1. Introduction
Entamoeba histolytica is a single cell protozoan parasite that causes amoebiasis in humans. It is one of the leading causes of parasitic disease burden in tropical regions and developing countries where hygiene and sanitation is limited (Aguilar-Diaz et al., Reference Aguilar-Diaz, Carrero, Arguello-Garcia, Laclette and Morales-Montor2011; Morf & Singh, Reference Morf and Singh2012). Calcium binding proteins are involved in erythrophagocytosis that characterises invasive amoebasis, and in the associated modulation of the actin cytoskeleton dynamics (Christy & Petri, Reference Christy and Petri2011; Somlata et al., Reference Somlata, Agrahari, Babuta, Bhattacharya and Bhattacharya2012). Somlata & Bhattacharya (Reference Somlata and Bhattacharya2011) and Somlata et al. (Reference Somlata, Agrahari, Babuta, Bhattacharya and Bhattacharya2012) showed that the initiation of erythrophagocytosis in E. histolytica depends on C2-domain containing protein kinase (EhC2PK), actin and calcium binding proteins (EhCaBP1 and EhCaBP3). EhCaBP3 shares homology with calmodulin (Aslam et al., Reference Aslam, Bhattacharya and Bhattacharya2012; Rout et al., Reference Rout, Padhan, Barnwal, Bhattacharya and Chary2011), and is present both in the nucleus as well as the cytoplasm of the parasite (Rout et al., Reference Rout, Padhan, Barnwal, Bhattacharya and Chary2011). Chemical depletion of cytoplasmic calcium in E. histolytica caused the localisation of a nuclear protein, EhCaBP6, to the cytoplasm (Verma et al., Reference Verma, Murmu, Gourinath, Bhattacharya and Chary2017) suggesting the presence of calcium dependent nuclear transport in the protozoan.
2. Objective
We examined the possibility that EhCaBP3 may be a nuclear shuttling protein capable of Ca2+ dependent nuclear import similar to calmodulin (CaM) in higher eukaryotes. CaM-dependent nuclear import was initially reported by Sweitzer and Hanover (Sweitzer et al., Reference Sweitzer, Love and Hanover2000), where CaM was shown to be able to facilitate nuclear import under conditions where high intracellular Ca2+ inhibited conventional Ran-dependent nuclear transport. We used over-expression of EhCaBP3 in transfected mammalian cells to determine its localisation, followed by investigation of change in localisation on increased cytosolic calcium.
3. Methods
3.1. Cell culture, transfection
COS-7 cells (CRL-1651, American Type Culture Collection) were grown as previously (Ghildyal et al., Reference Ghildyal, Ho, Dias, Soegiyono, Bardin, Tran, Teng and Jans2009) and used for transfection of plasmids using Lipofectamine. Where indicated, cells were treated 18 h post-transfection with 1 μM Thapsigargin (Calbiochem, La Jolla, CA, USA) for 6 h before imaging (Kaur & Jans, Reference Kaur and Jans2011).
3.2. Plasmid Constructs
The sequence for EhCaBP3 (Amoeba Database) was synthesised and cloned into the pMK-RQ shuttle vector (Life Technologies, GeneArt) and subcloned into the Gateway™ compatible pEPI-DESTC vector (Ghildyal et al., Reference Ghildyal, Ho, Dias, Soegiyono, Bardin, Tran, Teng and Jans2009) for expression in mammalian cells as GFP-EhCaBP3. Plasmids encoding GFP alone (Ghildyal et al., Reference Ghildyal, Ho, Wagstaff, Dias, Barton, Jans, Bardin and Jans2005), GFP-SRY-wt, GFP-SRY-R133W (GFP-SRY with arginine at position 133 mutated to tryptophan) and GFP-SRY-R76P (GFP-SRY with arginine at position 76 mutated to proline) have been described previously (Kaur & Jans, Reference Kaur and Jans2011).
3.3. Quantitative Confocal laser scanning microscopy
Transfected cells were imaged live in serum free FluoroBrite DMEM, using Nikon Ti Eclipse confocal laser-scanning microscope with Nikon 60x/1.40 oil immersion lens (Plan Apo VC OFN25 DIC N2; optical section of 0.5 μm) and the NIS Elements AR software. Data from four individual scans was averaged to obtain the final images (Walker et al., Reference Walker, Younessi, Fulcher, McCuaig, Thomas, Bardin, Jans and Ghildyal2013). Image analysis of digitized CLSM images was performed as previously (Shahriari et al., Reference Shahriari, Wei and Ghildyal2018). Data sets represent mean +/− SEM from at least 30 cells from three independent experiments.
3.5. Statistical Analysis
Mean and standard error of the mean values were calculated for each dataset, and significant differences in mean values determined by ANOVA; p < 0.05 was accepted as significant. All statistical analysis was performed with GraphPad Prism software.
4. Results
4.1. GFP-EhCaBP3 accumulates in the nucleus of eukaryotic cells
GFP-EhCaBP3 was present in both the nucleus and cytoplasm of COS-7 cells transfected to express the protein, with obvious accumulation in the nucleus relative to the cytoplasm (Fig. 1A, images labelled ‘no treatment’). Some GFP-EhCaBP3 was also localised to the plasma membrane, to structures in the cytoplasm resembling microfilaments and formed punctate structures in some cells. In the same experiment, GFP alone was present diffused through the whole cell as expected. GFP diffuses freely across the nuclear envelope and is commonly found distributed almost equally throughout the whole cell (Ghildyal et al., Reference Ghildyal, Ho, Dias, Soegiyono, Bardin, Tran, Teng and Jans2009; Shahriari et al., Reference Shahriari, Wei and Ghildyal2018; Yu et al., Reference Yu, Shahriari, Li and Ghildyal2016). Image analysis of the digital images (Fig. 1B, columns labelled ‘no add’) confirmed that GFP-EhCaBP3 was accumulated in nuclei relative to cytoplasm of cells in which it was expressed (Fn/c = 2.96 ± 0.62), while GFP was present diffused in the whole cell (Fn/c = 1.57 ± 0.07).
EhCaBP3 is localised in the nucleus and cytoplasm and has a calcium dependent nuclear transport
4.2. GFP-EhCaBP3 has calcium sensitive nuclear import
Thapsigargin treatment results in increased intracellular calcium due to release from endoplasmic reticulum (ER) and nuclear envelope (NE) stores (Thastrup et al., Reference Thastrup, Cullen, Drobak, Hanley and Dawson1990). GFP-EhCaBP3 became significantly less nuclear on treatment with Thapsigargin (Fig. 1A, images labelled ‘Thapsigargin’) compared to in its absence. Quantitative image analysis for Fn/c confirmed this finding (Fig. 1B, columns labelled ‘Thaps’) with a statistically significant reduction in Fn/c on treatment compared to no treatment (p = 0.0009). Thapsigargin treatment had no effect on the subcellular localisation of GFP, as expected. Interestingly, GFP-EhCaBP3 appeared to be associated with cytoskeletal components under conditions of increased cytosolar calcium (Fig. 1A, image labelled GFP-EhCaBP3, Thapsigargin; note the cytosolar network localisation).
Cells transfected with GFP-SRY or its mutants (GFP-R76P, GFP-R133W) were used to control for Thapsigargin activity; SRY has two nuclear localisation signals (NLSs), one modulated by calcium and the other dependent on Importin-β (Sudbeck & Scherer, Reference Sudbeck and Scherer1997). GFP-SRY-wt has both NLSs, GFP-SRY-R76P has the Importin-β dependent NLS only, while GFP-SRY-R133W has the calcium dependent NLS only. GFP-SRY-wt was highly accumulated in the nucleus while -R76P and -R133W less so as expected, due to presence of only one NLS (Fig. 1C, compare images labelled ‘no treatment’). As expected (Kaur & Jans, Reference Kaur and Jans2011), treatment with Thapsigargin resulted in a statistically significant change in nuclear-cytoplasmic distribution of GFP-SRY-wt (p = 0.0002) and GFP-SRY-R133W (p = 0.0025) but had no effect on the subcellular localisation of GFP-SRY-R76P (Fig. 1C, compare images labelled ‘Thapsigargin’); quantitative image analysis for Fn/c confirmed this finding (Fig. 1D). Data presented are representative of three independent experiments.
5. Conclusions
Our data show that nuclear transport of EhCaBP3 is calcium dependent. Eukaryotic calcium dependent nuclear transport is mediated by CaM (Sweitzer & Hanover, Reference Sweitzer and Hanover1996) and is conserved from yeast to humans (Hanover et al., Reference Hanover, Love and Prinz2009). A classical CaM has not been identified in E. histolytica with the closest homolog being EhCaBP3 (Aslam et al., Reference Aslam, Bhattacharya and Bhattacharya2012). Taken together with a recent study (Verma et al., Reference Verma, Murmu, Gourinath, Bhattacharya and Chary2017) that showed that a calcium binding protein in E. histolytica, EhCaBP6, is a nuclear–cytoplasmic shuttling protein with calcium-dependent nuclear transport, our study strongly suggests the existence of a calcium modulated nuclear transport pathway in E. histolytica. Whole genome analysis of E. histolytica has shown the presence of putative protein components of the nuclear trafficking machinery. Indeed, our bioinformatics investigations predict the presence of all the core components of Ran dependent nuclear transport (Gwairgi & Ghildyal, Reference Gwairgi and Ghildyal2018).
Taken together with the data presented in the current study, accumulating evidence strongly suggests the presence of complex nuclear transport pathways in E. histolytica, similar to higher eukaryotes.
Acknowledgements
We thank David A. Jans (Monash University, Melbourne, Australia) for the gift of the plasmids encoding SRY proteins and input into experimental design. We acknowledge the support of Alok Bhattacharya and Sudha Bhattacharya (Jawaharlal Nehru University, New Delhi, India) throughout this study.
Conflicts of Interest
KW and RG declare no conflicts.
Author Contributions
KW performed the experiments and generated the figure. RG supervised the work and the experimental design and wrote the manuscript.
Financial Support
The work described in this manuscript was partly funded through an Australia India Strategic Research Fund Senior Visiting Fellowship and a Centre for Research in Therapeutic Solutions Seed Grant to RG.
Data Availability Statement
All data associated with this study are included in the manuscript. Reagents developed during this study are available from the corresponding author on request.
Open access
Comments
Comments to the Author: The authors expressed the calmodulin-like calcium binding protein EhCaBP3 was expressed in human background, and with carefully chosen controls demonstrated that localizes in the nucleus utilizing a calcium-dependent pathway. However, three points must be addressed:
1. The title of the paper should reflect that EhCaBP3 was tested in human background.
2. The introduction should give proper credit to EhCaBP precedent (Verma et al., 2017), not only discussing such previous findings.
3. Since the image is very well contrasted, marking at least one cell membrane could help untrained eyes to interpret Figure 1C.