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Split-Halotag® Imaging Assay for Sophisticated Microscopy of Protein-Protein

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Split-Halotag® Imaging Assay for Sophisticated Microscopy of Protein-Protein bioRxiv preprint doi: https://doi.org/10.1101/2020.06.08.139378; this version posted February 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Split-HaloTag® Imaging Assay for Sophisticated Microscopy of Protein-Protein 2 Interactions in planta 3 Rieke Minner-Meinen1, Jan-Niklas Weber1, Andreas Albrecht2, Rainer Matis2, Maria 1 1 1 1 3 4 Behnecke , Cindy Tietge , Stefan Frank , Jutta Schulze , Henrik Buschmann , Peter Jomo 5 Walla2, Ralf-R. Mendel1, Robert Hänsch1,4* and David Kaufholdt1 6 [email protected], [email protected], [email protected], r.matis@tu- 7 bs.de, [email protected], [email protected], [email protected], 8 [email protected], [email protected], [email protected], 9 [email protected], [email protected], [email protected] 10 1Institut für Pflanzenbiologie, Technische Universität Braunschweig, Humboldtstrasse 1, D- 11 38106 Braunschweig, Germany 12 2Institut für Physikalische und Theoretische Chemie, Technische Universität Braunschweig, 13 Hagenring 30.023c, D-38106 Braunschweig, Germany 14 3Botany Department, Universität Osnabrück, Barbara Str. 11, 49076 Osnabrück Germany 15 4Center of Molecular Ecophysiology (CMEP), College of Resources and Environment, 16 Southwest University, Tiansheng Road No. 2, 400715 Chongqing, Beibei District, P.R. China 17 *author for correspondence: Tel: +49-(0)531-391-5867 18 Fax: +49-(0)531-391-8128 19 E-Mail: [email protected] 20 21 Date of submission: 2020-02-02 22 Word count total: 5918 23 Number of Tables: 0 24 Number of Supplementary Tables: 2 25 Number of Figures: 3 26 Number of Supplementary Figures: 2 27 Running title: Split-HaloTag® Imaging Assay bioRxiv preprint doi: https://doi.org/10.1101/2020.06.08.139378; this version posted February 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 28 Abstract 29 An ever-increasing number of intracellular multi-protein networks have been identified in 30 plant cells. Split-GFP based protein-protein interaction assays combine the advantages of in 31 vivo interaction studies in a native environment with additional visualisation of protein 32 complex localisation. Due to its simple protocols, it has become one of the most frequently 33 used methods. However, standard fluorescent proteins entail several drawbacks for 34 sophisticated microscopy. 35 With the HaloTag® system, these drawbacks can be overcome as this reporter forms covalent 36 irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in 37 adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we 38 established the Split-HaloTag® imaging assay in plants which is based on the reconstitution of 39 a functional HaloTag® protein upon protein-protein interaction and subsequent covalent 40 binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using 41 well-characterised interactions as an example for protein-protein interaction at cellular 42 structures: the molybdenum cofactor biosynthesis complex anchoring to filamentous actin. 43 Additionally, a specific interaction was visualised with subdiffractional polarisation microscopy 44 in a more distinctive manner as example for sophisticated imaging. 45 Split-GFP and Split-HaloTag® can complement one another as Split-HaloTag® represents an 46 alternative option and an addition to the large toolbox of in vivo methods. Therefore, this 47 promising new Split-HaloTag® imaging assay provides a unique and sensitive approach for 48 more detailed characterization of protein-protein interaction with specific microscopic 49 techniques such as 3D-imaging, single molecule tracking and super-resolution microscopy. 50 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.08.139378; this version posted February 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 51 Keywords 52 advanced microscopy, cytoskeleton, photostable fluorescent dyes, Gateway cloning, 53 Molybdenum cofactor biosynthesis complex, polarisation microscopy, protein-protein 54 interaction, Split-HaloTag® imaging assay 55 Abbreviations 56 BiFC: bimolecular fluorescence complementation; diAcFAM: diacetyl derivative of fluorescein; 57 FP: fluorescent protein; SPoD: Super-resolution by polarisation demodulation; TMR: 58 Tetramethylrhodamine bioRxiv preprint doi: https://doi.org/10.1101/2020.06.08.139378; this version posted February 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 59 Introduction 60 An ever-increasing number of protein networks has been identified in plants (Zitnik et al., 61 2019). Therefore, understanding the cellular biology of substrate channelling pathways 62 requires the characterisation of protein-protein interactions (PPIs) in their native 63 environment. A broad spectrum of in vivo methods has been employed to analyse PPIs such 64 as bimolecular fluorescence complementation (BiFC) belonging to the group of protein 65 fragment complementation assays (Struk et al., 2019). Basically, two non-fluorescent reporter 66 fragments of a fluorescent protein (FP) are fused genetically to putative interaction partners 67 and an interaction between the two allows formation of a bimolecular fluorescent complex 68 (Kerppola, 2008). Consequently, BiFC not only allows detection of PPIs but also visualisation 69 and localisation of the protein complex (Bhat et al., 2006). Furthermore, using FPs results in 70 fluorescence signals without an invasive insertion of exogenous chemical compounds into the 71 cell. Therefore, due to its simple protocols BiFC has become one of the most popular and 72 frequently used method to study PPIs in plant cells (Kudla & Bock, 2016). 73 Conventional light and fluorescence microscopy are diffraction-limited to a resolution limit of 74 approx. 200 nm in the lateral (x-y) and about 600 nm in the axial (z) direction (Cremer and 75 Masters, 2013). Many subcellular structures are smaller which hampers their detailed 76 observation (Huang et al., 2009). To circumvent these restrictions, advanced fluorescence 77 imaging methods such as single molecule detection, subdiffractional polarisation imaging or 78 super-resolution microscopy (SRM) techniques have been developed to improve the 79 resolution and to allow studying molecular processes more detailed (Moerner and Kador 80 1989; Orrit and Bernard, 1990; Bode et al., 2008; Holleboom et al., 2014; Liao et al., 81 2010/2011; Godin et al., 2014; Loison et al., 2018; Camacho et al., 2019). However, for such 82 advanced imaging techniques fluorescent dyes with high stability and brightness are needed 83 (Banaz et al., 2018), which is hard to realise by standard FPs as they show low quantum 84 efficiency, blinking behaviour, a high photobleaching rate during long-term observations 85 (Reck-Petersen et al., 2006), photoswitching (Morisaki and McNally, 2014) as well as the 86 tendency to form oligomers (Miyawaki et al., 2003). 87 Self-labelling enzyme tags such as the HaloTag® have been shown to overcome these 88 drawbacks of FPs and are suitable for such microscopy methods and super-resolution imaging bioRxiv preprint doi: https://doi.org/10.1101/2020.06.08.139378; this version posted February 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 89 (Grimm et al., 2015; Lee et al., 2010). The HaloTag® System (Promega, 90 https://www.promega.de/) is based on the bacterial haloalkane dehalogenase DhaA (EC 91 3.8.1.5) from Rhodococcus rhodochrous (Liss et al., 2015). The tag was modified to form 92 covalent irreversible bonds with synthetic chloralkane ligands (Los et al., 2008; England et al., 93 2015). This covalent bond is formed rapidly under physiological environments and remains 94 intact even under stringent conditions (Los and Wood, 2007). In addition, HaloTag® proteins 95 keep their monomeric structure, so the tag will not lead to oligomerisation of protein fusion 96 partners (Banaz et al., 2018). Numerous ligands are available for the HaloTag® including 97 different fluorescent dyes with extended spectral range, photostability and membrane 98 permeability such as the red fluorescent rhodamine derivative TMR (tetramethylrhodamine), 99 the green fluorescent Oregon Green or the yellow fluorescent diacetyl derivative of 100 fluorescein DiAcFAM. Furthermore, dyes can be varied in their dosages to either label all or 101 only a few molecules which is needed for single-molecule tracking approaches. Reck-Peterson 102 and colleagues (2006) for example used HaloTag® and a TMR ligand successfully to label 103 dynein in sea urchin axonemes and tracked single molecules with a precision of a few 104 nanometres
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