Parker, R. (2009). Eukaryotic stress granules: The ins and outs of translation. Mol Cell 36(6), 932–941.CrossRefGoogle ScholarPubMed
Dang, Y., Kedersha, N., Low, W.-K., Romo, D., Gorospe, M., Kaufman, R., Anderson, P. & Liu, J.O. (2006). Eukaryotic initiation factor 2alpha-independent pathway of stress granule induction by the natural product pateamine A. J Biol Chem 281(43), 32870–32878.CrossRefGoogle ScholarPubMed
Didiot, M.-C., Subramanian, M., Flatter, E., Mandel, J.-L. & Moine, H. (2009). Cells lacking the fragile X mental retardation protein (FMRP) have normal RISC activity but exhibit altered stress granule assembly. Mol Biol Cell 20(1), 428–437.Google Scholar
Elvira, G., Wasiak, S., Blandford, V., Tong, X.K., Serrano, A., Fan, X., del Rayo Sanchez-Carbente, M., Servant, F., Bell, A.W., Boismenu, D., Lacaille, J.C., McPherson, P.S., DesGroseillers, L., Sossin, W.S., Elvira, G., Wasiak, S., Blandford, V., Tong, X.-K., Serrano, A., Fan, X., del Rayo Sanchez-Carbente, M., Servant, F., Bell, A.W., Boismenu, D., Lacaille, J.-C., McPherson, P.S., DesGroseillers, L. & Sossin, W.S. (2006). Characterization of an RNA granule from developing brain. Mol Cell Prot 5(4), 635–651.Google Scholar
Eulalio, A., Frohlich, K.S., Mano, M., Giacca, M. & Vogel, J. (2011). A candidate approach implicates the secreted salmonella effector protein SpvB in P-body disassembly. PLoS One 6(3), e17296. Google Scholar
Fujimura, K., Suzuki, T., Yasuda, Y., Murata, M., Katahira, J. & Yoneda, Y. (2010). Identification of importin alpha1 as a novel constituent of RNA stress granules. Biochim Biophys Acta 1803(7), 865–871.CrossRefGoogle ScholarPubMed
Gallouzi, I.-E., Brennan, C.M., Stenberg, M.G., Swanson, M.S., Eversole, A., Maizels, N. & Steitz, J.A. (2000). HuR binding to cytoplasmic mRNA is perturbed by heat shock. Proc Natl Acad Sci USA 97 (7), 3073–3078.Google Scholar
Gilks, N., Kedersha, N., Ayodele, M., Shen, L., Stoecklin, G., Dember, L.M. & Anderson, P. (2004). Stress granule assembly is mediated by prion-like aggregation of TIA-1. Mol Biol Cell 15(12), 5383–5398.CrossRefGoogle ScholarPubMed
Kayali, F., Montie, H.L., Rafols, J.A. & DeGracia, D.J. (2005). Prolonged translation arrest in reperfused hippocampal cornu Ammonis 1 is mediated by stress granules. Neuroscience 134(4), 1223–1245.Google Scholar
Kedersha, N. & Anderson, P. (2002). Stress granules: Sites of mRNA triage that regulate mRNA stability and translatability. Biochem Soc Trans 30(Pt 6), 963–969.CrossRefGoogle ScholarPubMed
Kedersha, N. & Anderson, P. (2007). Mammalian stress granules and processing bodies. Method Enzymol 431, 61–81.CrossRefGoogle ScholarPubMed
Kedersha, N. & Anderson, P. (2009). Regulation of translation by stress granules and processing bodies. Prog Mol Biol Trans Sci 90, 155–185.Google Scholar
Kedersha, N., Stoecklin, G., Ayodele, M., Yacono, P., Lykke-Andersen, J., Fritzler, M.J., Scheuner, D., Kaufman, R.J., Golan, D.E., Anderson, P., Kedersha, N., Stoecklin, G., Ayodele, M., Yacono, P., Lykke-Andersen, J., Fritzler, M.J., Scheuner, D., Kaufman, R.J., Golan, D.E. & Anderson, P. (2005). Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J Cell Biol 169(6), 871–884.Google Scholar
Kedersha, N.L., Gupta, M., Li, W., Miller, I. & Anderson, P. (1999). RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules. J Cell Biol 147(7), 1431–1442.Google Scholar
Kim, B., Cooke, H.J. & Rhee, K. (2012). DAZL is essential for stress granule formation implicated in germ cell survival upon heat stress. Development 139(3), 568–578.Google Scholar
Kimball, S.R., Horetsky, R.L., Ron, D., Jefferson, L.S. & Harding, H.P. (2003). Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes. Am J Physiol Cell Physiol 284(2), C273–C284.CrossRefGoogle ScholarPubMed
Kodiha, M., Banski, P. & Stochaj, U. (2009a). Interplay between MEK and PI3 kinase signaling regulates the subcellular localization of protein kinases ERK1/2 and Akt upon oxidative stress. FEBS Lett 583(12), 1987–1993.Google Scholar
Kodiha, M., Banski, P. & Stochaj, U. (2011). Computer-based fluorescence quantification: A novel approach to study nucleolar biology. BMC Cell Biol 12(1), 25.Google Scholar
Kodiha, M., Brown, C.M. & Stochaj, U. (2008a). Analysis of signaling events by combining high-throughput screening technology with computer-based image analysis. Sci Signal 1(37), pl2. Google Scholar
Kodiha, M., Tran, D., Morogan, A., Qian, C. & Stochaj, U. (2009b). Dissecting the signaling events that impact classical nuclear import and target nuclear transport factors. PLoS One 4(12), e8420. Google Scholar
Kodiha, M., Tran, D., Qian, C., Morogan, A., Presley, J.F., Brown, C.M. & Stochaj, U. (2008b). Oxidative stress mislocalizes and retains transport factor importin-alpha and nucleoporins Nup153 and Nup88 in nuclei where they generate high molecular mass complexes. Biochim Biophys Acta 1783(3), 405–418.Google Scholar
Lachke, S.A., Alkuraya, F.S., Kneeland, S.C., Ohn, T., Aboukhalil, A., Howell, G.R., Saadi, I., Cavallesco, R., Yue, Y., Tsai, A.C.H., Nair, K.S., Cosma, M.I., Smith, R.S., Hodges, E., Alfadhli, S.M., Al-Hajeri, A., Shamseldin, H.E., Behbehani, A., Hannon, G.J., Bulyk, M.L., Drack, A.V., Anderson, P.J., John, S.W.M. & Maas, R.L. (2011). Mutations in the RNA granule component TDRD7 cause cataract and glaucoma. Science 331(6024), 1571–1576.CrossRefGoogle ScholarPubMed
Mazroui, R., Sukarieh, R., Bordeleau, M.-E., Kaufman, R.J., Northcote, P., Tanaka, J., Gallouzi, I. & Pelletier, J. (2006). Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation. Mol Biol Cell 17(10), 4212–4219.Google Scholar
McDonald, K.K., Aulas, A., Destroismaisons, L., Pickles, S., Beleac, E., Camu, W., Rouleau, G.A. & Vande Velde, C. (2011). TAR DNA-binding protein 43 (TDP-43) regulates stress granule dynamics via differential regulation of G3BP and TIA-1. Human Mol Gen 20(7), 1400–1410.Google Scholar
McInerney, G.M., Kedersha, N.L., Kaufman, R.J., Anderson, P. & Liljestrom, P. (2005). Importance of eIF2alpha phosphorylation and stress granule assembly in alphavirus translation regulation. Mol Biol Cell 16(8), 3753–3763.Google Scholar
Miller, L.C., Blandford, V., McAdam, R., Sanchez-Carbente, M.R., Badeaux, F., DesGroseillers, L. & Sossin, W.S. (2009). Combinations of DEAD box proteins distinguish distinct types of RNA: Protein complexes in neurons. Mol Cell Neurosci 40(4), 485–495.Google Scholar
Mokas, S., Mills, J.R., Garreau, C., Fournier, M.-J., Robert, F., Arya, P., Kaufman, R.J., Pelletier, J. & Mazroui, R. (2009). Uncoupling stress granule assembly and translation initiation inhibition. Mol Biol Cell 20(11), 2673–2683.Google Scholar
Mollet, S., Cougot, N., Wilczynska, A., Dautry, F., Kress, M., Bertrand, E. & Weil, D. (2008). Translationally repressed mRNA transiently cycles through stress granules during stress. Mol Biol Cell 19(10), 4469–4479.CrossRefGoogle ScholarPubMed
Montero, H. & Trujillo-Alonso, V. (2011). Stress granules in the viral replication cycle. Viruses 3(11), 2328–2338.Google Scholar
Olanow, C.W. & McNaught, K. (2011). Parkinson's disease, proteins, and prions: Milestones. Movement Disord 26(6), 1056–1071.Google Scholar
Rangan, P., DeGennaro, M., Jaime-Bustamante, K., Coux, R.-X., Martinho, R.G. & Lehmann, R. (2009). Temporal and spatial control of germ-plasm RNAs. Curr Biol 19(1), 72–77.CrossRefGoogle ScholarPubMed
Rowley, A.H., Baker, S.C., Shulman, S.T., Garcia, F.L., Fox, L.M., Kos, I.M., Crawford, S.E., Russo, P.A., Hammadeh, R., Takahashi, K. & Orenstein, J.M. (2008). RNA-containing cytoplasmic inclusion bodies in ciliated bronchial epithelium months to years after acute Kawasaki disease. PLoS One 3(2), e1582. Google Scholar
Sanchez-Carbente, M.D.R. & Desgroseillers, L. (2008). Understanding the importance of mRNA transport in memory. Prog Brain Res 169, 41–58.CrossRefGoogle ScholarPubMed
Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7), 671–675.Google Scholar
Stoecklin, G., Stubbs, T., Kedersha, N., Wax, S., Rigby, W.F.C., Blackwell, T.K. & Anderson, P. (2004). MK2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and ARE-mRNA decay. EMBO J 23(6), 1313–1324.Google Scholar
Thomas, M.G., Loschi, M., Desbats, M.A. & Boccaccio, G.L. (2011). RNA granules: The good, the bad and the ugly. Cell Signal 23(2), 324–334.CrossRefGoogle ScholarPubMed
Thomson, T. & Lasko, P. (2005). Tudor and its domains: Germ cell formation from a Tudor perspective. Cell Res 15(4), 281–291.Google Scholar
Thomson, T., Liu, N., Arkov, A., Lehmann, R. & Lasko, P. (2008). Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins. Mech Develop 125(9-10), 865–873.CrossRefGoogle ScholarPubMed
Tourriere, H., Chebli, K., Zekri, L., Courselaud, B., Blanchard, J.M., Bertrand, E. & Tazi, J. (2003). The RasGAP-associated endoribonuclease G3BP assembles stress granules. J Cell Biol 160(6), 823–831.Google Scholar
Vertii, A., Hakim, C., Kotlyarov, A. & Gaestel, M. (2006). Analysis of properties of small heat shock protein Hsp25 in MAPK-activated protein kinase 2 (MK2)-deficient cells. J Biol Chem 281(37), 26966–26975.Google Scholar
White, J.P., Cardenas, A.M., Marissen, W.E. & Lloyd, R.E. (2007). Inhibition of cytoplasmic mRNA stress granule formation by a viral proteinase. Cell Host Microbe 2(5), 295–305.Google Scholar
Zhang, J., Okabe, K., Tani, T. & Funatsu, T. (2011). Dynamic association-dissociation and harboring of endogenous mRNAs in stress granules. J Cell Sci 124, 4087–4095.Google Scholar
Zhang, J.-H., Chung, T.D.Y. & Oldenburg, K.R. (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2), 67–73.Google Scholar
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