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Inward budding and endocytosis of membranes regulated by de novo designed peptides Qiuhong Yu, Jianbo Sun, Siqi Huang, Haojing Chang, Qingwen Bai, Yong-Xiang Chen, and Dehai Liang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00882 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018
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Langmuir
Inward budding and endocytosis of membranes regulated by de novo designed peptides Qiuhong Yua, Jianbo Suna, Siqi Huangb, Haojing Changa, Qingwen Baia,Yong-Xiang Chenb,*, Dehai Lianga,* a
Beijing National Laboratory for Molecular Sciences and the Key Laboratory of Polymer Chemistry and Physics and Ministry of Education, College of Chemistry and Molecular Engineering b Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China KEYWORDS: membranes, inward budding, endocytosis, peptides
ABSTRACT: Protein-mediated endocytosis of membrane is a key event in biological system. The mechanism, however, is still not clear. Using a de novo designed bola-typed peptide KKKLLLLLLLLKKK (K3L8K3) as a protein mimic, we studied how it induced giant unilamellar vesicle (GUV) to form inward buds or endocytosis at varying conditions. Results show that the inward budding is initiated as the charged lipids are neutralized by K3L8K3, which results in a negative spontaneous curvature. If the charged lipids have unsaturated tails, the buddings are slim fibrils, which can further wrap into a spherical structure. In the case of saturated charged lipids, the buddings are rigid tubules, stable in the studied time period. The unsaturated lipid to saturated lipid ratio in the mother membrane is another key parameter governing the shape and dynamics of the buds. A complete endocytosis is observed when K3L8K3 is attached with a hydrophobic moiety, suggesting that hydrophobic interaction helps the buds detaching from the mother membrane. The molecules in the surrounding medium, such as negatively charged oligonucleotides, are engulfed into the GUV via endocytosis pathway induced by K3L8K3. Our study provides a novel strategy for illustrating the endocytosis mechanism by using peptides of simple sequence.
1. INTRODUCTION Cell is the fundamental structure and functional unit in all living organisms.1 The plasma membrane surrounding the cell not only segregates the intracellular content from the extracellular environment, but also plays an important role in material transport, energy conversion, and signal transfer between cells and the environment.2 In physiological environment, the plasma membrane undergoes morphological changes all the time, which is related to the cell functions, such as movement, division, extension of neuronal arbors, and vesicle trafficking.3 Among these morphological changes, endocytosis is one of the most important processes. It is generally accepted that endocytosis includes two categories: phagocytosis and pinocytosis. The latter occurs in all cells via several underlying mechanisms, such as micropinocytosis,4 clathrin-mediated endocytosis,5 and caveolae-mediated endocytosis.6 Many animal viruses enter the cell by taking advantage of the cell’s endocytic pathways mentioned above.7 It is also reported that the nanocarriers used for drug or gene delivery are internalized also via clathrin- or caveolae-mediated endocytosis.8 The study on the endocytosis mechanism not only gains insight into the cell functions but also helps to develop new approaches to block the entry of viruses and to develop drug/gene carriers with high performance. However, most of the endocytosis mechanisms have not been fully elucidated yet. Clathrin-mediated endocytosis is the most studied mechanism. It is reported that the invagination of the membrane is caused by the assembly of clathrin triskelions, which can form
a curved polygonal web.9 This inward budding of membranes is achieved on giant unilamellar vesicles (GUVs) by encapsulation of PEG/dextran solution,10,11 adsorption DNA,12 fatty acids,13 or phospholipid-binding protein Annexin A2.14 Quantitative theories focusing on morphological changes of vesical membranes have also been developed.15-18 The shape transition of vesicles is determined by the area-to-volume ratio,19 as well as the competition between curvature energy.20 Inward budding occurs as the area of the outer monolayer is larger than that of the inner monolayer, together with a reduction in volume. This prediction has been proven by experiments on dimyristoyl phosphatidylcholine (DMPC) vesicle in pure water via heating treatment.21 The formation of microdomains in the bilayer also induces a vesicle to change its morphology.22,23 Several models concerning the dynamics of microdomains and the corresponding budding mechanisms have been put forward, but still need to be experimentally verified.24-30 Most of the cell endocytosis pathways involve proteins, which take part in the specific receptor-ligand interactions before or during endocytosis.31-33 However, the structure and dynamics of proteins involved in endocytosis are complicated. It is difficult to reveal the roles of these proteins played in the endocytosis pathway at molecular level. A strategy named ‘divide and conquer’ has been proposed,34 in which the protein can be divided into peptides of known sequence and secondary structure.35,36 Our previous study showed that a 14-mer peptide formed by only lysine and leucine could induce GUV to leak, burst, or undergo shape transitions.37 It has also been reported
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that small antimicrobial peptides were able to cause the membrane to form buds.38 In this work, we designed a bola-typed peptide KKKLLLLLLLLKKK (abbreviated as K3L8K3) and studied how it induced GUV to form inward buddings or endocytosis. The GUV is composed of DOPC/DPPC/cholesterol, a combination commonly used to mimic biomembrane,39 as well as negatively charged lipids DOPG or DPPG. The cationic amphiphilic peptide K3L8K3 can interact with GUV via both electrostatic interaction and hydrophobic interaction, mimicking the receptor-ligand interaction between protein and cell membrane. Results show that the presence of K3L8K3 can effectively cause inward budding and endocytosis of GUV under certain conditions. The shape of the budding is not necessarily spherical. It could be fibrous or tubular shape depending on the lipid components in GUV, as well as the location of charged lipids. The substances in the environment can be internalized via endocytosis in the presence of K3L8K3. 2.MODELS AND METHODS Materials 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-di-(9Zoctadecenoyl)-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-racglycerol) (DPPG), the fluorescent lipids 1,2-dipalmitoyl-snglycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DPPE-Rho), and cholesterol (chol) were purchased from Avanti Polar Lipids, purity >99%. Calcein and perylene were purchased from Sigma. A 21-nt ss-oligo (MW = 6,387) of random sequence with or without Cy5 fluorescent tag was purchased from Invitrogen Inc. K3L8K3 (AcKKKLLLLLLLLKKK-amide) (>98%) was purchased from GL Biochem Ltd (Shanghai, China). FAM-labelled K3L8K3 was synthesized and purified in the lab by following a standard solid-phase peptide synthesis procedure.40 Milli-Q water (18.2 MΩ.cm) was used in all the experiments. Preparation and observation of GUVs GUVs were prepared by using a modified protocol of electroformation.41,42 The chamber is formed by two ITO glasses (50 mm × 50 mm × 1.1 mm) and a PDMS washer with the thickness of 2 mm. The phospholipid mixtures (total concentration: 3.75 mg/mL) with a known component ratio in 95% chloroform/5% acetonitrile were spin-coated at 800 rpm for 60 s onto the surface of one of the ITO glasses. These two ITO glass slides were then held together by binder clips. After incubated at 50°C for 10 min, the chamber was filled with 0.10 M sucrose solution which was preheated to 50°C. A 2.0 V (peak-to-peak), 10 Hz AC electric field was applied immediately. After 4 hours, the GUVs were detached from the electrode by applying a 3.0 V (peak-to-peak), 5 Hz AC field for 1 hour. Besides GUVs, giant multilamellar vesicles, vesicles with low stability and small size, and even solid spherical structures were generated by electroformation method, especially in the presence of charged lipids. Only GUVs with 1030 µm in diameter were used to study the deformations. The GUVs were viewed by Laser Scanning Confocal Microscope (LSCM, A1R-si, Nikon, Japan) for image taking or video shooting. 30 min after preparation, 10 µL of GUV suspension in 0.10 M sucrose was added to 200 µL solution containing 0.10 M glucose with or without 50 µM calcein. No
salts are introduced in this study. Because the sucrose solution was denser than the glucose solution, the GUV aliquot sank to the bottom of the chamber, which was convenient for in situ observation of the processes. The images of GUVs at the equatorial plane were collected, and the dynamical behavior was monitored after the addition of K3L8K3. The zero time point is set as the moment of adding peptide into GUV. The excitation wavelengths were 638 nm, 543 nm, and 488 nm for Cy5-21nt DNA, DPPE-Rho, and calcein, respectively. The exact lipid concentration in GUV was determined by modified Bartlett method.43 The final concentration was 0.15 mM. Preparation of Large Unilamellar Vesicle (LUV) The LUVs with the same lipid composition as GUVs were prepared, separately, by using Bangham method.44 In brief, lipids were added into a 25 mL pyriform flask with Teflon beads inside. The organic solvent was evaporated at 50°C under vacuum. After being dried overnight, the lipid film was rehydrated with Tris buffer (10 mM Tris, pH 7.4) at 50°C on a rotary evaporator for one hour. The resulting stock solution was then extruded through a polycarbonate filter with 100 nm pore size at 50°C for 21 times by using a mini-extruder (Avanti Polar Lipids, Inc.). The final concentration of the stock solutions was determined by modified Bartlett method.43 The stock solution was then diluted to 10 µM in Tris buffer. Circular Dichroism Spectroscopy (CD) The secondary structure of peptides in LUVs was studied by a mixture containing 100 µM lipids and 20 µM peptide, the same lipid/peptide ratio employed in the GUV experiment. CD spectra assay was conducted from 190 to 250 nm at 37°C using a JASCO J-810 spectrometer (Jasco, Tokyo, Japan). A cuvette of 1.0 cm path length was used. All the measurements were repeated three times. The background value of bulk solution without peptide was subtracted to account for instrumental differences. 3. RESULTS AND DISCUSSION 3.1 Effect of charge density The strong electrostatic interaction between the positively charged K3L8K3 and the negatively charged GUV exhibits a profound effect on the deformation of vesical structures. The concentration of K3L8K3 in all the studies was fixed at 1.5 µM, with varied membrane charge density of GUVs. The GUV containing 15% charged DOPG keeps basically intact in the first 30 s in the presence of 1.5 µM K3L8K3 (Fig. 1A), while the one containing 20% DOPG transforms into a toroidal shape under the same conditions (Fig. 2B). When the content of DOPG increases to 25%, the GUV bursts into pieces (Fig. 2C) in 30 s. In order to monitor the morphological transformation process of GUV in the presence of K3L8K3, the content of the charged lipid is fixed at 15% in the following studies.
Figure 1. Morphology of the GUVs containing (A) DOPG: DOPC: DPPC: chol=15: 45: 15: 25, (B) DOPG: DOPC: DPPC: chol=20: 40: 15: 25, and (C) DOPG: DOPC: DPPC: chol=25:
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Langmuir 35: 15: 25 in mole ratio in the presence of 1.5 µM K3L8K3 at 30 sec. The membrane of GUVs is labeled with DPPE-Rho (red). The inside of GUV is black when no dye molecules migrate in. Scale bar 10 µm.
3.2 Deformation of GUVs by K3L8K3 It is known that the GUV containing mixed lipids of different transition points exhibits lateral phase separation or lipid demixing.45-48 DPPC and cholesterol form liquid-ordered phase, and DOPC and DOPG form liquid-crystalline phase.48 The addition of cholesterol into the membrane not only increases the lateral diffusion rate of phospholipids but also facilitates flip-flops and releases the local stresses induced by bending deformations.49,50 The size of the domains and the interface between domains could be related to the deformation of the membrane. To testify this hypothesis, we prepared GUVs consisting of different ratios of saturated and unsaturated lipids. As listed in Table 1, the contents of charged lipids and cholesterol are fixed at 15% and 25%, respectively, while the ratios of unsaturated lipids (DOPC+DOPG) to saturated lipids (DPPG+DPPC) are ranged from 0 to infinite. The charged lipids are located in the domains either rich in unsaturated lipids (DOPG) or rich in saturated lipids (DPPG). The GUVs with the charged lipids as DOPG and DPPG are named as “O” and “P”, respectively, followed by the molar ratio of unsaturated to saturated lipids.
3B and Movie S2). As the content of unsaturated lipids in the membrane increases, the fibrils experience a faster transformation into a sphere right after a quick deformation of the mother vesical, as observed in O6-1 (Fig. 3C and Movie S3). In O1-0, the GUV composed of pure unsaturated lipids, no deformation or formation of fibrils is observed. Instead, dozens of spherical inward buds are observed simultaneously (Fig. 2E). These buds are highly mobile on the surface of the membrane (Movie S4). Besides, when the GUV contains more unsaturated lipids in its membrane, it witnesses a decrease in the radius of the budding (from 3.0 µm in O3-2 to no more than 1.3 µm in O1-0), as indicated by Fig. 4.
Table 1: components of the lipids in the GUV Name
Vesicle composition
O/P
O1-4
DOPG: DPPC: chol=15: 60: 25
1/4
O3-2
DOPG: DOPC: DPPC: chol=15: 30: 30: 25
3/2
O4-1
DOPG: DOPC: DPPC: chol=15: 45: 15: 25
4/1
O6-1
DOPG: DOPC: DPPC: chol=15: 50: 10: 25
6.5/1
O1-0
DOPG: DOPC: chol=15: 60: 25
1/0
P0-1
DPPG: DPPC: chol=15: 60: 25
0/1
P1-4
DPPG: DOPC: DPPC: chol=15: 15: 45: 25
1/4
P3-2
DPPG: DOPC: DPPC: chol=15: 45: 15: 25
3/2
P4-1
DPPG: DOPC: chol=15: 60: 25
4/1
Fig. 2 compares the deformation of the “O” series GUVs in the presence of 1.5 µM K3L8K3 at 37oC. No prominent change is observed in the studied period for O1-4 (Fig. 2A). While in O3-2 GUV, some thin and soft fibrils quickly appear (Fig. 3A1), followed by a substantial deformation of the vesicle (Fig. 3A2, 3A3). The transverse dimensions of the fibrils are beyond the resolution of optical microscopy. However, green fluorescence representing calcein molecules is observable inside the fibrils in the magnified snapshot (Fig. 2B). Because the calcein molecules are added beforehand only outside the vesicles, it suggests that the fibrils are thin tubules generated via inward budding. These fibrils are highly flexible and eventually transform into a solid globule (Fig. 3A4 and Movie S1). Meanwhile, the GUV recovers its original shape as a smooth and spherical vesicle. Compared with O3-2, O4-1 exhibits similar behaviors under the same conditions, but with a less prominent structural deformation of the mother vesicle, and a much faster transformation of fibrils into spheres (Fig.
Figure 2. Membrane deformation of the “O” series GUVs. Fluorescent intensity is determined using the built-in program in confocal microscope. The membrane of GUV is labeled with DPPERho (red) (Top row) and the surrounding solution contains calcein (green) (Second row). The third row magnifies the shape engulfed by GUV, and the corresponding intensity profile is shown in the bottom row (x-axis illustrates the length in µm and y-axis illustrates the intensity in a.u.). K3L8K3 concentration: 1.5 µΜ, 37oC. Scale bar, 10 µm.
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B2
B3
B4
112 s
148 s
194 s
202 s
C1
C2
C3
C4
110 s
120 s
126 s
134 s
Figure 3. Membrane deformation of O3-2(A), O4-1(B) and O61(C) over time. K3L8K3 concentration: 1.5 µΜ, 37oC. Scale bar, 10 µm.
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The transformation of tubules into spherical structures has also been reported on supported lipid bilayers (SLBs) 51,52 and GUVs 53,54, in which the antimicrobial peptides cause an outgrowth of fibrils or tubules. The fibrils transform into spherical structures under certain condition, but its mechanism is still unclear.55 In our study, the lipid bilayer of GUV is labeled by DPPE-Rho. The fluorescence intensity profile across the endocytic spherical structure shows that the intensity on the border is stronger but not zero inside (Fig. 4B and 4C), suggesting that it is not pure vesicles. The serial snapshots (Fig. 5, Movie S2) shows that the spherical structure and fibrils are interconvertible via reversible wrapping-dewrapping process. The persistence length of the fibril should be in the order of 10-50 µm if it is cylindrical, 10,56 too rigid to wrap into an aggregate less than 3 µm in diameter. It is highly possible that the fibrils are necklace-like tube. The mobile membrane surface facilitates the wrapping of the fibrils, making it looks like a vesicle under fluorescence microscope. In fact, the fluorescence intensity inside the “vesicle” is almost the same as the GUV’s border (Fig. 4A).
Figure 5. The sequential change of the fibrils with time. The spherical structure is formed by the wrapping of the fibrils and can also dewrap into fibrils (e.g., A6 to A7), scale bar, 10 µm.
Figure 6 shows behaviors of the “P” series GUVs in the presence of K3L8K3. Except for the P0-1, the “P” series GUVs studied have the same O/P ratio as that of their “O” series counterparts, but differ in charged domains with totally opposite degree of saturation. For the GUV mainly composed of saturated lipids, such as P0-1 and P1-4, the vesical structure remains basically intact at the studied conditions (Fig. 6A and 6B), similar to that of O1-4 (Fig. 2A). The inward budding of tubular structures is observed in P3-2 GUV (Fig. 6C), accompanied with a long-range fluctuation of GUV membrane (Fig. 8A). The transverse dimension of the tubule is about 1.6 µm (Fig. 7B), much larger than that of the fibrils generated in the GUV in “O” series (Fig. 7A). The tubules, with a length order of more than 15 µm, do not transform into spherical structures in the studied time period (Fig. 8A, Movie S5). In the case of P4-1, which is composed of mainly unsaturated lipids, the GUV membrane exhibits a heavy fluctuation in the presence of K3L8K3, as indicated by the wrinkle on the membrane under microscope (Fig. 6D). Only spherical structure is observed inside the P4-1 GUV (Figure 8B and Movie S6), which is similar to the behavior of O1-0 that resembles in membrane saturation (Fig. 2E). However, the radius of the sphere in P4-1 is about 2.6 µm (Fig. 7C), at least twice larger than those inside O1-0. The analysis on fluorescence intensity shows that the spherical structure is not pure vesicle either (Fig. S1).
Figure 4. Size of the endocytic buds of O3-2(A), O4-1(B), O61(C) and O1-0(D) are estimated in the first column. The engulfed shape is magnified in the second column and the fluorescent intensity along the solid white arrow across the endocytic buds are shown in the third column. The x-axis in column 3 illustrates the length in µm and the y-axis illustrates the intensity in a.u, scale bar, 10 µm.
A1
A2
A3
A4
112 s
118 s
122 s
138 s
A5
A6
A7
A8
148 s
154 s
166 s
182 s
Figure 6. Membrane deformation of the “P” series GUVs. Fluorescent intensity was determined using the built-in program in Confocal. The membrane of GUV is labeled with DPPE-Rho (red) (top row) and the surrounding solution contains calcein (green) (Second row). The third row magnifies the shape engulfed by GUV, and the corresponding intensity profile is shown in the bottom row (x-axis of length in µm and y-axis of intensity). K3L8K3 concentration: 1.5 µΜ, 37oC. Scale bar, 20 µm. Scale bar, 10 µm.
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Langmuir Since the movement of the daughter vesicle is not correlated with the mother GUV (Movie S7), and the daughter vesicle maintained a uniform hollow spherical structure with time (Fig.9), it should be fully detached from the O4-1membrane instead of attaching to the mother membrane via a tube underneath. Such process is different from the inward budding of fibrils followed by transformation to spherical structure in the presence of K3L8K3 under the same conditions (Fig.3B), suggesting that the hydrophobic FAM generates extra effect on the endocytosis of GUVs. In the case of O3-2, FAM-K3L8K3 peptide can also strongly stick on the membrane surface without causing any deformation of GUV (Fig. S2).
Figure 7. The endocytic fibrils and buds of O3-2(A), P3-2(B) and P4-1(C) are magnified in the second column and the size of the engulfed shape are estimated. A1
A2
A3
A1
A2
A3
A4
B1
B2
B3
B4
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136
160
196
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Time (sec) 164 s
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170 s
176 s
B1
B2
B3
B4
140 s
142 s
158 s
164 s
Figure 9. Snapshots of O4-1GUV in the presence of FAM-K3L8K3. The green fluorescence shows the distribution of FAM-K3L8K3, with the arrow indicating the vesicle formed by endocytosis (A1A4). The red fluorescence shows the membrane of the GUV. Scale bar, 10 µm. The experiment is conducted by using the same laser power.
Figure 8. Membrane deformation of the P3-2(A) and P4-1(B) over time. K3L8K3 concentration: 1.5 µΜ, 37oC. Scale bar, 10 µm.
3.3 Distribution and secondary structure of K3L8K3 To understand the role of K3L8K3 during the inward budding process, we labeled K3L8K3 by a fluorescence probe, FAM, and monitored its distribution during the deformation of O4-1. Calcein is removed from the environment because its fluorescence interferes with that of FAM. Fig. 9 shows how the FAMK3L8K3 interacts with O4-1 GUV at the same conditions used in Fig. 2C. The green fluorescence indicates the distribution of FAM-K3L8K3. At the early stage, FAM-K3L8K3 molecules accumulate on the GUV surface (Fig. 9A1, Movie S7), followed by endocytosis of a spherical vesicle with FAM-K3L8K3 on its membrane. The fluorescence intensity on the GUV membrane keeps increasing with time, while the fluorescence on the vesicle inside GUV is relatively stable after a slight increase at the early stage (Fig. 9A1-A4). These snapshots indicate that FAM- K3L8K3 can strongly attach to the GUV surface, and the endocytosis of GUV occurs at the early stage, where the GUV surface has not been fully covered by FAMK3L8K3. In case the coating is dominant, no endocytosis occurs, and the GUVs maintain their shape, which explains that only a portion of GUVs (Table S1) undergo the deformation, while the rest are intact. The red fluorescence from DPPE-Rho (Fig. 9B1-B4) indicates that the lipid membrane is stable during the whole endocytosis process. Fig. 10 compares the fluorescence intensity of FAM-K3L8K3 on GUV membrane and on the endo membrane. The intensity of the endo membrane is lower than that on GUV membrane as the endocytosis occurs. The difference could be 7 times in about 270 s.
Figure 10. Intensity statistics of FAM fluorescence of the GUV membrane and inner endo globule membrane using the built-in program in confocal microscope.
CD experiments were conducted to determine the secondary structure of K3L8K3 when interacting with lipid membranes. Since the deformation of GUV is time-dependent, the CD spectra at 5 min and 20 min were recorded (Fig. S3). The contents of α-helix and β-sheet were calculated using CDpro software (SELCON3 method). K3L8K3 is prone to take a conformation of α-helix while interacting with the GUV that can undergo inward budding or endocytosis, such as O4-1 and P41 (Fig. 11A). In all studied cases, the content of α-helix keeps increasing with time, correspondingly with a decrease in that of β-sheet (Fig.11B). Since a-helix is formed by intra-chain hydrogen bonding, while β-sheet is formed by inter-chain hydrogen bonding, the absorption of K3L8K3 on the GUV surface via electrostatic interaction increases its local concentration, facilitating the formation of β-sheet via aggregation. The insertion of K3L8K3 inside the lipid bilayer, mainly driven by hy-
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drophobic interaction, can tune K3L8K3 structure from β-sheet to α-helix.
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dynamic, may also make some contribution to the inward budding of the membrane.
Figure 11. Content of α-helix (A) and β-sheet (B) of K3L8K3 interacting with different LUVs. 3.4 Mechanism of inward budding and endocytosis Table 2 summarizes the deformation of different GUVs in the presence of K3L8K3. Control experiments show that no inward budding occurs if cholesterol or charged lipid is absent (Fig. S4, Fig. S5). Fluorescence images with perylene as the probes57, which preferentially labelling Lo phases, show that nanoscopic phase separation47,48,58 occurs in the presence of K3L8K3, which is reasonable considering that the experiments are conducted at 37oC, close to the transition temperature (about 41oC) of DPPC, but far above that (about -20oC) of DOPC. The presence of charged lipids also has the tendency to suppress the macroscopic phase separation59. Theoretical studies propose that the budding of multicomponent membrane is determined by the interaction parameter between different components, the spontaneous curvature, the bending rigidity, and the fraction of different components.60 Only negative spontaneous curvature can lead to inward budding.30 Negative curvature can be induced in the presence of peptides.61-63 In our system, the attachment of K3L8K3 on the membrane causes profound effects on the interaction parameter, the spontaneous curvature, the bending rigidity, as well as the redistribution of the components. Driven by electrostatic interaction, the K3L8K3 molecules quickly attach to the negatively charged membrane surface in the very beginning. As the concentration increases with time, K3L8K3 starts to aggregate on the membrane surface (Fig. 12A). The peptides form β-sheet inside the aggregates, while they stay mainly as α-helix on the interface between the aggregate and the lipid membrane. The aggregation of K3L8K3 is accompanied with a redistribution of the charged lipid, DOPG or DPPG (Fig. 12B). The neutralization of DOPG or DPPG reduces the headgroup-headgroup repulsion, resulting in a negative spontaneous curvature in the membrane segment (Fig. 12C). Inward budding or endocytosis occurs (Fig.12D) with time. Further accumulation of peptides results in the formation of a thick protection layer on the membrane, which hinders the deformation of GUVs. The βsheet to α-helix transition suggests that K3L8K3 also interacts with the bilayer via hydrophobic interactions. The length of the leucine block is about 1.2 nm in α-helix conformation, far less than the thickness (about 4 nm) of the membrane. Since K3L8K3 is bola type, the leucine block interacts with the membrane in folded form, which cut the effective length into halves. Therefore, the bola typed K3L8K3 mainly stay on or slightly below the membrane-water interface, and cannot insert into the bilayer. Otherwise, it would generate a positive curvature and suppress the inward budding. The hydrophobic interaction in the membrane-water interface, which is highly
Figure 12. A cartoon schematically showing the deformation of membrane caused by the attachment of K3L8K3. (A) The amount of K3L8K3 attached on the membrane is time dependent, so does its secondary structures. (B) The peptides form β-sheet inside the aggregates, while they stay mainly as α-helix on the interface between the aggregate and the lipid membrane. (C) Redistribution and neutralization of the charged lipids leads to a negative spontaneous curvature, followed by inward budding (D). Table 2. Deformation of GUVs in the presence of K3L8K3 GUV
O-series Charged lipid: DOPG P-series Charged lipid: DPPG
Name
Unsaturated lipid (%)
Deformation
O1-4
20%
In tact
O3-2
60%
Fibrils to globule
O4-1
80%
Fibrils to spheres
O6-1
87%
Fibrils to spheres
O1-0
100%
Spherical buds
P0-1
0%
In tact
P1-4
20%
In tact
P3-2
60%
Tubules
P4-1
80%
Spherical buds
Because the inward budding starts from the charged lipids, the property of the charge lipids is one of the key parameters governs the budding process. Unsaturated DOPG is more mobile than saturated DPPG at 37oC, it is easier to form bud even at smaller size patch domain. The redistribution of DPPG or DOPG to form a patch domain on the membrane in the presence of K3L8K3 is a prerequisite for inward budding. For the membrane composed of mainly saturated lipids, the redistribution of DPPG or DOPG is hindered at temperature below the transition point. The GUV maintains its structure, as demonstrated by O1-4 and P1-4. The incorporation of unsaturated lipids not only facilitates the mobility of charged lipids but also decreases the bending rigidity of the membrane. Therefore, the component ratio (O/P) is another key parameter governing the budding process. In the case of DOPG, the inward bud consists of mainly unsaturated lipids, and the diameter of the bud is smaller due to its flexibility. For the GUV at lower O/P ratios, such as O3-2, the liquid ordered phase is dominant in the membrane. The distance between the charged patch (dA) is larger (Fig. 13A), and the budding can grow longer, forming
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Langmuir a fibril. Due to the same reason, a long-range membrane fluctuation is observed during the budding process (Fig. 3A). Since the component of fibril is mainly unsaturated lipids, which is different from the mother membrane, the fibrils tend to wrap into one phase when encountered inside the vesicle (Fig. 13A). With increasing the O/P ratio in the GUV membrane, inward budding becomes easier as the diameter of buds and the distance between adjacent buds (dB) become smaller. The lateral movement of DOPG is faster as well. In the case of O1-0, which is formed entirely by unsaturated lipids, multiple small buds occur simultaneously in the presence of K3L8K3, and they wrap into one spherical structure before grow to a long fibril (Fig. 13B). Therefore, the growth of buds into fibrils and their wrapping into spherical structure is controlled by the O/P ratio in the mother membrane and the bending rigidity of the budding membrane. An optimal O/P ratio exists at which the buds can grow extremely long, O4-1 seems the case in our “O” series GUV.
The above results are based on freshly (30 min) prepared GUVs. Since the vesicle structures are kinetically stable,64 incubation of the GUV in a water bath for a long time induces some of the GUVs to burst by themselves. Even though the ratio of saturated to unsaturated lipids is kept constant during mixing, the eventual component in each GUV could be different during the electroformation process. For those composed mainly of saturated lipids, no inward budding occurs as indicated in Table 2, which explains the result that the GUV with higher saturated lipid ratio during mixing has a higher number of intact GUVs in the presence of K3L8K3 (Table S1). 3.5 Engulf of particles via endocytosis as induced by K3L8K3 Both the inward budding and the endocytosis induced by K3L8K3 should be able to engulf environmental particles into GUV. To testify this hypothesis, we choose cy5-labeled 21nt oligonucleotide as the probe and monitored its entrance into O4-1 in the presence of K3L8K3. Because both compounds are negatively charged, O4-1 and 21nt oligonucleotide repel each other when mixed. However, an endocytosis of a clear vesicle occurs in O4-1 in the presence of K3L8K3 (Fig. 14A and B). Fluorescence analysis shows that the intensity within the budded vesicle is about 30% higher than that of the background of the same size, suggesting that 21nt oligonucleotides are included in the budded vesicle. The full detachment of the bud is quite different from the deformation of O4-1 when only K3L8K3 exists (Fig. 3B). One possible explanation is that oligonucleotide and K3L8K3 form certain complexes with hydrophobic domains, inducing the endocytosis of GUV by following a similar mechanism as FAM-K3L8K3. Some free 21nt oligonucleotide molecules, as indicated by the red fluorescence, enters GUV as endo vesicle (Fig. 14). A1
A2
A3
A4
142 s
164 s
192 s
230 s
B1
B2
B3
B4
The flexibility is denoted by color as >
>
>
>
>
>
Figure 13. A cartoon schematically showing the inward budding of GUV with DOPG (A and B) and DPPG (C and D) as the charged lipids. The color denotes the rigidity of the membranes.
These principles are also applicable to the “P” series GUVs in the presence of K3L8K3. The major difference is that the budding starts with DPPG and the budding membrane is mainly saturated lipids. The high bending rigidity results in a very large diameter of inward buds (Fig. 13C), which grow into a tubule with extremely high persistence length. At low O/P ratios, the migration of DPPG is also deteriorated, which significantly decreases the number of inward buds. The wrapping of the tubules into spherical structures is hindered due to the higher persistence length and lower number (Fig. 13C). At higher O/P ratios, multiple inward budding also occurs. However, the liquid disordered mother membrane, which is softer than the bud membrane, fluctuates heavily (Fig. 13D), which prevents the bud from growing much longer. The detachment of the buds in the presence of FAM- K3L8K3 is not clear at current stage.
Figure 14. Carriage of oligonucleotide into the GUV during endocytosis. Oligonucleotide is labeled by Cy5 fluorescent tag (red). The membrane of GUV is labeled by DPPE-Rho (orange). The white dashed circle and arrow in (B) indicate the endocytic vesicle inside the mother vesicle, while a blue dashed circle in B4 indicates the background of same size as the endocytic vesicle. Scale bar, 10 µm.
4 CONCLUSIONS Using de novo designed peptide K3L8K3 and GUV of mixed lipids as a model system, we achieved inward budding and endocytosis of the membrane at varying conditions. The budding initiates as the charges on the lipids are neutralized by K3L8K3, which results in a negative spontaneous curvature. The property (saturated or unsaturated) of the charged lipid, the component ratio, and the redistribution of lipids in mother membrane determine the shape of the inward buds as well as the dynamics of the buds with time. A complete endocytosis is
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observed when K3L8K3 is attached with a hydrophobic moiety. The molecules in the surrounding medium, such as negatively charged oligonucleotides, are engulfed into the GUV via endocytosis pathway in the presence of K3L8K3. Even though the detailed mechanism on endocytosis is not clear at current stage, results show that extra hydrophobic interaction makes a significant contribution to the detachment of the bud from mother membrane. Our study provides a novel strategy for inducing inward budding and endocytosis of membranes by de novo designed peptides, and illustrates how the mechanism of endocytosis can be revealed in model systems. The conclusions are helpful to understand the processes in living organism.
ASSOCIATED CONTENT Supporting Information. Additional experimental data on P4-1 and O3-2. The movies on the behavior of O3-2(Movie S1),O41(Movie S2), O6-1(Movie S3), O1-0(Movie S4), P32(Movie S5), P4-1(Movie S6) in the presence of K3L8K3. The movie on O4-1in the presence of FAM-K3L8K3 (Movie S7), and the movie on O4-1 in the presence of K3L8K3 and Cy5-21nt oligonucleotide (Movie S8) This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author *Dehai Liang, email:
[email protected] *Yong-Xiang Chen, email:
[email protected] Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. /
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT Financial support of this work from the National Natural Science Foundation of China (21574002) and Beijing Natural Science Foundation (2171001) is gratefully acknowledged.
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Table of Content
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