Measurement of mechanical properties of naked cell membranes using atomic force microscope puncture test

Journal Pre-proof Measurement of mechanical properties of naked cell membranes using atomic force microscope puncture test Yan Shi, Mingjun Cai, Lulu Zhou, Hongda Wang PII:

S0039-9140(19)31270-6

DOI:

https://doi.org/10.1016/j.talanta.2019.120637

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TAL 120637

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Talanta

Received Date: 25 October 2019 Revised Date:

3 December 2019

Accepted Date: 8 December 2019

Please cite this article as: Y. Shi, M. Cai, L. Zhou, H. Wang, Measurement of mechanical properties of naked cell membranes using atomic force microscope puncture test, Talanta (2020), doi: https:// doi.org/10.1016/j.talanta.2019.120637. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Measurement of Mechanical Properties of Naked Cell Membranes Using Atomic Force Microscope Puncture Test Yan Shi1, Mingjun Cai1, Lulu Zhou2 and Hongda Wang1,*

1

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P.R. China 2

University of Chinese Academy of Sciences, Beijing 100049, China

*

Correspondence: [email protected]

Keywords: membrane mechanical properties, naked cell membranes, force-distance curves, atomic force microscope, Young’s modulus

Abstract Cell membrane defines the physical boundary of the cell, maintains their shape and volume, and meanwhile dominates the cells response to mechanical force. There are many limitations in the mechanical analysis of naked cell membrane, such as cell condition, the supporting of cytoskeleton and cytoplasm. To reduce these influences, non-supported membrane was prepared on high-ordered pore array silicon substrate. Rupture forces and Young’s modulus of three non-supported membranes, red blood cell membrane, somatic cell (MDCK) membrane and phospholipids membrane, are obtained quantitatively using AFM puncture tests. Results indicate that the sequence of both rupture forces and Young’s modulus of the three membranes is MDCK cell membrane>red blood cell membrane>phospholipids membrane. The determinant of naked cell membrane’s mechanical properties is the constituent of membrane itself, including the quantity and distribution of membrane proteins. Focused on the distribution of membrane proteins, two proposed models of cell membrane—the semi-mosaic model of red blood cell membrane and the Protein Layer-Lipid-Protein Island (PLLPI) model of nucleated mammalian cell membrane can be used to explain the different mechanical properties of naked cell membranes. 1. Introduction Cell membrane is the first barrier of cell, which protects cell from its environment and is involved in a variety of cellular processes such as cell adhesion, ion conductivity and cell signalling. In addition, cell membrane is responsible for the mechanical communication with the extracellular matrix (ECM) and neighboring cells. These mechanical interaction regulate the processes of cell adhesion, migration, proliferation and differentiation [1]. Moreover, intracellular signaling pathways can also be activated by the mechanics changes of cell membrane. Taking Hippo pathway as an example, it determines the proliferation and death of cells through the contact inhibition mechanism [2]. Mechanics change of cell membrane is also observed in many diseases, for the instance of that the increasing of red blood cell stiffness was proved to be involved in pulmonary and cordis disease [3, 4], and the process of

cancer was also accompanied with the stiffness changes [5, 6]. Cells are in the position of different micro-mechanical environments, while membrane tension, bending and membrane proteins' physiological functions help in the maintenance of these mechanical environments. The process of vesicle trafficking, exocytosis and endocytosis, was proved to be regulated by membrane stretching [7, 8], and the bending or tension of membrane was also involved in the migration, division of cell and the maintenance of its morphology and polarity [9]. Cell membrane is densely embedded with membrane proteins, which performed special functions, and varied from different types of cell membranes. Several membrane proteins responding in the changes of lateral membrane tension are acted as mechanosensitive channels, while other membrane proteins with particularly shaped domains sense or induce membrane curvature [10]. Additionally, membrane proteins linking cell plasma membrane to the cytoskeleton directly affect the membrane mechanical properties [11]. Above all, the types and the distributions of membrane proteins play crucial roles in the performance of membrane mechanical properties. The new cell membrane models can explain the instinct reason why the distribution of membrane proteins affects the mechanical properties of cell membranes. On the basis of the fluid mosaic model, we proposed the semi-mosaic model of red blood cell membrane. This model suggests that the ectoplasmic side of red blood cell membrane is smooth and the transmembrane proteins do not penetrate the lipid bilayer, whereas the cytoplasmic side is covered with a large amount of proteins [12]. In addition, our group put forward a new model of nucleated mammalian cell membrane named as Protein Layer-Lipid-Protein Island (PLLPI) model, which proved the differences in membrane protein distribution in the ectoplasmic side of cell membrane compared with red blood cell membrane. Membrane proteins in ectoplasmic side of nucleated mammalian cell membrane form a dense packed layer with smooth surface on the lipid bilayer [13]. These membrane characters are adapt to their physiological functions, such as smooth outer membrane is necessary for the red blood cells to flow in the vessels, while more outer membrane proteins help somatic cells to have stronger adhesion between themselves to form tissues and organs, etc.

To date, many groups have made use of different techniques to study cell mechanical properties, such as optical tweezers [14, 15], micropipettes aspiration [16], magnetic twisting and pulling cytometry [17, 18], optical stretching rheometry [19], magnetic bead [20], and atomic force microscope (AFM) [21-25]. The high sensitivity on force and deformation of AFM makes it a powerful tool for quantitative analysis of cell mechanics properties. In the application of AFM, the elastic constants of cells in different physiological [26, 27] and pathological processes [28-30] have been studied, and the obvious mechanical changes can even contribute to disease diagnosis [31, 32]. Cell itself, however, is considered as a complex organism. Directly mechanical measurement on cell surface is influenced by many factors, such as cell condition, cytoplasmic and substrate supporting, and the interaction position on cells (the stress distribution of the nucleus, organelles, or edge of cells were different). Non-supported membrane on high-ordered pore array silicon substrate is an ideal choice to reduce the interference mentioned above, and obtain mechanical parameters of naked cell membranes. In the absence of other support and tension, the composition of the cell membrane itself would be considered as the primary factor influencing the mechanical properties, as different quantities and distributions of membrane proteins may directly affect the mechanical properties of naked cell membranes. In order to confirm this opinion, AFM puncture tests were performed on different types of membranes spreading on high-ordered pore array silicon substrate in this paper. Through the process of membrane indentation to rupture event occurred, the rupture force and Young’s modulus were examined quantitatively on the three membranes: the red blood cell membrane, somatic cell (MDCK) membrane and phospholipid membrane. These results prove that different mechanical properties of the cell membrane were closely related to the quantity and distribution of membrane proteins. In the aspect of mechanical response, the semi-mosaic model of red blood cell membrane and PLLPI model of nucleated mammalian cell membrane were proved. 2. Experimental Section

2.1 Reagents and chemicals High-ordered pore array silicon substrates from AMMT (Advanced Micromachining Tools GmbH, Frankenthal, Germany) with pore radius of 800 nm and pore depth of 1 µm were used as the substrates for the puncture tests. Prior to use, the substrates need silanization

for

a

well

adsorption

of

cell

membranes.

Chicken

egg

phosphatidylcholine (Egg PC) and E. coli phosphatidylglycerol (E. coli PG) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). 2.2 Silanization of high-ordered pore array silicon substrates High-ordered pore array silicon substrates (pSi) were immersed in the mixture of acid lotion (H2SO4: H2O2=3:1) for 60 min to clean the surfaces. After washing with water and ethanol respectively, the substrates were exposed to O2 and UV wave for another 20 min, and then dried in the desiccator for 60 min. These clean substrates and two small containers were located on the bottom of the vacuum dryer with the followed piping of argon gas. After 5 min, 50 µL of APTES (3-aminopropyltriethoxysilane 99%, Sigma Aldrich, st. Louis, MO) and 15 µL of DIPEA (N,N-diisopropylethylamine 99%, distilled, Sigma Aldrich, st. Louis, MO) were added into the two small containers, respectively. Argon gas was then injected into a sealed vacuum dryer for another 5 minutes, and the silanization process would be completed in 4 hours. The APTES modified high-ordered pore array silicon substrates (APTES-pSi) were stored in the desiccator and used within one week. 2.3 Preparation of non-supported naked cell membranes on high-ordered pore array silicon substrates The human red blood cells were collected from healthy volunteers, 100 µL fresh blood (type B) was mixed into 1 mL 150 mM PBS buffer solution (137 mM NaCl, 2 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.5). After 5 cycles of centrifugal washing to re-dissolving, 100 µL suspended red blood cells were dropped onto APTES-pSi substrate for 60 min adsorption. To obtain the naked cell membrane, the adsorbed red blood cells were sheared open by a fast stream of hypotonic buffer (6.845 mM NaCl, 0.135 mM KCl, 0.405 mM Na2HPO4·7H2O, 0.075 mM KH2PO4,

pH7.4) through a syringe at an angle of 20°. With the function of hypotonic buffer and the shear force, the red blood cells were broken, and most of the cytoplasm along with nucleus was blown away, leaving a layer of membrane on the APTES-pSi substrate. In order to further exclude the effect of cell skeleton, the membranes were finally treated with high salt buffer (2 M NaCl, 2.7 mM KCl, 1 mM Na2HPO4·7H2O, 1.5 mM KH2PO4, pH 7.2) for 30 min at room temperature just before puncture tests with AFM in PBS buffer. For somatic cells, we chose Madin-Darby canine kidney cells (MDCK cells, purchased from Shanghai Institutes for Biological Sciences) as a representative, which are incubated with MEM medium containing 10% calf serum (Gibco, USA) and 0.1% penicillin/streptomycin (Invitrogen) at 37°C with 5% CO2. Usually, the cells need to be cultured for 2 or 3 days to achieve 75% coverage on the APTES-pSi substrate. The preparation of naked MDCK cell membranes was operated as the red blood cell membrane mentioned above. 2.4 Preparation of phospholipid membrane In order to simulate normal cell membranes, desired phospholipid powders were mixed under certain ratios (PC/PG=7:3, w/w) to form vesicle firstly. Phospholipid mixtures were dissolved in chloroform, dried by N2 flow, and then vacuumed overnight to remove the trace of organic solvents. Phospholipid film was hydrated in 10 mM HEPES and 150 mM NaCl buffer (pH 7.4) and extensively stirred above the phase-transition temperature of phospholipid, and then adsorbed on APTES-pSi for 60 min for the formation of one or two bilayers lipid membrane coating pSi samples. 2.5 AFM puncture test Puncture tests were carried out on AFM7500 (Agilent Technologies, Chandler, AZ) in Contact mode. Feedback system makes it possible to acquire the accurate location where force curves were obtained. AFM equipped with a top-down optical microscope interfaced with a charge-coupled device (CCD) camera, which was utilized to observe the sample surface and further to position the cantilever on a certain region. The probes (MLCT, SNL) with silicon nitride cantilevers were purchased from Bruker Corporation and Veeco Instruments (Germany). For

topography, SNL-10-C cantilever was used with nominal spring constant of 0.32 N/m and the tip radii of 20 nm. For puncture tests, MLCT-E cantilever was applied with nominal spring constant of 0.1 N/m, and the tip radii of 20 nm. Exact spring constants of the cantilevers were determined by the thermal noise method just before each puncture test. Before each experiment, the tips were cleaned in the mixture of acid lotion (H2SO4: H2O2=3:1) for 60 min. After washing with water and ethanol respectively, the tips were exposed to O2 and UV wave for another 20 min, and then stored in PBS solution until used. Prior to the force curves acquiring, the samples were fast scanned with 128×128 lines in order to estimate the regions covered with membranes. To keep the force curve collected in the center of each hole, the area of interest was zoomed with the scan size of 6.4 µm (integer multiple of the pore radius), and ensured the entire holes in this area (holes without membrane coated may help in this process). The volume parameter was set as 8×8 in the Force Volume model of AFM software, which was equal to the pore number, to make sure the force curve collected in the center of each hole. Then force curves were acquired about 200-1000 curves on each membrane sample for statistical analysis, and the tip was changed after every 20000 curves to reduce the influence of tip contamination and tip abrasion. 2.6 Young’s modulus calculation On the basis of the puncture tests, Young’s modulus is calculated by Hertz model, which describes the elastic deformation of the two objects in contact under load [33, 34]. It characterizes the relationship between applied force and indentation depth. When the shape of AFM tip can be approximated to a paraboloid, the force as a function of indentation is described by equation (1):



F = √′δ 

(1)

where δ is the indentation depth, R is the tip radius of curvature, E' is the reduced Young’s modulus of the tip-sample system defined as

=



 



+

 



(2)

where µsample is Poisson ratios of cell membrane (0.5 in this equation). Etip, Esample is the Young’s moduli of tip and sample, respectively. If Esample≪Etip (in the case of

Si3N4 tips, the Young’s modulus is 150 GPa) [35], E' can be simplified as: ′ =

  

(3)

Combined with our experiment, we made some modification following the works of professor Nikkhah [36] and Darling [37], the indentation depth δ is commonly interpreted as the difference between piezo movement and cantilever deflection, which are both related to the contact point (z0, d0). However, in our experiment, the indentation depth δ should be expressed as the difference of the contact point and the rupture event occurred point (z, d), because of that the mechanical properties of the membranes after the rupture event are no longer belong to the region of the elastic deformation. The coordinate of contact point (z0, d0) was obtained by the AtomicJ software [38], the distributions of Young’s modulus and standard errors of the three membranes were calculated by Gaussian fitting with the same loading rate of 2 µm/s. 3. Results and Discussion 3.1 AFM puncture tests and rupture forces determination AFM puncture tests were repeatedly carried out in the center of the membrane-covered pores to measure the mechanical response of the membranes. Force-distance curves on non-supported membrane region were irregular in shape (Figure 1). During the approaching process, after initial contact of the tip with the membrane, the vertical pressure is generated, the membrane area increases, the membrane is indented until ruptures, and then the cantilever relaxes until it touches the side wall of the pore. This significant peak clearly shows the rupture event of non-supported membranes and can be used to investigate the mechanical properties of different membranes. Taking MDCK cell membrane as an example, the morphology of non-supported membrane on high-ordered pore array silicon substrate was displayed in Figure 2. Cross section data of the red solid line demonstrated the depth of uncovered pores was about 500 nm, while the membrane covered pores was about 100 nm in depth. Due to the diameter of the tip bottom is larger than that of the pore, the tip cannot reach the bottom of the pore, so that the depth of the pore was not accurately examined. Before

puncture tests, we scanned the membrane to evaluate the regions covered with membrane (Figure 2C), then force-distance curves were measured on each membrane covered pore, resulting in the rupture of the membrane covering pores (Figure 2D). The typical force-distance curves and calculated rupture forces of the three membranes were clearly shown in Figure 3. In order to acquire accurate statistical analysis, mechanical data was collected on different membrane covered pores or with different probes to avoid the impact of locations and probes. For red blood cell membrane, the membrane structure is relatively simple, proteins of intramembrane were less than those of somatic cell. Therefore, we believe that the rupture force of red blood cell membrane is smaller than that of MDCK cell membrane. Experimental results confirmed our conjecture that the rupture forces of red blood cell membrane and MDCK cell membrane were about 0.48±0.02 nN and 0.99±0.02 nN, respectively. For the simulated cell membrane, the rupture forces of phospholipids membranes are 0.043±0.001 nN and 0.107±0.002 nN, respectively. The two peaks in Figure 3H could be explained as the one or two bilayers of phospholipid membranes produced with the adsorption of vesicles. When vesicles solution was added onto substrates, bilayer membrane was formed through a thinning process, while the two bilayers membrane was developed with the whole vesicle adsorption (Figure 3I). So, the force of the second peak (0.107±0.002 nN) was about twice as that of the first peak (0.043±0.001 nN). The different rupture forces of the three membranes may closely relate to the quantity and distribution of membrane proteins, which could be explained from their own membrane structure. Based on the classic fluid mosaic model, we proposed the semi-mosaic model of red blood cell membrane, which suggested that proteins were partly set within the lipid bilayer rather than protruding out of the outer cell surface, and the membrane proteins are mainly located on the cytoplasmic side of membranes (Figure 3C) [12]. As described in PLLPI model of nucleated mammalian cell membrane, proteins on the ectoplasmic side of the cell membrane form a closely packed protein layer on phospholipid bilayer, resulting in a smooth outer membrane surface. The proteins on the cytoplasmic side of the cell membrane aggregate in

cholesterol-rich microregions, which is conducive to energy conversion and signal transduction (Figure 3F) [13]. Due to the larger amount of the membrane proteins and their dense packed distribution on MDCK cell membrane, the rupture forces of MDCK cell membrane is larger than it of red blood cell membrane. As the contrast, the rupture force of phospholipid membrane is the lowest one, because of no protein layer and intramembrane protein exist on the membrane. 3.2. Young’s modulus calculation On the basis of the puncture tests, we also investigated the elastic properties of the three membranes through Young's modulus calculation. As shown in Figure. 4, the Young’s modulus of the three membranes was clearly depicted, the values were 2.12±0.23 kPa for red blood cell membrane, 3.59±0.29 kPa for MDCK cell membrane, and 0.34±0.03 kPa/1.24±0.03 kPa for one/two bilayer phospholipid membrane, respectively. Compared with MDCK cell membrane, the Young’s modulus of the red blood cell membrane is smaller, which means the red blood cell membrane is softer. To meet their physiological function, the quantity and types of membrane proteins of red blood cell are much less than those of MDCK cell [39], resulting in the weak contribution for the resistance of rupture force and the lower Young’s moduli. For the bilayer phospholipid membrane, the Young’s modulus was much lower than it of the red blood cell membrane or MDCK cell membrane. The two peaks in the Young’s modulus distribution histogram of phospholipid membrane were consistent with those of rupture force (Figure 4C), which was due to the one or two bilayers of the lipid membrane derived from the vesicles. One possible mechanism contributing to the lowest Young’s modulus of bilayer phospholipid membranes is that no protein existed on lipid membrane, and the only lateral interaction between phospholipid molecules maintains the stiffness of the membrane. The small amount of proteins on red blood cell membrane determined its higher stiffness than that of phospholipid membrane, while somatic cell membranes with a large number of proteins and the closely packed protein layer have much higher stiffness. These conclusions are complementary to the rupture force of the three membranes, which proves that the mechanical properties of

membranes are closely related to the quantity, distribution and interaction of proteins in the membranes. 4. Conclusion The mechanical properties measurement of cell membranes on whole cell is usually influenced by the support of the substrate, cytoplasm, the cell condition and the different stress location. Non-supported membranes on high-ordered pore array silicon substrate can help to reduce or eliminate these effects, and obtain the mechanical properties of naked membranes. Results indicate that the sequences of both rupture forces and Young’s modulus of the three membranes is MDCK cell membrane>red blood cell membrane>phospholipids membrane. Compared with MDCK cells, membrane proteins of red blood cells is less in quantity and not distributed on the ectoplasmic side of cell membrane, resulting in the lower rupture force than that of MDCK cells. Additionally, the simulated cell membrane-the phospholipid membranes have no intramembrane proteins or dense protein layer, so that the rupture force is the lowest. The elastic character reflected by Young's modulus further verifies this conclusion. Therefore, we conclude that the quantity, distribution of membrane proteins play an important role in the mechanical properties of non-supported membranes. These results proved the semi-mosaic model of red blood cell membrane and PLLPI model of nucleated mammalian cell membrane in the aspect of mechanical properties. Acknowledgements This work was financially supported by National Key R&D Program of China (No. 2017YFA0505300 to HW), National Natural Science Foundation of China (No. 21727816, 21721003, 21525314 to HW, No. 21703231 to JJ, No. 21907089 to YS) and the Natural Science Foundation of Jilin Province, China (No. 20170101196JC to MC).

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Figure Captions Figure 1 Scheme of AFM puncture test on membrane covered high ordered pore array silicon substrate, and the typical force-distance curve of the rupture event. In the approaching parts, the tip is far away from the membrane at the beginning (position 1); when tip gets very close to the cell surface, the contact point is not as clear as it is on the force curve of solid surface (position 2); a further push makes the cantilever to bend upwards, and the rupture event occurred (position 3); Instantaneously, the cantilever relaxes and the force curve drops back to the base line (position 4), then the continuous diagonal line represents that the tip touches the side-wall of the pore (position 5). Figure 2 AFM topography of (A) high-ordered pore array silicon substrate; (B) MDCK cell membrane on APTES-pSi imaged in air with the cross section data. The membrane-covered and uncovered pores can be readily distinguished. The height profile taken from the red solid line demonstrates that a membrane covered pore is indented only 100 nm, while the cantilever tip reaches a depth of nearly 500 nm in an uncovered pore, and a pore radius of 800 nm; MDCK membrane before (C) and after (D) puncture test on high-ordered pore array silicon substrate. Figure 3 Typical force-distance curves, the rupture forces distribution histograms with Gauss fitting and the structural schemes of the three non-supported membranes. A, B and C for red blood cell membranes; D, E and F for MDCK cell membranes; G, H and I for phospholipids membranes. C, F is the front view of semi-mosaic model of red blood cell membrane12 and PLLPI model of nucleated mammalian cell membranes.13 I is the formation of one or two bilayers phospholipid membranes derived from vesicles. Reproduced with permission.12 Copyright 2010, Elsevier; Reproduced with permission.13 Copyright 2014, Elsevier. Figure 4 The Young’s modulus distribution histograms obtained between the contact point and the rupture event on pSi substrate for (A) red blood cell membrane (RBCM), (B) MDCK cell membrane (MDCK) and (C) lipid membrane (LIPID). (D) Box plots of modulus values for the three membranes. The boxes mean the main distribution (50%), with the vertical lines through boxes representing the overall distribution and the horizontal lines in every box representing the average values.

Figure 1

Figure 2

Figure 3

Figure 4

Mechanical properties of naked cell membrane are obtained on non-supported cell membranes prepared on high-ordered pore array silicon substrate. Rupture forces and Young’s modulus of red blood cell membrane, MDCK cell membrane and phospholipid membrane are measured using AFM puncture tests. The mechanical properties of naked cell membrane are determined by the quantity and distribution of membrane proteins.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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