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Discipline
Medical, Biological
Keywords
Early Endosome
Polyethylenimine
Lysosome
Transfection Protocol
Observation Type
Standalone
Nature
Standard Data
Submitted
Apr 28th, 2017
Published
Dec 18th, 2017
  • Abstract

    Transfection of cells using polyethylenimine (PEI) polymers is a widely used technique for gene insertion and exogenous protein expression that offers several advantages over virus or lipid-based transfection methods. PEI facilitates endocytosis of DNA into the cell, release of DNA from the endolysosomal system and protection from nucleases. Although PEI is extensively investigated for application in research and therapy, the mechanism of its release from the endosomal system has remained a point of debate. The most prevailing model states that PEI causes endosomal rupture, resulting in release of PEI-DNA complexes into the cytosol. Hence, the use of PEI transfection should be applied with great care in studies on the endosomal system. Here we study the effect of PEI transfections on the endolysosomal system. Using fluorescent and electron microscopy we find a decrease in early endosomes after PEI transfection. Adapting the PEI transfection protocol by including a chase time after initial uptake of the PEI-DNA polyplexes rescues this phenotype without affecting transfection efficiency. Our data result in an adapted protocol for PEI transfection that can be used in studies involving the endolysosomal system.

  • Figure
  • Introduction

    Exogenous expression of (tagged) proteins is a commonly used method in Life Science studies. For example, to localize proteins using fluorescence or electron microscopy when endogenous protein levels are too low for detection or when there are no specific antibodies available. Also functional assays, for example, luciferase assays or co-immunoprecipitations, often require over-expression of (tagged) proteins to obtain sufficient levels for quantitative measurements. The transfection method used to express the protein of interest should have minimal effect on the cells to allow unbiased measurements. However, transfection protocols pose a number of strains onto cells in order to deliver foreign DNA to the nucleus and to escape the cells natural defense mechanisms against incorporation of non-self DNA. Polyethylenimine (PEI) is a synthetic polymer agent for nucleic acid delivery in vitro and in vivo. Since its initial discovery as a transfection agent it has become one of the most widely used gene transfer agents. The reasons for PEI’s popularity are: i) PEI is not expensive and can be stored for several years. ii) PEI is able to transfect many different cell lines with high efficiency. iii) The protocol to use PEI is simple. iv) PEI can be chemically modified to adapt it for special transfection procedures. These advantages make PEI a favored reagent for gene transfections in biochemistry and cell biology research (Reviewed in ), as well as a research target for the application of gene therapy.

    The efficiency of PEI in gene delivery relies on its chemical characteristics. PEI is a cationic polymer that efficiently binds to nucleic acids via charged amino groups. Despite extensive research, however, the details on the mechanism by which PEI transfects cells is still unclear. The PEI-DNA complexes, called polyplexes, condense into particles with a cationic surface, which can interact with anionic proteoglycans that are abundantly present on the cell surface. These particles are endocytosed via clathrin-dependent as well as clathrin-independent endocytosis. After endocytosis, the polyplexes need to escape from the endocytic system to the cytosol in order to reach the nucleus. The generally accepted mechanism for endosomal release is the “proton sponge” hypothesis.

    Endocytosis encompasses the transfer of endocytosed cargo from early endosome (EEs) to late endosomes (LEs) known as endosomal maturation. During endosomal maturation, the pH inside endosomes is lowered by resident ATPase proton pumps that actively translocate protons into the endosomes. This causes the PEI polymers to be protonated, inhibiting acidification of the endosomal lumen. The proton sponge hypothesis poses that since there is no change in pH, the ATPase proton pumps will continue to translocate protons into the endosomes, resulting in an accumulation of protons inside endosomes causing an influx of water and chloride ions. This causes osmotic swelling of endosomes leading to disruption of their membrane and consequent release of the PEI-DNA polyplexes to the cytosol. After release into the cytosol, a portion of the PEI-DNA polyplexes enters the nucleus, either via the nuclear pore complex or after dismantling of the nuclear envelope during cell division.

    Studies involving the endolysosomal system are often combined with protein expression to label distinct compartments, for example with fluorescent Rab5 or RAB7 to label EEs or LEs, respectively. However, since PEI transfections may cause endosomal membrane rupture, this imposes a problem for studying the endolysosomal system in combination with PEI-mediated transfection. Here we test to what extent the endolysosomal system is affected by PEI-mediated transfections. We show that PEI transfections specifically affect EEs and provide a protocol to overcome these PEI-induced artefacts.

  • Objective

    In this study, we aimed to establish the effect of PEI-mediated transfection on the endolysosomal system and optimize the PEI transfection protocol to circumvent PEI-induced artifacts.

  • Results & Discussion

    Transfected PEI-DNA polyplexes accumulate in the endosomal system

    Endocytosis starts with vesicles that bud off from the plasma membrane (PM) and fuse with an early endosome (EE). From EEs, endocytosed cargo can be recycled to the PM via recycling endosomes (REs)or proceed to late endosomes (LEs) and lysosomes for degradation. To track PEI-DNA polyplexes throughout the endolysosomal system, we transfected HeLa cells overnight with a GFP construct using a well-established PEI-based transfection protocol with a DNA:PEI ratio of 1:5 (protocol A). After overnight transfection, cells were fixed and prepared for electron microscopy (EM) to assess the cellular localization of the PEI-DNA polyplexes, which by EM are visible as dense aggregates of approximately 60–100 nm. Using this protocol, we could readily identify PEI-DNA polyplexes in EEs, LEs and lysosomes (Fig. 1A). The polyplexes appeared as individual particles (Fig. 1A, arrows left panel), or larger accumulations consisting of multiple particles. The proton sponge hypothesis predicts that PEI-mediated transfection causes endosomal swelling prior to polyplex release. To study if PEI indeed causes an increased endosomal size, we measured the size of endosomes after PEI-mediated transfection and control cells. Endocytosed PEI polyplexes prevent the morphological characterization of endosome subtype by morphology, therefore all endosomes were measured. This revealed a small yet significant increase in endosome size after transfection, indicating that endosomal swelling indeed occurred (Fig. 1A, graph). These data show that endocytosed PEI-DNA polyplexes traverses the entire endolysosomal system to lysosomes and induce swelling of endosomal compartments. PEI-mediated membrane rupture could occur at both early and late stages of endosomal maturation.

    PEI transfection leads to a reduced early endosomal population

    To test whether PEI-mediated transfection affects the composition of the endolysosomal system we used markers for specific endosomal compartments and quantified their distribution after PEI-mediated transfection. HeLa cells were transfected overnight with GFP using protocol A, after which cells were fixed and labeled for the early endosomal marker EEA1 or the LE/lysosomal marker LAMP-1. In addition, cells were incubated with the fluorescent endocytic marker dextran for 2 h before fixation to mark the entire endolysosomal system. Subsequent analysis revealed that EEA1 and dextran fluorescence was decreased in the transfected GFP-positive cells compared to the non-transfected cells in the wild-type condition, whereas LAMP-1 was not affected (Fig. 1B). Quantification showed that EEA1 and dextran were reduced 2-fold in transfected cells compared to non-treated cells, while LAMP-1 did not change significantly (Fig. 1B, graph). Decreased EEA1 fluorescence indicates a reduction in the number of EEs, or a reduction in recruitment of EEA1 to endosomes. By contrast, LAMP-1-positive LEs/lysosomes were not affected by the transfection. Reduced levels of dextran indicate that either endocytosis is slowed in transfected cells or disruption of endosomal membranes causes leaking of fluorescent dextran out of the compartment. Together these results indicate that the endolysosomal system is disrupted after PEI-mediated transfection and primarily affects EEs.

    Lysosome integrity is not affected after PEI-mediated transfection

    According to the proton sponge hypothesis, PEI-DNA polyplexes escape the endolysosomal pathway through membrane rupture. Several studies suggest that permeabilization occurs at the lysosome, while our data indicate PEI-mediated transfection affects endosomes. Permeabilization of the lysosomal membrane can lead to release of lysosomal enzymes from the lysosomal lumen to the cytoplasm, which ultimately can cause cell death. To study lysosomal integrity after PEI-mediated transfection, we used a galectin3 based assay that detects lysosomal membrane permeabilization. In short, the stably transfected mAzami-green (mAG)-conjugated galectin3 protein (described in) is localized to the cytoplasm and nucleus in steady state conditions. However, upon disruption of the lysosomal membrane, galectin3 translocates into the lysosome and binds to β-galactosides in the glycocalix. This causes the appearance of fluorescent mAG-galectin3 puncta, which correspond to damaged lysosomes. We used stably transfected mAG-galectin3 HeLa cells to test lysosomal membrane permeabilization (LMP) after PEI-mediated transfection of a mCherry-construct using protocol A. We also included a positive control where we induced lysosomal leakage by using a lipid reagent. Both in control cells and cells transfected with protocol A, the mAG-galectin3 localization was primarily cytosolic. In contrast, clearly visible puncta were observed in the positive control (Fig. 1C). Quantification of mAG-galectin3 puncta per cell confirmed that PEI transfection does not increase the number or size of mAG-galectin3 puncta (Fig. 1C, graph). We conclude from these data that PEI transfection does not cause significant lysosomal membrane permeabilization.

    The early endosome population recovers after a pulse-chase PEI transfection protocol

    Our findings show that after overnight PEI transfection the population of EEA1-marked EEs is decreased (Fig. 1B). Since EEs are critical compartments for sorting and signaling of a wide variety of proteins, distortion of EE function is a highly unwanted side-effect in studies that require transfection. We reasoned that the impact of PEI on EE function might be restored over time and therefore developed a pulse-chase transfection protocol to increase the recovery time after transfection. To this end, cells were transfected for 8 h, washed to remove surplus PEI-DNA polyplexes and chased overnight with fresh medium. We refer to this transfection protocol as protocol B. Cells were then fixed and prepared for analysis by EM or IF microscopy. By EM, PEI-DNA polyplexes were observed throughout the endolysosomal system (Fig. 1D), similar as was observed in protocol A. Analysis of the endosomal size revealed a similar amount of endosomal swelling as protocol A (Fig. 1D, graph). However, quantification of the EEA1 signal by IF showed that protocol B restored EEA1 fluorescence area to control levels (Fig. 1E). These data show that the disruption of the endosomal population is restored by the chase period in protocol B.

    The pulse-chase protocol has identical transfection efficiency

    A prerequisite for use of our adapted pulse-chase transfection protocol B is an efficient transfection yield. To test this we used FACS analysis to determine GFP expression after protocol A or B (Fig. 1F). This showed that the transfection efficiency of HeLa cells was identical in the original (A) and adapted (B) protocol (Fig. 1F, graph). Together our data show that the effect of PEI-mediated transfection on the endolysosomal system is temporary and that cells are able to restore their endosomal system. Our adapted transfection protocol incorporates time for restoring endosome function while maintaining high expression levels of the transfected protein.

    In this study we use fluorescence and electron microscopy to study the effect of PEI transfection on the endolysosomal system, using HeLa cells as a model system. We show that a widely used transfection protocol for PEI caused a significant decrease in EEA1-positive EE numbers. By contrast, LAMP-1-positive LE/lysosome numbers and integrity were unaffected. Importantly, adapting the standard overnight transfection protocol to a pulse-chase protocol restored the number of EEs to wild-type levels while retaining a high level of transfection activity. From these data, we conclude that PEI-mediated transfections particularly affect EEs but that this effect is temporary and can be reversed. We, therefore, recommend an adapted transfection protocol for studies that require an intact endolysosomal system in PEI transfected cells.

    Escape from the endolysosomal system is an essential step for successful transfection. For cationic polymers such as PEI, the proton sponge model for endosomal escape indicates that rupture of endosomal membranes is needed for transfer of DNA from the endosomal lumen to the cytoplasm. By EM we found PEI-DNA polyplexes in early and late endosomes as well as lysosomes, which is in agreement with earlier observations. Notably, despite the presence of PEI-DNA polyplexes in lysosomes, we observed no change in LE/lysosome number and no lysosomal membrane permeabilization could be detected. This indicates that lysosomes are not affected by PEI, implicating that PEI-DNA polyplexes that reach lysosomes will be degraded rather than transferred to the cytosol. An explanation for this is that lysosomes have heavily glycosylated limiting membranes that protect them from hydrolase activityand may protect them from rupture.

    We revealed a significant decrease in EEA1 fluorescence signal after PEI-mediated transfection. One possible explanation is that the number of EEA1-positive EEs is decreased. If osmotic rupture disrupts endosomal membranes, it is likely that damaged endosomes are cleared from the cytosol by autophagy, a mechanism that is known to clear damaged endosomes and lysosomes. Alternatively, or additionally, disruption of the endosomal membrane could prevent recruitment of effector proteins such as EEA1 to EEs. In addition to decreased levels of EEA1, we found a significant decrease in dextran levels after PEI-mediated transfection. This can be explained by either a decrease in endocytosis or by leakage of dextran from damaged endosomes to the cytosol. Since PEI-DNA polyplexes are endocytosed themselves, and we could detect polyplexes in EEs after transfection (Fig. A), it is likely that the latter explanation causes the decrease in dextran fluorescence. Dextran leaking into the cytosol is diluted, resulting in loss of fluorescent signal and a decrease in fluorescent puncta.

    Other commonly used transfection methods that require endosomal escape are the use of lipid-based reagents or calcium phosphate precipitates (CCPs). CCPs induce endosomal damage, upregulating autophagy resulting in major alterations of the endolysosomal system after transfection. CCP-based transfection protocols could therefore also potentially profit from a protocol that includes a chase period. By contrast, the destabilizing effect of the lipid complexes on the endolysosomal system is less damaging since they do not cause osmotic swelling or membrane rupture, but back-fuse with the limiting membrane of endosomal compartments, causing only minor disruptive effects on endosomal membrane integrity. However, in studies that require an intact endolysosomal system the use of any transfection method that requires endosomal escape could potentially benefit from a protocol that includes a chase period to allow restoration of endosome numbers.

    In summary, our data show that PEI-induced alterations to the endolysosomal system are limited to the early endocytic compartments and transient in nature. Application of an adapted protocol that includes a chase period is an easy tool to circumvent this disruption.

  • Conclusions

    By EM we show that endocytosed PEI-DNA polyplexes are found in early and late endosomes, as well as lysosomes.

    PEI-mediated transfection causes a minor swelling of endosomes.

    PEI-mediated transfection disrupts the endolysosomal system of HeLa cells and primarily affects EEs.

    PEI-mediated transfection does not cause significant lysosomal membrane permeabilization in HeLa cells.

    The disruptive effect of PEI-mediated transfection on the endolysosomal system is reversible.

    Adapting the transfection protocol to incorporate a chase period results in the restoration of endosome numbers while maintaining high expression levels of the transfected protein.

  • Methods

    Cell culture

    Hela cells (ATCC clone ccl-2) were cultured in Dulbecco’s modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 2 mM L-Glutamin, 100 U/mL penicillin and 100 µg/mL streptomycin (complete DMEM) at 37°C and 5% CO2.

    Antibodies and reagents

    Mouse-anti-human EEA1 (BD Transduction Lab), mouse-anti-human LAMP-1 CD107a (BD Pharmingen), Fluorescent secondary antibodies were obtained from Invitrogen, Fluorescent 10.000 MW dextran-Alexa Fluor 568 (Invitrogen), linear 40 kDa polyethylenimine (PEI) (Polysciences inc.), effectene transfection reagent (Qiagen).

    Transfection protocols

    HeLa cells were transfected with Pcdna3.2-GFP cDNA using linear 40 kDa polyethylenimine (Polysciences inc.) with a DNA:PEI ratio of 1:5 using protocol described in. In the standard protocol, the transfection mix was added to the HeLa cells for 16 h. In the adapted protocol cells were washed 3 times and chased for 16 h with 37°C complete DMEM after being subjected to the PEI-DNA mixture for 8 h. Effectene transfection reagent (Qiagen) was used as a positive control for lysosomal leakage assay using a 5 times higher concentration of effectene than the manufacturer’s protocol.

    Immunofluorescence microscopy

    HeLa cells grown on sterile glass coverslips were washed with ice-cold PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at RT (room temperature). Then, cells were permeabilized using 0,1% Triton-X100 in PBS for 5 min and blocked for 15 min using PBS supplemented with 1% BSA. Cells were labeled with primary antibodies diluted in blocking buffer at RT for 1 h, washed, and labeled with fluorescent secondary antibodies for 30 min in the dark. After labeling the cells were washed and mounted using Prolong Gold antifade reagent with DAPI. Cells were imaged on a Deltavision wide field microscope using a 100X/1.4NA immersion objective. Widefield pictures were deconvolved using Softworx software and analyzed using FIJI.

    Electron microscopy

    Cells grown on 6 cm dishes were fixed in 2% wt/vol PFA, 2.5% wt/vol GA in Na-cacodylate buffer (Karnovsky fixative) for 2 h at RT. Subsequently, the Karnovsky fixative was replaced with 0.1 M Na-cacodylate buffer, pH 7.4. Post-fixation before Epon embedding was performed with 1% wt/vol OsO4, 1.5% wt/vol K3Fe(III)(CN)6 in 0.065 M Na-cacodylate for 2 h at 4°C. Subsequently, the cells were stained with 0.5% uranyl acetate for 1 h at 4°C, dehydrated with ethanol, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate. Images were taken on a TECNAI T12 electron microscope.

    Lysosomal membrane permeabilization assay

    The galectin3-based assay detects lysosomal membrane permeabilization as described in. HeLa cells were stably transfected with the mAzami-green (mAG)-conjugated galectin3 construct (described in, Addgene Plasmid #62734) using lentiviral transduction. In steady state conditions, mAG-Gal3 is localized to the cytoplasm and nucleus. Upon lysosomal membrane permeabilization (LMP) galectin3 is able to bind N-acetyllactosamine-containing glycans present on the inner membrane of lysosomes, leading to an accumulation selectively in damaged lysosomes. Effectene transfection reagent (Qiagen) was used as a positive control for lysosomal leakage assay using 5 times higher concentration of Effectene than the manufacturer’s protocol. Of note, recommended use of Effectene transfection reagent does not induce significant lysosomal leakage.

    FACS analysis

    Transfection efficiency was measured by FACS using the GFP signal after PEI transfection of PCDNA3.2-GFP in HeLa cells using 20.000 cells analyzed on a BD CANTO-II FACS.

  • Acknowledgements

    We thank Ann De Mazière and George Posthuma for assistance with the electron micrographs and René Scriwanek for preparation of EM figures.

  • Ethics statement

    Not applicable.

  • References
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    Matters15.5/20

    An adapted protocol to overcome endosomal damage caused by polyethylenimine (PEI) mediated transfections

    Affiliation listing not available.
    Abstractlink

    Transfection of cells using polyethylenimine (PEI) polymers is a widely used technique for gene insertion and exogenous protein expression that offers several advantages over virus or lipid-based transfection methods. PEI facilitates endocytosis of DNA into the cell, release of DNA from the endolysosomal system and protection from nucleases. Although PEI is extensively investigated for application in research and therapy, the mechanism of its release from the endosomal system has remained a point of debate. The most prevailing model states that PEI causes endosomal rupture, resulting in release of PEI-DNA complexes into the cytosol. Hence, the use of PEI transfection should be applied with great care in studies on the endosomal system. Here we study the effect of PEI transfections on the endolysosomal system. Using fluorescent and electron microscopy we find a decrease in early endosomes after PEI transfection. Adapting the PEI transfection protocol by including a chase time after initial uptake of the PEI-DNA polyplexes rescues this phenotype without affecting transfection efficiency. Our data result in an adapted protocol for PEI transfection that can be used in studies involving the endolysosomal system.

    Figurelink

    Figure 1. PEI mediated transfection reduces the number of early endosomes.

    (A) Electron micrographs of HeLa cells transfected using PEI. DNA-PEI polyplexes are visible as dense particles (arrows). EE= early endosome, LE= late endosome, Ly= lysosome, N= nucleus, PM= plasma membrane. Bars 200 nm. Right panel, endosomal size quantification from >30 randomly selected cells from 3 separate transfections. Endosome size is increased after PEI-mediated transfection using protocol A. Error bars represent the standard deviation of the mean (SD).

    (B) Immunofluorescence analysis of HeLa cells labeled for endogenous EEA1 or LAMP-1 are incubated with dextran-Alexa568 for 2 h. Non-transfected (upper panels) or PEI transfection with protocol A (lower panels). Insets show the GFP signal after transfection. Bars 10 µM. The graph shows the quantification of >30 cells from 3 separate experiments, measuring fluorescence signal area corrected for cell area. Error bars represent the SD.

    (C) HeLa cells stably expressing mAG-galectin3 were transfected using PEI or a treated with a lysosomal leakage positive control. Non-treated or PEI transfected cells show only a few galectin3 puncta. Positive control cells show many intense puncta of galectin3 indicating lysosomal permeabilization. Bars 10 µM. Quantification of the galectin3 puncta above cytosolic fluorescence level from >30 cells from 3 separate experiments reveals a significant increase in puncta in the positive control, but not in PEI transfected cells. Error bars represent SD.

    (D) Electron micrograph showing cells transfected with PEI using the adapted protocol B. DNA-PEI polyplexes are present in EEs, LEs and lysosomes. EE= early endosome, LE= late endosome, Ly= lysosome, M= mitochondrium, PM= plasma membrane. Bars 200 nm. Endosome size quantification using pictures taken from >30 randomly selected cells from 3 separate experiments shows that endosome size is not changed in protocol B in contrast to protocol A. Error bars represent SD.

    (E) Quantification of HeLa cells labeled for endogenous EEA1 transfected using the adapted protocol B shows a rescue in the early endosomal population compared to the standard protocol A. The adapted protocol B restores the EEA1 signal to control levels. The graph shows the quantification of >30 cells over 3 separate experiments using fluorescence signal area corrected for cell area. Error bars represent SD.

    (F) FACS analysis of the GFP signal in the PEI transfected HeLa cells shows that transfection efficiency is similar using the standard protocol (PEI-A) or adapted protocol (PEI-B). Quantification of GFP positive (Q2) versus total cells shows a transfection efficiency of ~75% using protocol A, which is equal to the adapted protocol B. Error bars represent SD.

    Introductionlink

    Exogenous expression of (tagged) proteins is a commonly used method in Life Science studies. For example, to localize proteins using fluorescence or electron microscopy when endogenous protein levels are too low for detection or when there are no specific antibodies available. Also functional assays, for example, luciferase assays or co-immunoprecipitations, often require over-expression of (tagged) proteins to obtain sufficient levels for quantitative measurements. The transfection method used to express the protein of interest should have minimal effect on the cells to allow unbiased measurements. However, transfection protocols pose a number of strains onto cells in order to deliver foreign DNA to the nucleus and to escape the cells natural defense mechanisms against incorporation of non-self DNA[1]. Polyethylenimine (PEI) is a synthetic polymer agent for nucleic acid delivery in vitro and in vivo. Since its initial discovery as a transfection agent[2] it has become one of the most widely used gene transfer agents. The reasons for PEI’s popularity are: i) PEI is not expensive and can be stored for several years. ii) PEI is able to transfect many different cell lines with high efficiency. iii) The protocol to use PEI is simple. iv) PEI can be chemically modified to adapt it for special transfection procedures. These advantages make PEI a favored reagent for gene transfections in biochemistry and cell biology research (Reviewed in [3]), as well as a research target for the application of gene therapy[4][5].

    The efficiency of PEI in gene delivery relies on its chemical characteristics. PEI is a cationic polymer that efficiently binds to nucleic acids via charged amino groups[2]. Despite extensive research, however, the details on the mechanism by which PEI transfects cells is still unclear. The PEI-DNA complexes, called polyplexes, condense into particles with a cationic surface, which can interact with anionic proteoglycans that are abundantly present on the cell surface[6][7][8]. These particles are endocytosed via clathrin-dependent as well as clathrin-independent endocytosis[9]. After endocytosis, the polyplexes need to escape from the endocytic system to the cytosol in order to reach the nucleus. The generally accepted mechanism for endosomal release is the “proton sponge” hypothesis[10].

    Endocytosis encompasses the transfer of endocytosed cargo from early endosome (EEs) to late endosomes (LEs) known as endosomal maturation. During endosomal maturation, the pH inside endosomes is lowered by resident ATPase proton pumps that actively translocate protons into the endosomes. This causes the PEI polymers to be protonated, inhibiting acidification of the endosomal lumen. The proton sponge hypothesis poses that since there is no change in pH, the ATPase proton pumps will continue to translocate protons into the endosomes, resulting in an accumulation of protons inside endosomes causing an influx of water and chloride ions. This causes osmotic swelling of endosomes leading to disruption of their membrane and consequent release of the PEI-DNA polyplexes to the cytosol[11][12]. After release into the cytosol, a portion of the PEI-DNA polyplexes enters the nucleus, either via the nuclear pore complex or after dismantling of the nuclear envelope during cell division[13].

    Studies involving the endolysosomal system are often combined with protein expression to label distinct compartments, for example with fluorescent Rab5 or RAB7 to label EEs or LEs, respectively[14][15]. However, since PEI transfections may cause endosomal membrane rupture, this imposes a problem for studying the endolysosomal system in combination with PEI-mediated transfection. Here we test to what extent the endolysosomal system is affected by PEI-mediated transfections. We show that PEI transfections specifically affect EEs and provide a protocol to overcome these PEI-induced artefacts.

    Objectivelink

    In this study, we aimed to establish the effect of PEI-mediated transfection on the endolysosomal system and optimize the PEI transfection protocol to circumvent PEI-induced artifacts.

    Results & Discussionlink

    Transfected PEI-DNA polyplexes accumulate in the endosomal system

    Endocytosis starts with vesicles that bud off from the plasma membrane (PM) and fuse with an early endosome (EE)[16]. From EEs, endocytosed cargo can be recycled to the PM via recycling endosomes (REs)[17]or proceed to late endosomes (LEs) and lysosomes for degradation[18][19][20]. To track PEI-DNA polyplexes throughout the endolysosomal system, we transfected HeLa cells overnight with a GFP construct using a well-established PEI-based transfection protocol[21] with a DNA:PEI ratio of 1:5 (protocol A). After overnight transfection, cells were fixed and prepared for electron microscopy (EM) to assess the cellular localization of the PEI-DNA polyplexes, which by EM are visible as dense aggregates of approximately 60–100 nm[22]. Using this protocol, we could readily identify PEI-DNA polyplexes in EEs, LEs and lysosomes (Fig. 1A). The polyplexes appeared as individual particles (Fig. 1A, arrows left panel), or larger accumulations consisting of multiple particles. The proton sponge hypothesis predicts that PEI-mediated transfection causes endosomal swelling prior to polyplex release. To study if PEI indeed causes an increased endosomal size, we measured the size of endosomes after PEI-mediated transfection and control cells. Endocytosed PEI polyplexes prevent the morphological characterization of endosome subtype by morphology, therefore all endosomes were measured. This revealed a small yet significant increase in endosome size after transfection, indicating that endosomal swelling indeed occurred (Fig. 1A, graph). These data show that endocytosed PEI-DNA polyplexes traverses the entire endolysosomal system to lysosomes and induce swelling of endosomal compartments. PEI-mediated membrane rupture could occur at both early and late stages of endosomal maturation.

    PEI transfection leads to a reduced early endosomal population

    To test whether PEI-mediated transfection affects the composition of the endolysosomal system we used markers for specific endosomal compartments and quantified their distribution after PEI-mediated transfection. HeLa cells were transfected overnight with GFP using protocol A, after which cells were fixed and labeled for the early endosomal marker EEA1 or the LE/lysosomal marker LAMP-1. In addition, cells were incubated with the fluorescent endocytic marker dextran for 2 h before fixation to mark the entire endolysosomal system. Subsequent analysis revealed that EEA1 and dextran fluorescence was decreased in the transfected GFP-positive cells compared to the non-transfected cells in the wild-type condition, whereas LAMP-1 was not affected (Fig. 1B). Quantification showed that EEA1 and dextran were reduced 2-fold in transfected cells compared to non-treated cells, while LAMP-1 did not change significantly (Fig. 1B, graph). Decreased EEA1 fluorescence indicates a reduction in the number of EEs, or a reduction in recruitment of EEA1 to endosomes. By contrast, LAMP-1-positive LEs/lysosomes were not affected by the transfection. Reduced levels of dextran indicate that either endocytosis is slowed in transfected cells or disruption of endosomal membranes causes leaking of fluorescent dextran out of the compartment. Together these results indicate that the endolysosomal system is disrupted after PEI-mediated transfection and primarily affects EEs.

    Lysosome integrity is not affected after PEI-mediated transfection

    According to the proton sponge hypothesis, PEI-DNA polyplexes escape the endolysosomal pathway through membrane rupture. Several studies suggest that permeabilization occurs at the lysosome[11][12][10], while our data indicate PEI-mediated transfection affects endosomes. Permeabilization of the lysosomal membrane can lead to release of lysosomal enzymes from the lysosomal lumen to the cytoplasm, which ultimately can cause cell death[23][24]. To study lysosomal integrity after PEI-mediated transfection, we used a galectin3 based assay that detects lysosomal membrane permeabilization[25]. In short, the stably transfected mAzami-green (mAG)-conjugated galectin3 protein (described in[26]) is localized to the cytoplasm and nucleus in steady state conditions. However, upon disruption of the lysosomal membrane, galectin3 translocates into the lysosome and binds to β-galactosides in the glycocalix. This causes the appearance of fluorescent mAG-galectin3 puncta, which correspond to damaged lysosomes. We used stably transfected mAG-galectin3 HeLa cells to test lysosomal membrane permeabilization (LMP) after PEI-mediated transfection of a mCherry-construct using protocol A. We also included a positive control where we induced lysosomal leakage by using a lipid reagent. Both in control cells and cells transfected with protocol A, the mAG-galectin3 localization was primarily cytosolic. In contrast, clearly visible puncta were observed in the positive control (Fig. 1C). Quantification of mAG-galectin3 puncta per cell confirmed that PEI transfection does not increase the number or size of mAG-galectin3 puncta (Fig. 1C, graph). We conclude from these data that PEI transfection does not cause significant lysosomal membrane permeabilization.

    The early endosome population recovers after a pulse-chase PEI transfection protocol

    Our findings show that after overnight PEI transfection the population of EEA1-marked EEs is decreased (Fig. 1B). Since EEs are critical compartments for sorting and signaling of a wide variety of proteins, distortion of EE function is a highly unwanted side-effect in studies that require transfection. We reasoned that the impact of PEI on EE function might be restored over time and therefore developed a pulse-chase transfection protocol to increase the recovery time after transfection. To this end, cells were transfected for 8 h, washed to remove surplus PEI-DNA polyplexes and chased overnight with fresh medium. We refer to this transfection protocol as protocol B. Cells were then fixed and prepared for analysis by EM or IF microscopy. By EM, PEI-DNA polyplexes were observed throughout the endolysosomal system (Fig. 1D), similar as was observed in protocol A. Analysis of the endosomal size revealed a similar amount of endosomal swelling as protocol A (Fig. 1D, graph). However, quantification of the EEA1 signal by IF showed that protocol B restored EEA1 fluorescence area to control levels (Fig. 1E). These data show that the disruption of the endosomal population is restored by the chase period in protocol B.

    The pulse-chase protocol has identical transfection efficiency

    A prerequisite for use of our adapted pulse-chase transfection protocol B is an efficient transfection yield. To test this we used FACS analysis to determine GFP expression after protocol A or B (Fig. 1F). This showed that the transfection efficiency of HeLa cells was identical in the original (A) and adapted (B) protocol (Fig. 1F, graph). Together our data show that the effect of PEI-mediated transfection on the endolysosomal system is temporary and that cells are able to restore their endosomal system. Our adapted transfection protocol incorporates time for restoring endosome function while maintaining high expression levels of the transfected protein.

    In this study we use fluorescence and electron microscopy to study the effect of PEI transfection on the endolysosomal system, using HeLa cells as a model system. We show that a widely used transfection protocol for PEI[21] caused a significant decrease in EEA1-positive EE numbers. By contrast, LAMP-1-positive LE/lysosome numbers and integrity were unaffected. Importantly, adapting the standard overnight transfection protocol to a pulse-chase protocol restored the number of EEs to wild-type levels while retaining a high level of transfection activity. From these data, we conclude that PEI-mediated transfections particularly affect EEs but that this effect is temporary and can be reversed. We, therefore, recommend an adapted transfection protocol for studies that require an intact endolysosomal system in PEI transfected cells.

    Escape from the endolysosomal system is an essential step for successful transfection[27]. For cationic polymers such as PEI, the proton sponge model for endosomal escape indicates that rupture of endosomal membranes is needed for transfer of DNA from the endosomal lumen to the cytoplasm. By EM we found PEI-DNA polyplexes in early and late endosomes as well as lysosomes, which is in agreement with earlier observations[10][12]. Notably, despite the presence of PEI-DNA polyplexes in lysosomes, we observed no change in LE/lysosome number and no lysosomal membrane permeabilization could be detected. This indicates that lysosomes are not affected by PEI, implicating that PEI-DNA polyplexes that reach lysosomes will be degraded rather than transferred to the cytosol. An explanation for this is that lysosomes have heavily glycosylated limiting membranes that protect them from hydrolase activity[19][20]and may protect them from rupture.

    We revealed a significant decrease in EEA1 fluorescence signal after PEI-mediated transfection. One possible explanation is that the number of EEA1-positive EEs is decreased. If osmotic rupture disrupts endosomal membranes, it is likely that damaged endosomes are cleared from the cytosol by autophagy, a mechanism that is known to clear damaged endosomes and lysosomes[28][29]. Alternatively, or additionally, disruption of the endosomal membrane could prevent recruitment of effector proteins such as EEA1 to EEs. In addition to decreased levels of EEA1, we found a significant decrease in dextran levels after PEI-mediated transfection. This can be explained by either a decrease in endocytosis or by leakage of dextran from damaged endosomes to the cytosol. Since PEI-DNA polyplexes are endocytosed themselves, and we could detect polyplexes in EEs after transfection (Fig. A), it is likely that the latter explanation causes the decrease in dextran fluorescence. Dextran leaking into the cytosol is diluted, resulting in loss of fluorescent signal and a decrease in fluorescent puncta.

    Other commonly used transfection methods that require endosomal escape are the use of lipid-based reagents or calcium phosphate precipitates (CCPs). CCPs induce endosomal damage, upregulating autophagy resulting in major alterations of the endolysosomal system after transfection[28]. CCP-based transfection protocols could therefore also potentially profit from a protocol that includes a chase period. By contrast, the destabilizing effect of the lipid complexes on the endolysosomal system is less damaging since they do not cause osmotic swelling or membrane rupture, but back-fuse with the limiting membrane of endosomal compartments[30][31][32], causing only minor disruptive effects on endosomal membrane integrity. However, in studies that require an intact endolysosomal system the use of any transfection method that requires endosomal escape could potentially benefit from a protocol that includes a chase period to allow restoration of endosome numbers.

    In summary, our data show that PEI-induced alterations to the endolysosomal system are limited to the early endocytic compartments and transient in nature. Application of an adapted protocol that includes a chase period is an easy tool to circumvent this disruption.

    Conclusionslink

    By EM we show that endocytosed PEI-DNA polyplexes are found in early and late endosomes, as well as lysosomes.

    PEI-mediated transfection causes a minor swelling of endosomes.

    PEI-mediated transfection disrupts the endolysosomal system of HeLa cells and primarily affects EEs.

    PEI-mediated transfection does not cause significant lysosomal membrane permeabilization in HeLa cells.

    The disruptive effect of PEI-mediated transfection on the endolysosomal system is reversible.

    Adapting the transfection protocol to incorporate a chase period results in the restoration of endosome numbers while maintaining high expression levels of the transfected protein.

    Methodslink

    Cell culture

    Hela cells (ATCC clone ccl-2) were cultured in Dulbecco’s modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 2 mM L-Glutamin, 100 U/mL penicillin and 100 µg/mL streptomycin (complete DMEM) at 37°C and 5% CO2.

    Antibodies and reagents

    Mouse-anti-human EEA1 (BD Transduction Lab), mouse-anti-human LAMP-1 CD107a (BD Pharmingen), Fluorescent secondary antibodies were obtained from Invitrogen, Fluorescent 10.000 MW dextran-Alexa Fluor 568 (Invitrogen), linear 40 kDa polyethylenimine (PEI) (Polysciences inc.), effectene transfection reagent (Qiagen).

    Transfection protocols

    HeLa cells were transfected with Pcdna3.2-GFP cDNA using linear 40 kDa polyethylenimine (Polysciences inc.) with a DNA:PEI ratio of 1:5 using protocol described in[21]. In the standard protocol, the transfection mix was added to the HeLa cells for 16 h. In the adapted protocol cells were washed 3 times and chased for 16 h with 37°C complete DMEM after being subjected to the PEI-DNA mixture for 8 h. Effectene transfection reagent (Qiagen) was used as a positive control for lysosomal leakage assay using a 5 times higher concentration of effectene than the manufacturer’s protocol.

    Immunofluorescence microscopy

    HeLa cells grown on sterile glass coverslips were washed with ice-cold PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at RT (room temperature). Then, cells were permeabilized using 0,1% Triton-X100 in PBS for 5 min and blocked for 15 min using PBS supplemented with 1% BSA. Cells were labeled with primary antibodies diluted in blocking buffer at RT for 1 h, washed, and labeled with fluorescent secondary antibodies for 30 min in the dark. After labeling the cells were washed and mounted using Prolong Gold antifade reagent with DAPI. Cells were imaged on a Deltavision wide field microscope using a 100X/1.4NA immersion objective. Widefield pictures were deconvolved using Softworx software and analyzed using FIJI.

    Electron microscopy

    Cells grown on 6 cm dishes were fixed in 2% wt/vol PFA, 2.5% wt/vol GA in Na-cacodylate buffer (Karnovsky fixative) for 2 h at RT. Subsequently, the Karnovsky fixative was replaced with 0.1 M Na-cacodylate buffer, pH 7.4. Post-fixation before Epon embedding was performed with 1% wt/vol OsO4, 1.5% wt/vol K3Fe(III)(CN)6 in 0.065 M Na-cacodylate for 2 h at 4°C. Subsequently, the cells were stained with 0.5% uranyl acetate for 1 h at 4°C, dehydrated with ethanol, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate. Images were taken on a TECNAI T12 electron microscope.

    Lysosomal membrane permeabilization assay

    The galectin3-based assay detects lysosomal membrane permeabilization as described in[25]. HeLa cells were stably transfected with the mAzami-green (mAG)-conjugated galectin3 construct (described in[26], Addgene Plasmid #62734) using lentiviral transduction. In steady state conditions, mAG-Gal3 is localized to the cytoplasm and nucleus. Upon lysosomal membrane permeabilization (LMP) galectin3 is able to bind N-acetyllactosamine-containing glycans present on the inner membrane of lysosomes, leading to an accumulation selectively in damaged lysosomes. Effectene transfection reagent (Qiagen) was used as a positive control for lysosomal leakage assay using 5 times higher concentration of Effectene than the manufacturer’s protocol. Of note, recommended use of Effectene transfection reagent does not induce significant lysosomal leakage.

    FACS analysis

    Transfection efficiency was measured by FACS using the GFP signal after PEI transfection of PCDNA3.2-GFP in HeLa cells using 20.000 cells analyzed on a BD CANTO-II FACS.

    Acknowledgementslink

    We thank Ann De Mazière and George Posthuma for assistance with the electron micrographs and René Scriwanek for preparation of EM figures.

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    Not applicable.

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

    Referenceslink
    1. Marika Ruponen, Paavo Honkakoski, Seppo Rönkkö,more_horiz, Arto Urtti
      Extracellular and intracellular barriers in non-viral gene delivery
      Journal of Controlled Release, 93/2003, pages 213-217 DOI: 10.1016/j.jconrel.2003.08.004chrome_reader_mode
    2. O Boussif, F Lezoualc'H, M A Zanta, M D Mergny, D Scherman, B Demeneix, J P Behr
      A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.
      Proceedings of the National Academy of Sciences, 92/1995, pages 7297-7301 DOI: 10.1073/pnas.92.16.7297chrome_reader_mode
    3. Neuberg Patrick, Kichler Antoine
      Recent Developments in Nucleic Acid Delivery with Polyethylenimines
      Nonviral Vectors for Gene Therapy - Lipid- and Polymer-based Gene Transfer, 2014, pages 263-288 DOI: 10.1016/b978-0-12-800148-6.00009-2chrome_reader_mode
    4. Yin Hao, Kanasty Rosemary L., Eltoukhy Ahmed A.,more_horiz, Anderson Daniel G.
      Non-viral vectors for gene-based therapy
      Nature Reviews Genetics, 15/2014, pages 541-555 DOI: 10.1038/nrg3763chrome_reader_mode
    5. Kay Mark A.
      State-of-the-art gene-based therapies: the road ahead
      Nature Reviews Genetics, 12/2011, pages 316-328 DOI: 10.1038/nrg2971chrome_reader_mode
    6. K A Mislick, J D Baldeschwieler
      Evidence for the role of proteoglycans in cation-mediated gene transfer.
      Proceedings of the National Academy of Sciences, 93/1996, pages 12349-12354 DOI: 10.1073/pnas.93.22.12349chrome_reader_mode
    7. Paris, Sébastien, Burlacu, Alina, Durocher, Yves
      Opposing Roles of Syndecan-1 and Syndecan-2 in Polyethyleneimine-mediated Gene Delivery
      Journal of Biological Chemistry, 283/2008, pages 7697-7704 DOI: 10.1074/jbc.m705424200chrome_reader_mode
    8. Kopatz Idit, Remy Jean-Serge, Behr Jean-Paul
      A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin
      The Journal of Gene Medicine, 6/2004, pages 769-776 DOI: 10.1002/jgm.558chrome_reader_mode
    9. von Gersdorff Katharina, Sanders Niek N., Vandenbroucke Roosmarijn,more_horiz, Ogris Manfred
      The Internalization Route Resulting in Successful Gene Expression Depends on both Cell Line and Polyethylenimine Polyplex Type
      Molecular Therapy, 14/2006, pages 745-753 DOI: 10.1016/j.ymthe.2006.07.006chrome_reader_mode
    10. Benjaminsen Rikke V, Mattebjerg Maria A, Henriksen Jonas R,more_horiz, Andresen Thomas L
      The Possible “Proton Sponge” Effect of Polyethylenimine (PEI) Does Not Include Change in Lysosomal pH
      Molecular Therapy, 21/2013, pages 149-157 DOI: 10.1038/mt.2012.185chrome_reader_mode
    11. Bieber Thorsten, Meissner Wolfgang, Kostin Sawa,more_horiz, Elsasser Hans-Peter
      Intracellular route and transcriptional competence of polyethylenimine–DNA complexes
      Journal of Controlled Release, 82/2002, pages 441-454 DOI: 10.1016/s0168-3659(02)00129-3chrome_reader_mode
    12. Merdan Thomas, Kunath Klaus, Fischer Dagmar,more_horiz, Kissel Thomas
      Pharmaceutical Research, 19/2002, pages 140-146 DOI: 10.1023/a:1014212630566chrome_reader_mode
    13. Brunner Sylvia, Fürtbauer Elke, Sauer Thomas,more_horiz, Wagner Ernst
      Overcoming the Nuclear Barrier: Cell Cycle Independent Nonviral Gene Transfer with Linear Polyethylenimine or Electroporation
      Molecular Therapy, 5/2002, pages 80-86 DOI: 10.1006/mthe.2001.0509chrome_reader_mode
    14. Sönnichsen Birte, de Renzis Stefano, Nielsen Erik,more_horiz, Zerial Marino
      Distinct Membrane Domains on Endosomes in the Recycling Pathway Visualized by Multicolor Imaging of Rab4, Rab5, and Rab11
      The Journal of Cell Biology, 149/2000, pages 901-914 DOI: 10.1083/jcb.149.4.901chrome_reader_mode
    15. Rink Jochen, Ghigo Eric, Kalaidzidis Yannis, Zerial Marino
      Rab Conversion as a Mechanism of Progression from Early to Late Endosomes
    16. Doherty Gary J., McMahon Harvey T.
      Mechanisms of Endocytosis
      Annual Review of Biochemistry, 78/2009, pages 857-902 DOI: 10.1146/annurev.biochem.78.081307.110540chrome_reader_mode
    17. Grant Barth D., Donaldson Julie G.
      Pathways and mechanisms of endocytic recycling
      Nature Reviews Molecular Cell Biology, 10/2009, pages 597-608 DOI: 10.1038/nrm2755chrome_reader_mode
    18. Huotari Jatta, Helenius Ari
      Endosome maturation
      The EMBO Journal, 30/2011, pages 3481-3500 DOI: 10.1038/emboj.2011.286chrome_reader_mode
    19. Saftig Paul, Klumperman Judith
      Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function
      Nature Reviews Molecular Cell Biology, 10/2009, pages 623-635 DOI: 10.1038/nrm2745chrome_reader_mode
    20. Klumperman J., Raposo G.
      The Complex Ultrastructure of the Endolysosomal System
      Cold Spring Harbor Perspectives in Biology, 6/2014, pages a016857-a016857 DOI: 10.1101/cshperspect.a016857chrome_reader_mode
    21. Longo Patti A., Kavran Jennifer M., Kim Min-Sung, Leahy Daniel J.
      Transient Mammalian Cell Transfection with Polyethylenimine (PEI)
      Methods in Enzymology, 2013, pages 227-240 DOI: 10.1016/b978-0-12-418687-3.00018-5chrome_reader_mode
    22. Choosakoonkriang Sirirat, Lobo Brian A., Koe Gary S.,more_horiz, Middaugh C.Russell.
      Biophysical Characterization of PEI/DNA Complexes
      Journal of Pharmaceutical Sciences, 92/2003, pages 1710-1722 DOI: 10.1002/jps.10437chrome_reader_mode
    23. Aits S., Jaattela M.
      Lysosomal cell death at a glance
      Journal of Cell Science, 126/2013, pages 1905-1912 DOI: 10.1242/jcs.091181chrome_reader_mode
    24. Ellegaard A.-M., Groth-Pedersen L., Oorschot V.,more_horiz, Jaattela M.
      Sunitinib and SU11652 Inhibit Acid Sphingomyelinase, Destabilize Lysosomes, and Inhibit Multidrug Resistance
      Molecular Cancer Therapeutics, 12/2013, pages 2018-2030 DOI: 10.1158/1535-7163.mct-13-0084chrome_reader_mode
    25. Aits Sonja, Kricker Jennifer, Liu Bin,more_horiz, Jäättelä Marja
      Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay
      Autophagy, 11/2015, pages 1408-1424 DOI: 10.1080/15548627.2015.1063871chrome_reader_mode
    26. D’astolfo Diego S., Pagliero Romina J., Pras Anita,more_horiz, Geijsen Niels
      Efficient Intracellular Delivery of Native Proteins
    27. The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids
      FEBS Letters, 414/1997, pages 187-192 DOI: 10.1016/s0014-5793(97)00973-3chrome_reader_mode
    28. Chen X., Khambu B., Zhang H.,more_horiz, Yin X.-M.
      Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes
      Journal of Biological Chemistry, 289/2014, pages 11162-11174 DOI: 10.1074/jbc.m113.531855chrome_reader_mode
    29. Hung Yu-Hsien, Chen Lily Man-Wen, Yang Jin-Yi, Yuan Yang Wei
      Spatiotemporally controlled induction of autophagy-mediated lysosome turnover
      Nature Communications, 4/2013 DOI: 10.1038/ncomms3111chrome_reader_mode
    30. O Zelphati, F C Szoka
      Mechanism of oligonucleotide release from cationic liposomes.
      Proceedings of the National Academy of Sciences, 93/1996, pages 11493-11498 DOI: 10.1073/pnas.93.21.11493chrome_reader_mode
    31. Xu Yuhong, Szoka Francis C.
      Mechanism of DNA Release from Cationic Liposome/DNA Complexes Used in Cell Transfection
      Biochemistry, 35/1996, pages 5616-5623 DOI: 10.1021/bi9602019chrome_reader_mode
    32. Zhou Xiaohuai, Huang Leaf
      DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action
      Biochimica et Biophysica Acta (BBA) - Biomembranes, 1189/1994, pages 195-203 DOI: 10.1016/0005-2736(94)90066-3chrome_reader_mode
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