Inducible membrane permeabilization by ... - ACS Publications

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Article

Inducible membrane permeabilization by attenuated lytic peptides: A new concept for accessing cell interiors through ruffled membranes Misao Akishiba, and Shiroh Futaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00156 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 7, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Inducible membrane permeabilization by attenuated lytic peptides: A new concept for accessing cell interiors through ruffled membranes

Misao Akishiba and Shiroh Futaki* Institute for Chemical Research, Kyoto University Uji, Kyoto 611-0011, Japan.

KEYWORDS: Intracellular delivery; macropinocytosis; transient membrane perturbation

1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract

A variety of mid-sized and large biomolecules have been used as tools to explore fundamental biological questions. However, such molecules are often cell-impermeable and thus unable to attain sufficient access to the cell interior. This inhibits their ability to yield analytical data about the cell interior or modify the cellular events. We have recently developed a peptide, engineered from a natural hemolytic peptide, named L17E. Substantial cytosolic delivery of biomacromolecules, including antibodies, was attained in the presence of this peptide. In this study, detailed analysis of the modes of action of L17E was conducted, elucidating that a large fraction of the cytosolic translocation of biomacromolecules is accomplished in the presence of L17E within 5 min. L17E stimulates actin polymerization and induces a dynamic structural alteration of cell membranes, resulting in a ruffled appearance. Studies using macropinocytosis inhibitors and proteins that control endosome maturation raises the possibility that the transient permeabilization of ruffled cell membranes, rather than the rupture of endosomal membranes, is the crucial mechanism for facile cytosolic translocation of biomacromolecules in the presence of L17E. Our results provide a distinct concept of intracellular delivery, different from direct translocation through cell membranes or endocytic uptake followed by endosomal escape. This method of permeabilization via membrane ruffling provides a novel concept in intracellular delivery.


Page 2 of 41

Page 3 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Recent developments in molecular biology and cell biology have revealed the networks of molecular interplays in living cells. Attaining a profound understanding of the various molecular interactions, and the development of means with which to modify them, has become a major focus in chemistry. The challenges to this end have resulted in a new and extraordinarily broad research area. Numerous molecules, including mid-sized and large biomimetic molecules, have been being developed through these efforts, resulting in considerable scientific and therapeutic advancements. These molecules usually reside in cytosol or in other organelles, including the nucleus and mitochondria. However, their size often precludes sufficient access to the cell interior, which can decrease their effectiveness in modifying cellular events. The simplest way for a molecule to reach the cell interior is direct delivery through the cell membrane (plasma membrane). However, the passage of large, exogenous molecules typically requires pore formation or membrane rupture. Alternatively, endocytic pathways have been employed to transport large molecules into cells.1-5 Endocytosis is an energy-dependent cellular uptake system in which exogenous molecules are delivered into the cell while encapsulated in vesicular compartments, i.e., endosomes. Preferential perturbation of endosomal membranes, without perturbing the cell membrane, is then needed to empty the contents of the endosomes into the cytosol. A close examination of the endosomal uptake of bioactive molecules and their liberation into the cytosol, i.e., endosomal escape, should provide useful guidelines for the design of novel delivery systems. However, compared with the number of reports describing the development of delivery systems, there have been few studies pursuing the fundamental aspects of endosomal delivery.6-9



We recently reported a system for intracellular protein delivery using a peptide named L17E.10 Exposing cells to biomacromolecules in the presence of L17E yielded salient cytosolic distributions of the biomacromolecule. Most notably, antibodies [immunoglobulin G (IgG), ~150 kDa] were readily delivered into the cytosol with the aid of L17E, where they continued to exert their functions of target recognition and signal modulation. L17E contains the motif of a cationic amphiphilic spider toxin peptide (M-lycotoxin; M-LCTX). Replacing the Leu residue at position 17 with a negatively charged Glu markedly attenuated the lytic activity of the peptide on cell membranes. The original intent of this substitution was to show that the protonation of Glu at endosomal pH, which tends to be lower than that of the surroundings, should result in the recovery of its membrane-rupture activity. This, we hypothesized, would result in the selective perturbation of endosomal membranes. However, unexpectedly, L17E did not show an apparent pH sensitivity in liposomal dye-leakage assays. Instead, it preferentially perturbed membranes containing acidic phospholipids over those comprised of neutral, zwitterionic phospholipids. Since endosomal membranes are generally rich in negatively charged lipids,11,12 while cell membranes are mostly composed of neutral lipids, we deduced that L17E would preferentially perturb endosomal membranes. Additionally, an L17E-dependent increase in cell numbers, showing cytosolic localization of extracellular biomacromolecules, was observed. This phenomenon was absent under treatments with a representative macropinocytosis inhibitor, EIPA [5-(N-ethyl-Nisopropyl)amiloride].13 Therefore, the induction of macropinocytosis, which results in a significant increase in the uptake of extracellular materials, may explain the effectiveness of L17E. This study provides a detailed analysis of the modes of action of L17E and furthers our mechanical understanding of cellular uptake. This understanding lends itself to the rational design of more sophisticated delivery systems.


Page 4 of 41

Page 5 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In the presence of L17E, a considerable fraction of biomacromolecules (10 kDa dextran and IgG) were able to reach the cytosol after 5 min. Unlike physicochemical methods that induce membrane rupture, the observed cytosolic translocation was fast and energy-dependent, suggesting the involvement of cellular machinery. Careful dissection of the various stages of macropinocytosis using inhibitors and other biological techniques suggests the importance of L17E-mediated membrane ruffling preceding membrane fusion and macropinosome formation. A large proportion of cytosolic translocation can be accomplished during this stage via membrane permeation, as opposed to endosomal escape (Fig. 1A). In this paper, we propose that membrane permeation by L17E, along with dynamic structural alterations of ruffled membranes, allows for transient influx of biomacromolecules into the cell interior with no notable cytotoxicity. This method of membrane permeabilization constitutes a distinct class of intracellular delivery routes alongside direct penetration through the cell membrane (i.e., passive diffusion or pore formation), and endocytic uptake followed by endosomal escape.



Experimental Section Materials All reagents including salts, inhibitors and incubation media were obtained from Sigma-Aldrich or Wako unless otherwise specified. L17E (IWLTALKFLGKHAAKHEAKQQLSKL-CONH2) and M-lycotoxin (M-LCTX, IWLTALKFLGKHAAKHLAKQQLSKL-CONH2) were prepared as reported.10 Peptides were dissolved in dimethyl sulfoxide (DMSO) to yield final DMSO concentration (1%) in culture media unless otherwise specified. Dextran (70 kDa) labeled with tetramethylrhodamine (Dex70-TMR) and labeled with fluorescein (Dex70-FL), and dextran (10 kDa) labeled with Alexa Fluor 488 (Dex10-Alexa), anti-mouse IgM antibodies (Alexa Fluor 488 labeled), Annexin V, stromal cell-derived factor (SDF)-1α, di-4-ANEPPDHQ, and heatinactivated bovine serum were all purchased from Invitrogen. Immunoglobulin G (IgG, human) was purchased from Wako and labeled with Alexa Fluor 488 5-SDP ester (Invitrogen) as reported.10 Anti-phosphatidyl-inositol bisphosphate (PIP2) antibodies (2C11, ab11039) were purchased from Abcam.

Cell Culture HeLa cells (human epithelial carcinoma cell line) obtained from The European Collection of Authenticated Cell Cultures (ECACC) (93021013) were cultured in α-minimum essential medium (α-MEM) supplemented with 10% (v/v) heat-inactivated bovine serum (BS) [α-MEM(+)]. The cells were maintained at 37°C in a humidified 5% CO2 incubator, and are passaged every 3-4 days.


Page 6 of 41

Page 7 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Plasmid To construct mammalian expression vector [mCherry–WT] encoding mCherry-Rab5 (WT), the DNA fragment encoding the Rab5(WT) sequence was amplified from the EGFP-Rab5 kindly provided

by

Prof.

H.-W.

Shin

using

following

primer

AGATCTCGAGCTCAAATGGCTAGTCGAGGCGCAAC-3'

pairs;

and

TCCGGTGGATCCCGGGTACCGTCGACTGCAGTTAGTTACTAC-3'.

Forward: Reverse:

5'5'-

This fragment was

assembled into pmCherry-C1 vector (Clontech) using Gibson assembly System (New England Biolabs). Prior to assembly, pmCherry-C1 vector (Clontech) was linearized by PCR using following

primer

pairs:

5'-TTGAGCTCGAGATCTGAGTCCGG-3'

and

5'-

CCGGGATCCACCGGATCTAGA-3'. Rab5 mutant vectors (mCherry–S34N and mCherry–Q79L) were constructed by introducing a site-directed mutation onto mCherry-Rab5(WT) plasmid as follows. Two kinds of DNA fragments for each vector, including the flanking N-and C-terminal regions of Rab5 coding sequence including the mutated site were amplified by PCR from mCherry-Rab5(WT) plasmid using following

primer

pairs:

for

S34N

GTTGGACATCACCTCCCACAACGAG-3'

mutation,

(mCherry-C1-F)

Forward:

and

Reverse:

5'5'-

GCACTAGGCTATTTTTGCCAACAG-3', Forward: 5'-TTGGCAAAAATAGCCTAGTGC-3' and Reverse: 5'-CCTCTACAAATGTGGTATGG-3' (SV40pA-R) / for Q79L mutation, Forward: mCherry-C1-F

and

Reverse:

5'-TGGTATCGTTCAAGACCAGCTGT-3',

Forward:

5'-

ACAGCTGGTCTTGAACGATACCA-3' and Reverse: SV40pA-R. These fragments were spliced into a single DNA fragment by PCR using the primer pair: Forward: mCherry-C1-F and Reverse: SV40pA-R. This fragment was digested using the XhoI and BamHI and inserted into mCherryRab(WT) vector. 7 ACS Paragon Plus Environment


Observation of membrane ruffling HeLa cells (1.5 × 105 cells) were plated onto 35-mm glass-bottomed dishes (Iwaki) and were transfected with Lifeact–mCherry plasmid kindly provided by Prof. T. Itoh using Lipofectamine LTX (Invitrogen) according to the manufacturer’s instructions. One day after transfection, cells were washed twice with phosphate-buffered saline without containing Ca2+ and Mg2+ [PBS(–)], incubated with serum-free α-MEM [α-MEM(–)] (100 µL) and allowed to settle for 10 min in an Olympus MI-IBC-IF micro-chamber (humidified with 5% CO2 at 37°C) attached to the microscope stage. The actin structure in live cells was observed using a 60 × objective (oil, NA 1.35). After time-lapse imaging in the absence of L17E for 10 min, L17E (60 µM) in α-MEM(–) (50 µL) was added to the incubation medium to yield an L17E concentration of 20 µM. Timelapse imaging was continued for another 10 min. The time of L17E addition was defined as time = 0. An aqueous L17E solution was employed for this experiment as the stock solution.

Cellular uptake of macromolecules and microscopic observation For pulse-chase experiments (Figs. 2A and B), cells (2.0 × 105 cells) were seeded in 35 mm glass-bottom dishes (Iwaki) and were cultured at 37˚C in a humidified 5% CO2 atmosphere up to 90-100% confluence. Cells were washed twice with PBS(–) and were then incubated with 200 µg mL–1 Dex10-Alexa in the presence of L17E (40 µM) in α-MEM(–) (200 µL) at 37˚C for 5 min. After incubation, the cells were washed twice with PBS and incubated in α-MEM(+) at 37˚C for 5, 15, 30 and 60 min. Dextran cellular uptake was analyzed via live-cell imaging using an FV1000 Olympus confocal laser scanning microscope (CLSM) using 40 × objective. The numbers of cells


Page 8 of 41

Page 9 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


bearing diffused dextran signals in total >350 cells in >10 visual fields were counted for each sample. For analyzing the cytosolic translocation of Dex10-Alexa just after L17E addition (Fig. S2), cells were preincubated in α-MEM(+) containing Hoechst 33342 at 37˚C for 5 min to stain nucleus. After washing, cells were treated with Dex10-Alexa (200 µg mL–1) in α-MEM(–) containing L17E (40 µM) (200 µL) for 1, 3 or 5 min prior to CLSM observation. The numbers of cells bearing cytosolic dextran signals were determined in terms colocalization of Dex10-Alexa and Hoechst 33342 signals. Totally, > 400 cells from > 10 visual fields were counted for each sample. To evaluate the effect of NaN3 (Fig. 2C), cells were washed twice with PBS(–) and incubated at 37 °C for 15 min in PBS supplemented with 1 mM of MgCl2 and 1 mM of CaCl2 [PBS(+)] containing 10 mM NaN3 and 20 mM deoxy-D-glucose. The media were removed and fresh PBS(+) media containing 40 µM L17E and 200 µg mL–1 Dex10-Alexa with 10 mM NaN3 and 20 mM deoxy-D-glucose were added. The cells were then incubated for 5 min. For the 4°C experiment (Fig. 2D), cells were preincubated at 4°C for 30 min. Cells were then washed with cold PBS(–) and incubated with Dex10-Alexa (200 µg mL–1) in the presence of L17E (40 µM) in α-MEM(–) (200 µL) at 4°C for 5 min. After incubation, the cells were washed twice with PBS(–) and incubated in α-MEM(+) containing Hoechst 33342 to stain nucleus for 5 min at 37˚C. Cytosolic distribution of dextran in live cells was then analyzed using CLSM. For the inhibitor experiments, cells were preincubated in α-MEM(–) containing inhibitors at the following concentration [Cytochalasin D (CytoD), 5 µM; 5-(N-ethyl-N-isopropyl)amiloride (EIPA), 100 µM; wortmannin (500 nM); methyl-β-cyclodextrin (MβCD), 2mM] for 30 min. The media were removed and fresh α-MEM(–) (200 µL) containing 40 µM L17E and 200 µg mL–1 Dex10-Alexa as well as corresponding inhibitor were added and the cells were incubated at 37 °C



for 5 min. Control samples were run alongside experimental samples. After incubation, the cells were washed twice with PBS(–) and incubated in α-MEM(+) containing Hoechst 33342 to stain nucleus for 5 min at 37˚C. Observation by CLSM (40 × objective) followed. The numbers of cells bearing cytosolic dextran signals were determined in terms colocalization of Dex10-Alexa and Hoechst 33342 signals. Totally, > 350 cells from > 10 visual fields were counted for each sample. For the microscopic observation of 70 kDa dextran and IgG (Figs. 1D and S3), Cells were washed twice with PBS(–) and were then incubated with tetramethylrhodamine (TMR)-labeled dextran (70 kDa) (Dex70-TMR, 1 mg mL–1) or human IgG labeled with Alexa Fluor 488 (IgGAlexa, 500 µg mL–1) in the presence of L17E (40 µM) in serum-free or serum-supplemented αMEM (200 µL) at 37˚C for 5 min. After incubation, the cells were washed twice with PBS(–) and incubated in α-MEM(+) containing Hoechst 33342 to stain nucleus for 5 min at 37˚C. Cellular uptake was analyzed via live-cell imaging using CLSM.

Flow cytometric analysis of cellular uptake Cells (4.0 × 104 cells) were seeded in 24 well microplate (Iwaki) and were cultured at 37˚C in a humidified 5% CO2 atmosphere up to 90-100% confluence. Cells were washed twice with PBS(– ) and treated with dextran [Dex70-TMR (1 mg mL–1) (Fig. 1C) or FITC-labeled dextran (70 kDa) (Dex70-FL, 1 mg mL–1) (Fig. S9)] in α-MEM(–) (200 µL) in the presence of a L17E (40 µM) or SDF-1α (100 nM). Before the treatment of SDF-1α (Fig. S9), cells were cultured in α-MEM(–) overnight for starvation. After washing with PBS(–), the cells were treated with 0.01% trypsin for 10 min at 37˚C. The cells were collected, washed with PBS(–) and subjected to flow cytometry analysis using Attune NxT (Invitrogen).


Page 10 of 41

Page 11 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Flow cytometric analysis of cell-surface lipid exposure HeLa cells (8.0 × 104 cells) were plated onto 24 well microplate (Iwaki) and were cultured at 37°C in a humidified 5% CO2 incubator up to 90-100% confluence. Cells were washed twice with PBS(–) and were then incubated in PBS(+) (200 µL) containing 20 mM D-glucose [or 10 mM NaN3 and 20 mM deoxy-D-glucose (for ATP-depletion)] for 15 min. The media were removed and fresh PBS(+) (200 µL) containing 40 µM L17E as well as 20 mM D-glucose [or NaN3 and deoxy-D-glucose] were added and the cells were incubated for 5 min. Cells were washed twice with PBS(–) and incubated with 0.01% trypsin at 37 ˚C for 10 min, collected and washed with PBS(–). Control experiments in the absence of L17E were similarly conducted. For staining cell surface PIP2, cells were resuspended in 50 µL PBS(–) containing 1% bovine serum albumin (BSA) and 0.1% NaN3 and primary antibodies reagent (10 µL, 1/10 dilution). Thirty minutes after incubation at room temperature (~25°C), cells were washed with PBS(–). Treatment with secondary antibodies [anti-mouse IgM antibodies (Alexa Fluor 488 labeled)] was performed in the same way as primary antibodies. For staining cell surface PS, cells were resuspended in 100 µL Annexin binding buffer (Invitrogen) and Annexin V (5 µL) was then added. Fifteen minutes after incubation at room temperature, cells were diluted in Annexin binding buffer (300 µL). After staining each lipid, cells were subjected to flow cytometry analysis using Attune NxT. Analysis was conducted on 10,000 gated events per sample.

Di-4-ANEPPDHQ imaging and analysis of GP values Membrane fluidity was analyzed using Di-4-ANEPPDHQ as previously described.14,15 Briefly, HeLa cells were washed twice with PBS(–), then incubated with 5 µM di-4-ANEPPDHQ in α-



MEM(–) (100 µL) and allowed to settle for 10 min in an Olympus MI-IBC-IF micro-chamber. Imaging was performed using an FV-1000 confocal microscope equipped with a standard 60 × objective (oil, NA1.35). A 488-nm laser was used to excite di-4-ANEPPDHQ. L17E (60 µM) in α-MEM(–) (50 µl) was added. In this experiment, DMSO was not contained in the medium. Values for GP were calculated using a pixel basis according to 𝐼510 - 550 - 𝐼630 - 670

GP = 𝐼510 - 550 + 𝐼630 - 670 where I510-550 and I630-670 is the fluorescence intensities between 510–550 nm and 630–670 nm, respectively. In order to obtain GP distributions and pseudo-colored GP images, a macro designed by Owen et al. via ImageJ 1.49n (NIH, Bethesda, MD) was applied.15

LDH release assay The lactate dehydrogenase (LDH) release assay to assess the plasma membrane integrity was performed as previously reported.16 Briefly, HeLa cells (1.0 × 104 cells) were seeded into 96 well microplates (Iwaki). The cells were washed with PBS(–) twice and treated with peptides in αMEM(–) (50 µL) at 37°C for 5 or 60 min. The incubation media were collected and mixed with assay solution of LDH-Cytotoxic Test (Wako). Thirty minutes after incubation at room temperature (~25°C), absorbance at 492 nm was measured. The released LDH was calculated as 100 % when the cells were treated with 0.2 % (w/v) Triton X-100 in α-MEM(–) (50 µL) at 37°C for 5 or 60 min.

Transmission electron microscopy (TEM) 12 ACS Paragon Plus Environment

Page 12 of 41

Page 13 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cells (2.4 × 105 cells) were seeded in 35 mm cell culture dish (Iwaki) and were cultured at 37˚C in a humidified 5% CO2 atmosphere up to 90-100% confluence. Cells were washed twice with PBS(–) and were then incubated with 5 nm gold nanoparticles (1/10 dilution, streptavidinconjugated, Cytodiagnostics) in the presence of L17E (40 µM) in α-MEM(–) (1 mL) for 5 min at 37˚C. After incubation, the cells were washed twice with PBS(–). The cells were fixed in 0.1 M phosphate buffered 2% glutaraldehyde, and subsequently post-fixed with 2% osmium tetra-oxide for 2 hours in the ice bath. Then, the specimens were dehydrated in a graded ethanol and embedded in the epoxy resin. Ultrathin sections were obtained by ultramicrotome technique. Ultrathin sections stained with uranyl acetate for 15 min and lead staining solution for 5 min were submitted to TEM observation (HITACHI H-7600 at 100kV).



Results and Discussion L17E induces actin rearrangement Macropinocytosis is an actin-driven cellular uptake mechanism that serves to direct extracellular fluid into cells. Except for certain, specific cells, such as dendritic cells and macrophages, macropinocytosis is transiently induced by external stimuli and is accompanied by actin rearrangement, membrane ruffling, and eventual membrane fusion to form macropinosomes.17,18 To understand the relationship between macropinocytosis and the L17E-mediated translocation of biomacromolecules, we conducted a time-course study of actin rearrangement and cellular uptake of 70-kDa dextran as a model macromolecule for macropinosome-based uptake.17,19 The cytosolic translocation of dextran occurred in the presence of L17E after only 5 min and was synchronized with actin rearrangement. Actin filaments in live cells were visualized without disrupting the actin cytoskeleton using an actin-binding motif fused with a fluorescent protein (Lifeact-mCherry).20 Actin rearrangement before and after the addition of L17E were monitored in real time using confocal laser scanning microscopy (CLSM). Veil-like extension and dynamic rearrangement of actin structures were observed at the periphery of cells, suggesting ruffling of the cell membrane.21 This was observed within 2 min after exposing the cells to L17E (Fig. 1B arrowheads, Videos 1 and 2). Flow cytometric analyses also showed a 50% increase in the cellular uptake of tetramethylrhodamine (TMR)-labeled dextran (70 kDa) (Dex70-TMR) after incubating with L17E for 5 min (Fig. 1C). These results support the role of L17E in the induction of actin rearrangement and macropinocytosis.


Page 14 of 41

Page 15 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. (A) A proposed mode of action of L17E allowing facile translocation of biomacromolecules into the cytosol. Cell-surface interactions with L17E stimulate the reorganization of cytoskeletal actin, resulting in ruffled cell membranes. Transient permeabilization of the ruffled membranes allows for the cytosolic translocation of extracellular biomacromolecules. (B) (upper panels) Live cell images of an actin cytoskeleton (visualized using Lifeact-mCherry) 3.5 min before (left) and after (right) the addition of L17E to yield final concentration of 20 µM (t = 0). Scale bars, 25 µm. The boxed area in the right panel represents a typical area yielding a veil-like actin structure (lamellipodia) induced by L17E (see also the corresponding area in the left panel before the addition of L17E). Arrowheads indicate areas 15 ACS Paragon Plus Environment


yielding lamellipodia. The lower panels show enlarged images of the boxed areas in the upper panels; see also Videos 1 and 2, which show the changes in actin structure 10 min before and after the addition of L17E. (C) Increased Dex70-TMR (a macropinosome marker) uptake was observed after treatment with L17E (40 µM) for 5 min. The results are presented as the mean ± standard error of the mean (SEM) (n = 3). ***, p < 0.005 (Student's t-test). (D) Cells in the absence or presence of L17E (40 µM) after 5 min. In the presence of L17E, diffuse cytosolic Dex70-TMR signals were observed in addition to large punctate signals suggestive of macropinosome entrapment. Scale bars, 25 µm.

The terms “endocytosis” and “endosomes” is sometimes employed to specifically denote clathrin-mediated endocytosis (CME) and the vesicular compartments formed by CME, differentiating from macropinocytosis and macropinosomes, respectively. Since macropinocytosis is a form of the clathrin-independent endocytosis, we described “endocytosis” in this article as a general mechanism of the energy-dependent cellular uptake systems, which include both CME and macropinocytosis. Although there are many uncertainties in the intracellular fates of macropinosomes, these are in general considered to be fused with early endosomes to allow further maturation, similarly as in the case of vesicular compartments taken up into cells via other types of endocytosis including CME. The rupture of endosomal membranes can be detected using a fusion protein of galectin-3 with enhanced green fluorescent protein (EGFP).22 Galectin-3 is a marker of damaged endosomal membranes. Galectin is a lectin showing affinity for -galactosides. Since significant amounts of cell surface proteoglycans are incorporated into endosomes, the disruption of endosomal membranes leads to accumulation of galectin-3-EGFP in endosomes when the fusion protein is expressed in the cytosol. Entrapment of cell surface proteoglycans into macropinosomes has also been suggested.23 Endosomes bearing accumulated galectin-3-EGFP can be observed as puncta of intense fluorescence (Fig. S1, arrowheads). Cells that had been treated with L17E exhibited significant and highly fluorescent puncta. Similar results were obtained with Lipofectamine 2000, 16 ACS Paragon Plus Environment

Page 16 of 41

Page 17 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a representative transfection agent based on cationic liposomes. In contrast, no punctate galectin3-EGFP fluorescence was observed in the absence of L17E. CLSM observations of cells treated with Dex70-TMR in the presence of L17E yielded relatively large punctate or dot-like Dex70TMR signals, suggesting the presence of macropinosomes. However, diffuse Dex70-TMR signals were observed simultaneously in the cytosol (Fig. 1D). This suggests that the dextran was translocated into the cytosol in the very early stages of macropinocytosis.



L17E stimulates cellular responses to allow immediate cytosolic translocation of biomacromolecules In the presence of L17E, a considerable fraction of biomacromolecules reaches the cytosol within a few minutes. A pulse-chase experiment was conducted to determine the process that allows for this immediate translocation. Dextran (10 kDa), labeled with Alexa Fluor 488 (Dex10Alexa), was employed as a model macromolecule. Nuclear pores allow the passage of macromolecules under ~30–40 kDa.24 Therefore, the cytosolic translocation of 10-kDa dextran should yield nuclear localization. The populations of cells bearing cytosolic dextran signals were thus analyzed in terms of nuclear localization of Dex10-Alexa (see Experimental Section for details). Prior to CLSM analyses, cells were treated with Dex10-Alexa in the presence of 40 µM L17E for 1, 3, or 5 min. Although faint, significant cytosolic fluorescence from Dex10-Alexa was observed in ~30% of the cells after only 1 min. Prolonged incubation yielded increases in both the number of cells exhibiting fluorescence and the intensity of the fluorescence signal. Surprisingly, up to 40–50% of the cells exhibited cytosolic localization of Dex10-Alexa after incubation with L17E for 5 min (Figs. S2A and B). As previously reported,10 no marked decrease in cytosolic distribution was observed when the cells were similarly incubated with Dex10-Alexa and L17E in serum-containing media for 5 min (Fig. S2C). A pulse-chase experiment was conducted to examine the timing of endosomal escape or cytosolic translocation of Dex10-Alexa. Cells were pretreated with Dex10-Alexa in the presence of 40 µM L17E for 5 min and washed with PBS(−) to remove any extracellular peptides. The cells were then incubated in a medium without L17E. The cytosolic distribution of Dex10-Alexa was analyzed 5, 15, 30, and 60 min after washing (Fig. 2A). Figure 2B shows the number of cells 18 ACS Paragon Plus Environment

Page 18 of 41

Page 19 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


exhibiting a diffuse cytosolic distribution of Dex10-Alexa. Notably, there were no significant differences in the percentages of cells containing cytosolic Dex10-Alexa after 5 min of incubation compared to baseline. If significant amounts of Dex10-Alexa had remained in endosomes after cell washing, the number of cells exhibiting cytosolic Dex10-Alexa would have increased. Therefore, these data suggest that endosomal maturation is not required for L17E-mediated translocation of Dex10-Alexa. Rapid translocation in the presence of L17E was also observed with antibodies (IgG, human, ~150 kDa) in both the presence and absence of serum (Fig. S3).

Figure 2. The L17E-mediated uptake of Dex10-Alexa into the cytosol is energy-dependent and occurs within 5 min. (A) Pulse-chase experiment: HeLa cells were treated with Dex10-Alexa (200 µg mL–1) in the presence of L17E (40 µM) in serum free α-MEM [α-MEM(–)] for 5 min prior to washing. The cells were then incubated in a fresh serum containing α-MEM [α-MEM(+)] for 5, 15, 30 and 60 min, and subjected to CLSM observation. Scale bars, 50 µm. (B) The percentage of cells from (A) showing cytosolic Dex10-Alexa. The data are presented as the mean ± SEM (n = 4). One-way analysis of variance (ANOVA) showed no significant differences. (C, D) Complete absence of cytosolic Dex10-Alexa was observed after treatment with Dex10-Alexa (200 µg mL– 1) in the presence of L17E (40 µM) for 5 min under ATP-depleted conditions (C) and at 4˚C (D). Scale bars, 50 µm.



One hypothesis for the immediate cytosolic translocation of Dex10-Alexa is L17E-mediated pore formation on the cell membrane. L17E originates from a lytic peptide, M-LCTX, having a cationic amphiphilic structure. However, the observed results cannot be attained through simple, physicochemical methods of pore formation. The presence of Dex10-Alexa in the cytosol was energy-dependent such that no cytosolic Dex10-Alexa was observed under ATP-depleted conditions, i.e., incubation with sodium azide25 or at 4°C18 (Fig. 2C and D). In addition, lactate dehydrogenase (LDH) assays indicated very little damage to cell membranes following L17E treatment (Fig. S4). LDH is a cytosolic enzyme and the release of cellular LDH via cell membrane damage can be determined by measuring the LDH activity of a culture supernatant.16 No significant leakage of LDH from cells was detected following cell incubation in 40 µM L17E for 5 or 60 min. Conversely, cell cultures treated with 20 µM M-LCTX showed considerable amounts of free LDH.

The majority of L17E-mediated cytosolic translocation is accomplished prior to endosomal maturation We further assessed the contribution of endosomal maturation to the L17E-mediated cytosolic translocation of Dex10-Alexa. Studies using Rab5 and its mutants suggested that L17E-mediated cytosolic translocation does not require endosomal maturation (Fig. 3). Here, Rab5 is a key protein regulating the formation of early endosomes and their maturation.26 Involvement of Rab5 in maturation of macropinosomes has also been reported.27 The constitutively inactive Rab5 mutant (S34N) inhibits the membrane fusion involved in early endosome formation. The constitutively active mutant (Q79L) prevents the further fusion of early endosomes to other vesicular compartments to generate late endosomes (Fig. 3A).6,7


Page 20 of 41

Page 21 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cells expressing fusion proteins of wild type (WT) Rab5 and its S34N and Q79L mutants with mCherry (mCherry–WT, –S34N, and –Q79L, respectively) were treated with Dex10-Alexa for 5 min. The degree of cellular localization was determined by CLSM (Fig. 3B). As previously reported,6,7 signals from mCherry-WT indicate localization within a vesicular compartment, i.e., early endosomes (Fig. 3B). In the case of mCherry–S34N, small, vesicle-like structures stained with mCherry–S34N were observed. This suggests the inhibition of Rab5-mediated early endosome formation. In cells expressing mCherry–Q79L, swollen vesicles stained with mCherry– Q79L were observed. This suggests the accumulation of lipids into early endosomes and their enlargement by the suspension of endosomal maturation. As expected, the observed cellular localization of Rab5 mutants confirms the distortion of Rab5. Conversely, there was little difference in the efficacy of cytosolic translocation or the percentage of cells yielding diffuse cytosolic Dex10-Alexa fluorescence when in the presence of L17E for 5 min, although there were differences in the patterns of cytosolic labeling due to morphological changes in endosomal structures (Fig. 3B, C). These results strongly suggest that most L17E-mediated cytosolic translocation of macromolecules occurs before their reaching early endosomes. Furthermore, this is accomplished in a few minutes.



This idea is supported by transmission electron microscopy (TEM) observations. Cells were treated with avidin-conjugated gold nanoparticles (5 nm) in the presence of L17E for 5 min. The cell samples were then fixed, sliced, and subjected to TEM observation. In the absence of L17E, gold nanoparticles were found in the cells in vesicular compartments (Figs. S5A and B). This

suggests that these particles are taken up into cells within 5 min and trapped within endosomes. In cells treated with L17E, the gold nanoparticles had been released into the cytosol. No vesicle-like structures were observed around the gold nanoparticles (Fig. S5C).

Figure 3. L17E-mediated cytosolic translocation does not require endosomal maturation. (A) A schematic shows the inhibitory effects of Rab5 mutants on endocytosis. (B) No significant 22 ACS Paragon Plus Environment

Page 22 of 41

Page 23 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


inhibition of L17E-mediated Dex10-Alexa uptake was observed in cells expressing Rab5 (WT) or its mutants. Scale bars, 25 µm. (C) The percentage of cells in (B) containing cytosolic Dex10Alexa. The data are presented as the mean ± SEM (n = 4). **, p < 0.01, N/S means of no statistically significant difference. (one-way ANOVA followed by Dunnett’s post hoc test).



L17E-mediated cytosolic delivery requires membrane ruffling leading to macropinocytosis The above results suggest that the induction of actin reorganization and macropinocytosis by L17E allows for the cytosolic translocation of Dex10-Alexa during the very early stages of macropinocytosis. Close examination of the initial steps of macropinocytosis using specific inhibitors suggest that the majority of Dex10-Alexa enters the cytosol after the induction of membrane ruffling but prior to membrane fusion or closure, which completes macropinosome formation (Fig. 4). The initiation of macropinocytic uptake can be described as two stages, actin polymerization followed by membrane ruffling and the ruffle closure into macropinosomes (Fig. 4A). Cytochalasin D (CytoD), EIPA, and wortmannin are classical macropinocytosis inhibitors.28 CytoD is the inhibitor for actin polymerization, which is largely related to macropinocytosis induction process. EIPA shows broad inhibition for uptake of macromolecules. One proposed mechanism of EIPA is inhibiting the formation of ruffled membranes, i.e., membrane upthrust in accordance with actin polymerization, where acidification of membrane peripherals occurs via local proton influx through Na+-H+ exchange.29 Finally wortmannin inhibits membrane fusion prior to macropinosome formation but does not suppress the induction of membrane ruffling.30 While the cytosolic influx of Dex10-Alexa was strongly inhibited by treatment with cytochalasin D or EIPA (Fig. 4B, C), no inhibition was observed with wortmannin treatments (Fig. 4D). Additionally, the inhibition of cytosolic Dex10-Alexa translocation by EIPA and CytoD was also observed in the cells expressing Rab5 mutants (Fig. S6), further ensuring that the major internalization route of Dex10-Alexa is through the ruffled membranes as suggested in Fig. 4 and that the Rab5 mutants are not capable of internalizing Dex10-Alexa via other mechanism.


Page 24 of 41

Page 25 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The inhibition of Dex10-Alexa uptake was closely correlated with the inhibition of membrane ruffling, but less so with macropinosome formation. Live imaging of cells treated with L17E in the presence of EIPA showed an absence of membrane ruffling (Fig. S7, upper panels). Conversely, membrane ruffling was observed with cells treated with L17E in the presence of wortmannin (Fig. S7, middle and lower panels). Wortmannin inhibits membrane fusion prior to macropinosome formation but does not suppress the induction of membrane ruffling.28,30 The slight increase in Dex10-Alexa uptake when exposed to wortmannin may also be explained by the retention of ruffled membranes, which would be permeable to Dex10-Alexa. We therefore hypothesize that L17E-mediated cytosolic delivery occurs concurrently with membrane ruffling. Note that the cytosolic translocation can be accomplished by the L17E interaction with membranes that undergo a dynamic structural alteration. In the absence of inhibitors, the above steps of engulfment take place continuously, and there must be fluctuation of membrane structures even after the ruffle closure followed by macropinosome formation. Therefore, the above inhibitor study does not exclude possibility that some part of cytosolic translocation may be attained by the leakage from macropinosomes at very early stage of macropinosomes.



Figure 4. Membrane ruffling induced by L17E allows the cytosolic translocation of biomacromolecules. (A) The different stages of membrane ruffling and macropinocytosis can be differentiated with selective inhibitor treatments. (B–D) The effects of inhibitors on the cytosolic translocation of Dex10-Alexa. The data are presented as the mean ± SEM [n = 4 (C) and n = 6 (B, D)]. *, p < 0.05, ***, p < 0.005 (Student's t-test). (E) The GP value of cell membranes is reduced following exposure to L17E to yield final concentration of 20 µM. Fluorescence micrographs show pseudo-color images of GP values obtained 1 min before and 5 min after L17E treatment. The box plots represent the mean ± SEM [n = 10 (L17E), 8 (no peptide treatment, NP)]. **, p

Inducible membrane permeabilization by ... - ACS Publications

Recommend Documents