CAPPI: A Cytoskeleton-Based Localization Assay Reports Protein-Protein Interaction in Living Cells by Fluorescence Microscopy

Lv, Sulian; Miao, Huiying; Luo, Ming; Li, Yinxin; Wang, Qiaomei; Lee, Yuh‐Ru Julie; Liu, Bo

doi:10.1016/j.molp.2017.09.006

Cited by 11 publications

(11 citation statements)

References 10 publications

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“…We show that rerouting various proteins to distinct subcellular localizations is feasible in our system. Other 'anchor-away' techniques such as NTA, CAPPI and MILo (Dixon and Lim, 2010;Kaplan--Levy et al, 2014;Lv et al, 2017) are limited in terms of subcellular rerouting options due to their direct fusion with the anchor. By contrast, KSP easily combines with multiple anchors without the need to re-clone fusion constructs.…”

Section: Rapamycin-induced Fkbp-frb Heterodimerization Is Feasible In Plantamentioning

confidence: 99%

“…This type of 'drag-away' technique has already been explored for PPIs in plant research. For instance, the Nuclear Translocation Assay (NTA), the Cytoskeleton-based localization Assay for Protein-Protein Interaction (CAPPI), and the Modification of Intracellular Localization (MILo) function via a similar approach (Dixon and Lim, 2010;Kaplan--Levy et al, 2014;Lv et al, 2017). Nevertheless, the above tools are not conditional, and their subcellular targeting options reported so far are limited.…”

Section: Introductionmentioning

confidence: 99%

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Visualizing protein–protein interactions in plants by rapamycin-dependent delocalization

et al. 2021

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Identifying protein-protein interactions (PPI) is crucial for understanding biological processes. Many PPI tools are available, yet only some function within the context of a plant cell. Narrowing down even further, only a few tools allow complex multi-protein interactions to be visualized. Here, we present a conditional in vivo PPI tool for plant research that meets these criteria. Knocksideways in plants (KSP) is based on the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations of other PPI systems. This in vivo tool does not require spatial proximity of the bait and prey fluorophores and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore the visualization of the proteins in the absence of rapamycin acts as an internal control. We used KSP to confirm previously identified interactions in Nicotiana benthamiana leaf epidermal cells. Furthermore, the scripts that we generated allow the interactions to be quantified at high throughput. Finally, we demonstrate that KSP can easily be used to visualize complex multi-protein interactions. KSP is therefore a versatile tool with unique characteristics and applications that complements other plant PPI methods.

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Section: Rapamycin-induced Fkbp-frb Heterodimerization Is Feasible In Plantamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Visualizing protein–protein interactions in plants by rapamycin-dependent delocalization

et al. 2021

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“…The readout of these colocalization assays is usually the translocation of one protein upon its association with a second distinctly localized protein (e.g., localization to a membrane, the nucleus, or granules). “Cytoskeleton-based assay for protein-protein interaction” (CAPPI), “membrane recruitment assay” (MeRA), and “knocksideways in plants” (KSP) are powerful translocation assays developed for use with plant cells (Grefen et al, 2008; Lv et al, 2017; Winkler et al, 2021). “Nuclear translocation assay” (NTA), “emerging circle of interactive proteins at specific endosomes” (ECLIPSE), and “protein interactions from imaging of complexes after translocation” (PICT) are assays, which require the addition of a compound (e.g., rapamycin) to monitor the translocation upon PPI (Dixon and Lim, 2010; Gallego et al, 2013; Lee et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

ReLo is a simple and quick colocalization assay to identify and characterize direct protein-protein interactions

Salgania

Metz

Jeske

2022

Preprint

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Characterizing protein-protein interactions (PPIs) is fundamental for understanding biochemical processes. Many methods have been established to identify and study direct PPIs; however, screening and investigating PPIs involving large and poorly soluble proteins remain challenges. As a result, we developed ReLo, a simple cell culture-based method to detect and investigate interactions in a cellular context. ReLo allows both the identification of protein domains that mediate the formation of complexes and screening of interfering point mutations. ReLo is sensitive to drugs that mediate or interfere with a specific interaction. Furthermore, protein conformation- and protein arginine methylation-dependent interactions can be studied. Importantly, ReLo can be used for specifically detecting direct but not indirect PPIs and can be applied for describing the binding topology of subunits within multiprotein complexes. Because of these attributes, ReLo is a simple, quick and versatile tool for identifying and studying binary PPIs, as well as for characterizing multisubunit complexes.

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“…The successful applications of this method to cytosolic proteins Atg8 and Atg8-interacting proteins, transmembrane proteins Atg9 and Ctl1, nuclear proteins Xrc4 and Lig4, components of the Atg1 complex, and components of the two PtdIns3K complexes demonstrate that the Pil1 co-tethering assay is an effective tool that can be broadly used to detect protein-protein interactions. In other organisms, imaging-based colocalization assays similar in principle but different in design have been used for the detection of binary protein-protein interactions (Blanchard et al, 2006;Gallego et al, 2013;Herce et al, 2013;Lv et al, 2017;Miller et al, 2007;Yurlova et al, 2014;Zolghadr et al, 2008). In this study, we expanded the applications of this class of assays to the detection of ternary and quaternary protein-protein interactions.…”

Section: Discussionmentioning

confidence: 99%

Visual detection of binary, ternary, and quaternary protein-protein interactions in fission yeast by Pil1 co-tethering assay

Liu

Zhao

et al. 2021

Preprint

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Protein-protein interactions are vital for executing nearly all cellular processes. To facilitate the detection of protein-protein interactions in living cells of the fission yeast Schizosaccharomyces pombe, here we present an efficient and convenient method termed the Pil1 co-tethering assay. In its basic form, we tether a bait protein to mCherry-tagged Pil1, which forms cortical filamentary structures, and examine whether a GFP-tagged prey protein colocalizes with the bait. We demonstrate that this assay is capable of detecting pairwise protein-protein interactions of cytosolic proteins, transmembrane proteins, and nuclear proteins. Furthermore, we show that this assay can be used for detecting not only binary protein-protein interactions, but also ternary and quaternary protein-protein interactions. Using this assay, we systematically characterized the protein-protein interactions in the Atg1 complex and in the phosphatidylinositol 3-kinase (PtdIns3K) complexes and found that Atg38 is incorporated into the PtdIns3K complex I via an Atg38-Vps34 interaction. Our data show that this assay is a useful and versatile tool and should be added to the routine toolbox of fission yeast researchers.

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CAPPI: A Cytoskeleton-Based Localization Assay Reports Protein-Protein Interaction in Living Cells by Fluorescence Microscopy

Cited by 11 publications

References 10 publications

Visualizing protein–protein interactions in plants by rapamycin-dependent delocalization

Visualizing protein–protein interactions in plants by rapamycin-dependent delocalization

ReLo is a simple and quick colocalization assay to identify and characterize direct protein-protein interactions

Visual detection of binary, ternary, and quaternary protein-protein interactions in fission yeast by Pil1 co-tethering assay

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