BACKGROUND
cell-permeable or cell-penetrating peptides  are considered a promising method to deliver macromolecular cargoes into live cells across lipid bilayers.
these peptides commonly bear stretches of basic amino acids.
many cpps have been shown to deliver oligonucleotides or sirna efficiently across cell membrane with or without covalent conjugation  <cit> .
as for the delivery of proteins, expression and purification of cpp-fusion protein in a single step produces more stable form of cargo for in vitro and in vivo uses.
the protein transduction domain  from tat protein of the human immunodeficiency virus , is known to deliver large proteins up to ~ <dig> kda into cells in the form of fusion protein  <cit> .
thus, it has been in the forefront of protein delivery and is being widely used to deliver various proteins for functional experiments  <cit> .
other cpps have been tested for their efficiency for protein delivery by cross-linking, by simple mixing, or as a fusion protein form  <cit> .
while the list of applicable cpps is expanding, recent works have been focused on identifying the mechanisms of cellular uptake of cpps.
these studies generally utilize arrays of endocytosis inhibitors to identify specific endocytic pathway involved in the uptake  <cit> .
these works provided evidence that the uptake of tat ptd, polyarginine, and other peptides is dependent on the lipid rafts-mediated macropinocytosis, "the cell-drinking" process.
mpg is a designed cpp comprised of two independent domains  <cit> .
the first  <dig> amino acids of the n-terminus is derived from glycine-rich region of the viral gp <dig>  <cit>  and the hydrophilic c-terminus from nuclear localization signal  of the sv <dig> large t antigen  <cit> .
the original mpg peptide is acetylated at the n-terminus and synthesized with a cysteamide group at the c-terminus.
this form was used to deliver sirna or oligonucleotide and the effectiveness was proven in several cell systems  <cit> .
a recent report showed that the initial interaction of mpg peptide with the cell surface uses negatively charged glycosaminoglycans.
furthermore, the mechanism of mpg peptide-mediated delivery of nucleic acids seems to involve rac1-dependent remodeling of actin network within the cell  <cit> .
however, the potential for mpg as a carrier of protein cargoes has not been investigated.
the present investigation was initiated to identify cpps which can be effectively used for the protein delivery in the form of cpp-fusion proteins.
we chose  <dig> known cpp sequences and prepared cpp-egfp fusion proteins  <cit> .
our initial screening unveiled the efficient transduction of modified mpg-egfp fusion proteins into various cell lines and thus we focused on identifying the mechanism of cellular uptake of mpg-egfp.
we report herein that the uptake of mpg-fusion proteins utilizes specific endocytic pathway which is sensitive to amiloride and partially dependent on dynamins.
RESULTS
high transduction efficiency of mpg-egfp fusion protein in mammalian cells
we initially chose  <dig> cpps that have not been used to deliver protein cargoes in the form of fusion proteins , and prepared cpp-egfp fusion proteins.
these cpps, in peptide forms, had all been shown effective in delivering oligonucleotides or nucleic acids into cells when used as a mixture.
as n-terminal tags of egfp recombinant proteins, however, most of these cpps did not seem to enter cells efficiently .
among the tested cpps, mpg-egfp entered the cells and exhibited a punctate vesicular pattern of egfp fluorescence .
efficient transduction of mpg-egfp was confirmed in various cell lines, including an3ca, 293t, nih3t <dig>  f <dig>  bv <dig>  and ht <dig>  treatment of mpg-egfp at  <dig>   <dig>  or  <dig> μg/ml for more than  <dig> hr did not cause any significant cytotoxicity .
representative figures are shown in figure 1a.
intracellular localization of mgp-egfp was mostly cytoplasmic vesicular patterns reminiscent of endosomal vesicles.
since the fixation of cells reportedly affects the subcellular localization of cpps  <cit> , we compared the subcellular localization of mpg-egfp in live and fixed cells.
as shown in figure 1a, no significant redistribution of mpg-egfp signal in the cells was noted.
to examine if mpg-egfp proteins are present within endosomal vesicles, we stained the mpg-egfp-treated hela cells with a general fluorescence marker of endocytosis fm4- <dig>  <cit> .
as shown in figure 1b, most of the mpg-egfp overlaps with the endosomal staining of fm4- <dig> .
localization of mpg-egfp in intracellular vesicles was further confirmed by the vesicle fractionation procedure.
by using the differential centrifugation, the vesicle fractionation procedure separates the particulate fractions containing most of the subcellular vesicles from the cytosolic supernatant.
as shown in figure 1c, the vesicle fraction of the mpg-egfp treated cells showed a clear immunoreactive egfp, confirming the vesicular localization of mpg-egfp.
the immunoreactive egfp was detected in the cytosolic fraction at a low intensity.
the result shows that mpg-egfp proteins are mainly localized in endosomes.
the confocal live imaging clearly demonstrated that mpg-egfp signals are present within multiple endosomes.
these vesicles dynamically move around and a small number of them fuses or disappears during several minutes of observation .
in our work, two different mpg sequences were adapted from published works  <cit>  and are shown in table  <dig> as mpg <dig> and mpg <dig>  these mpg sequences do not contain the linker region and thus are modified from the original sequence.
the seventh amino acid is tryptophan in mpg <dig> and phenylalanine in mpg <dig>  this modification of w to f at the seventh position was reported to enhance nuclear localization of the mixed cargo  <cit> .
both forms, without significant difference, showed effective transduction and exhibited similar cellular distribution and signal intensity .
therefore, we used mpg1-egfp in all the subsequent experiments .
rapid internalization of mpg-egfp depends on endocytosis
internalization of mpg-egfp occurs within  <dig> min and the signal gradually intensifies until about  <dig> min of incubation .
to eliminate the possibility that mpg-egfp adheres to the cell surface, we washed mpg-egfp-treated cells with the acid buffer for  <dig> sec before observing under a fluorescence microscope in all experiments.
the acid wash did not significantly reduce the fluorescence signals of mpg-egfp, suggesting that the observed signal is not an artifact produced by mpg-egfp adhering to the cell surface.
to address if the entry of mpg fusion protein depends on endocytosis, we examined the entry of mpg-egfp at 4°c when general endocytosis is blocked.
mpg-egfp was added to bv <dig> cells or hela cells at  <dig> μg/ml.
bv <dig> cells were co-treated with fm4- <dig>  as shown in figure 2b, the uptake of mpg-egfp was significantly reduced in both cell lines at 4°c, showing only a weak surface binding in some cells.
fm4- <dig> staining was also absent at 4°c in bv <dig> cells, suggesting efficient blockade of endocytosis in our experimental condition.
this result suggests that an energy-dependent endocytic pathway is responsible for the transduction of mpg-egfp.
in the next series of experiments, we investigated which pathway of endocytosis is associated with the uptake of the mpg fusion protein.
effects of endocytosis inhibitors on the entry of mpg-egfp
endocytosis occurs in cells using multiple pathways, such as clathrin-mediated, caveolae-mediated, lipid rafts-mediated endocytosis, and macropinocytosis  <cit> .
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  <cit> .
inhibitors were added to the cells  <dig> min prior to the addition of mpg-egfp and were maintained at the same concentrations for the duration of the experiments .
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,  <dig> mm amiloride treatment effectively reduced mpg-egfp uptake as shown in figure 3a.
methyl-β-cyclodextrin  depletes cholesterol from the plasma membrane, and is used as an inhibitor of the caveolae-mediated endocytosis and macropinocytosis  <cit> .
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 .
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 .
mpg-egfp uptake is independent of caveolins but partly dependent on the gtpase activity of dynamins
to examine if caveolin-mediated endocytosis is involved in the transduction of mpg-egfp, we used the human t lymphocyte jurkat cells which is known to lack caveolin- <dig>  <cit> .
egfp or mpg-egfp was added to the cells at  <dig> μg/ml for  <dig> hr and cells were processed for facs analysis after the acid wash. as shown in figure 4a, the transduction efficiency of mpg-egfp in jurkat cells was above 60%.
although the overall efficiency of mpg-egfp transduction in jurkat cells was not as high as in adherent cells, the successful entry of mpg-egfp shows that this transduction was not dependent on the caveolin-mediated endocytosis.
dynamin gtpase is required for several types of endocytosis including clathrin-mediated endocytosis  <cit> .
transferrin is a marker of dynamin-dependent endocytosis.
thus, we first examined if transferrin is co-localized with mpg-egfp within cells.
as shown in figure 4b, the vesicles containing mpg-egfp partially overlapped with those of transferrin-alex fluor  <dig>  showing a yellow fluorescence.
amiloride treatment did not seem to affect the endocytosis of transferrin but most of mpg-containing vesicles disappeared by this treatment.
some vesicles with both transferrin and mpg were noted.
the observation suggests that dynamins may be partly involved in the transduction of mpg-egfp.
thus, we tested this hypothesis by using the dominant-negative form of dynamin- <dig>  dynk44a.
dynk44a, a dominant-negative form blocking the gtpase activity of dynamin i, has been widely used to elucidate the involvement of these proteins in endocytosis  <cit> .
this form is capable of blocking the gtpase activity of both dynamin- <dig> and dynamin- <dig>  <cit> .
dynamin- <dig> is a neuron-specific form, while dynamin- <dig> is ubiquitously expressed  <cit> .
we overexpressed dynk44a in hela cells and added  <dig> μg/ml mpg-egfp and  <dig> μg/ml transferrin .
expression of dynk44a was detected by an antibody specific to dynamin- <dig>  <cit> .
as shown in figure 5a, overexpression of dynk44a in hela cells blocks the entry of transferrin effectively.
while mpg-egfp entry is shown in dynk44a-expressing hela cells, the number of vesicular structures in these cells was notably reduced .
furthermore, amiloride treatment in dynk44a-expressing hela cells almost completely abolished mgp-egfp uptake, suggesting that the transduction of the mpg fusion protein utilizes unique pathway which is amiloride-sensitive and dynamin-dependent.
blockade of a lysosomal pathway intensifies the accumulation of mpg-egfp in endosomes
chloroquine is an inhibitor which blocks the lysosomal pathway of protein degradation  <cit> .
we co-treated chloroquine and mpg-egfp to hela cells for  <dig> 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  <dig> 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.
uptake of mpg-egfp in mouse eggs
cpp is an excellent tool for a short-term regulation of protein expression.
as we showed herein, mpg fusion protein may be a useful tool to deliver proteins to achieve short-term regulation of protein expression.
we tested if mpg fusion protein can be used in mammalian eggs for this purpose.
so far, the transduction efficiency of any cpp-fusion protein has not been tested in mammalian oocytes.
we obtained mouse eggs by the ovary puncture and removed zona pellucidae with the acid tyrode solution .
denuded eggs were treated with control egfp or mpg-egfp recombinant protein at  <dig> μg/ml.
as shown in figure  <dig>  the treatment of egfp did not give any fluorescence signal, while mpg-egfp-treated eggs showed strong signal around the ooplasmic periphery.
as in the cultured mammalian cells,  <dig> mm amiloride treatment disturbed the uptake of mpg-egfp, suggesting that similar endocytic pathway is operative in the mouse egg.
whether mpg fusion protein can be used to deliver proteins to eggs without adverse effects requires further investigation.
discussion
in a peptide form, mpg is highly effective in delivering oligonucleotides, plasmid dna, sirnas, and peptides via a non-covalent complex formation  <cit> .
unlike other cpps, the uptake of mpg-nucleic acid complex was not inhibited by the treatment of cytochalasin b  <cit> , and this result suggests that the transduction of mpg peptide is independent of macropinocytosis.
cpp tag at the n-terminal of a full-length protein would pose an entirely different conformation in the solution.
thus, we aimed to investigate if a similar or distinct cellular pathway of transduction is used for the uptake of the mpg-fusion protein.
the macropinocytosis is an actin-dependent endocytic pathway which takes up solutes and fluid by the membrane ruffling and fusion  <cit> .
several arginine-rich cpps, such as hiv tat, antp, rev, and vp <dig> are shown to be dependent on the lipid rafts-mediated macropinocytosis, as their uptake is inhibited by cytochalasin d, mβcd, and amiloride  <cit> .
tat-cre, in a fusion protein form, also used a similar endocytic pathway, and the release of this fusion protein from macropinosomes can be improved by the addition of tat peptides conjugated with the influenza virus hemagglutinin fusogenic motif  <cit> .
we found that, unlike tat-fusion protein and arginine-rich cpps, mpg-egfp is likely to use a specific endosomal pathway which is sensitive only to amiloride among other endocytosis inhibitors we used.
since the uptake of mpg-egfp is resistant to actin depolymerization, the macropinocytosis is ruled out.
likewise, caveolae and cholesterol both are not required for the transduction of mpg-egfp, suggesting that lipid rafts are not associated with the uptake.
notably, the overexpression of the mutant form of dynamin could partially reduce mpg-egfp uptake in hela cells, suggesting that the gtpase activity of dynamins may be involved in the transduction.
a recent report showed that the transduction of vp22-egfp fusion protein was similarly dependent on dynamins, but the uptake of vp22-egfp is sensitive to mβcd and cytod  <cit> .
thus, the transduction pathway is seemingly distinct.
collectively, our results indicate that the transduction of mpg-egfp seems to utilize a less well-characterized endocytic pathway  <cit>  and suggest that the transduction of mpg-egfp is dependent on more than singular endocytic pathway, as amiloride and dynamin mutant are both able to reduce the transduction efficiency.
one unique feature of mpg-egfp transduction is that the sequestration of plasma membrane cholesterol increased cytosolic labeling of this fusion protein .
as the cholesterol depletion has a negative effect on the transduction of many cpps including tat-fusion protein, this observation further supports that mpg-fusion protein utilizes a distinct endocytic pathway.
enhanced labeling of a fluorescence-tagged octaarginine peptide in the presence of mβcd has been reported and it was suggested that the entry of this peptide depends on more fluidic region of the plasma membrane with less cholesterol  <cit> .
whether this interpretation can be applied to the case of mpg-fusion protein requires further investigation.
the mechanisms of endocytosis are diverse and new information is continuously being added.
the amiloride-sensitive pathway that mpg-egfp depends on may be somewhat associated with clathrin- and caveolin-independent endocytosis.
however, the mechanisms of this pathway mostly remain unknown  <cit> .
one novel cpp, namely c105y, has been reported to use a clathrin- and caveolin-independent endocytosis  <cit> .
the amiloride-sensitive uptake of mpg-egfp seems to be reminiscent of what was previously described as one type of the endocytic uptake of chlamydiae  <cit> .
this intracellular bacterium utilizes multiple endocytic pathways, one pathway being the cytochalasin d-resistant and amiloride-sensitive pathway.
this is very similar to what we observed herein with the mpg-fusion protein.
no other information regarding this pathway is available to delineate the molecular characteristics.
a recent report evaluating the entry of the human papillomavirus type  <dig> showed that this virus utilizes tetraspanin-enriched microdomains, another example of clathrin- and caveolin-independent endocytosis  <cit> .
more elaborate follow-up to dissect the endocytic pathway of mpg-egfp could serve as a tool to characterize this pathway.
the gtpase dynamin is involved in multiple forms of endocytosis, including caveolae-mediated, clathrin-mediated, and some clathrin- and caveolae-independent endocytic pathway  <cit> .
therefore, our result showing a partial reduction of mpg-egfp transduction after the overexpression of the dynk44a mutant cannot pinpoint which pathway is utilized by the transduction of this fusion protein.
since the intracellular localization of mpg-egfp to some extent overlaps with that of transferrin, clathrin-mediated endocytosis may be involved.
however, clear dissection on the role for dynamins in cpp transduction warrants further investigation.
as in the case of mpg peptide  <cit> , whether the initial interaction of mpg-fusion protein with the cell surface also utilizes negatively charged glycosaminoglycans needs further work.
likewise, studies on the interaction of mpg-fusion protein with lipid membrane will address if mpg-fusion protein bears a similar affinity towards lipids as mpg peptide  <cit> .
we previously showed that mpg-egfp is effectively transduced into the human amnion-derived mesenchymal stem cells  <cit> .
this system may also be applicable to the oocyte system as a short-term manipulation of protein expression, as we show herein.
CONCLUSIONS
in this work, we show that mpg fusion protein utilizes an endocytic pathway which is amiloride-sensitive and partially dynamin-dependent, which is entirely different from other widely used cpps.
thus, we suggest that 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.
furthermore, the mpg fusion protein system can be used to target and deliver cargos to certain subcellular compartments.
and it also may be a useful tool to explore the mechanism of previously uncharacterized type of endocytosis.

 cell-permeable or cell-penetrating peptides are considered a promising method to deliver macromolecular cargoes into live cells across lipid bilayers. these peptides commonly bear stretches of basic amino acids. many cpps have been shown to deliver oligonucleotides or sirna efficiently across cell membrane with or without covalent conjugation. as for the delivery of proteins, expression and purification of cpp-fusion protein in a single step produces more stable form of cargo for in vitro and in vivo uses. the protein transduction domain from tat protein of the human immunodeficiency virus, is known to deliver large proteins up to ~ kda into cells in the form of fusion protein. thus, it has been in the forefront of protein delivery and is being widely used to deliver various proteins for functional experiments. other cpps have been tested for their efficiency for protein delivery by cross-linking, by simple mixing, or as a fusion protein form. while the list of applicable cpps is expanding, recent works have been focused on identifying the mechanisms of cellular uptake of cpps. these studies generally utilize arrays of endocytosis inhibitors to identify specific endocytic pathway involved in the uptake. these works provided evidence that the uptake of tat ptd, polyarginine, and other peptides is dependent on the lipid rafts-mediated macropinocytosis, "the cell-drinking" process. mpg is a designed cpp comprised of two independent domains. the first amino acids of the n-terminus is derived from glycine-rich region of the viral gp and the hydrophilic c-terminus from nuclear localization signal of the sv large t antigen. the original mpg peptide is acetylated at the n-terminus and synthesized with a cysteamide group at the c-terminus. this form was used to deliver sirna or oligonucleotide and the effectiveness was proven in several cell systems. a recent report showed that the initial interaction of mpg peptide with the cell surface uses negatively charged glycosaminoglycans. furthermore, the mechanism of mpg peptide-mediated delivery of nucleic acids seems to involve rac1-dependent remodeling of actin network within the cell. however, the potential for mpg as a carrier of protein cargoes has not been investigated. the present investigation was initiated to identify cpps which can be effectively used for the protein delivery in the form of cpp-fusion proteins. we chose known cpp sequences and prepared cpp-egfp fusion proteins. our initial screening unveiled the efficient transduction of modified mpg-egfp fusion proteins into various cell lines and thus we focused on identifying the mechanism of cellular uptake of mpg-egfp. we report herein that the uptake of mpg-fusion proteins utilizes specific endocytic pathway which is sensitive to amiloride and partially dependent on dynamins.  high transduction efficiency of mpg-egfp fusion protein in mammalian cells we initially chose cpps that have not been used to deliver protein cargoes in the form of fusion proteins, and prepared cpp-egfp fusion proteins. these cpps, in peptide forms, had all been shown effective in delivering oligonucleotides or nucleic acids into cells when used as a mixture. as n-terminal tags of egfp recombinant proteins, however, most of these cpps did not seem to enter cells efficiently. among the tested cpps, mpg-egfp entered the cells and exhibited a punctate vesicular pattern of egfp fluorescence. efficient transduction of mpg-egfp was confirmed in various cell lines, including an3ca, 293t, nih3t f bv and ht treatment of mpg-egfp at or μg/ml for more than hr did not cause any significant cytotoxicity. representative figures are shown in figure 1a. intracellular localization of mgp-egfp was mostly cytoplasmic vesicular patterns reminiscent of endosomal vesicles. since the fixation of cells reportedly affects the subcellular localization of cpps, we compared the subcellular localization of mpg-egfp in live and fixed cells. as shown in figure 1a, no significant redistribution of mpg-egfp signal in the cells was noted. to examine if mpg-egfp proteins are present within endosomal vesicles, we stained the mpg-egfp-treated hela cells with a general fluorescence marker of endocytosis fm4-. as shown in figure 1b, most of the mpg-egfp overlaps with the endosomal staining of fm4-. localization of mpg-egfp in intracellular vesicles was further confirmed by the vesicle fractionation procedure. by using the differential centrifugation, the vesicle fractionation procedure separates the particulate fractions containing most of the subcellular vesicles from the cytosolic supernatant. as shown in figure 1c, the vesicle fraction of the mpg-egfp treated cells showed a clear immunoreactive egfp, confirming the vesicular localization of mpg-egfp. the immunoreactive egfp was detected in the cytosolic fraction at a low intensity. the result shows that mpg-egfp proteins are mainly localized in endosomes. the confocal live imaging clearly demonstrated that mpg-egfp signals are present within multiple endosomes. these vesicles dynamically move around and a small number of them fuses or disappears during several minutes of observation. in our work, two different mpg sequences were adapted from published works and are shown in table as mpg and mpg these mpg sequences do not contain the linker region and thus are modified from the original sequence. the seventh amino acid is tryptophan in mpg and phenylalanine in mpg this modification of w to f at the seventh position was reported to enhance nuclear localization of the mixed cargo. both forms, without significant difference, showed effective transduction and exhibited similar cellular distribution and signal intensity. therefore, we used mpg1-egfp in all the subsequent experiments. rapid internalization of mpg-egfp depends on endocytosis internalization of mpg-egfp occurs within min and the signal gradually intensifies until about min of incubation. to eliminate the possibility that mpg-egfp adheres to the cell surface, we washed mpg-egfp-treated cells with the acid buffer for sec before observing under a fluorescence microscope in all experiments. the acid wash did not significantly reduce the fluorescence signals of mpg-egfp, suggesting that the observed signal is not an artifact produced by mpg-egfp adhering to the cell surface. to address if the entry of mpg fusion protein depends on endocytosis, we examined the entry of mpg-egfp at 4°c when general endocytosis is blocked. mpg-egfp was added to bv cells or hela cells at μg/ml. bv cells were co-treated with fm4- as shown in figure 2b, the uptake of mpg-egfp was significantly reduced in both cell lines at 4°c, showing only a weak surface binding in some cells. fm4- staining was also absent at 4°c in bv cells, suggesting efficient blockade of endocytosis in our experimental condition. this result suggests that an energy-dependent endocytic pathway is responsible for the transduction of mpg-egfp. in the next series of experiments, we investigated which pathway of endocytosis is associated with the uptake of the mpg fusion protein. effects of endocytosis inhibitors on the entry of mpg-egfp endocytosis occurs in cells using multiple pathways, such as clathrin-mediated, caveolae-mediated, lipid rafts-mediated endocytosis, and macropinocytosis. 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. inhibitors were added to the cells min prior to the addition of mpg-egfp and were maintained at the same concentrations for the duration of the experiments. 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, mm amiloride treatment effectively reduced mpg-egfp uptake as shown in figure 3a. methyl-β-cyclodextrin depletes cholesterol from the plasma membrane, and is used as an inhibitor of the caveolae-mediated endocytosis and macropinocytosis. 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. 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. mpg-egfp uptake is independent of caveolins but partly dependent on the gtpase activity of dynamins to examine if caveolin-mediated endocytosis is involved in the transduction of mpg-egfp, we used the human t lymphocyte jurkat cells which is known to lack caveolin-. egfp or mpg-egfp was added to the cells at μg/ml for hr and cells were processed for facs analysis after the acid wash. as shown in figure 4a, the transduction efficiency of mpg-egfp in jurkat cells was above 60%. although the overall efficiency of mpg-egfp transduction in jurkat cells was not as high as in adherent cells, the successful entry of mpg-egfp shows that this transduction was not dependent on the caveolin-mediated endocytosis. dynamin gtpase is required for several types of endocytosis including clathrin-mediated endocytosis. transferrin is a marker of dynamin-dependent endocytosis. thus, we first examined if transferrin is co-localized with mpg-egfp within cells. as shown in figure 4b, the vesicles containing mpg-egfp partially overlapped with those of transferrin-alex fluor showing a yellow fluorescence. amiloride treatment did not seem to affect the endocytosis of transferrin but most of mpg-containing vesicles disappeared by this treatment. some vesicles with both transferrin and mpg were noted. the observation suggests that dynamins may be partly involved in the transduction of mpg-egfp. thus, we tested this hypothesis by using the dominant-negative form of dynamin- dynk44a. dynk44a, a dominant-negative form blocking the gtpase activity of dynamin i, has been widely used to elucidate the involvement of these proteins in endocytosis. this form is capable of blocking the gtpase activity of both dynamin- and dynamin-. dynamin- is a neuron-specific form, while dynamin- is ubiquitously expressed. we overexpressed dynk44a in hela cells and added μg/ml mpg-egfp and μg/ml transferrin. expression of dynk44a was detected by an antibody specific to dynamin-. as shown in figure 5a, overexpression of dynk44a in hela cells blocks the entry of transferrin effectively. while mpg-egfp entry is shown in dynk44a-expressing hela cells, the number of vesicular structures in these cells was notably reduced. furthermore, amiloride treatment in dynk44a-expressing hela cells almost completely abolished mgp-egfp uptake, suggesting that the transduction of the mpg fusion protein utilizes unique pathway which is amiloride-sensitive and dynamin-dependent. blockade of a lysosomal pathway intensifies the accumulation of mpg-egfp in endosomes chloroquine is an inhibitor which blocks the lysosomal pathway of protein degradation. we co-treated chloroquine and mpg-egfp to hela cells for 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 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. uptake of mpg-egfp in mouse eggs cpp is an excellent tool for a short-term regulation of protein expression. as we showed herein, mpg fusion protein may be a useful tool to deliver proteins to achieve short-term regulation of protein expression. we tested if mpg fusion protein can be used in mammalian eggs for this purpose. so far, the transduction efficiency of any cpp-fusion protein has not been tested in mammalian oocytes. we obtained mouse eggs by the ovary puncture and removed zona pellucidae with the acid tyrode solution. denuded eggs were treated with control egfp or mpg-egfp recombinant protein at μg/ml. as shown in figure the treatment of egfp did not give any fluorescence signal, while mpg-egfp-treated eggs showed strong signal around the ooplasmic periphery. as in the cultured mammalian cells, mm amiloride treatment disturbed the uptake of mpg-egfp, suggesting that similar endocytic pathway is operative in the mouse egg. whether mpg fusion protein can be used to deliver proteins to eggs without adverse effects requires further investigation. discussion in a peptide form, mpg is highly effective in delivering oligonucleotides, plasmid dna, sirnas, and peptides via a non-covalent complex formation. unlike other cpps, the uptake of mpg-nucleic acid complex was not inhibited by the treatment of cytochalasin b, and this result suggests that the transduction of mpg peptide is independent of macropinocytosis. cpp tag at the n-terminal of a full-length protein would pose an entirely different conformation in the solution. thus, we aimed to investigate if a similar or distinct cellular pathway of transduction is used for the uptake of the mpg-fusion protein. the macropinocytosis is an actin-dependent endocytic pathway which takes up solutes and fluid by the membrane ruffling and fusion. several arginine-rich cpps, such as hiv tat, antp, rev, and vp are shown to be dependent on the lipid rafts-mediated macropinocytosis, as their uptake is inhibited by cytochalasin d, mβcd, and amiloride. tat-cre, in a fusion protein form, also used a similar endocytic pathway, and the release of this fusion protein from macropinosomes can be improved by the addition of tat peptides conjugated with the influenza virus hemagglutinin fusogenic motif. we found that, unlike tat-fusion protein and arginine-rich cpps, mpg-egfp is likely to use a specific endosomal pathway which is sensitive only to amiloride among other endocytosis inhibitors we used. since the uptake of mpg-egfp is resistant to actin depolymerization, the macropinocytosis is ruled out. likewise, caveolae and cholesterol both are not required for the transduction of mpg-egfp, suggesting that lipid rafts are not associated with the uptake. notably, the overexpression of the mutant form of dynamin could partially reduce mpg-egfp uptake in hela cells, suggesting that the gtpase activity of dynamins may be involved in the transduction. a recent report showed that the transduction of vp22-egfp fusion protein was similarly dependent on dynamins, but the uptake of vp22-egfp is sensitive to mβcd and cytod. thus, the transduction pathway is seemingly distinct. collectively, our results indicate that the transduction of mpg-egfp seems to utilize a less well-characterized endocytic pathway and suggest that the transduction of mpg-egfp is dependent on more than singular endocytic pathway, as amiloride and dynamin mutant are both able to reduce the transduction efficiency. one unique feature of mpg-egfp transduction is that the sequestration of plasma membrane cholesterol increased cytosolic labeling of this fusion protein. as the cholesterol depletion has a negative effect on the transduction of many cpps including tat-fusion protein, this observation further supports that mpg-fusion protein utilizes a distinct endocytic pathway. enhanced labeling of a fluorescence-tagged octaarginine peptide in the presence of mβcd has been reported and it was suggested that the entry of this peptide depends on more fluidic region of the plasma membrane with less cholesterol. whether this interpretation can be applied to the case of mpg-fusion protein requires further investigation. the mechanisms of endocytosis are diverse and new information is continuously being added. the amiloride-sensitive pathway that mpg-egfp depends on may be somewhat associated with clathrin- and caveolin-independent endocytosis. however, the mechanisms of this pathway mostly remain unknown. one novel cpp, namely c105y, has been reported to use a clathrin- and caveolin-independent endocytosis. the amiloride-sensitive uptake of mpg-egfp seems to be reminiscent of what was previously described as one type of the endocytic uptake of chlamydiae. this intracellular bacterium utilizes multiple endocytic pathways, one pathway being the cytochalasin d-resistant and amiloride-sensitive pathway. this is very similar to what we observed herein with the mpg-fusion protein. no other information regarding this pathway is available to delineate the molecular characteristics. a recent report evaluating the entry of the human papillomavirus type showed that this virus utilizes tetraspanin-enriched microdomains, another example of clathrin- and caveolin-independent endocytosis. more elaborate follow-up to dissect the endocytic pathway of mpg-egfp could serve as a tool to characterize this pathway. the gtpase dynamin is involved in multiple forms of endocytosis, including caveolae-mediated, clathrin-mediated, and some clathrin- and caveolae-independent endocytic pathway. therefore, our result showing a partial reduction of mpg-egfp transduction after the overexpression of the dynk44a mutant cannot pinpoint which pathway is utilized by the transduction of this fusion protein. since the intracellular localization of mpg-egfp to some extent overlaps with that of transferrin, clathrin-mediated endocytosis may be involved. however, clear dissection on the role for dynamins in cpp transduction warrants further investigation. as in the case of mpg peptide, whether the initial interaction of mpg-fusion protein with the cell surface also utilizes negatively charged glycosaminoglycans needs further work. likewise, studies on the interaction of mpg-fusion protein with lipid membrane will address if mpg-fusion protein bears a similar affinity towards lipids as mpg peptide. we previously showed that mpg-egfp is effectively transduced into the human amnion-derived mesenchymal stem cells. this system may also be applicable to the oocyte system as a short-term manipulation of protein expression, as we show herein.  in this work, we show that mpg fusion protein utilizes an endocytic pathway which is amiloride-sensitive and partially dynamin-dependent, which is entirely different from other widely used cpps. thus, we suggest that 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. furthermore, the mpg fusion protein system can be used to target and deliver cargos to certain subcellular compartments. and it also may be a useful tool to explore the mechanism of previously uncharacterized type of endocytosis. 
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. we show that mpg-egfp fusion proteins efficiently enter various mammalian cells within a few minutes and are co-localized with fm4- a general marker of endosomes. the entry of mpg-egfp is inhibited by amiloride, but cytochalasin d and methyl-β-cyclodextrin did not inhibit the entry, suggesting that macropinocytosis is not involved in the transduction. 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. mpg is a cell-permeable peptide with proven efficiency to deliver macromolecular cargoes into cells. the results show that the transduction of mpg fusion protein utilizes endocytic pathway which is amiloride-sensitive and partially dynamin-dependent. overexpression of a mutant form of dynamin partially reduced the transduction of mpg-egfp. 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. we examined transduction of mpg-egfp fusion protein by live imaging, flow cytometry, along with combination of cell biological and pharmacological methods. the transduction of mpg-egfp occurs rapidly and is inhibited at a low temperature.
 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.  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- 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-β-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.  the results show that the transduction of mpg fusion protein utilizes endocytic pathway 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. 