Membrane Tubulation in Lipid Vesicles Triggered by the Local

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Membrane Tubulation in Lipid Vesicles Triggered by the Local Application of Calcium Ions Baharan Ali Doosti,# Weria Pezeshkian,⊥ Dennis S. Bruhn,⊥ John H. Ipsen,⊥ Himanshu Khandelia,⊥ Gavin D. M. Jeffries,# and Tatsiana Lobovkina*,# #

Department of Chemistry and Chemical Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden Center for Biomembrane Physics (MEMPHYS), Department of Physics, Chemistry and Pharmacy (FKF), University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark

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S Supporting Information *

ABSTRACT: Experimental and theoretical studies on ion−lipid interactions predict that binding of calcium ions to cell membranes leads to macroscopic mechanical effects and membrane remodeling. Herein, we provide experimental evidence that a point source of Ca2+ acting upon a negatively charged membrane generates spontaneous curvature and triggers the formation of tubular protrusions that point away from the ion source. This behavior is rationalized by strong binding of the divalent cations to the surface of the charged bilayer, which effectively neutralizes the surface charge density of outer leaflet of the bilayer. The mismatch in the surface charge density of the two leaflets leads to nonzero spontaneous curvature. We probe this mismatch through the use of molecular dynamics simulations and validate that calcium ion binding to a lipid membrane is sufficient to generate inward spontaneous curvature, bending the membrane. Additionally, we demonstrate that the formed tubular protrusions can be translated along the vesicle surface in a controlled manner by repositioning the site of localized Ca2+ exposure. The findings demonstrate lipid membrane remodeling in response to local chemical gradients and offer potential insights into the cell membrane behavior under conditions of varying calcium ion concentrations.

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and rigidity13,15,17,18 and triggers vesicular aggregation,19,20 in addition to membrane fusion mediation18,20,21 and spontaneous curvature induction.22,23 To date, the majority of experimental studies introduce calcium ions to lipid membranes in bulk solution, which results in a homogeneous distribution of ions over the membrane surface. However, the utilization of a localized concentration gradient has been shown to alter membrane behavior compared to bulk exposure, leading to local perturbation. For example, crista-like membrane protrusions were observed during local proton stimulation in giant vesicles, which were not present in bulk pH changes.24 Additionally, it was shown that localized exposure of Ca2+ to flat negatively charged double lipid bilayer leads to protrusive spreading in lipid membranes.25 Herein we demonstrate that the membrane of a negatively charged GUV, locally exposed to Ca2+, forms tubular protrusions, a process which is not observed when the bulk calcium concentration is increased. This membrane tubulation is described as the response to the trans-bilayer mismatch of surface charge density upon calcium ion binding from one side of the bilayer. Molecular dynamics simulations were performed on a lipid bilayer model, which demonstrated that heterogeneous calcium ion binding can be sufficient to induce inward

INTRODUCTION Calcium is an important biological agent involved in many cellular processes, ranging from cell division to apoptosis,1−3 including molecular transport,1 endo- and exocytosis,4 and communication between cells.5 The principles of calcium ion interaction with the cell membrane can be elucidated using artificial lipid bilayers to mimic multicomponent biological membranes. Examples of these artificial systems include giant unilamellar lipid vesicles (GUVs), lipid nanotubes, and supported bilayers. The lipid composition of these model membranes is engineered in such a manner to contain selected amounts of important biological lipids, tuning the ion−lipid interactions. Often, these lipids are phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) or phosphatidic acid (PA). The PS lipids are of particular interest due to their role in signaling, coagulation, and cell behavior under stress conditions and apoptosis.6 Due to the ubiquitous occurrence of calcium in many cellular processes, the response of lipid membranes to ion binding has been studied extensively using complementary techniques such as 1H NMR,7 X-ray diffraction,8 thermodynamic techniques,9 and spectroscopic studies,10 in addition to molecular dynamics simulations.11,12 Experimental and theoretical studies have shown that binding of calcium ions to the lipid membrane results in lipid dehydration and tighter packing of the head groups and the hydrophobic regions of the lipids.11,13−16 This lateral membrane reorganization increases membrane tension © XXXX American Chemical Society

Received: April 28, 2017 Revised: September 7, 2017 Published: September 14, 2017 A

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Figure 1. Experimental setup and typical examples of spherical and undulating vesicles. (A) A schematic representation of the experimental setup. The giant unilamellar vesicle (GUV) is coupled to a multilamellar vesicle (MLV) and deposited on a glass surface in a 1 cm sized buffer droplet (not drawn). The ion gradient is established at a distance of 3−4 μm from the membrane surface by applying a positive pressure (p) through the tapered glass micropipet filled with the ion solution. The illustration is not drawn to scale. (B−D) Typical example of a spherical GUV at different time points. (E−G) Typical example of a GUV with visible thermal undulations in the membrane at different time points. The black arrows highlight the visibly deformed areas showing fluctuations in the membrane. The fluorescence images are enhanced and inverted to improve visualization of the GUV membranes. KCl (5 mM). The micropipet was used to serve as a local point-source for the ion injection (injection pressure 20−25 hPa). Upon application of a positive pressure, the content of the micropipet was slowly released into the solution creating an ion gradient. The positioning of glass micropipets to vesicles was controlled by a PatchStar micromanipulator (Scientifica, Uckfield, UK). A microinjection system (Eppendorf Femtojet) was used for point-wise application of the ions. The micropipet was positioned at a distance of 3−4 μm from the vesicle surface. Microscopy Observation and Data Analysis. An inverted fluorescence microscopy system (Leica DM IRB, Wetzlar, Germany), equipped with a 100× oil immersion objective (Leica, 100×, 1.4 NA, oil immersion), was used for observation during investigation, and a 488 nm laser line (Cobolt MLD-488 nm) enabled excitation of the ATTO488 fluorophore. A camera (Prosilica Ex 1920, Allied Vision Technologies GmbH, Thuringia, Germany) and a custom-made script in Labview 2009 (National Instruments) was used to collect the data. Fluorescence images and movies were edited and improved using NIH ImageJ software and VirtualDub 1.10.4. See Supporting Information, section S1A, for the details. The simulation figures were produced using Matlab, Inkscape 0.91, and VMD 1.9.3. Simulation Methods. Coarse grained molecular dynamics simulations were performed using GROMACS 4.6.730−32 software and the Martini force field.33,34 The size of the systems and the simulation time limited us to use a coarse grained model. Each system consists of two identical and symmetric bilayers to create two solvent compartments (Figure 4A,B). They each consist of 646 lipids, with 80% DOPC (dioleoylphosphatidylcholine) and 20% DOPS or 60% DOPC and 40% DOPS (Supporting Information, section S2B, Table S1). The negative charges from DOPS are balanced by adding Na+ equally between the two water compartments. Every tenth water particle is replaced by an “antifreeze particle” in accordance with Marrink et al.34 Additionally, in one system Na+ and Cl− are added in the middle compartment (Figure 4B) to reach a concentration of around 0.5 M while in the other Ca2+ and Cl− is added (Figure 4A) to reach around 0.25 M Ca2+ (or 0.5 M Cl−). This guarantees the same amount of positive charge in the central compartment. For each system, 20 simulations were performed to minimize statistical uncertainties in the estimated quantities. Each simulation was run for 2 μs with a time step of 30 fs. The final 1 μs was used for

spontaneous curvature and bend the membrane. Additionally, we reveal that formed tubular protrusions can be translated around the GUV membrane in the direction of increasing calcium ion concentration, by moving the source of ions along the vesicle surface.

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EXPERIMENTAL SECTION

Chemicals. Soybean Polar Lipid Extract (SPE) and 1,2-dioleoyl-snglycero-3-phospho-L-serine (DOPS) were purchased from Avanti Polar Lipids, Inc. (Alabaster, USA). Chloroform, KCl, NaCl, and 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) solution (1 M) were obtained from Sigma-Aldrich (Missouri, USA). CaCl2 was purchased from KEBO lab (Sweden). MgCl2 (MgCl2·6H20) was purchased from Sharlau Chemie S.A. (Spain). 1,2-Dioleoyl-sn-glycero3-phosphoethanolamine with the fluorophore ATTO-488 (ATTO488DOPE) was purchased from ATTO-TEC (Germany). Giant Vesicle Preparation. The giant lipid vesicles were composed of SPE consisting of PE (phosphatidylethanolamine) 22.1%, PI 18.4%, PC 45.7%, PA 6.9%, other lipids 6.9% with or without addition of 20 wt % of DOPS. Both lipid mixtures included the fluorescently labeled lipid ATTO488-DOPE (1 wt %). The DOPS molecules were added to the lipid mixture to mimic the cytoplasmic side of the cell plasma membrane.26,27 Giant vesicles were prepared using a dehydration−rehydration method28 with modifications.29 First, small unilamellar vesicles (SUVs) were produced as previously described,25 after which, a droplet of the SUV solution (5 μL) was placed on a glass coverslip and dehydrated in a vacuum desiccator for 20 min. The dry lipid film was placed for 4 min at room temperature. Then, 50 μL of 10 mM HEPES buffer (pH 7.4) was used to rehydrate the dry lipid film for 5 min to form vesicles. The vesicle solution was thereafter transferred onto a new glass coverslip holding a 300 μL droplet of HEPES buffer, and vesicles were allowed settle and adhere to the glass surface for 30 min. Local Injection of Ions. Tapered glass micropipets (GC100TF-10, Clark Electromedical Instruments, Reading, UK) were pulled on a P2000 CO2 laser-puller instrument (Sutter Instruments, Novato, USA) resulting in a glass capillary with a tip diameter of 0.2−0.3 μm. The tapered glass micropipets were filled with HEPES buffer supplemented with CaCl2 (1, 2, or 5 mM), MgCl2 (5 mM), NaCl (7.5 or 75 mM), or B

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Langmuir analysis. The temperature and pressure were kept constant at 310 K and 1 bar, respectively, using the velocity-rescale thermostat35 and the Parrinello−Rahman barostat.36 All other settings are in line with the Martini standards.33,34 For a translational invariant, symmetric and continuous bilayer in a simulation box with periodic boundary conditions, the bilayer curvature does not appear as visible geometric bending. The nonzero spontaneous curvature will appear as a lateral average force asymmetry that can be observed by asymmetry in the lateral pressure profile. Therefore, we have calculated the spontaneous curvature from the simulations using the κc0 = ∫ zπ(z) dz 12,37,38 where κ is the bending rigidity of the bilayer, c0 is the spontaneous curvature, z is the axis perpendicular to the plane of the bilayer, starting from the bilayer center, and π(z) is the so-called lateral pressure profile, defined as π(z) = PL(z) − PN(z), where PL(z) = (Pxx(z) + Pyy(z))/2 is the in-plane pressure and PN(z) is the normal pressure. The lateral pressure profile is calculated using the method of Vanegas et al.43,44 The bending rigidity of the bilayers was determined from the spectrum of the bilayer shape fluctuations that takes the form k T ⟨|u(q)|2 ⟩ = 4 B 2 for large wavelengths, where u(q) is the Fourier

We utilized two standard membrane compositions, SPE/ DOPS and SPE, in order to address the effect of local calcium application to the GUV membranes at various ionic concentrations and at two different morphological regimes, (i) spherical GUVs and (ii) thermally undulating GUVs. The interaction of Ca2+ with spherical SPE/DOPS vesicles results in bending of the membrane, and the formation of dense and closely packed tubular-like structures that protrude into the lumen of the GUVs. In a typical experiment, the membrane tubulation begins with the accumulation of membrane material at the application site, which can be observed by an increase of fluorescence and small membrane indentations (Figure 2A,B).

κq + σq

transform of the bilayer midsurface in the Monge representation, kB is the Boltzmann constant, T is the temperature, and σ is the membrane tension.39,40 In order to capture the large wavelength membrane fluctuations, for each composition of lipids and ions concentration, we have simulated a system with a single large bilayer patch (Supporting Information, section S2B, Table S1, last 4 rows. For more details on the bending rigidity calculations, see Supporting Information, section S2A, Figure S3).

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RESULTS AND DISCUSSION GUVs are an appropriate model system for studying the properties of cell membranes due to their close resemblance to mammalian plasma membranes, including size and lipid composition, and their ability to be integrated with established interrogation schemes, such as micromanipulation and electroporation.41,42 Within our study, GUVs were prepared as described previously28 in such a manner to be connected to multilamellar vesicles (MLVs), required as a source of lipids for the tube formation.43 A schematic illustration of the MLV− GUV system is shown in Figure 1A. This vesicle platform has been used in previous studies for mimicking cell tubular structures,41 exocytosis, and membrane interaction with polymers.44,45 The morphology of the prepared GUVs (typically 5−15 μm in diameter) was studied using fluorescence microscopy; the majority (∼75%) were found to be spherical, as illustrated in Figure 1B−D (see also Supporting Information, Movie 1). However, a fraction of the prepared GUVs (∼25%) were undergoing visible thermal undulations, further referred to as undulating, thus having a lower membrane tension, likely as a result of an excess of membrane material (Figure 1E−G, Supporting Information, Movie 2). For both vesicle populations, we assumed homogeneous lipid distribution with zero spontaneous curvature. Injection of one solution into the buffer leads to diffusional spreading, resulting in a concentration gradient from tip to the vesicle. To probe for this diffusional spread of ions, we used 0.1 mM fluorescein solution and monitored the fluorescence intensity profile away from the pipet tip. By measuring the normalized intensity values between the micropipet tip and the vesicle surface, we estimated a concentration decrease of approximately 75% from the initial value (i.e., from 0.1 mM to 0.025 mM) at the membrane surface from a distance of ∼3 μm. The concentration of the ions mentioned further in the text refers to the ion concentration within the micropipet, unless it is otherwise stated.

Figure 2. Formation of tubular protrusions in SPE/DOPS GUVs triggered by the local action of Ca2+ and Mg2+ ions. (A) The MLV− GUV system prior to Ca2+ (5 mM) gradient introduction. (B) Increase in fluorescence intensity of the membrane and formation of small tubular protrusions directed inside the GUV upon Ca2+ injection. (C) After an additional 3 s of exposure to Ca2+ ions, the tubulation area increases in size, and the tubular protrusions become longer. The schematic drawings on the right from the fluorescence images are simplified illustrations of formation and growth of the tubular protrusions. The images are not drawn to the scale. (D) An undulating GUV exposed to Ca2+ gradient (1 mM). Thin, long, and sparse tubules are formed at the site of Ca2+ injection. The dashed black lines outline the GUV membrane. (E) An undulating GUV exposed to a Mg2+ gradient (5 mM), which forms sparse tubular protrusions directed inside the GUV. The pipet tip in panels D and E is out of frame of the image. The fluorescence images are enhanced and inverted to improve visualization of the tubular protrusions.

Holding the micropipet close to the membrane surface while injecting Ca2+ promotes the growth of tubules (Figure 2C). These formed tubular protrusions are directed away from the membrane, inside the vesicles, that is, the membrane bends away from the ion binding site. The protrusions can reach a length of 3−5 μm after ∼5 s and continue to grow upon Ca2+ C

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Figure 3. Translation of membrane tubular structures along the GUV surface by moving the point source of calcium ions. (A) The MLV−GUV system prior to calcium exposure (5 mM). (B) Tubular protrusions are formed by the injection of the calcium ions. (C,D) Translating the micropipet around the vesicles surface, resulting in the entire tubulation area moving in the direction of the pipet tip. The fluorescence images are enhanced and inverted to aid in tubular protrusions visualization.

supply. They appear closely packed and dynamic and fluctuate within the vesicle, a concise video of which can be found in the Supporting Information, Movie 3. The spherical SPE/DOPS vesicles were exposed to various local Ca2+ concentrations, 1, 2, and 5 mM. Tubular protrusions formed using both 2 and 5 mM concentrations, but not at 1 mM. This indicates a threshold concentration at which protrusions are formed. We further examined undulating SPE/DOPS vesicles using the same concentration range (1, 2, and 5 mM Ca2+), and formation of tubular protrusions was observed at all three concentrations. The difference in membrane behavior at lower calcium ion concentration, when compared to the spherical GUVs, highlights the importance of the excess membrane material, which likely aids in the formation of the protrusions. The observed protrusions were generated in a shorter period of time, within 1−2 s, while appearing sparse in comparison with spherical vesicles. These lower densities of protrusions allowed the individual tubes to be resolved using fluorescence microscopy (Figure 2D, Supporting Information, Movie 4). The length of the individual tubules was measured to be 3−5 μm, similar to those produced in spherical vesicles. To elucidate the influence of DOPS molecules on the process of membrane tubulation, we excluded them from the lipid mixture and tested the SPE-only vesicles with local application of 5 mM of Ca2+. The undulating vesicles responded to calcium ion stimulation in a manner similar to the SPE/DOPS composition, forming loosely packed tubular protrusions (Supporting Information, Movie 7). However, the local application of calcium ions did not result in tubules for spherical vesicles, indicating that formation of the tubular protrusions for such vesicles is DOPS-dependent. To further investigate the role of calcium in the formation of tubular protrusions, another divalent cation, Mg2+, was tested. We observed that the spherical GUVs (both SPE/DOPS and SPE) exposed to 5 mM of Mg2+ did not produce tubular protrusions. In contrast, for undulating vesicles, the tubular protrusions were similar to those generated by Ca2+ ions, Figure 2E, Supporting Information, Movies 5 and 8. This indicates that the ion size plays a role in the force required to bend the membrane. The role of the exposure gradient was probed by conducting experiments where Ca2+ or Mg2+ was introduced to the vesicle solution in the bulk preparation, to a final concentration of 5 mM for each cation. In these instances, tubular protrusions were not observed. The vesicles (both SPE/DOPS and SPE) instead ruptured resulting in lipid patches adhered to the glass

surface (Supporting Information, section S1B, Figure S1). However, it should be noted that if GUV rupture could be prevented, it would allow investigation of the formation of tubular protrusions at bulk exposure. An example of such an experiment would include placing the MLV−GUV vesicle system on a microfabricated pillar, thereby limiting the adhesion of the lipid membrane to a surface. The effect of valency on the formation of the tubular protrusions was also probed, exposing the vesicles to monovalent cations (Na+, K+). No observable tubulation was present at concentrations of 7.5 or 75 mM of Na+ and 5 mM of K+, thus excluding the role of osmotic stress. The influence of hydrodynamic flow was excluded by performing control experiments where the micropipet was filled with pure HEPES buffer, which resulted in no observable effect on the membrane. Intrigued by the fact that tubular protrusions were induced in lipid vesicles by the local action of cations, we explored the possibility to translocate these tubules within the membrane by controlling the Ca2+ ion concentration profile at the membrane surface. By carefully translating the micropipet around the spherical SPE/DOPS vesicle surface (taking care to maintain the same distance between the vesicle and the pipet tip), we were able to guide the tubular protrusions in a contactless manner toward the pipet tip, that is, in the direction of increasing Ca2+ concentration (Figure 3, Supporting Information, Movie 6). Such a translational movement of the formed tubular protrusions is likely accompanied by the formation of new tubules at the new location of the calcium ion source, as we demonstrated in Supporting Information, section S1C, Figure S2. The observed controlled translation of tubular protrusions can have important implications for further understanding the mechanisms of lipid mobility and distribution within cell membranes, which can be guided by spatiotemporal calcium ion oscillations in the vicinity of the membrane. The tubular protrusions that form within the vesicles are reminiscent of the ones that are produced in model membranes upon localized proton flow,24 protein−protein crowding effect,46 binding of bacterial toxic proteins, for example, Shiga and cholera toxin47,48 or pore-forming protein perforin49 (at homogeneous concentrations). Interestingly, a generic mechanism is often shared among these phenomena, that is, a small increment of local curvature is induced by the inclusions (proteins)50,51 and the curvature inducing inclusions cluster and colocalize.52,53 This leads to a buildup of spontaneous D

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Figure 4. Molecular dynamics simulations of DOPC/DOPS bilayers under Ca2+ and Na+ ions gradients. (A, B) Snapshots for the 20%/Na+ and 20%/Ca2+ systems, respectively. The two bilayers create two solvent compartments to impose different ionic concentrations on each side of a bilayer. (Blue sphere) Na+ ions, (green sphere) Ca2+ ions, and lipids are shown in stick representation. Water particles and Cl− ions are not shown for clarity. (C) Averaged pressure profiles for each system. Asymmetry in the profile indicates spontaneous curvature and is more pronounced for systems containing Ca2+, as indicated in the inset. (D) Total charge density of the systems. The negative surface charge density of one side of the bilayer is effectively neutralized for systems containing Ca2+. (E) Charge densities of Na+ and Ca2+ for each system (Note, Ca2+ exists in only two systems). Notice, panels C, D, and E present the results for only one bilayer in the system. Ca2+ ions are localized on the surface of the bilayer, effectively neutralizing the surface charge density of the bilayer.

away from the Ca2+ ions; consistent with the experimental results (Figure 4C). However, for the 20%/Na+ system, the curvature was solely due to thermally driven deformation of the membrane surface, fluctuating around zero. This is also consistent with the experimental findings. Upon increase of the DOPS concentration to 40%, the radius of curvature of 40%/Ca2+ system decreased to 22 ± 4 nm (corresponding to 255 pN/μm spontaneous tension), while for the 40%/Na+ system, no spontaneous curvature was observed (Figure 4C). Ca2+ tends to bind to the surface of the bilayer and effectively neutralize the negative charge density of the bilayer (see Figure 4D, E). The adsorbed charge between two monolayers is 0.61 e/nm2 and 0.67 e/nm2 for 20%/Ca2+ and 40%/Ca2+ systems respectively, while this value is around 0.25 e/nm2 for both Na+ systems (Supporting Information, section S2C, Table S2). The resulting large asymmetry in the Ca2+ systems causes spontaneous membrane bending.55 Although some Na+ ions also bind to the membrane surface (Figure 4E), they only make a small reduction in the bilayer surface charge density (Figure 4D). Therefore, Na+ fails to induce large membrane curvature. Nevertheless, we should notice that this phenomenon described here is generic and is not limited only to the case Ca2+ and Na+. In general, multivalent counterions tend to bind strongly to the surface56 of the charged membrane and can neutralize the surface charge density,57 while the distribution of the monovalent counterions is more extended around charged surfaces (large Gouy−Chapman length).58 Our results suggest

tension (σs = 2κCm 2 , where Cm is the spontaneous mean curvature) that subsequently drives the formation of membrane tubular protrusions. Therefore, a sufficiently large domain with a small spontaneous curvature is enough for tubulation formation.54 In order to understand how Ca2+ binding induces spontaneous curvature to a charged bilayer, we have performed coarse grained molecular dynamics simulations of bilayers containing DOPC and two different concentrations of DOPS lipids (20% or 40%). These model systems contain two identical and symmetric bilayers to impose different ionic concentrations on each side of a bilayer (Figure 4A,B). The negative charge from the DOPS lipids is countered by adding Na+ evenly between the two solvent compartments. Additionally, in the middle solvent compartment, we insert Ca2+ and Cl− in one system (we refer to this system as 20%/Ca2+) and Na+ and Cl− in another (referred to as 20%/Na+). Spontaneous curvature of the bilayers was found by calculating the first moment of the pressure profile (see Simulation Methods) that is nonzero only for asymmetric profiles (Figure 4C). Figure 4C clearly shows that for all systems containing Ca2+ the pressure profile is asymmetric while for Na+ systems the profile is relatively symmetric. The results show that for the Ca2+ system with 20% DOPS concentration (20%/Ca2+), a local membrane curvature is induced with a curvature radius (inverse of the spontaneous mean curvature, 1/Cm) of 52 ± 12 nm (corresponding to 45 pN/μm spontaneous tension) directed E

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fluctuations of intracellular calcium ion concentration can vary by orders of magnitude, these concentration bursts may be sufficient to generate spontaneous curvature and lead to membrane deformations. Calcium ion variations may also directly regulate lipid mobility and lateral membrane reorganization, whereby membrane components sense the Ca2+ profile within the microenvironment. Biological cells contain a wide variety of charged molecular species, which may screen or magnify the Ca2+ ion effect, the contribution of each could be dissected by using our approach.

that the spontaneous curvature induced by divalent ion adsorption is directly related to the amount of adsorbed ions (Figure 4D,E). Therefore, the larger ions that have lower adsorption19 will induce a smaller spontaneous curvature and subsequently a lower spontaneous tension. This explains the different behaviors of Mg2+ and Ca2+ in the formation of the tubular protrusions. Notice, if there is no asymmetry in the Ca2+ exposure, the binding of Ca2+ ions induces lipid packing, instead of membrane bending (Supporting Information, section S2D, Table S3). Additionally, we ranked the effect of Ca2+, Mg2+, and Na+ ions according to their ability to generate tubular protrusions in the SPE/DOPS vesicles, such that Ca2+ > Mg2+ > Na+, where no membrane tubulation by Na+ was observed. We compared this ranking to the Hofmeister series, that is, a series in which the ions are empirically ranked with regards to the effect of these ions on, for example, the solubility of proteins in aqueous solution. According to the Hofmeister series, these cations are ranked as Na+ > Mg2+ > Ca2+.59 Interestingly, in our study on formation of tubular protrusions, the ranking is pointing in the direction of the reverse Hofmeister series. Screening of other cations according to their ability to induce formation of tubular protrusions would allow us to address the complete Hofmeister series.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.7b01461. Images of lipid vesicles exposed to Ca2+ and Mg2+, details on the microscopy image adjustments, and simulation details (PDF) Example of a SPE/DOPS giant lipid vesicle with spherical morphology (AVI) Example of a SPE/DOPS giant lipid vesicle with undulating morphology (AVI) Spherical SPE/DOPS giant lipid vesicle exposed to a Ca2+ gradient (5 mM), showing formation and growth of tubular protrusions directed inward in the GUV (AVI) Undulating SPE/DOPS giant lipid vesicle exposed to Ca2+ gradient (1 mM) (AVI) Undulating SPE/DOPS giant lipid vesicle exposed to Mg2+gradient (5 mM), showing formation of long and sparse tubular protrusions upon local Mg2+ stimulation (AVI) Formation and directed movement of tubular protrusions along the GUV surface in SPE/DOPS giant lipid vesicle exposed to Ca2+ ions (5 mM) (AVI) Undulating SPE giant lipid vesicle exposed to Ca2+ gradient (5 mM), showing formation of long and sparse tubular protrusions at the site of Ca2+ injection (AVI) Undulating SPE giant lipid vesicle exposed to Mg2+ gradient (5 mM), showing formation of long and sparse tubular protrusions at the site of Mg2+injection (AVI)

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SUMMARY AND CONCLUSIONS Our results demonstrate that application of calcium ions to the membrane of giant lipid vesicles triggers membrane bending and tubulation, which can only be observed under conditions of local membrane stimulation. In particular, we show that the localized binding of Ca2+ to the outer membrane of negatively charged giant lipid vesicles generates inward spontaneous curvature sufficient to induce membrane bending, leading to the formation of tubular protrusions directed inside the vesicles. Lipid vesicles of two different compositions (SPE/DOPS and SPE) and two different morphological regimes (spherical and thermally undulating GUVs) were tested. We observed that within vesicles having spherical morphology, the tubular protrusions can only be observed in DOPS-enriched membranes. However, for undulating vesicles, the membrane protrusions were formed for both lipid mixtures, as well as in response to another divalent cation, Mg2+. The observed inward spontaneous curvature and membrane bending can best be described as a response of the bilayer to a large reduction in the surface charge density of one membrane leaflet upon binding of divalent cations. A charged bilayer has a strong repulsive coulomb interaction (a positive Maxwell stress) in both monolayers. Divalent cations bind strongly to the surface of the charged bilayer and neutralize the surface charge density of the bound monolayer. This asymmetry in the stress causes the membrane to bend. Molecular dynamics simulations, examining the effect of Ca2+ binding, demonstrated that sufficient force can be generated to induce spontaneous curvature, bending the membrane, leading to the formation of tubular protrusions, which agreed well with our experimental findings. The demonstrated lipid membrane behavior upon local application of Ca2+ offers unique insights into possible mechanisms of cell membrane remodeling, including that ion−lipid interaction alone can lead to the membrane deformations and generate highly curved tubular protrusions. These curved membrane regions are of fundamental importance in many cellular processes such as cell division, endo- and exocytosis, and trafficking of proteins and lipids between cellular compartments. Considering that cytosolic

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Baharan Ali Doosti: 0000-0001-9149-0751 Weria Pezeshkian: 0000-0001-5509-0996 Dennis S. Bruhn: 0000-0003-1362-4747 Tatsiana Lobovkina: 0000-0002-8923-1328 Author Contributions

B.A.D. and T.L. conceived the study. B.A.D., G.D.M.J., and T.L. participated in the experimental study. W.P., D.S.B., J.H.I., and H.K. performed the theoretical study. B.A.D., W.P., and D.S.B. are the major contributors to the experimental (B.A.D.) and theoretical work (W.P., D.S.B.). 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. F

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ACKNOWLEDGMENTS The authors acknowledge A-S. Cans for informative discussions. We also thank the Swedish Research Council (Grant 21220109) and Area of Advance Nano at Chalmers University of Technology (Grant 21420010) for funding. Simulations for the work described in this paper were performed using resources provided by the DeiC National HPC Centre, University of Southern Denmark.

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DOI: 10.1021/acs.langmuir.7b01461 Langmuir XXXX, XXX, XXX−XXX