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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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Transduction of MPG-EGFP is dependent on an amiloride-sensitive endocytic pathway. A. Flow cytometric analysis of the MPG-EGFP uptake in the presence of various endocytosis inhibitors. The following concentrations were used: 10 μM cytochalasin D, 4 mM amiloride, and 5 mM MβCD. An inhibitor or DMSO (vehicle, 0.2%) was added to HeLa cells 30 min prior to the addition of MPG-EGFP, except for amiloride, which was added 10 min or 30 min prior to the addition of MPG-EGFP. MPG-EGFP at 40 μg/ml was treated to cells for 30 min in the presence of an inhibitor (see the diagram in B.). All cells were washed with the acid buffer before the analysis. Note the decreased MPG-EGFP signal in the amiloride-treated cells. MβCD treatment increased the intensity of EGFP signal significantly. These experiments were repeated at least three times with similar results and one representative set of data is shown. B. Confocal live images of HeLa cells treated with an endocytosis inhibitor as indicated. Photomicrographs were taken at 40X and zoomed in 2X. C. A barogram showing the averaged transduction efficiencies in the presence of various inhibitors. Errors bars represent the standard deviations. C, no protein added; EGFP, 40 μg/ml EGFP protein; MPG-EGFP, 40 μg/ml MPG-EGFP; -, no drug added; D, 0.2% DMSO (vehicle); A10, 4 mM amiloride for 10 min; A30, 4 mM amiloride for 30 min; C10, 10 μM cytochalasin D; M5, 5 mM MβCD.
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Figure 3: Transduction of MPG-EGFP is dependent on an amiloride-sensitive endocytic pathway. A. Flow cytometric analysis of the MPG-EGFP uptake in the presence of various endocytosis inhibitors. The following concentrations were used: 10 μM cytochalasin D, 4 mM amiloride, and 5 mM MβCD. An inhibitor or DMSO (vehicle, 0.2%) was added to HeLa cells 30 min prior to the addition of MPG-EGFP, except for amiloride, which was added 10 min or 30 min prior to the addition of MPG-EGFP. MPG-EGFP at 40 μg/ml was treated to cells for 30 min in the presence of an inhibitor (see the diagram in B.). All cells were washed with the acid buffer before the analysis. Note the decreased MPG-EGFP signal in the amiloride-treated cells. MβCD treatment increased the intensity of EGFP signal significantly. These experiments were repeated at least three times with similar results and one representative set of data is shown. B. Confocal live images of HeLa cells treated with an endocytosis inhibitor as indicated. Photomicrographs were taken at 40X and zoomed in 2X. C. A barogram showing the averaged transduction efficiencies in the presence of various inhibitors. Errors bars represent the standard deviations. C, no protein added; EGFP, 40 μg/ml EGFP protein; MPG-EGFP, 40 μg/ml MPG-EGFP; -, no drug added; D, 0.2% DMSO (vehicle); A10, 4 mM amiloride for 10 min; A30, 4 mM amiloride for 30 min; C10, 10 μM cytochalasin D; M5, 5 mM MβCD.

Mentions: Endocytosis occurs in cells using multiple pathways, such as clathrin-mediated, caveolae-mediated, lipid rafts-mediated endocytosis, and macropinocytosis [23]. By using various inhibitors of endocytosis, we determined which endocytic pathway is associated with MPG-EGFP uptake. Cytochalasin D, an inhibitor of actin polymerization, blocks actin-dependent macropinocytosis. Amiloride is a sodium channel blocker and is known to block macropinocytosis [24]. Inhibitors were added to the cells 30 min prior to the addition of MPG-EGFP and were maintained at the same concentrations for the duration of the experiments (Figure 3B). When cytochalasin D was added prior to MPG-EGFP treatment, most of cells became round due to the depolymerization of actin filaments but the distinct vesicular pattern of MPG-EGFP remained unchanged. In contrast, 4 mM amiloride treatment effectively reduced MPG-EGFP uptake as shown in Figure 3A. Methyl-β-cyclodextrin (MβCD) depletes cholesterol from the plasma membrane, and is used as an inhibitor of the caveolae-mediated endocytosis and macropinocytosis [25]. Both of these pathways are known to be dependent on cholesterol-rich lipid rafts. Surprisingly, MβCD treatment significantly increased the fluorescence intensity of MPG-EGFP (Figure 3B). Collectively, these results show that the uptake of MPG-EGFP seems to utilize amiloride-sensitive endocytic pathway, but is not dependent on the organization of actin filaments or cholesterol. These experiments were repeated several times with similar results (Figure 3C).


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)

Transduction of MPG-EGFP is dependent on an amiloride-sensitive endocytic pathway. A. Flow cytometric analysis of the MPG-EGFP uptake in the presence of various endocytosis inhibitors. The following concentrations were used: 10 μM cytochalasin D, 4 mM amiloride, and 5 mM MβCD. An inhibitor or DMSO (vehicle, 0.2%) was added to HeLa cells 30 min prior to the addition of MPG-EGFP, except for amiloride, which was added 10 min or 30 min prior to the addition of MPG-EGFP. MPG-EGFP at 40 μg/ml was treated to cells for 30 min in the presence of an inhibitor (see the diagram in B.). All cells were washed with the acid buffer before the analysis. Note the decreased MPG-EGFP signal in the amiloride-treated cells. MβCD treatment increased the intensity of EGFP signal significantly. These experiments were repeated at least three times with similar results and one representative set of data is shown. B. Confocal live images of HeLa cells treated with an endocytosis inhibitor as indicated. Photomicrographs were taken at 40X and zoomed in 2X. C. A barogram showing the averaged transduction efficiencies in the presence of various inhibitors. Errors bars represent the standard deviations. C, no protein added; EGFP, 40 μg/ml EGFP protein; MPG-EGFP, 40 μg/ml MPG-EGFP; -, no drug added; D, 0.2% DMSO (vehicle); A10, 4 mM amiloride for 10 min; A30, 4 mM amiloride for 30 min; C10, 10 μM cytochalasin D; M5, 5 mM MβCD.
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Figure 3: Transduction of MPG-EGFP is dependent on an amiloride-sensitive endocytic pathway. A. Flow cytometric analysis of the MPG-EGFP uptake in the presence of various endocytosis inhibitors. The following concentrations were used: 10 μM cytochalasin D, 4 mM amiloride, and 5 mM MβCD. An inhibitor or DMSO (vehicle, 0.2%) was added to HeLa cells 30 min prior to the addition of MPG-EGFP, except for amiloride, which was added 10 min or 30 min prior to the addition of MPG-EGFP. MPG-EGFP at 40 μg/ml was treated to cells for 30 min in the presence of an inhibitor (see the diagram in B.). All cells were washed with the acid buffer before the analysis. Note the decreased MPG-EGFP signal in the amiloride-treated cells. MβCD treatment increased the intensity of EGFP signal significantly. These experiments were repeated at least three times with similar results and one representative set of data is shown. B. Confocal live images of HeLa cells treated with an endocytosis inhibitor as indicated. Photomicrographs were taken at 40X and zoomed in 2X. C. A barogram showing the averaged transduction efficiencies in the presence of various inhibitors. Errors bars represent the standard deviations. C, no protein added; EGFP, 40 μg/ml EGFP protein; MPG-EGFP, 40 μg/ml MPG-EGFP; -, no drug added; D, 0.2% DMSO (vehicle); A10, 4 mM amiloride for 10 min; A30, 4 mM amiloride for 30 min; C10, 10 μM cytochalasin D; M5, 5 mM MβCD.
Mentions: Endocytosis occurs in cells using multiple pathways, such as clathrin-mediated, caveolae-mediated, lipid rafts-mediated endocytosis, and macropinocytosis [23]. By using various inhibitors of endocytosis, we determined which endocytic pathway is associated with MPG-EGFP uptake. Cytochalasin D, an inhibitor of actin polymerization, blocks actin-dependent macropinocytosis. Amiloride is a sodium channel blocker and is known to block macropinocytosis [24]. Inhibitors were added to the cells 30 min prior to the addition of MPG-EGFP and were maintained at the same concentrations for the duration of the experiments (Figure 3B). When cytochalasin D was added prior to MPG-EGFP treatment, most of cells became round due to the depolymerization of actin filaments but the distinct vesicular pattern of MPG-EGFP remained unchanged. In contrast, 4 mM amiloride treatment effectively reduced MPG-EGFP uptake as shown in Figure 3A. Methyl-β-cyclodextrin (MβCD) depletes cholesterol from the plasma membrane, and is used as an inhibitor of the caveolae-mediated endocytosis and macropinocytosis [25]. Both of these pathways are known to be dependent on cholesterol-rich lipid rafts. Surprisingly, MβCD treatment significantly increased the fluorescence intensity of MPG-EGFP (Figure 3B). Collectively, these results show that the uptake of MPG-EGFP seems to utilize amiloride-sensitive endocytic pathway, but is not dependent on the organization of actin filaments or cholesterol. These experiments were repeated several times with similar results (Figure 3C).

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