Planta DOI 10.1007/s00425-017-2789-2
COMMENTARY
Subcellular localization and function of 2LIM proteins in plants and humans Céline Hoffmann1 · Josh Brown‑Clay1 · Clément Thomas1
Received: 4 September 2017 / Accepted: 29 September 2017 © Springer-Verlag GmbH Germany 2017
In their recent review article published in Planta, Srivastava and Verma (2017) comprehensively summarise 25 years of research on plant LIM proteins, with a focus on their biological functions and potential for translational applications in crop improvement. The present commentary extends the discussion on the subcellular localization of two-LIM domain proteins (2LIMs), with the objective to further delineate the key questions that need to be answered, as well as the hurdles that investigators may have to overcome. The 2LIMs belong to a unique LIM protein subfamily which is found in both plants and vertebrates. In the latter, they are referred to as cysteine-rich proteins or CRPs (Weiskirchen and Gunther 2003). In addition to their similar structural organization and dual cytoplasmic and nuclear distribution, 2LIMs and CRPs share common functionalities, such as the capacity to crosslink actin filaments (AFs) (Hoffmann et al. 2014b, 2016; Tran et al. 2005), and to regulate gene transcription (Chang et al. 2003, 2007; Kawaoka et al. 2000; Moes et al. 2013). Accordingly, we also draw attention to those studies on CRPs relevant to the question of 2LIM nuclear-cytoplasmic partitioning. Our current knowledge of plant 2LIM subcellular localization mostly relies on studies employing fluorescent protein-fused 2LIMs proteins expressed under the control of a strong promoter. Such studies usually report the presence of exogenously expressed 2LIMs in both cytoplasmic and nuclear compartments. Very few studies employing antibodies raised against 2LIMs support that endogenous 2LIMs * Clément Thomas [email protected] 1
Laboratory of Experimental Cancer Research, Department of Oncology, Luxembourg Institute of Health, 84 Val, Fleuri, Luxembourg, Luxembourg
have a preferential nuclear or cytoplasmic localization in specific cell types and/or at specific developmental stages (Han et al. 2013; Mundel et al. 2000). Therefore, the strong promoters used to express recombinant 2LIMs and/or the fused fluorescent moiety likely provide an inaccurate view of the physiological subcellular distribution of endogenous 2LIMs, and alter 2LIM intracellular trafficking. Also, capturing the precise location of 2LIMs in the cytoplasm using specific antibodies has been shown to be fairly challenging. To our best knowledge, there is no immunolabelling analysis confirming the prominent association of 2LIMs with AFs observed with fluorescently-tagged proteins. Noticeably, such association is disrupted by detergent-containing rhodamine–phalloidin buffers and poorly conserved by standard fixation procedures (Thomas et al. 2006). To complicate matters further, plant AFs are notoriously difficult to preserve, making the elaboration of an effective fixation procedure a real headache. Mechanical stress regulates human CRP targeting to specific subcellular locations. Myocyte contractility promotes endogenous CRP3 translocation into the nucleus and nucleolus, where it activates myogenic transcription factors and ribosomal protein synthesis, respectively (Boateng et al. 2007, 2009; Kong et al. 1997). However, the exact physiological localization and function of CRP3 in the cytoplasmic compartment remains a matter of debate (Gunkel et al. 2010). Depending on the antibodies used, CRP3 was localized either diffusely in the cytoplasm or in association with diverse sarcomeric structures, including costamers, intercalated discs, the Z-disc and I band regions. Recently, cotton WLIM1a was shown to respond to hydrogen peroxide by translocating to the nucleus, where it induces the expression of genes involved in secondary wall formation (Han et al. 2013). Thus, both CRPs and plant 2LIMs mediate stress-induced
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changes in gene expression. Because most stresses promote cytoskeletal rearrangements, such rearrangements may conceivably weaken the interaction of 2LIMs with AFs, and thereby facilitate their translocation to the nucleus which is assumed to be driven by a (yet putative) nuclear localization signal. In support of such a scenario, experimentally-induced actin depolymerization has been shown to increase the nuclear levels of GFP-fused WLIM2 (Moes et al. 2013). However, further investigations are required to establish how stress regulates 2LIM expression and subcellular localization. Another interesting matter relevant to 2LIM cytoplasmicnuclear shuttling is the state of protein oligomerization. Both 2LIMs and CRPs self-associate along cytoplasmic actin structures, and this was argued to be required for AF crosslinking (Boateng et al. 2007; Hoffmann et al. 2014a, b). Nuclear translocation of CRP3 is associated with a shift toward the monomeric form (Boateng et al. 2007; Paudyal et al. 2016). Whether the oligomerization status of 2LIMs dictates their targeting to specific subcellular compartments or vice versa is an important question to be addressed. As reviewed by Srivastava and Verma, our knowledge of 2LIM biology has considerably increased since they were discovered about 25 years ago. It has become clear that 2LIMs combine two main functions. They regulate AF organization and dynamics in the cytoplasm and gene expression in the nucleus. The molecular mechanisms underlying each function have been characterized to a large extent, although some specific aspects remain to be clarified, especially regarding the way 2LIMs regulate target gene transcription. Also, detailed characterization of 2LIM promoters should significantly contribute to reveal 2LIM functions in stress responses (Srivastava and Verma 2015). The subsequent, pressing question to be addressed is how 2LIM cytoskeletal and nuclear functions are intertwined and affect each other. Because of their simple domain organization, 2LIMs represent attractive models to better understand the cross-talk between the cytoskeleton and nucleus. Although fluorescently-tagged proteins have significantly contributed to the characterization of 2LIM functions in living cells, weaker, ideally endogenous promoters should be used instead of the conventional strong constitutive promoters, and investigators should remain cautious with the use of fluorescent, GFP-fused proteins, especially considering the small size of 2LIMs. Although the use of complementary techniques to reliably determine localization and oligomeric status will require more work of investigators, the extra effort seems worth it, given the potential translational impact of this family of proteins. Author contribution statement CT wrote the manuscript with assistance from CH and JBC. All authors edited and revised the manuscript.
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Compliance with ethical standards Conflict of interest The authors declare no conflict of interest. Financial support The work in Thomas’s lab is supported by the Luxembourg Cancer Foundation (FC/2016/02) and the Luxembourg National Research Fund (FNR; C16/BM/11297905). Joshua Brown Clay is recipient of a Postdoctoral fellowship from Belgium FNRS (7.4512.16).
References Boateng SY, Belin RJ, Geenen DL, Margulies KB, Martin JL, Hoshijima M, de Tombe PP, Russell B (2007) Cardiac dysfunction and heart failure are associated with abnormalities in the subcellular distribution and amounts of oligomeric muscle LIM protein. Am J Physiol Heart Circ Physiol 292(1):H259–H269 Boateng SY, Senyo SE, Qi L, Goldspink PH, Russell B (2009) Myocyte remodeling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein. J Mol Cell Cardiol 47(4):426–435 Chang DF, Belaguli NS, Iyer D, Roberts WB, Wu SP, Dong XR, Marx JG, Moore MS, Beckerle MC, Majesky MW, Schwartz RJ (2003) Cysteine-rich LIM-only proteins CRP1 and CRP2 are potent smooth muscle differentiation cofactors. Dev Cell 4(1):107–118 Chang DF, Belaguli NS, Chang J, Schwartz RJ (2007) LIM-only protein, CRP2, switched on smooth muscle gene activity in adult cardiac myocytes. Proc Natl Acad Sci USA 104(1):157–162 Gunkel S, Linke WA, Heineke J, Hilfiker-Kleiner D, Knoll R (2010) Response to Gehmlich et al. Letter to the Editor of the Journal of Molecular and Cellular Cardiology Regarding “MLP: a stress sensor goes nuclear”. J Mol Cell Cardiol 48(2):426–427 Han LB, Li YB, Wang HY, Wu XM, Li CL, Luo M, Wu SJ, Kong ZS, Pei Y, Jiao GL, Xia GX (2013) The dual functions of WLIM1a in cell elongation and secondary wall formation in developing cotton fibers. Plant Cell 25(11):4421–4438 Hoffmann C, Moes D, Dieterle M, Neumann K, Moreau F, Tavares Furtado A, Dumas D, Steinmetz A, Thomas C (2014a) Live cell imaging reveals actin-cytoskeleton-induced self-association of the actin-bundling protein WLIM1. J Cell Sci 127(Pt 3):583–598 Hoffmann C, Moreau F, Moes M, Luthold C, Dieterle M, Goretti E, Neumann K, Steinmetz A, Thomas C (2014b) Human muscle LIM protein dimerizes along the actin cytoskeleton and crosslinks actin filaments. Mol Cell Biol 34(16):3053–3065 Hoffmann C, Mao X, Dieterle M, Moreau F, Al Absi A, Steinmetz A, Oudin A, Berchem G, Janji B, Thomas C (2016) CRP2, a new invadopodia actin bundling factor critically promotes breast cancer cell invasion and metastasis. Oncotarget 7(12):13688–13705 Kawaoka A, Kaothien P, Yoshida K, Endo S, Yamada K, Ebinuma H (2000) Functional analysis of tobacco LIM protein Ntlim1 involved in lignin biosynthesis. Plant J 22(4):289–301 Kong Y, Flick MJ, Kudla AJ, Konieczny SF (1997) Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD. Mol Cell Biol 17(8):4750–4760 Moes D, Gatti S, Hoffmann C, Dieterle M, Moreau F, Neumann K, Schumacher M, Diederich M, Grill E, Shen WH, Steinmetz A, Thomas C (2013) A LIM domain protein from tobacco involved in actin-bundling and histone gene transcription. Mol Plant 6(2):483–502 Mundel C, Baltz R, Eliasson A, Bronner R, Grass N, Krauter R, Evrard JL, Steinmetz A (2000) A LIM-domain protein from sunflower is localized to the cytoplasm and/or nucleus in a wide variety of
Planta tissues and is associated with the phragmoplast in dividing cells. Plant Mol Biol 42(2):291–302 Paudyal A, Dewan S, Ikie C, Whalley BJ, de Tombe PP, Boateng SY (2016) Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure. J Physiol 594(12):3287–3305 Srivastava V, Verma PK (2015) Genome wide identification of LIM genes in Cicer arietinum and response of Ca-2LIMs in development, hormone and pathogenic stress. PLoS One 10(9):e0138719 Srivastava V, Verma PK (2017) The plant LIM proteins: unlocking the hidden attractions. Planta. doi:10.1007/s00425-017-2715-7
Thomas C, Hoffmann C, Dieterle M, Van Troys M, Ampe C, Steinmetz A (2006) Tobacco WLIM1 is a novel F-actin binding protein involved in actin cytoskeleton remodeling. Plant Cell 18(9):2194–2206 Tran TC, Singleton C, Fraley TS, Greenwood JA (2005) Cysteine-rich protein 1 (CRP1) regulates actin filament bundling. BMC Cell Biol 6(1):45 Weiskirchen R, Gunther K (2003) The CRP/MLP/TLP family of LIM domain proteins: acting by connecting. BioEssays 25(2):152–162
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COMMENTARY
Subcellular localization and function of 2LIM proteins in plants and humans Céline Hoffmann1 · Josh Brown‑Clay1 · Clément Thomas1
Received: 4 September 2017 / Accepted: 29 September 2017 © Springer-Verlag GmbH Germany 2017
In their recent review article published in Planta, Srivastava and Verma (2017) comprehensively summarise 25 years of research on plant LIM proteins, with a focus on their biological functions and potential for translational applications in crop improvement. The present commentary extends the discussion on the subcellular localization of two-LIM domain proteins (2LIMs), with the objective to further delineate the key questions that need to be answered, as well as the hurdles that investigators may have to overcome. The 2LIMs belong to a unique LIM protein subfamily which is found in both plants and vertebrates. In the latter, they are referred to as cysteine-rich proteins or CRPs (Weiskirchen and Gunther 2003). In addition to their similar structural organization and dual cytoplasmic and nuclear distribution, 2LIMs and CRPs share common functionalities, such as the capacity to crosslink actin filaments (AFs) (Hoffmann et al. 2014b, 2016; Tran et al. 2005), and to regulate gene transcription (Chang et al. 2003, 2007; Kawaoka et al. 2000; Moes et al. 2013). Accordingly, we also draw attention to those studies on CRPs relevant to the question of 2LIM nuclear-cytoplasmic partitioning. Our current knowledge of plant 2LIM subcellular localization mostly relies on studies employing fluorescent protein-fused 2LIMs proteins expressed under the control of a strong promoter. Such studies usually report the presence of exogenously expressed 2LIMs in both cytoplasmic and nuclear compartments. Very few studies employing antibodies raised against 2LIMs support that endogenous 2LIMs * Clément Thomas [email protected] 1
Laboratory of Experimental Cancer Research, Department of Oncology, Luxembourg Institute of Health, 84 Val, Fleuri, Luxembourg, Luxembourg
have a preferential nuclear or cytoplasmic localization in specific cell types and/or at specific developmental stages (Han et al. 2013; Mundel et al. 2000). Therefore, the strong promoters used to express recombinant 2LIMs and/or the fused fluorescent moiety likely provide an inaccurate view of the physiological subcellular distribution of endogenous 2LIMs, and alter 2LIM intracellular trafficking. Also, capturing the precise location of 2LIMs in the cytoplasm using specific antibodies has been shown to be fairly challenging. To our best knowledge, there is no immunolabelling analysis confirming the prominent association of 2LIMs with AFs observed with fluorescently-tagged proteins. Noticeably, such association is disrupted by detergent-containing rhodamine–phalloidin buffers and poorly conserved by standard fixation procedures (Thomas et al. 2006). To complicate matters further, plant AFs are notoriously difficult to preserve, making the elaboration of an effective fixation procedure a real headache. Mechanical stress regulates human CRP targeting to specific subcellular locations. Myocyte contractility promotes endogenous CRP3 translocation into the nucleus and nucleolus, where it activates myogenic transcription factors and ribosomal protein synthesis, respectively (Boateng et al. 2007, 2009; Kong et al. 1997). However, the exact physiological localization and function of CRP3 in the cytoplasmic compartment remains a matter of debate (Gunkel et al. 2010). Depending on the antibodies used, CRP3 was localized either diffusely in the cytoplasm or in association with diverse sarcomeric structures, including costamers, intercalated discs, the Z-disc and I band regions. Recently, cotton WLIM1a was shown to respond to hydrogen peroxide by translocating to the nucleus, where it induces the expression of genes involved in secondary wall formation (Han et al. 2013). Thus, both CRPs and plant 2LIMs mediate stress-induced
13
Vol.:(0123456789)
Planta
changes in gene expression. Because most stresses promote cytoskeletal rearrangements, such rearrangements may conceivably weaken the interaction of 2LIMs with AFs, and thereby facilitate their translocation to the nucleus which is assumed to be driven by a (yet putative) nuclear localization signal. In support of such a scenario, experimentally-induced actin depolymerization has been shown to increase the nuclear levels of GFP-fused WLIM2 (Moes et al. 2013). However, further investigations are required to establish how stress regulates 2LIM expression and subcellular localization. Another interesting matter relevant to 2LIM cytoplasmicnuclear shuttling is the state of protein oligomerization. Both 2LIMs and CRPs self-associate along cytoplasmic actin structures, and this was argued to be required for AF crosslinking (Boateng et al. 2007; Hoffmann et al. 2014a, b). Nuclear translocation of CRP3 is associated with a shift toward the monomeric form (Boateng et al. 2007; Paudyal et al. 2016). Whether the oligomerization status of 2LIMs dictates their targeting to specific subcellular compartments or vice versa is an important question to be addressed. As reviewed by Srivastava and Verma, our knowledge of 2LIM biology has considerably increased since they were discovered about 25 years ago. It has become clear that 2LIMs combine two main functions. They regulate AF organization and dynamics in the cytoplasm and gene expression in the nucleus. The molecular mechanisms underlying each function have been characterized to a large extent, although some specific aspects remain to be clarified, especially regarding the way 2LIMs regulate target gene transcription. Also, detailed characterization of 2LIM promoters should significantly contribute to reveal 2LIM functions in stress responses (Srivastava and Verma 2015). The subsequent, pressing question to be addressed is how 2LIM cytoskeletal and nuclear functions are intertwined and affect each other. Because of their simple domain organization, 2LIMs represent attractive models to better understand the cross-talk between the cytoskeleton and nucleus. Although fluorescently-tagged proteins have significantly contributed to the characterization of 2LIM functions in living cells, weaker, ideally endogenous promoters should be used instead of the conventional strong constitutive promoters, and investigators should remain cautious with the use of fluorescent, GFP-fused proteins, especially considering the small size of 2LIMs. Although the use of complementary techniques to reliably determine localization and oligomeric status will require more work of investigators, the extra effort seems worth it, given the potential translational impact of this family of proteins. Author contribution statement CT wrote the manuscript with assistance from CH and JBC. All authors edited and revised the manuscript.
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Compliance with ethical standards Conflict of interest The authors declare no conflict of interest. Financial support The work in Thomas’s lab is supported by the Luxembourg Cancer Foundation (FC/2016/02) and the Luxembourg National Research Fund (FNR; C16/BM/11297905). Joshua Brown Clay is recipient of a Postdoctoral fellowship from Belgium FNRS (7.4512.16).
References Boateng SY, Belin RJ, Geenen DL, Margulies KB, Martin JL, Hoshijima M, de Tombe PP, Russell B (2007) Cardiac dysfunction and heart failure are associated with abnormalities in the subcellular distribution and amounts of oligomeric muscle LIM protein. Am J Physiol Heart Circ Physiol 292(1):H259–H269 Boateng SY, Senyo SE, Qi L, Goldspink PH, Russell B (2009) Myocyte remodeling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein. J Mol Cell Cardiol 47(4):426–435 Chang DF, Belaguli NS, Iyer D, Roberts WB, Wu SP, Dong XR, Marx JG, Moore MS, Beckerle MC, Majesky MW, Schwartz RJ (2003) Cysteine-rich LIM-only proteins CRP1 and CRP2 are potent smooth muscle differentiation cofactors. Dev Cell 4(1):107–118 Chang DF, Belaguli NS, Chang J, Schwartz RJ (2007) LIM-only protein, CRP2, switched on smooth muscle gene activity in adult cardiac myocytes. Proc Natl Acad Sci USA 104(1):157–162 Gunkel S, Linke WA, Heineke J, Hilfiker-Kleiner D, Knoll R (2010) Response to Gehmlich et al. Letter to the Editor of the Journal of Molecular and Cellular Cardiology Regarding “MLP: a stress sensor goes nuclear”. J Mol Cell Cardiol 48(2):426–427 Han LB, Li YB, Wang HY, Wu XM, Li CL, Luo M, Wu SJ, Kong ZS, Pei Y, Jiao GL, Xia GX (2013) The dual functions of WLIM1a in cell elongation and secondary wall formation in developing cotton fibers. Plant Cell 25(11):4421–4438 Hoffmann C, Moes D, Dieterle M, Neumann K, Moreau F, Tavares Furtado A, Dumas D, Steinmetz A, Thomas C (2014a) Live cell imaging reveals actin-cytoskeleton-induced self-association of the actin-bundling protein WLIM1. J Cell Sci 127(Pt 3):583–598 Hoffmann C, Moreau F, Moes M, Luthold C, Dieterle M, Goretti E, Neumann K, Steinmetz A, Thomas C (2014b) Human muscle LIM protein dimerizes along the actin cytoskeleton and crosslinks actin filaments. Mol Cell Biol 34(16):3053–3065 Hoffmann C, Mao X, Dieterle M, Moreau F, Al Absi A, Steinmetz A, Oudin A, Berchem G, Janji B, Thomas C (2016) CRP2, a new invadopodia actin bundling factor critically promotes breast cancer cell invasion and metastasis. Oncotarget 7(12):13688–13705 Kawaoka A, Kaothien P, Yoshida K, Endo S, Yamada K, Ebinuma H (2000) Functional analysis of tobacco LIM protein Ntlim1 involved in lignin biosynthesis. Plant J 22(4):289–301 Kong Y, Flick MJ, Kudla AJ, Konieczny SF (1997) Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD. Mol Cell Biol 17(8):4750–4760 Moes D, Gatti S, Hoffmann C, Dieterle M, Moreau F, Neumann K, Schumacher M, Diederich M, Grill E, Shen WH, Steinmetz A, Thomas C (2013) A LIM domain protein from tobacco involved in actin-bundling and histone gene transcription. Mol Plant 6(2):483–502 Mundel C, Baltz R, Eliasson A, Bronner R, Grass N, Krauter R, Evrard JL, Steinmetz A (2000) A LIM-domain protein from sunflower is localized to the cytoplasm and/or nucleus in a wide variety of
Planta tissues and is associated with the phragmoplast in dividing cells. Plant Mol Biol 42(2):291–302 Paudyal A, Dewan S, Ikie C, Whalley BJ, de Tombe PP, Boateng SY (2016) Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure. J Physiol 594(12):3287–3305 Srivastava V, Verma PK (2015) Genome wide identification of LIM genes in Cicer arietinum and response of Ca-2LIMs in development, hormone and pathogenic stress. PLoS One 10(9):e0138719 Srivastava V, Verma PK (2017) The plant LIM proteins: unlocking the hidden attractions. Planta. doi:10.1007/s00425-017-2715-7
Thomas C, Hoffmann C, Dieterle M, Van Troys M, Ampe C, Steinmetz A (2006) Tobacco WLIM1 is a novel F-actin binding protein involved in actin cytoskeleton remodeling. Plant Cell 18(9):2194–2206 Tran TC, Singleton C, Fraley TS, Greenwood JA (2005) Cysteine-rich protein 1 (CRP1) regulates actin filament bundling. BMC Cell Biol 6(1):45 Weiskirchen R, Gunther K (2003) The CRP/MLP/TLP family of LIM domain proteins: acting by connecting. BioEssays 25(2):152–162
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