Recombination-induced tag exchange to track old and new proteins

Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2009; 107(1):64-8. DOI: 10.1073/pnas.0911164107
Source: PubMed


The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequence after a hormone-induced activation of Cre recombinase. The time-controlled tag switch provides a unique ability to detect and separate old and new proteins in time and space, which opens up opportunities to investigate the dynamic behavior of proteins. We validated the technology by determining exchange of endogenous histones in chromatin by biochemical methods and by visualizing and quantifying replacement of old by new proteasomes in single cells by microscopy. RITE is widely applicable and allows probing spatiotemporal changes in protein properties by multiple methods.

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    • "Leeuwen, Netherlands Cancer Institute, Amsterdam, Netherlands; Verzijlbergen et al., 2010) were sequentially replaced by mCherry and GFP genes, respectively . ORFs encoding NSP1 and NIC96 with their cognate promoters and 3 UTRs were inserted into pRS416 (Sikorski and Hieter, 1989) to generate PLPC19 and PLPC20. "
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    ABSTRACT: Nuclear pore complexes (NPCs) are essential protein assemblies that span the nuclear envelope and establish nuclear-cytoplasmic compartmentalization. We have investigated mechanisms that control NPC number in mother and daughter cells during the asymmetric division of budding yeast. By simultaneously tracking existing NPCs and newly synthesized NPC protomers (nups) through anaphase, we uncovered a pool of the central channel nup Nsp1 that is actively targeted to the bud in association with endoplasmic reticulum. Bud targeting required an intact actin cytoskeleton and the class V myosin, Myo2. Selective inhibition of cytoplasmic Nsp1 or inactivation of Myo2 reduced the inheritance of NPCs in daughter cells, leading to a daughter-specific loss of viability. Our data are consistent with a model in which Nsp1 releases a barrier that otherwise prevents NPC passage through the bud neck. It further supports the finding that NPC inheritance, not de novo NPC assembly, is primarily responsible for controlling NPC number in daughter cells.
    The Journal of Cell Biology 10/2013; 203(2):215-32. DOI:10.1083/jcb.201305115 · 9.83 Impact Factor
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    • "Thereby, RITE can be combined with proteomics methods, genomics methods, or DNA-based highthroughput screens (Verzijlbergen et al. 2010; De Vos et al. 2011; Radman-Livaja et al. 2011; Verzijlbergen et al. 2011). The fluorescent tags in the RITE cassettes can be applied to measure protein dynamics in single cells by live imaging (Verzijlbergen et al. 2010; Hotz et al. 2012; Menendez-Benito et al. 2013) and further expanded toward high-throughput genetic screening. The suite of RITE cassettes enables a flexible design for many applications. "
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    ABSTRACT: Proteins are not static entities. They are highly mobile and their steady state levels are achieved by a balance between ongoing synthesis and degradation. The dynamic properties of a protein can have important consequences for its function. For example, when a protein is degraded and replaced by a newly synthesized one, post-translational modifications are lost and need to be reincorporated in the new molecules. Protein stability and mobility are also relevant for duplication of macromolecular structures or organelles, which involves coordination of protein inheritance with the synthesis and assembly of newly synthesized proteins. To measure protein dynamics we recently developed a genetic pulse-chase assay called Recombination-Induced Tag Exchange (RITE). RITE has been successfully used in Saccharomyces cerevisiae to measure turnover and inheritance of histone proteins, to study changes in post-translational modifications on aging proteins, and to visualize the spatiotemporal inheritance of protein complexes and organelles in dividing cells. Here we describe a series of successful RITE cassettes that are designed for biochemical analyses, genomics studies, as well as single cell fluorescence applications. Importantly, the genetic nature and the stability of the tag-switch offer the unique possibility to combine RITE with high-throughput screening for protein dynamics mutants and mechanisms. The RITE cassettes are widely applicable, modular by design, and can therefore be easily adapted for use in other cell types or organisms.
    G3-Genes Genomes Genetics 05/2013; 3(8). DOI:10.1534/g3.113.006213 · 3.20 Impact Factor
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    • "Based on existing knowledge and reagents, we developed a dual-color histone labeling system, similar to a previously reported strategy (Verzijlbergen et al. 2010). Using this system, preexisting histones can be precisely distinguished from newly synthesized histones because an irreversible DNA recombination induced the old-to-new histone switch (Tran et al. 2012). "
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    ABSTRACT: It has long been known that epigenetic changes are inheritable. However, except for DNA methylation, little is known about the molecular mechanisms of epigenetic inheritance. Many types of stem cells undergo asymmetric cell divisions to generate self-renewed stem cells and daughter cells committed for differentiation. Still, whether and how stem cells retain their epigenetic memory remain questions to be elucidated. During the asymmetric division of Drosophila male germline stem cell (GSC), our recent studies revealed that the preexisting histone 3 (H3) are selectively segregated to the GSC, whereas newly synthesized H3 deposited during DNA replication are enriched in the differentiating daughter cell. We propose a two-step model to explain this asymmetric histone distribution. First, prior to mitosis, preexisting histones and newly synthesized histones are differentially distributed at two sets of sister chromatids. Next, during mitosis, the set of sister chromatids that mainly consist of preexisting histones are segregated to GSCs, while the other set of sister chromatids enriched with newly synthesized histones are partitioned to the daughter cell committed for differentiation. In this review, we apply current knowledge about epigenetic inheritance and asymmetric cell division to inform our discussion of potential molecular mechanisms and the cellular basis underlying this asymmetric histone distribution pattern. We will also discuss whether this phenomenon contributes to the maintenance of stem cell identity and resetting chromatin structure in the other daughter cell for differentiation.
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