Response of cells to osmotic pressure and control of water permeability of biomembranes are very important for cells to survive. There have been many ... Lipidomics - Biomembranes - Sugar - Glass - Pressure control - Biological materials - Microscopy - Equations - Permeability measurement - Volume measurement - biomembranes - cellular biophysics - lipid bilayers - water permeability - lipid membranes - GUV - osmotic pressure - biomembranes - micropipet aspiration - actin cytoskeletons - liquid-ordered phase - actin filaments - water efflux - chemical potential - nonequilibrium condition
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Water permeability of lipid membranes of GUVs and its dependence on actin cytoskeletons inside the GUVs 2
Takuya Yoshitani,land Masahito Yamazaki" 2 'Department ofPhysics, Faculty of Science, Shizuoka University, Shizuoka, 422-8529, Japan
Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka, 422-8529, Japan
Abstract: Response of cells to osmotic pressure and control of water permeability of biomembranes are very important for cells to survive. There have been many studies using cells, but their details remain unknown. In this study, we investigated water permeability of various lipid membranes of GUVs by the micropipet aspiration technique, and then its dependence on actin cytoskeletons inside the GUVs.
First, we measured of water permeability of various lipid
membranes. When we transferred a single GUV fixed by a micropipet using a small aspiration pressure into a buffer with a higher osmolarity, the volume of the GUV decreased due to
actin filaments and actin/filamin A-gel in cells can change water permeability of biomembranes or not. In this study, we investigated water permeability of various lipid membranes of GUVs by the micropipet aspiration technique, and then its dependence on actin cytoskeletons inside the GUVs. 2. MATERIALS AND METHODS 2.1 Materials
the water efflux. We determined the decrease in the GUV volume by the increase in aspiration length in the micropipet. We analyzed quantitatively the time course of volume change of the GUV on the basis of the theoretical equation, and obtain water permeability of the lipid membrane. The average value
Dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and dioleoylphosphatidylglycerol (DOPG) were purchased from Avanti Polar Lipids
of lipid membranes. For example, the water permeability of DPPC/cholesterol (6/4, molar ratio) membrane in a liquidordered phase was 1.3 ± 0.1 gm/s, which was much smaller
Pure Chemical Industry Ltd. (Japan). Monomeric actin (G-actin) was extracted from acetone powder of rabbit skeleton muscle .
of water permeability of DOPC membrane in the La, phase 45± 4 m/s. The water permeability depended greatly on kinds
than that of DOPC membrane.
Second, we investigated the effect of actin filaments on
water permeability of lipid membranes. We succeeded in
filaments. The water permeability of the GUV containing the oe than aha that htoOf DOJPC/DOJPG OCDP actinactin~~~~~~~~~~~~~ filaments was fiaet lower
(1/1)-GUVs containing 1 mg/mL actin monomers (no polymerization). This result indicates that the actin filaments reduced the water efflux through the GUV when an osmotic pressure was applied on the GUV. It can be explained by the decrease in chemical potential of water induced by the actin filaments under the nonequilibrium condition due to the osmotic pressure. We discuss the mechanism of the effect of the actin filaments on the osmotic response of the GUV.
Response of cells to osmotic pressure and control of water permeability of biomembranes are very important for cells to survive. There have been many studies using cells, but their details remain unknown. It is reported that actin filaments reduced the osmotic pressure-induced water flow through a semipermeable membrane [1,2]. However, it is well not known whether
Inc. (lase mtergA, SA. onprea CA2CA,17 As. was purchased Ua). Bovine serum albumin (BSA) was purchased from Wako
2.2. Formation and microscopic observation of GUVs GUVs of lipid membranes were prepared in a buffer by
. . . the natural swelling of a dry lipid film at 37 0C as follows [e.g., 4]. One hundred tl of 1 mM DOPC or DPPC/chol (6/4: molar ratio) in chloroform were placed in a glass vial (5 ml) and dried under a stream of N2 gas to produce a thin, o
homogeneous lipid film. The solvent was completely removed by placing the bottle containing the dry lipid film in a vacuum desiccator connected to a rotary vacuum pump for more than 12 h. Next, 10 tL water was added into this glass vial, and the mixture was incubated at 45 °C for 10 min (prehydration). The hydrated lipid film was then incubated with 0.1 M sucrose solution for 2-3 h at 37 'C. A 10 Vtl aliquot of the GUV solution (containing 0. IM sucrose solution; internal solution) was diluted into 290 Ul of 0.1iM glucose solution and then transferred into a hand-made microchamber. A slide glass and micropipets were coated with 0.1 00O (w/v) BSA in 0.1 M glucose solution. We observed GUVs using an inverted phase contrast, differential interference contrast (DIG) microscope (IX-71, Olympus, Tokyo) at 25 °C. Phase contrast images and DIG
images of GUVs were recorded using a CCD camera with a hard disk. (A) To prepare DOPC/DOPG-GUVs containing actin filaments, a following preparation method was used. 50 _tl of 1mM phospholipid mixtures (DOPC/DOPG/A23 187= 49/49/2b molar ratio) in chloroform in a small lass bottle (1..8 ml) was dried by N2 gas, and then in a vacuum desiccator connected to a rotary vacuum pump for more than 12 h. After the prehydration, 220 Vtl of 1 mg/mL actin (B) in buffer G (2 mM Tris-HCI (pH 8.0), 0.2 mM CaCl2, 0.5 mM ATP, 10 mM DTT) containing 0.1 M sucrose was b added into the glass bottle, and it was incubated at 25 i C for 2 h. Then actin molecules outside GUVs were removed by ultrafiltration. The purified GUV solution was 25 times diluted with buffer M (2 mM Tris-HCI (pH 8.0), 0.2 mM CaCl2, 0.5 mM ATP, 10 mM DTT, 10 MM MgCl2) containing 0.07 M glucose, and incubated at 25 'C for 1.5 h. During this incubation, Mg2+ was transported into the inside of the GUVs by A23 187 and the polymerization of acin Figure 1: Measurement of the volume change of a GUV by the micropipet occurred.
2.3. Measurement of water permeability of lipid membranes.
To obtain water permeability of lipid membranes, we measured a volume change of a single GUV when it was transferred to a buffer with a higher osmolarity, using the micropipet aspiration method, and analyzed the time course of the volume change. This method is similar to the method developed by Olbrich et al. . At first a single GUV in a buffer was fixed by a micropipet using a small aspiration pressure (tension of the membrane is -1 mN/m) and then it was transferred into another buffer with a higher osmolarity. Since there is a small air gap between the chambers containing these different buffers, we used another larger size-micropipet filled with buffer to transfer the GUV between these chambers. As a buffer, we used 0.1 M glucose in water for GUVs of lipid membranes, and buffer M for GUVs containing actin filaments. The volume change of the GUV,AV, was determined by the change of the projection length of the GUV inside the micropipet (i.e., the aspiration length), ALp (Fig. 1) as follows,
AV V - Vo0 -Dp (Dv - Dp)ALp / 4
where V and V0 are the volume of the GUV at t = t, and t = 0, respectively (t is the time after the transfer of the GUV into another buffer), Dv is the diameter ofthe GUy, and Dp is the diameter of the micropipet. The flux of water molecules through the membrane, Jwater [mol/s.jtm2], iS proportional to the difference between the solute concentrations inside and outside of the GUy, AC [mol4im3], and its proportional constant is called the water permeability through the membrane, Qw [pim Is] as follows,
aspiration method. (A) A video micrograph of the GUV fixed at the micropipet in a buffer (i.e., initial state). DV is the diameter of the GUV, Dp is the diameter of the micropipet, Lo is the initial projection length of the GUV inside the micropipet. (B) A video micrograph of the GUV incubated in another buffer with a higher osmolarity for some time (i.e., 30 s). The
projection length Lp is increased, and its increment is ALP.
The flux of water volume through the membrane, Jv [Vtm/s] The flows, iS as follows, _ _ JV= VWJwater =-VwQwAC (3) where Vw [L/mol] is the molar volume of water. Thereby, the rate of volume change of the GUV is determined by,
where A is the area of the GUV. We can transform eq.4 into a dimensionless equation using the dimensionless volume, V*=V/Va. (where VGO is the final equilibrium volume of the GUV), and the final equilibrium solute concentration inside the GUV, Cin = Cn Vo /V0 as follows, dV -( A y V -18 d = -QC w * | dt VVO0)A V )
The solution of the differential eq. 5 is,
f(V) )(V* -1) exp(V) )(V -1) exp(-kt + V)
where k =AQWCOOVW / VOO,. We fitted a graph (f(V*) vs. t) using eq.6 to determine the value of Qw.
3.2 Water permeability ofDPPC/chol(6/4) membrane in the
liquid-ordered (lo) phase
Next, to investigate the dependence of water permeability on phase of lipid membranes, we measured water permeability of DPPC/cholesterol (6/4, molar ratio) membrane in a liquid-ordered (lo) phase . When we transferred a single DPPC/chol(6/4)-GUV into a buffer with a higher osmolarity (AC = 40 mM), the volume of the GUV decreased slowly and reached to an equilibrium volume more than 100 min (Fig. 3A,B). The average value of Qwapp of DPPC/chol membrane was 1.3 ± 0.2 jtm/s (n = 4) (Fig.3C), which was much smaller than that of DOPC membrane. This volume change was reversible; when the GUV was transferred into the original buffer after the volume decrease stopped, the volume increased to return to the original value. For the diffusion of water molecule in lipid membranes, it is important to produce free space for one water molecule in the membrane, especially in the hydrophobic core region, and also to move this space rapidly in the membrane. In lipid membranes in the L, phase, rapid conformational changes of acyl chains and also rapid lateral diffusion of lipid
3. RESULTS AND DISCUSSION .
phase First, we measured water permeability of DOPC membrane in the L, phase. When we transferred a single DOPC-GUV fixed by a micropipet using a small aspiration pressure into a buffer with a higher osmolarity (AC 20 mM), the volume of the GUV decreased due to the water efflux and reached to an equilibrium one less than 5 min (Fig.2A). We determined the decrease in the GUV volume by the increase in aspiration length in the micropipet. We transformed Fig.2A into a graph of the time course of f(V*) according to the definition of the left-side of eq.6, and fitted the experimental data to a theoretical curve of eq.6 to get water permeability of the DOPC membrane quantitatively (Fig. 2B). The best fitted value of Qwapp in Fig.2B was 50 jtm/s. We made the same experiments using several single GUVs, and obtained the average value of Qwapp of DOPC membrane, which was 48 ± 8 Vtm/s (n = 5). This volume change was reversible; when the GUV was transferred into the original buffer after the volume decrease stopped, the volume increased to the original value (Fig. 2A). 1.00
~ ~~ 000
2000 4000 ~~~~~~~~~~~0
~ ~ 3o
~~~0 U0 *0 U
0ou... 2000 4000 6000 ~~
Figure 2: (A) Volume change of a DOPC-GUV: first the GUV was transferred from 100 mM glucose solution to 120 mM\ glucose solution (in), and then the GUV was transferred into the original buffer (o). (B) f(V*) is a function of V*=V/VI, which can be determined by a theoretical analysis of the osmotic response ofthe GUV volume. The best fitting of the theoretical curve (eq.6) to the experimental data gives Qwapp =50 ptm/s.
glucose solution to 140 mM glucose solution. The increase in aspiration length in the micropipet (shown in arrows in each image) indicates the decrease in the GUV volume. (B) Volume change of a DPPC/chol (6/4)-GUy determined by the analysis of data in A. (C) f(V*) is a function of V*=V/VI, which can be determined by a theoretical analysis of the osmotic response of the GUV volume. The best fitting of the theoretical curve to the experimental data gives Qwapp =1.3 [[11/5.
molecules occur, and thereby the molecular motion and physical property of the hydrophobic core is almost the same as that of liquids. In contrast, in the lo phase of lipid membranes, acyl chains of phosphatidylcholine (PC) have high orientational order similar to that in the gel phase, and its conformational change is very small. Moreover, cholesterol induces the lateral attraction between PC and> cholesterol to increase the lateral packing of lipids in the membrane. Thereby, it is difficult to produce free spaces for water molecules in the hydrocarbon chain. These difference in physical properties of lipid membranes in these phases can explain the small water permeability of the lipid membrane in the lo phase compared with that in the La
>° osl 0.85
80 l (C)
3.3. Water permeability of the membrane DOPCIDOPG-GUV containing actin filaments
mM), the volume of the GUV decreased due to the water efflux and reached to an equilibrium one less than 5 min (Fig. 4B). The average value of Qwapp of the GUV containing the actin filaments was 35 ± 5 jtm/s (n =5) (Fig. 4C). In a control experiment, we measured water permeability of DOPC/DOPG (1/1)-GUVs containing 1 mg/mL actin monomers (no polymerization), and obtained the average value of Qwapp (46 ± 6 jtm/s) (n =5). This result indicates that the actin filaments reduced the water efflux through the GUV when an osmotic pressure was applied. We consider the mechanism of the effect of the actin filaments on the osmotic response of the GUV. We can reasonably consider that the actin filaments inside the GUV did not change the physical properties of its lipid membrane, and thereby, the water permeability of the lipid membrane did not change. From a physical point of view, the diffusion of water is driven by the difference between the chemical potential of water inside and outside the GUV, Aitw. We can rewrite the eq.3 into a following equation. containing th ciiaetsws3 (i
Q ~~~V \=V
WT RT W
The result that the water permeability of the GUV containing the actin filaments was lower than that of DOPC/DOPG (1/1)-GUVs containing 1 mg/mL actin monomers indicates that the actin filaments reduced the water efflux through the
200 300 400 500 600
We investigated the effect of actin filaments on water permeability of lipid membranes. We succeeded *in preparing DOPC/DOPG (1/1)-GUVs containing 1 mg/mL actin filaments using Mg2+-induced polymerization. DOPC/DOPG (1/1) membrane is in the L, phase. When we transferred a single DOPC/DOPG-GUV containing actin filaments fixed by a micropipet using a small aspiration
100 200 300 400 500 600 700 ~~~~~~~~~~~~~~~0
time ( s)
Figure 4: (A) DIC microscopic images of a DOPC/DOPG(1/1)-GUV containing 1 mg/mL actin filaments fixed at a micropipet when osmotic pressure was applied on the GUV. The time in each image indicates time after the GUV was transferred from 70 mM glucose solution (buffer M) to 90 mM glucose solution (buffer M). The increase in aspiration length in the
micropipet (shown in arrows in each image) indicates the decrease in the GUV volume. (B) Volume change of a DOPC/DOPG(1/1)-GUV containing I mg/mL actin filaments determined by the analysis of data in A.: first the GUV was transferred from 70 mM glucose solution to 90 mM glucose solution (m), and then the GUV was transferred into the original buffer (in). (C) f(V*) vs. time. The best fitting of the theoretical curve to the experimental data gives Qwapp=33 [tm/s.
GUV when an osmotic pressure was applied on the GUy. It explained by the decrease in chemical potential of water induced by the actin filaments under the nonequilibrium condition due to the osmotic pressure as can be
Q Aw - RT ( -
The mechanisms of the actin filaments-induced decrease in water chemical potential is described in detail in Ref. . In couple with the work done by the osmotic stress, the chemical potential of actin filaments increase, which induces the decrease in the chemical potential of water in the solution containing the actin filaments. Thereby, the response of the actin filaments to the osmotic pressure reduces the effect of the applied osmotic pressure.
In this study, we investigated water permeability of various lipid membranes of GUVs by the micropipet aspiration technique, and then its dependence on actin cytoskeletons inside the GUVs. First, we found that the average value of water permeability of DPPC/chol (6/4) membrane in the lo phase was 1.3 ± 0.2 !tm/s, which was much smaller than that of DOPC membrane in the L. phase. This result can be reasonably explained by the physical property of the lo phase membrane. Second, we found that the water permeability of the GUV containing the actin filaments was lower than that of DOPC/DOPG (1/1)-GUVs containing 1 mg/mL actin monomers (no polymerization). This result indicates that the actin filaments reduced the water efflux through the GUV when an osmotic pressure was applied. It can be explained by the decrease in chemical potential of water induced by the actin filaments under the nonequilibrium condition due to the osmotic pressure. However, further investigation on the effect of actin filaments on water flux through the membrane under the osmotic pressure (such as its dependence of actin concentration and its dependence on osmotic pressure) is indispensable near future. It is well known that actin cytoskeleton such as actin filaments and actin/filamin A gel play important roles in the mechanical stability of the plasma membranes and mechanical response of cells [e.g., 7,8]. We have proposed the importance of actin cytoskeleton in the evolution of cells . Bare cells without cytoskeletons are very weak when force is applied on them. For example, a weak force induced by the growth of protein crystals breaks DOPC-GUVs . Thus, for the survival of cells, the actin cytoskeleton must control the mechanical stability and the shape of plasma membranes. Similarly, bare cells without cytoskeletons are very weak under the osmotic pressure. They are easily ruptured. Our studies indicate that actin cytoskeleton also plays an important role in the evolution of cells in the response to the osmotic pressure. Acknowledgement
This work was supported in part by a Grant-in-Aid for Scientific Research in Priority Areas "System Cell Engineering by Multi-scale manipulation" (No. 18048020, & No. 20034023) from the Ministry of Education, Science, and Culture (Japan).
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