Labeling of Cytoskeletal Proteins in Living Cells Using Biotin Ligase Carrying a Fluorescent Protein

Nishi, Sairi; Yamamoto, Chihiro; Yoneda, Sawako; Sueda, Shinji

doi:10.2116/analsci.33.897

Cited by 7 publications

(5 citation statements)

References 20 publications

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“…11,12 By taking advantage of this unique property, we previously constructed fluorescent labeling systems for proteins in the living cells, in which the target proteins fused to BCCP are labeled by BPL carrying fluorophores. 13,14 In the present work, BPL was expressed as a fusion protein with a single transmembrane domain of the human platelet-derived growth factor receptor (TM) in living cells. BPL was attached to the C-terminus of the TM (Figure 1a), and in the cells expressing the resulting fusion protein, i.e., TM-BPL, the BPL moiety should be displayed on the surface of the membrane facing cytoplasm or nucleoplasm.…”

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confidence: 99%

“…In our method, the NE is labeled by localizing GFP on the INM based on a unique biotinylation reaction from the archaeon Sulfolobus tokodaii (Figure ). In biotinylation, biotin protein ligase (BPL) mediates the attachment of biotin to the specific lysine residue of its substrate protein, biotin carboxyl carrier protein (BCCP). , Biotinylation from S. tokodaii has a unique property in which BPL forms a stable complex with its product, the biotinylated BCCP. , By taking advantage of this unique property, we previously constructed fluorescent labeling systems for proteins in the living cells, in which the target proteins fused to BCCP are labeled by BPL carrying fluorophores. , In the present work, BPL was expressed as a fusion protein with a single transmembrane domain of the human platelet-derived growth factor receptor (TM) in living cells. BPL was attached to the C-terminus of the TM (Figure a), and in the cells expressing the resulting fusion protein, i.e., TM-BPL, the BPL moiety should be displayed on the surface of the membrane facing cytoplasm or nucleoplasm.…”

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confidence: 99%

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Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane

2018

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The nuclear envelope (NE) is a double membrane that segregates nuclear components from the cytoplasm in eukaryotic cells. It is well-known that the NE undergoes a breakdown and reformation during mitosis in animal cells. However, the detailed mechanisms of the NE dynamics are not yet fully understood. Here, we propose a method for the fluorescent labeling of the NE in living cells, which enables the tracing of the NE dynamics during cell division under physiological conditions. In our method, labeling of the NE is accomplished by fixing green fluorescent protein carrying the nuclear localization signal on the inner nuclear membrane based on a unique biotinylation reaction from the archaeon Sulfolobus tokodaii. With this method, we observed HeLa cells during mitosis by confocal laser scanning microscopy and succeeded in clearly visualizing the difference in the timing of the formation of the NE and the nuclear lamina.

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Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane

2018

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“…To observe detailed actin movement, we used structured illumination microscopy (SIM), which allowed us to observe it with high spatial resolution. 32 Additionally, in order to visualize actin dynamics, we used mCherry-tagged lifeact that is comprised of 17-amino-acid peptide, and specifically binds to filamentous actin structures 33,34 (Fig. 4A).…”

Section: Resultsmentioning

confidence: 99%

Membrane Dynamics Induced by a Phosphatidylinositol 3,4,5-Trisphosphate Optogenetic Tool

et al. 2018

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Membrane dynamic structures such as filopodia, lamellipodia, and ruffles have important cellular functions in phagocytosis and cell motility, and in pathological states, such as cancer metastasis. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) is a crucial lipid that regulates PIP3 dynamics. Investigations of how PIP3 is involved in these functions have mainly relied on pharmacological interventions, and therefore have not generated detailed spatiotemporal information concerning membrane dynamics upon PIP3 production. In the present study, we applied an optogenetic approach using the CRY2-CIBN system. Using this system, we revealed that local PIP3 generation induced directional cell motility and membrane ruffles in COS7 cells. Furthermore, combined with structured illumination microscopy (SIM), membrane dynamics were investigated with high spatial resolution. We observed PIP3-induced apical ruffles and unique actin fiber behavior in that a single actin fiber protruded from the plasma membrane was taken up into the plasma membrane without depolymerization. This system has the potential to investigate other high-level cell motility and dynamic behaviors, such as cancer cell invasion and wound healing with high spatiotemporal resolution, and could provide new insights of biological sciences for membrane dynamics.

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“…From a mechanistic perspective, hydrodynamic injections of 0.5 mL of tolonium chloride solutions revealed robust distributions of this pigment across the injected kidney and no visible traces within the right contralateral kidney. Additionally, the localization of the large molecular weight dextran molecules within the damaged tubular epithelium, illustrated the potential to facilitate the cellular internalization of exogenous plasmids of comparable size using this technique ( Corridon et al, 2013 ; Nishi et al, 2017 ). These macroscopic and microscopic evaluations provided sufficient evidence that the injection process could facilitate widespread and targeted delivery of the vector to the kidney.…”

Section: Discussionmentioning

confidence: 99%

Enhancing the expression of a key mitochondrial enzyme at the inception of ischemia-reperfusion injury can boost recovery and halt the progression of acute kidney injury

Corridon

2023

Front. Physiol.

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Hydrodynamic fluid delivery has shown promise in influencing renal function in disease models. This technique provided pre-conditioned protection in acute injury models by upregulating the mitochondrial adaptation, while hydrodynamic injections of saline alone have improved microvascular perfusion. Accordingly, hydrodynamic mitochondrial gene delivery was applied to investigate the ability to halt progressive or persistent renal function impairment following episodes of ischemia-reperfusion injuries known to induce acute kidney injury (AKI). The rate of transgene expression was approximately 33% and 30% in rats with prerenal AKI that received treatments 1 (T1hr) and 24 (T24hr) hours after the injury was established, respectively. The resulting mitochondrial adaptation via exogenous IDH2 (isocitrate dehydrogenase 2 (NADP+) and mitochondrial) significantly blunted the effects of injury within 24 h of administration: decreased serum creatinine (≈60%, p < 0.05 at T1hr; ≈50%, p < 0.05 at T24hr) and blood urea nitrogen (≈50%, p < 0.05 at T1hr; ≈35%, p < 0.05 at T24hr) levels, and increased urine output (≈40%, p < 0.05 at T1hr; ≈26%, p < 0.05 at T24hr) and mitochondrial membrane potential, Δψm, (≈ by a factor of 13, p < 0.001 at T1hr; ≈ by a factor of 11, p < 0.001 at T24hr), despite elevated histology injury score (26%, p < 0.05 at T1hr; 47%, p < 0.05 at T24hr). Therefore, this study identifies an approach that can boost recovery and halt the progression of AKI at its inception.

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Labeling of Cytoskeletal Proteins in Living Cells Using Biotin Ligase Carrying a Fluorescent Protein

Cited by 7 publications

References 20 publications

Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane

Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane

Membrane Dynamics Induced by a Phosphatidylinositol 3,4,5-Trisphosphate Optogenetic Tool

Enhancing the expression of a key mitochondrial enzyme at the inception of ischemia-reperfusion injury can boost recovery and halt the progression of acute kidney injury

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