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Transduction of the MPG-tagged fusion protein into mammalian cells and oocytes depends on amiloride-sensitive endocytic pathway.

Kwon SJ, Han K, Jung S, Lee JE, Park S, Cheon YP, Lim HJ - BMC Biotechnol. (2009)

Bottom Line: The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-beta-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction.Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP.MPG-EGFP transduction is also observed in the mammalian oocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Science & Technology IBST Konkuk University 1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, Korea. ksjpo47@hanmail.net

ABSTRACT

Background: MPG is a cell-permeable peptide with proven efficiency to deliver macromolecular cargoes into cells. In this work, we examined the efficacy of MPG as an N-terminal tag in a fusion protein to deliver a protein cargo and its mechanism of transduction.

Results: We examined transduction of MPG-EGFP fusion protein by live imaging, flow cytometry, along with combination of cell biological and pharmacological methods. We show that MPG-EGFP fusion proteins efficiently enter various mammalian cells within a few minutes and are co-localized with FM4-64, a general marker of endosomes. The transduction of MPG-EGFP occurs rapidly and is inhibited at a low temperature. The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-beta-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction. Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP. The partial blockade of MPG-EGFP transduction by a dynamin mutant is abolished by the treatment of amiloride. MPG-EGFP transduction is also observed in the mammalian oocytes.

Conclusion: The results show that the transduction of MPG fusion protein utilizes endocytic pathway(s) which is amiloride-sensitive and partially dynamin-dependent. Collectively, the MPG fusion protein could be further developed as a novel tool of "protein therapeutics", with potentials to be used in various cell systems including mammalian oocytes.

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Chloroquine effectively blocks the endolysosomal pathway of transduced MPG-EGFP. A. 100 μM chloroquine was added along with 40 μg/ml MPG-EGFP for 17 hr. Note the increased intensity of MPG-EGFP signal and enlarged vesicle. Confocal live images are shown at 80X. B. Vesicle fractionation was performed using HeLa cells treated with MPG-EGFP and chloroquine for 17 hr. Note that the vesicular uptake of MPG-EGFP intensified in the presence of chloroquine, and that the cytosolic fraction also contains a significant amount of MPG-EGFP. EGFP, 40 μg/ml EGFP; MPG-EGFP, 40 μg/ml MPG-EGFP; Chl 50, MPG-EGFP plus 50 μM chloroquine; Chl 100, MPG-EGFP plus 100 μM chloroquine; S, supernatant containing the cytosolic fraction; P, pellet containing the intracellular vesicles. The asterisk indicates that the band is a merged doublet due to the prolonged exposure, and the arrow points the expected size of MPG-EGFP. Western blotting was performed with anti-GFP antibody.
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Figure 6: Chloroquine effectively blocks the endolysosomal pathway of transduced MPG-EGFP. A. 100 μM chloroquine was added along with 40 μg/ml MPG-EGFP for 17 hr. Note the increased intensity of MPG-EGFP signal and enlarged vesicle. Confocal live images are shown at 80X. B. Vesicle fractionation was performed using HeLa cells treated with MPG-EGFP and chloroquine for 17 hr. Note that the vesicular uptake of MPG-EGFP intensified in the presence of chloroquine, and that the cytosolic fraction also contains a significant amount of MPG-EGFP. EGFP, 40 μg/ml EGFP; MPG-EGFP, 40 μg/ml MPG-EGFP; Chl 50, MPG-EGFP plus 50 μM chloroquine; Chl 100, MPG-EGFP plus 100 μM chloroquine; S, supernatant containing the cytosolic fraction; P, pellet containing the intracellular vesicles. The asterisk indicates that the band is a merged doublet due to the prolonged exposure, and the arrow points the expected size of MPG-EGFP. Western blotting was performed with anti-GFP antibody.

Mentions: Chloroquine is an inhibitor which blocks the lysosomal pathway of protein degradation [32]. We co-treated chloroquine and MPG-EGFP to HeLa cells for 17 hr to examine if the blockade of the endolysosomal pathway leads to the accumulation of the transduced MPG fusion protein. As shown in Figure 6A, chloroquine-treated cells show enlarged endosomes containing MPG-EGFP. Number of MPG-EGFP-containing vesicles seems to have increased as well. This observation was further explored by the vesicle fractionation experiment. Prolonged treatment of MPG-EGFP showed a lower band representing degradation products. Chloroquine treatment increased the intensity of MPG-EGFP band in the particulate fraction and, notably, the supernatant fraction in 100 μM chloroquine-treated cells contains a much higher level of MPG-EGFP than that in control cells. Collectively, the result shows that the blockade of the lysosomal pathway would increase the transduction efficiency and also lead to the increased release of MPG fusion proteins into the cytosol from the endosomes.


Transduction of the MPG-tagged fusion protein into mammalian cells and oocytes depends on amiloride-sensitive endocytic pathway.

Kwon SJ, Han K, Jung S, Lee JE, Park S, Cheon YP, Lim HJ - BMC Biotechnol. (2009)

Chloroquine effectively blocks the endolysosomal pathway of transduced MPG-EGFP. A. 100 μM chloroquine was added along with 40 μg/ml MPG-EGFP for 17 hr. Note the increased intensity of MPG-EGFP signal and enlarged vesicle. Confocal live images are shown at 80X. B. Vesicle fractionation was performed using HeLa cells treated with MPG-EGFP and chloroquine for 17 hr. Note that the vesicular uptake of MPG-EGFP intensified in the presence of chloroquine, and that the cytosolic fraction also contains a significant amount of MPG-EGFP. EGFP, 40 μg/ml EGFP; MPG-EGFP, 40 μg/ml MPG-EGFP; Chl 50, MPG-EGFP plus 50 μM chloroquine; Chl 100, MPG-EGFP plus 100 μM chloroquine; S, supernatant containing the cytosolic fraction; P, pellet containing the intracellular vesicles. The asterisk indicates that the band is a merged doublet due to the prolonged exposure, and the arrow points the expected size of MPG-EGFP. Western blotting was performed with anti-GFP antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748071&req=5

Figure 6: Chloroquine effectively blocks the endolysosomal pathway of transduced MPG-EGFP. A. 100 μM chloroquine was added along with 40 μg/ml MPG-EGFP for 17 hr. Note the increased intensity of MPG-EGFP signal and enlarged vesicle. Confocal live images are shown at 80X. B. Vesicle fractionation was performed using HeLa cells treated with MPG-EGFP and chloroquine for 17 hr. Note that the vesicular uptake of MPG-EGFP intensified in the presence of chloroquine, and that the cytosolic fraction also contains a significant amount of MPG-EGFP. EGFP, 40 μg/ml EGFP; MPG-EGFP, 40 μg/ml MPG-EGFP; Chl 50, MPG-EGFP plus 50 μM chloroquine; Chl 100, MPG-EGFP plus 100 μM chloroquine; S, supernatant containing the cytosolic fraction; P, pellet containing the intracellular vesicles. The asterisk indicates that the band is a merged doublet due to the prolonged exposure, and the arrow points the expected size of MPG-EGFP. Western blotting was performed with anti-GFP antibody.
Mentions: Chloroquine is an inhibitor which blocks the lysosomal pathway of protein degradation [32]. We co-treated chloroquine and MPG-EGFP to HeLa cells for 17 hr to examine if the blockade of the endolysosomal pathway leads to the accumulation of the transduced MPG fusion protein. As shown in Figure 6A, chloroquine-treated cells show enlarged endosomes containing MPG-EGFP. Number of MPG-EGFP-containing vesicles seems to have increased as well. This observation was further explored by the vesicle fractionation experiment. Prolonged treatment of MPG-EGFP showed a lower band representing degradation products. Chloroquine treatment increased the intensity of MPG-EGFP band in the particulate fraction and, notably, the supernatant fraction in 100 μM chloroquine-treated cells contains a much higher level of MPG-EGFP than that in control cells. Collectively, the result shows that the blockade of the lysosomal pathway would increase the transduction efficiency and also lead to the increased release of MPG fusion proteins into the cytosol from the endosomes.

Bottom Line: The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-beta-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction.Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP.MPG-EGFP transduction is also observed in the mammalian oocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Science & Technology IBST Konkuk University 1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, Korea. ksjpo47@hanmail.net

ABSTRACT

Background: MPG is a cell-permeable peptide with proven efficiency to deliver macromolecular cargoes into cells. In this work, we examined the efficacy of MPG as an N-terminal tag in a fusion protein to deliver a protein cargo and its mechanism of transduction.

Results: We examined transduction of MPG-EGFP fusion protein by live imaging, flow cytometry, along with combination of cell biological and pharmacological methods. We show that MPG-EGFP fusion proteins efficiently enter various mammalian cells within a few minutes and are co-localized with FM4-64, a general marker of endosomes. The transduction of MPG-EGFP occurs rapidly and is inhibited at a low temperature. The entry of MPG-EGFP is inhibited by amiloride, but cytochalasin D and methyl-beta-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction. Overexpression of a mutant form of dynamin partially reduced the transduction of MPG-EGFP. The partial blockade of MPG-EGFP transduction by a dynamin mutant is abolished by the treatment of amiloride. MPG-EGFP transduction is also observed in the mammalian oocytes.

Conclusion: The results show that the transduction of MPG fusion protein utilizes endocytic pathway(s) which is amiloride-sensitive and partially dynamin-dependent. Collectively, the MPG fusion protein could be further developed as a novel tool of "protein therapeutics", with potentials to be used in various cell systems including mammalian oocytes.

Show MeSH
Related in: MedlinePlus