Exploration of New Chromophore Structures Leads to the Identification of Improved Blue Fluorescent Proteins

Ai, Hui‐wang; Shaner, Nathan C.; Cheng, Zihao; Tsien, Roger Y.; Campbell, Robert E.

doi:10.1021/bi700199g

Cited by 291 publications

(270 citation statements)

References 23 publications

Supporting

Mentioning

267

Contrasting

Order By: Relevance

“…EBFP2-parkin was described previously 27 . EBFP2-PMP34 and EGFP2-p62 were constructed through PCR amplification of human genes from HeLa cell total cDNA, followed by its insertion into the EBFP2-Nuc (Addgene plasmid 14893) 28 and EGFP-C1 vectors. EGFP-vkskl plasmid was constructed through appending the DNA sequence encoding amino acids VKSKL to the EGFP-C1 plasmid.…”

Section: Methodsmentioning

confidence: 99%

Spatiotemporally controlled induction of autophagy-mediated lysosome turnover

et al. 2013

View full text Add to dashboard Cite

Lysosomes are the major degradative compartments within cells, harbouring a wide variety of hydrolytic enzymes within their lumen. Release of lysosomal hydrolases from lysosomes into the cell cytoplasm results in cell death. Here we report that damaged lysosomes undergo autophagic turnover. Using a light-induced lysosome impairing scheme that can be controlled spatially and temporally within a cell, we show that damaged lysosomes are selectively ubiquitinated, recruit autophagic proteins and are eventually incorporated into autolysosomes for degradation. We propose that autophagic removal of lysosomes, which we term lysophagy, is a surveillance mechanism that alleviates cells from the adverse effects of lysosomal damage. We envision our method to induce lysosomal damage will enable detailed molecular studies of the lysophagy pathway in the future.

show abstract

Section: Methodsmentioning

confidence: 99%

Spatiotemporally controlled induction of autophagy-mediated lysosome turnover

et al. 2013

View full text Add to dashboard Cite

show abstract

“…First, we added a small nuclear localization signal (NLS) from simian virus large T-antigen (Ai et al, 2007) at the C terminus of the PWL2:mCherry fusion (PWL2:mCherry:NLS, 44.5 kD); second, we added histone H1 (hH1) from Neurospora crassa as a nuclear targeting sequence between PWL2 and mCherry (PWL2:hH1:mCherry, 66.1 kD). At successful infection sites with uniform BAS4 outlining, PWL2: mCherry:NLS (n = 41) and PWL2:hH1:mCherry (n = 103) exhibited significant fluorescence in BICs and in nuclei of invaded host cells (Figures 7A, 7B, and 7D).…”

Section: Nuclear Targeting Of Fluorescent Effectors Facilitates Visuamentioning

confidence: 99%

“…Nuclear targeting reporters were constructed by cloning NLS (three tandem repeats of the nuclear localization signal from simian virus large T-antigen) or hH1 (histone H1 from Neurospora crassa) at the C or N terminus of mCherry, respectively. NLS was isolated from pEBFP2-Nuc (Ai et al, 2007) (Addgene plasmid 14893) and hH1 from pAM1293 obtained from Marc Orbach (University of Arizona). All the fusion constructs were cloned in binary vectors pBHt2 (Mullins et al, 2001) or pBGt (S. Kang, unpublished data), and their transcriptional and translational fusions were verified by DNA sequencing.…”

Section: Strains Plasmids and Fungal Transformationmentioning

confidence: 99%

Translocation ofMagnaporthe oryzaeEffectors into Rice Cells and Their Subsequent Cell-to-Cell Movement

et al. 2010

View full text Add to dashboard Cite

Knowledge remains limited about how fungal pathogens that colonize living plant cells translocate effector proteins inside host cells to regulate cellular processes and neutralize defense responses. To cause the globally important rice blast disease, specialized invasive hyphae (IH) invade successive living rice (Oryza sativa) cells while enclosed in host-derived extrainvasive hyphal membrane. Using live-cell imaging, we identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH. In each newly entered rice cell, effectors were first secreted into BICs at the tips of the initially filamentous hyphae in the cell. These tip BICs were left behind beside the first-differentiated bulbous IH cells as the fungus continued to colonize the host cell. Fluorescence recovery after photobleaching experiments showed that the effector protein PWL2 (for prevents pathogenicity toward weeping lovegrass [Eragrostis curvula]) continued to accumulate in BICs after IH were growing elsewhere. PWL2 and BAS1 (for biotrophyassociated secreted protein 1), BIC-localized secreted proteins, were translocated into the rice cytoplasm. By contrast, BAS4, which uniformly outlines the IH, was not translocated into the host cytoplasm. Fluorescent PWL2 and BAS1 proteins that reached the rice cytoplasm moved into uninvaded neighbors, presumably preparing host cells before invasion. We report robust assays for elucidating the molecular mechanisms that underpin effector secretion into BICs, translocation to the rice cytoplasm, and cell-to-cell movement in rice.

show abstract

“…The vectors used express a fluorescent protein under the control of the spleen focus-forming virus (SFFV) promoter. To complement the colors of the vectors LeGO-G2 (expressing EGFP, green) and LeGO-V2 (expressing Venus, yellow), 17 four other fluorescent proteins were cloned: namely, EBFP2 (blue; a gift from Robert Campbell) (plasmid 14891; Addgene), 36 T-Sapphire (violet excitable green; a gift from Oliver Griesbeck), 37 mOrange2 (orange; a gift from Lalita Ramakrishnan) (plasmid 30175; Addgene), 38 and dKatushka2 (red; a gift from Lalita Ramakrishnan) (plasmid 30181; Addgene). 39 The six LeGO vectors used for OBC are shown in Table 1.…”

Section: Lentiviral Vectorsmentioning

confidence: 99%

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

et al. 2017

View full text Add to dashboard Cite

Intratumoral heterogeneity has been identified as one of the strongest drivers of treatment resistance and tumor recurrence. Therefore, investigating the complex clonal architecture of tumors over time has become a major challenge in cancer research. We developed a new fluorescent "optical barcoding" technique that allows fast tracking, identification, and quantification of live cell clones in vitro and in vivo using flow cytometry (FC). We optically barcoded two cell lines derived from malignant glioma, an exemplary heterogeneous brain tumor. In agreement with mathematical combinatorics, we demonstrate that up to 41 clones can unambiguously be marked using six fluorescent proteins and a maximum of three colors per clone. We show that optical barcoding facilitates sensitive, precise, rapid, and inexpensive analysis of clonal composition kinetics of heterogeneous cell populations by FC. We further assessed the quantitative contribution of multiple clones to glioblastoma growth in vivo and we highlight the potential to recover individual viable cell clones by fluorescence-activated cell sorting. In summary, we demonstrate that optical barcoding is a powerful technique for clonal cell tracking in vitro and in vivo, rendering this approach a potent tool for studying the heterogeneity of complex tissues, in particular, cancer.

show abstract

Exploration of New Chromophore Structures Leads to the Identification of Improved Blue Fluorescent Proteins

Cited by 291 publications

References 23 publications

Spatiotemporally controlled induction of autophagy-mediated lysosome turnover

Spatiotemporally controlled induction of autophagy-mediated lysosome turnover

Translocation ofMagnaporthe oryzaeEffectors into Rice Cells and Their Subsequent Cell-to-Cell Movement

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

Contact Info

Product

Resources

About