Self-Assembly and Biogenesis of the Cellular Membrane are Dictated

Subscriber access provided by UNIV OF SOUTHERN INDIANA

B: Biomaterials and Membranes

Self-Assembly and Biogenesis of Cellular Membrane is Dictated by Membrane Stretch and Composition Akshata R. Naik, Eric R Kuhn, Kenneth T. Lewis, Keith M Kokotovich, Krishna R. Maddipati, Xuequn Chen, J. Heinrich K. Hörber, Douglas J Taatjes, Jeffrey Potoff, and Bhanu P Jena J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b04769 • Publication Date (Web): 19 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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

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

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

The Journal of Physical Chemistry

Self-Assembly and Biogenesis of Cellular Membrane is Dictated by Membrane Stretch and Composition

Akshata R. Naik1, Eric R. Kuhn1, Kenneth T. Lewis1, Keith M. Kokotovich1, Krishna R. Maddipati4, Xuequn Chen1, J.H.K. Hörber6, Douglas J. Taatjes7, Jeffrey J. Potoff5, Bhanu P. Jena1,2,3* 1

Department of Physiology; 2NanoBioScience Institute, 3Center for Molecular Medicine & Genetics, School of Medicine, Wayne State University, Detroit, MI 48201, USA; 4Department of Pathology, Lipidomics Core Facility, Wayne State University, Detroit, MI 48201, USA; 5 Department of Chemical Engineering & Materials Science, College of Engineering; Wayne State University, Detroit, MI 48201, USA 6 Department of Physics, University of Bristol, Bristol BS8 1TD, UK 7 Department of Pathology and Laboratory Medicine, Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, VT 05405, USA

*Correspondence: Bhanu P. Jena, Ph.D., D.Sc.; Telephone: 313-577-1532; Fax:313-9934177; E-mail: [email protected]

Running Title: Membrane biogenesis is dictated by stretch and composition

ACS Paragon Plus Environment

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

Page 2 of 29

2 ABSTRACT The cell plasma membrane is a highly dynamic organelle governing a wide range of cellular activities including ion transport, secretion, cell division, growth and development. The fundamental process involved in the addition of new membrane to preexisting plasma membrane however, is unclear. Here we report using biophysical, morphological, biochemical and molecular dynamic simulations, the selective incorporation of proteins and lipids from the cytosol into the cell plasma membrane dictated by membrane stretch and composition. Stretching of the cell membrane as a consequence of volume increase following incubation in a hypotonic solution, results in the incorporation of cytosolic proteins and lipids into the existing plasma membrane. Molecular dynamic simulations further confirm that increased membrane stretch result in the rapid insertion of lipids into the existing plasma membrane. Similarly, depletion of cholesterol from the cell plasma membrane selectively alters the incorporation of lipids into the membrane.


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


3 INTRODUCTION Biological membranes, in addition to delimiting the cell and its intracellular compartments, participate in a plethora of life-sustaining activities1,2. The addition of new membrane to existing cellular membranes may involve fusion of preformed proteoliposomes and or the incorporation of intracellular free lipids and proteins. It is reported that lipid bilayers with adhered lipid vesicles expand due to fusion of those vesicles when stretched laterally3,4. This observation is further supported by mathematical modeling of the plasma membrane which suggests trafficking of lipids into stretched membranes is utilized to relieve tension5. In addition, cell swelling has been reported during mitosis6,7 and mechanical forces are implicated in morphogenesis and development8. Membrane shrinkage at the furrow region, caused by the actomyosin contractile ring leading to cytokinesis, has long been long suggested2. This tension at the furrow has been demonstrated to occur in conjunction with the addition of new membrane at the polar regions of the cell9,10. Composition of the new membrane would differ from the parent membrane, since membrane growth in cytokinesis for example in the Drosophila embryo, occur in defined locations and ordered sequence by insertion of intracellular components10. Despite these studies, the fundamental process of addition of new membrane to preexisting cellular membranes is poorly understood. Biological membranes are nearly inelastic11,12 , yet must respond to rapid physiological changes in volume of the cell or individual intracellular compartments. Thus, in the case of hypertrophy, cells must either unfurl membrane invaginations or incorporate large amounts of new protein and lipid to accommodate the increase in surface area. Studies13,14 have demonstrated that isolated secretory vesicles are capable of rapid swelling despite their lack of membrane folds or intra-vesicular liposomes, suggesting that intra-vesicular lipids and or proteins incorporate into the existing vesicle membrane. In this study we report using biophysical, morphological, biochemical, and molecular dynamic simulations, the selective



Page 4 of 29

4 incorporation of proteins and lipids from the cytosol into the cell plasma membrane, dictated by membrane stretch and composition.

METHODS RBC isolation Sprague-Dawley rats weighing 100-120 g were used for the study. Blood was collected by cardiac puncture immediately following CO2-induced euthanasia of the animal. The blood sample was diluted in 20 volumes of phosphate buffered saline (PBS) pH 7.5, and centrifuged at 300 x g for 10 min at 4°C. The resulting pellet was resuspended in 2 volumes of PBS, and washed three times, followed by their final resuspension in 10 volumes of PBS prior to use. Scanning Electron Microscopy of RBCs Twenty-five micro liters of isolated RBC from rat blood were fixed in 2% paraformaldehyde 2% glutaraldehyde, rinsed with buffer and post-fixed in 1% OsO4 for 30 minutes at 4oC. Following rinses with 0.1M cacodylate buffer, the cells were spread onto 13 mm Thermonox cover slips coated with 0.2% Poly-L-lysine. Cells were allowed to settle and adhere to the surface of the coverslip for several days at 4°C, and then the sample was rinsed gently by dipping into 4 changes of Milli Q ddH2O. The cover slips were loaded into a cylindrical holder and dehydrated for 5 min each, in increasing concentrations of ethanol (35, 50, 70, 85, and 95%) followed by 4 5 min rinses in 100% ethanol. The sample was critical point dried and mounted onto one large aluminum specimen stub in puddles of liquid graphite, sputter coated with gold and palladium and imaged with a JSM 6060 SEM (JEOL USA, Peabody, MA) at 15 and 20kV, spot size 30, WD~6 mm at various magnifications of up to 28,000 x. Transmission Electron Microscopy of RBC


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


5 Transmission electron microscopy of RBCs was performed as described in a previously published procedure15,16. Briefly, cells were fixed in 2% glutaraldehyde/2% paraformaldehyde in ice-cold PBS for 24 h, washed with buffer, embedded in 2% SeaPrep agarose, followed by post-fixation for 1h at 4 C using 1% OsO4 in 0.1 M cacodylate buffer. The sample was then dehydrated in a graded series of ethanols, through propylene oxide in a microwave, and infiltrated and embedded in Spurr’s resin. Ultrathin sections were cut with a diamond knife, retrieved onto 200 mesh nickel thin-bar grids, and contrasted with alcoholic uranyl acetate and lead citrate. Grids were viewed with a JEOL 1400 transmission electron microscope (JEOL USA, Inc., Peabody, MA) operating at 80 kV, and digital images were acquired with an AMTXR611 11 mega pixel ccd camera (Advanced Microscopy Techniques, Danvers, MA).

Light microscopy The plasma membrane of isolated RBCs from rat blood was stained using 10 M of the Octadecyl rhodamine B (R18) dye (Life Technologies, Grand Island, NY). R18 is a lipophilic cation that has been extensively used as a membrane PBS, and fluorescent images of the RBCs at various time intervals acquired using an FSX100 Olympus microscope through a 100x objective lens (numerical aperture = 1.40). Photonic force microscopy The Photonic Force Microscope (PFM)

17

employed in these experiments uses nano- to

micrometer sized particles in solution as probes and measures the particle’s position relative to the geometric focus of the laser trap with nanometer and microsecond resolution along all three dimensions. In our set-up a single-gradient laser trap17 is used, which is part of an inverted microscope (Axiovert 35, Carl Zeiss, Oberkochen, Germany). The trapping laser is a Nd:YVO4 laser (1064 nm) (T20-B10-106Q, Spectra Physics, Darmstadt, Germany), which operates in



Page 6 of 29

6 cw-mode and can be directed by two scanning mirrors (M2, General Scanning, Munich,Germany). The oil immersion microscope objective (Plan-Neofluor 100X, NA 1.3, Carl Zeiss) focuses the light into the sample chamber. The laser focus is moved vertically by a piezo-driven objective lens (P721.00, Physik Instrumente, Waldbronn, Germany). The trap captures polystyrene beads in all three dimensions. The movement can be observed using the microscope equipped with differential interference contrast (DIC) optics. During the experiments the temperature of the sample chamber was held constant at 30 0C. The lateral position of the bead with respect to the laser focus is monitored by detection of the forward scattered laser light using a four-segment photodiode (S5981, Hamamatsu). The signal is amplified and digitized with an AdWin-transputer board (AD-Win 5F, Jäger Electronics, Lorsch, Germany). Data analysis was performed using IgorPro (WaveMetrics). Polystyrene spheres (Sigma-Aldrich) with radius of 200±10 nm suspended in phosphate buffered saline pH 7.4 were used. Determination of major lipids in RBC membranes Lipids from RBC membranes were extracted with methanol and methyl-tert-butyl ether (MTBE) according to published methods (18). Briefly, methanol (1.5 mL) containing 100 ng each of internal standards (diheptadecanoyl PC, diheptadecanoyl PE, diheptadecanoyl PS, diheptadecanoyl PA, diheptadecanoyl PG, monoheptadecanoyl glycerol, diheptadecanoyl glycerol,

tridiheptadecanoyl

glycerol,

1-palmitoyl(d31)-2-oleoyl-sn-glycero-3-

phosphoinositol, N-heptadecanoyl-ceramide-C18, N-heptadecanoyl-sphingomyelin-C18, and PAF-C16-d4) was added to RBC membrane followed by MTBE and mixed well. The mixture was left for 1 h at room temperature with occasional mixing. Water (1.5 mL) was added to the mixture, mixed thoroughly, and centrifuged (1000 x g) for 5 min to assist the separation of phases. The upper organic phase was collected to a clean glass tube. The lower aqueous phase was extracted twice (2 mL each time) with MTBE saturated with methanol and water (10:3:2.5


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


7 v/v) and the extracts were combined. The MTBE extracts were evaporated to dryness under a gentle stream of nitrogen and the residue was dissolved in LC-MS grade chloroform-methanol100 mM aqueous ammonium acetate (58:40:2 v/v). The reconstituted lipid extract was analyzed for lipids by mass spectrometry. Lipid extracts were directly infused into the TurboVIon source by a syringe pump at 10 µL/min and analyzed by QTRAP5500 mass spectrometer (ABSCIEX) using the Multiple Precursor Ion Scanning method as described earlier19,20. Mass analyzer conditions are as follows: for positive ion mode: Ionization Potential: 5500 V, Declustering Potential: 120 V, Entrance Potential: 9 V, Collision cell Exit Potential: 9 V and for the negative ion mode: Ionization Potential: -4500 V, Declustering Potential: -100 V, Entrance Potential: -10 V, Collision Cell Exit Potential: -10 V. Collision energy for each precursor ion scan was held at optimum. Data were analyzed for the identification of lipid species using LipidView software (ABSCIEX). Lipids were quantified against corresponding internal standards. Computation methods Simulations were performed on lipid bilayers containing 128 DPPC lipids. All interactions between lipids and between lipids and water were modeled with the MARTINI force field. The MARTINI force field maps four heavy atoms into a single coarse-grained interaction site21. To create the initial configuration for the coarse-grained simulations, an all-atom representation of the bilayer was created with CHARMM-GUI22. This all-atom atom representation was equilibrated for 50 ns using isobaric-isothermal ensemble molecular dynamics simulations at 330 K and 1.01 bar. A 2fs time step was used. For the all-atom simulations, the CHARMM force field, version 36, was used to describe the interactions between atoms23. A coarsegrained representation of the bilayer was then obtained from the final configuration of the allatom simulations using the CGTools plugin, version 0.1 in VMD 1.9.224. All simulations were performed with NAMD version 2.10. Coarse-grained simulations were performed in the NPT



Page 8 of 29

8 ensemble to determine the effect of bilayer stretching on the incorporation of free lipids into the membrane. The box volume was allowed to fluctuate independently in the x-y plane and the z coordinate axis. Lennard-Jones interactions were shifted smoothly to zero between 9 and 12 Å. A constant temperature of 323 K was maintained using Langevin dynamics with a damping coefficient of 5.0 ps-1. The pressure was maintained at 1.01 bar using the Nosé– Hoover method25,26, with an oscillation period of 2000 fs and piston decay period of 1000 fs. A 20fs time step was used for all calculations. Proteomics on RBC membrane protein band resolved using SDS-PAGE RBC membranes isolated from before (control) and following osmotic swelling (experimental) were solubilized and resolved in 12.5% SDS-PAGE. Following Coomassie Blue staining of the gel, the most prominently stained band that was expressed in the experimental membrane fraction and its corresponding control band were subjected to in-gel digestion, as previously published27. Matrix-assisted laser desorption ionization (MALDI) Mass spectrometry was performed using the Applied Biosystems (ABI) 4700 Proteomics Analyzer (TOF/TOF) in positive ion mode. As previously described28, a fraction of the tryptic peptides from each gel band was spotted onto a MALDI target plate for mass spectrometric analysis. Peptide mass fingerprints were collected for each well and the four most intense peaks above S/N of 60 were selected for MS/MS analysis. After the MS and MS/MS, spectra were processed using 4700 ExporerTM software (v2.0, Applied Biosystems). The monoisotopic peak lists generated in ABI’s GPS ExplorerTM v2.0, was submitted to the GPS ExplorerTM v2.0 search tool (based on MASCOT) for protein identities. The Non-redundant Protein Database, NCBInr, was searched using the following parameters for: 0 or 1 missed cleavage by trypsin, carboxyamidomethylation of cysteines as fixed modification, and methionine oxidations, N-


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


9 terminal protein acetylation, Pyro-glu (N-term E), Pyro-glu (N-term Q) as variable modifications. LC-MS/MS analysis and database search After a detergent removal procedure, tryptic peptides were separated by reverse phase chromatography (Magic C18 column, Michrom), followed by ionization with the ADVANCE ion source (Michrom), and then analyzed in an LTQ-XL mass spectrometer (Thermo Scientific). Six abundant species were fragmented with collision-induced dissociation. Data analysis was performed using Proteome Discoverer 1.1 (Thermo), which incorporated the Mascot algorithm (Matrix Science). The NCBI database was used against rat protein sequences and a reverse decoy protein database was run simultaneously for false discovery rate (FDR) determination. A duplicate porosome sample was also analyzed by nanoLC-MS/MS. In this case, the tryptic peptides were separated on a reversed-phase C18 column with a 90 min gradient using the Dionex UltimateTM HPLC system. Then the MS and MS/MS spectra were acquired on an Applied Biosystems QSTAR XL mass analyzer using information dependent acquisition mode. A MS scan was performed from m/z 400-1,500 for 1s followed by product ion scans on two most intense multiply charged ion peaks. Peak lists were submitted to the Mascot server to search against the NCBInr database for rat sequences with carbamidomethyl (C) used as a fixed modification and oxidation (M), N-acetylation (protein N terminus) as variable modifications. Secondary analysis of both the LTO XL and QSTAR XL were next performed using Scaffold (Proteome Software). A fixed modification of +57 on cysteine (carbamidomethylation) and variable modifications of +16 on methionine (oxidation) and +42 on protein n-terminus (acetylation) were included in the database search. Minimum protein identification probability was set at ≥95% with 2 unique peptides at 95% minimum peptide identification probability. SDS-PAGE and gel staining



Page 10 of 29

10 Equal protein amounts (10μg) of control and volume- induced stretched RBC membranes in Laemmli reducing sample preparation buffer were resolved on a 10% polyacrylamide gel. Gels were then stained with Coomassie Brilliant Blue and silver stain according to previously published procedures29 for protein visualization. PC: PS liposome preparation Broad-form PC: PS (70:30) liposomes were prepared either with or without cholesterol (10%) and were incubated with 5μg/mL TopFluor® Cardiolipin (Avanti polar lipids) for 30-40 minutes at room temperature. They were plated on 35 mm glass bottom microwell dishes to air dry and washed gently with PBS (1x) three times and imaged on FSX100 Olympus microscope through a 40x objective lens (numerical aperture 1.3) with illumination at 405 nm. Min6 cell culture Mouse pancreatic insulinoma (Min6) cells were grown to confluence in 100 x 13 mm Petri dishes in high- glucose (25mM) Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal calf serum, 50μM β-mercaptoethanol and 1% antibiotics (Penicillin and Streptomycin) according to published procedure23. Immunofluorescence microscopy was performed on Min6 cells grown to 60- 70% confluence on 35mm Petri dishes. Immunofluorescence on Min6 cells Min6 cells, grown on 35mm glass bottom petri dishes were designated either as control (treated with 1x PBS) or treated with 5mM methyl- beta-cyclodextrin (M-βCD) for 30-45 minutes to sequester cell plasma membrane cholesterol. To determine the distribution of SNARE protein SNAP-25 and Gαi3 in control and 5mM M-βCD treated Min6 cells, immunofluorescence studies were performed according to published procedures23. To determine the position of the cell nucleus, cells were exposed to DAPI nuclear stain (Molecular Probes, Life Technologies). SNAP 25 was stained with goat polyclonal α- SNAP 25 sc 7538 and Gαi3 was stained with


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


11 rabbit polyclonal α- Gαi3 sc 262, both at a 1:500 dilution. Secondary antibodies mouse antigoat AF 488 sc and donkey anti rabbit AF 594 at a dilution of 1:1000 were used. Images were acquired using an immunofluorescence FSX100 Olympus microscope through a ×100 objective lens (numerical aperture, 1.40) with illumination at 405, 488, or 647 nm. The coassociation of Gαi3 and SNAP-25 and their cellular distribution was determined by merging the fluorescent images using the software imageJ. Cytosol and membrane isolation from Min6 cells Min6 cells were grown to 70- 80% confluence on 100 x13 mm Petri dishes and divided onto two groups. One group was treated with 5mM M-βCD and the second group was treated with vehicle (1x PBS) for 30- 45 minutes. Both groups of cells were then subjected to subcellular fractionation to isolate their cytosol and membrane fractions. All fractionation steps were performed at 4 degrees Celsius. Briefly, cells were scraped and transferred into ice- cold fractionation buffer for 15 minutes (20 mM HEPES, 10mM KCl, 2mM MgCl2, 1mM EDTA, 1mM EGTA, 1mM DTT, 1mM PI cocktail) and passed through a 251/2 gage needle at least 10 times. Cell lysates were centrifuged at 720xg for 5 minutes to separate out the nuclei pellet. Supernatant obtained was centrifuged at 10,000xg for 5 minutes to pellet the mitochondria. Supernatant now contains the cytosol and membrane fraction. To isolate the membrane fraction, supernatant from the previous step was ultra-centrifuged at 100,000xg for 1 hour. After the membrane fraction was collected the cytosol was obtained as the supernatant.

RESULTS AND DISCUSSION Cell swelling incorporates cytosolic lipids and proteins into the plasma membrane To test the hypothesis that cytosolic proteins and lipids are selectively recruited for growth of the existing plasma membrane in cells, we used isolated rat red blood cells (RBC) (Figure 1). Similar to secretory vesicles, no organelles or membrane-bound vesicles are present within the



Page 12 of 29

12 rat RBC (Figure 1b), making them ideal models for determining the entry of cytosolic proteins and lipids into the plasma membrane. Stretching of the RBC plasma membrane was achieved by pulling on a membrane-adhered polystyrene bead using an optical laser trap (Figure 1c-1e), and by rapidly increasing cell volume upon subjecting the RBC to a hypotonic buffer solution (Figure 1f-1m). Membrane tethers in cells30,31 have been extensively investigated using optical tweezers, and studies suggest the contribution of cytoskeletal proteins in their formation in RBCs32. Membrane tethers are typically a few tens-of-nanometer thick tubes with lipid bilayer walls, playing a critical role in various physiological processes in the cell such as endoplasmic reticulum and Golgi dynamics, and for the storage of excess lipids. Pulling at 10-15 pN force on a 200 nm polystyrene bead adhered to the plasma membrane of rat RBC using laser tweezers (33) of a photonic force microscope, demonstrates the formation of membrane tethers nearly 150 nm in diameter and >6 m in length (Figure 1c-1e). As illustrated in the transmission electron micrograph (Figure 1b), rat RBC is devoid of either membrane folds or intracellular vesicles. Consequently, the rapid generation of excess membrane contributing to membrane tether elongation when pulled, suggests stretch-induced insertion of cytoplasmic lipids and proteins. Similarly, osmotic swelling of isolated RBC induced by incubation in hypotonic (70%) phosphate buffered saline (PBS), results in membrane stretching and growth. When swollen RBCs are returned to isotonic medium (100%) PBS, membrane pegs or protrusions appear, as observed in both light (Figure 1h, 1i, Figure S1) and electron micrographs (Figure 1j, 1m). The pegs represent the excess membrane generated as a consequence of swellinginduced stretch of the cell plasma membrane. Unlike a secretory vesicle, which will lose its spherical form following loss of intra-vesicular fluid, the RBC with its well-organized underlying cytoskeleton8, is able to return to its original toroidal shape and the excess membrane incorporated into the plasma membrane is pushed outward to form the observed tube-like protrusions or pegs.


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


13

Mass spectrometry confirms stretch-induced incorporation of new lipids and proteins into plasma membrane To determine if new proteins and lipids incorporate into the cell plasma membrane in RBC following swelling-mediated membrane stretch, control and stretched RBC membranes were resolved by SDS-PAGE and subjected to both silver and Coomassie Brilliant Blue staining. New bands appear in the stretched membrane samples by both staining methods (Figure 2a, 2b), confirming incorporation of cytosolic proteins into the stretched RBC plasma membrane. To further determine the incorporation of new proteins and possibly lipids into the existing cell plasma membrane following membrane stretch, both lipid and protein mass spectrometry were performed on isolated plasma membrane preparations from control RBC and RBC subjected to osmotic swelling (Figure 2c, 2d; Supporting Table I, II). Mass spectrometry demonstrates the presence of additional proteins and lipids in the stretched plasma membrane, significantly altering its composition. The insertion of new classes of lipids (nearly 30%), and new proteins (15%) from the cytosolic compartment of the RBC into the plasma membrane was demonstrated, that contributed to membrane growth. It is important to note that insertion of new lipids and proteins into the existing plasma membrane, and the consequent membrane growth is rapid in the case of RBCs, since water channels or aquaporins present at the plasma membrane allow for swift gradient-driven active transport of water into cells at a rate of approximately 3x109 water molecules/sec via each aquaporin channel34,36.

MD simulation supports stretch-induced lipid incorporation into plasma membrane To further understand the molecular mechanism of the insertion of lipids into existing membranes upon stretching, NPT molecular dynamic simulations were performed. In Figure 3a, the imposed tension is plotted as a function of log of the area per lipid. As expected, the



Page 14 of 29

14 data show linear behavior. Taking the slope of the best-fit line through the data produces an area compressibility modulus of KA = 234 mN/m, which is in close agreement with the experimental value of 231 mN/m16,17. To determine the effect of imposed tension on the incorporation of free lipids, a single DPPC lipid was placed near the bilayer surface, but fully solvated by water, as shown in Figure 3b,3c. Simulations were initiated for a range of interfacial tensions from 0 to 60 dyne/cm. For values of the interfacial tension between 10-50 dynes/cm, the free lipid was incorporated into the bilayer within 50-140 ns as shown in Figure 3b. For simulations performed at 0 tension and 5 dynes/cm, incorporation of the free lipid in the bilayer had not occurred until after 500 ns. These results support the premise that application of tension to the lipid bilayer enhances significantly the kinetics of free lipid incorporation into bilayer membranes.

Composition of existing membrane dictates the incorporation of proteins and lipids In addition to the cell plasma membrane, intracellular organelles such as the nucleus, Golgi apparatus, lysosome, or the endoplasmic reticulum, are all encased by membrane, the compositions of which are different and very tightly regulated37. The generation of additional membrane must therefore be strictly governed by the composition of the parent membrane in addition to being induced by stretch. We tested this hypothesis using live Min6 cells, an insulin secreting mouse insulinoma cell line that mimics pancreatic beta cells. Cells incubated with increasing concentrations of methyl beta cyclodextrin (M-βCD) to deplete cholesterol from the cell plasma membrane38 demonstrate loss in fluorescent PS uptake by the cell plasma membrane (Figure S2), while the uptake of PE and CL remain unchanged (Figures S2-S4). Selectivity in lipid species uptake is an interesting observation since it has been previously reported that cholesterol maintenance in the cytoplasmic leaflet of the plasma membrane requires the retention of PS39. The reduction of PS uptake upon depletion of cholesterol from


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


15 the membrane could be due to the fact that cholesterol is known to participate in cholesterolenriched membrane rafts resulting from lipid-lipid phase separation. Lipid phase separations are caused by distinct PS lipid-CHOL versus PG lipid-CHOL interactions. Similarly, PS are confined to the inner leaflet of the cell membrane by the ATP-dependent flippase enzyme ATP11C, counteracting the activity of ATP-independent scramblase. When cells are depleted of cholesterol, PS quickly appears at the outer surface, implying that cholesterol acts in the cell as a powerful scramblase inhibitor. The critical role of lipids on the presence and distribution of plasma membrane proteins is well documented, for example SNARE proteins are found to be embedded in cholesterol-rich lipid domains40. To test the impact of the loss of cholesterol on plasma membrane proteins, the presence and distribution of syntaxin, SNAP25, Gi3, and v-ATPase, was determined using immunochemical analysis (Figure 4). Extraction of cholesterol from the Min6 cells alters the distribution of SNAP-25 and Gi3 at the cell plasma membrane and further demonstrates that while there is a loss of syntaxin and SNAP25 at the cell plasma membrane, there is a gain in the relative amounts of Gi3 and v-ATPase immunoreactivity (Figure 4). It is interesting to note that both non-membrane and membrane proteins such as hemoglobin, carbonic anhydrase, clathrin, flotillin-2, lin-54 homolog, and TLR4 interactor are found associated with the membrane following stretch. The interaction of hemoglobin with both cytosolic and membrane proteins and lipids have been reported, and similarly, earlier studies show the association of some plasma membrane bicarbonate transporters with carbonic anhydrase enzymes to form bicarbonate transport metabolon to facilitate

metabolic

CO(2)-HCO(3)(-)

conversions

and

coupled

HCO(3)(-)

transport. Furthermore, the detection of membrane proteins such as clathrin, flotillin-2, lin-54 homolog, and TLR4 interactor following stretch, is a consequence of their selective incorporation into the plasma membrane and exceed detection threshold of mass spectrometry.



Page 16 of 29

16 These results demonstrate the critical role of membrane cholesterol in maintaining the concentration and distribution of t-SNAREs at the cell plasma membrane.

CONCLUSION The present study demonstrates using both experimental and computational approaches, that membrane stretch and composition govern the insertion of specific proteins and lipids into existing cellular membranes. These observations provide an explanation to a number of fundamental and intriguing cellular processes, among them the membrane-bound secretory vesicles that undergo rapid stretching of the vesicle membrane resulting from volume increase required for membrane fusion and the regulated release of intra-vesicular contents during secretion14, and the stretching of cellular membrane at various domains required for cell division.

SUPPORTING MATERIAL Supporting Materials and Methods and Figures are available on line

AUTHOR CONTRIBUTIONS BPJ. developed the idea for the study and designed the experiments. ARN. ERK. and KTL. conducted the experiments, prepared the biological samples, and analyzed the data. KRM. performed the lipid extraction and lipid mass spectrometry, and XC. performed the proteomics mass spectrometry. JHKH. and BPJ. performed the optical tweezer membrane pulling experiments. JJP. performed the molecular dynamic simulations, and DJT. performed the electron microscopy. All authors participated in discussions and writing of the manuscript.

SUPPORTING MATERIAL


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


17 Figures (S1–S4) and Tables I and II appear in the Supporting Material.

ACKNOWLEDGEMENTS This work was supported in part by the National Science Foundation grants EB00303, CBET1066661 (J.J.P and B.P.J); the National Institutes of Health grant S10RR027926 (K.R.M); the Wayne State University Interdisciplinary Biomedical Systems Fellowship (K.T.L. and A.R.N); and the Thomas C. Rumble Wayne State University Graduate Fellowship (K.T.L).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Bhanu P. Jena: 0000-0002-6030-8766 Conflict of Interest: The authors have nothing to declare.



Page 18 of 29

18

Figure 1. Stretching of the existing cell plasma membrane results in biogenesis of additional membrane. a, Isolated rat RBCs observed by scanning electron microscopy (Scale, 5 m) and b, transmission electron microscopy (Scale, 500 nm), demonstrating purity of the RBC preparation and the absence of intracellular membrane vesicles. c-e, Light micrographs demonstrating the generation of a membrane tether (yellow arrowheads) formed when an optically trapped 200 nm polystyrene bead adhered to the outer surface of a rat RBC membrane is pulled using the laser tweezers of the photonic force microscope to establish a nearly 6 m membrane tether (Scale, 4 m in c,d and 8 m in e). f, Isolated RBCs in isotonic PBS stained with the R18 Octadecyl Rhodamine B Chloride lipid dye, demonstrate plasma membrane staining. g,i, Exposure of RBC to hypotonic medium (70% PBS), followed by h,j, return to isotonic PBS solution, results in the formation of membrane protrusions, as observed using both light and scanning electron microscopy. k, Schematic drawing of the process of osmotic stretch-induced membrane biogenesis, and l,m, high resolution light l, and electron micrograph m showing protrusions at the RBC membrane.


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


19

Figure 2. Swelling-induced stretch of the rat RBC plasma membrane results in incorporation of intracellular proteins and lipids into the membrane. a,b, Control RBC membranes (C) and experimental (E) membranes obtained from RBC before (C) and following osmotic swelling (E), were solubilized and subjected to SDS-PAGE to resolve the proteins,



Page 20 of 29

20 and then staining of the resolved protein bands using the silver staining protocol a, and the Coomassie Blue staining b. Note the new bands (arrowheads) is observed in the silver-stained gel in the experimental membrane lane. The major band (arrowhead) observed in the Coomassie Blue-stained gel in the experimental and the corresponding band in the control lane were used for protein mass spectrometry d. The major lipid species in control (CONT) and experimental (EXP) RBC membranes and the protein profiles in the major digested band from the Coomassie Blue-stained gel are presented as heat maps in c and d respectively. c, Isolated Log10 of lipid signal intensity from tandem MS lipidomic analysis of stretched (EXP, right column) and unstretched (CONT, left column) RBC membranes are presented as a heat map, where negative values are shown in shades of brown, positive values in shades of blue, and undetected lipids in gray. The rows of the heat map show the log10 signal from individual lipid species; the hierarchical clustering dendrogram shown on the left sorts the lipids by similarity in their distribution in the CONT and EXP columns. Nearly 30% new lipids, and 15% new proteins are inserted into the parent membrane following osmosis-induced stretching (Tables I and II of Supporting Material).


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


21

Figure 3. Molecular dynamic simulations demonstrate membrane stretch-induced lipid insertion. a. Applied interfacial tension plotted as a function of the logarithm of the area per lipid. Predictions of molecular dynamics simulation (circles), and linear regression of the simulation data (solid line) is shown. The slope of the curve yields the area compressibility modulus. b. Increased membrane stretch results in the rapid insertion of lipids into the membrane. c. Snapshot from molecular dynamics simulations illustrating the initial configuration (far left), a typical intermediate state (middle), and after lipid incorporation into the bilayer (far right).



Page 22 of 29

22

Figure 4. Cholesterol depletion from Min6 cells alters the association-dissociation of both lipids and proteins in the cell plasma membrane. Immunofluorescent staining of Min6 cells using antibody for two membrane-associated protein SNAP25 and Gi3, demonstrate their altered distribution following the extraction of membrane cholesterol using 5mM M-CD b,e,f compared to control cells a,c,d scale bar = 10m. Control Min6 cells showing DAPI staining of the nucleus in blue (a), SNAP25 immunostaining in green, and Gi3 immunostaining in red. Note greater co-localization (white arrowheads show co-localization, while red arrowheads show separate green and red fluorescence) of SNAP25 and Gi3 at the cell plasma membrane in M-CD-treated Min6 cells in the inset within white square in g presented in f, compared to controls d, scale bar = 10m. Resolved membrane fractions from untreated (-MCD) and treated (+M-CD) Min6 cells, demonstrate the loss (red arrowheads) or gain (green


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


23 arrowheads) of proteins in coomassie g, and silver stained (h) SDS-PAGE. i, Western blot analysis of membrane proteins syntaxin, SNAP25, Gi3, and v-ATPase, further demonstrate changes in the presence of the proteins following extraction of cholesterol from the Min6 cell plasma membrane. Note while there is a loss of syntaxin and SNAP25, there is a gain in relative Gi3 and v-ATPase immunoreactivity in the plasma membrane of M-CD-treated Min6 cells.



Page 24 of 29

24 REFERENCES 1. Staykova, M., Holmes, D. P., Read, C., and Stone, H. A. Mechanics of surface area regulation in cells examined with confined lipid membranes. Proc, Natl. Acad. Sci. USA 2011, 108, 9084-9088.Coughlin, 2. Shigematsu, T., Koshiyama, K., Wada, S. Effects of Stretching Speed on Mechanical Rupture of Phospholipid/Cholesterol Bilayers: Molecular Dynamics Simulation. Sci. Reports 2015, 5, doi:10.1038/srep15369. 3. Atilla-Gokcumen, G. E., Muro, E., Relat-Goberna, J., Sasse, S., Bedigian, A., Coughlin, M.L., Garcia-Manyes, S., Eggert, U.S. Dividing Cells Regulate Their Lipid Composition and Localization. Cell 2014, 156, 428-439. 4. Gudejko, H. F. M., Alford, L. M., Burgess, D. R. Polar Expansion During Cytokinesis. Cytoskeleton (Hoboken, N.J.) 2012, 69, 1000-1009. 5. Fisher, J. L., Margulies, S. S. Modeling the effect of stretch and plasma membrane tension on Na+-K+-ATPase activity in alveolar epithelial cells. Am. J. Physiol. Lung cellular and molecular physiology 2007, 292, L40-53. 6. Zlotek-Zlotkiewicz, E., Monnier, S., Cappello, G., Le Berre, M., Piel, M. Optical volume and mass measurements show that mammalian cells swell during mitosis. J. Cell Biol. 2015, 211, 765-774. 7. Son, S., Kang, J.H., Oh, S., Kirschner, M.C., Mitchison, T.J., Manalis, S. Resonant microchannel volume and mass measurements show that suspended cells swell during mitosis. J. Cell Biol. 2015, 211, 757-763. 8. Heisenberg, C. P., Bellaiche, Y. Forces in tissue morphogenesis and patterning. Cell 2013, 153, 948-962. 9. Dan, K., Dan, J. C. Behavior of the Cell Surface during Cleavage: III. On the Formation of New Surface in the Eggs of Strongylocentrotus Pulcherrimus. Biol. Bull. 1940, 78, 486-501.


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


25 10. Lecuit, T., Wieschaus, E. Polarized insertion of new membrane from a cytoplasmic reservoir during cleavage of the Drosophila embryo. J. Cell Biol. 2000, 150, 849-860. 11. Evans, E. A. Bending elastic modulus of red blood cell membrane derived from buckling instability in micropipet aspiration tests. Biophys. J. 1983, 43, 27-30. 12. Waugh, R., Evans, E. A. Thermoelasticity of red blood cell membrane. Biophys. J. 1979, 26, 115-131. 13. Jena, B. P., Schneider, S. W., Geibel, J. P., Webster, P., Oberleithner, H., Sritharan, K. C. Gi regulation of secretory vesicle swelling examined by atomic force microscopy. Proc. Natl. Acad. Sci. USA. 1997, 94, 13317–13322. 14. Cho, S.–J., Sattar, A. K., Jeong, E. H., Satchi, M., Cho, J., Dash, S., Mayes, M. S., Stromer, M. H., Jena, B. P. Aquaporin 1 regulates GTP-induced rapid gating of water in secretory vesicles. Proc. Natl. Acad. Sci. USA 2002, 99, 4720-4724. 15. Hou, X., Lewis, K.T., Wu, Q., Wang, S., Chen, X., Flack, A., Mao, G., Taatjes, D.J., Sun, F., Jena, B.P. Proteome of the porosome complex in human airways epithelia: Interaction with the cystic fibrosis transmembrane conductance regulator (CFTR) J. Proteomics 2014, 96, 82-91. 16. Kovari, L.C., Brunzelle, J.S., Lewis, K.T., Cho, W.J., Lee, J-S., Taatjes, D.J., Jena, B.P. (2013) X-ray solution structure of the native neuronal porosome-synaptic vesicle complex. X-ray solution structure of the native neuronal poropsome-synaptic vesicle complex: Implications in neurotransmitter release. Micron 2014, 56, 37-43. 17. Pralle, A., Prummer, M., Florin, E. L., Stelzer, E. H., Horber, J. K. Three-dimensional high-resolution particle tracking for optical tweezers by forward scattered light. Microscopy Res. Tech. 1999, 44, 378-386.



Page 26 of 29

26 18. Matyash, V., Liebisch, G., Kurzchalia, T.V., Shevchenko, A., Schwudke, D. Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J. Lipid Res. 2008, 49, 1137-1146. 19. Schwudke, D., Oegema, J., Burton, L., Entchev, E., Hennich, J.T., Ejsing, C.S., Kurzchalia, T., Shevchanko, A. Lipid profiling by multiple precursor and neutral loss scanning driven by the data-dependent acquisition. Anal. Chem. 2006, 78, 585-595. 20. Ejsing, C. S., Duchoslav, E., Sampaio, J., Simons, K., Bonner, R., Thiele, C., Ekroos, K., Shevchanko, A. Automated Identification and Quantification of Glycerophospholipid Molecular Species by Multiple Precursor Ion Scanning Anal. Chem. 2006, 78, 6202-6214. 21. Marrink, S. J., Risselada, H.J., Yefimov, S., Tieleman, D.P., de Vries, A.H. The MARTINI Force Field: Coarse Grained Model for Biomolecular Simulations. J. Phys. Chem. B 2007, 111, 7812-7824. 22. Jo, S., Lim, J. B., Klauda, J. B., Im, W. CHARMM-GUI Membrane Builder for Mixed Bilayers and Its Application to Yeast Membranes. Biophys. J. 2009, 97, 50-58. 23. Klauda, J. B., Venable, R.M., Freites, J.A., O’Connor, J.W., Tobias, D.J., Mondragon-Ramirez, C., Vorobyov, I., MacKerell, A.D., Pastor, R.W. Update of the CHARMM all-atom additive force field for lipids: Validation on six lipid types. J. Phys. Chem. B 114:7830-7843. 24. Humphrey, W., Dalke, A., Schulten, K. 1996. VMD: Visulal molecular dynamics. J. Mol. Graphics Modell. 2010, 14, 33-38. 25. Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Kale, L., Schulten, K. Scalable molecular dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781-1802.


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


27 26. Martyna, G. J., Tobias, D. J., Klein, M. L. Constant pressure molecular dynamics algorithms. J. Chem. Phys. 1994, 101, 4177-4189. 27. Feller, S. E., Zhang, Y. H., Pastor, R. W., Brooks, B. R. Constant pressure molecular dynamics simulation: The Langevin piston method. J. Chem. Phys. 1995, 103, 46134621. 28. Chen, X., Ulintz, P.J., Simon, E.S., Williams, J.A., Andrews, P.C. Global Topology Analysis of Pancreatic Zymogen Granule Membrane Proteins. Mol. Cell Proteom. 2006, 5, 306-312. 29. Winkler, C., Denker, K., Wortelkamp, S., Sickmann, A. Silver- and Coomassiestaining protocols: detection limits and compatibility with ESI MS. Electrophoresis 2007, 28, 2095-2099. 30. Hochmuth, R. M., Mohandas, N., and Blackshear, P. L., Jr. Measurement of the elastic modulus for red cell membrane using a fluid mechanical technique. Biophys. J. 1973, 13, 747-762. 31. Dai, J., and Sheetz, M. P. Mechanical properties of neuronal growth cone membranes studied by tether formation with laser optical tweezers. Biophys. J. 1995, 68, 988-996. 32. Borghi, N., Brochard-Wyart, F. Tether extrusion from red blood cells: integral proteins unbinding from cytoskeleton. Biophys J. 2007, 93, 1369-1379. 33. Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., Chu, S. Observation of a single-beam gradient force optical trap for dielectric particles. Optics Lett. 1986, 11, 288-290. 34. Zeidel, M. L., Ambudkar, S. V., Smith, B. L., Agre, P. Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein. Biochemistry 1992, 31, 7436-7440.



Page 28 of 29

28 35. Preston, G. M., Jung, J. S., Guggino, W. B., Agre, P. The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel. J. Biol. Chem. 1993, 268, 17-20. 36. Kozono, D., Yasui, M., King, L. S., Agre, P. Aquaporin water channels: atomic structure molecular dynamics meet clinical medicine. J. Clinic. Invest. 2002, 109, 1395-1399. 37. Lewis, K. T., Maddipati, K. R., Naik, A. R., Jena, B. P. Unique Lipid Chemistry of Synaptic Vesicle and Synaptosome Membrane Revealed Using Mass Spectrometry. ACS Chem. Neurosci. 2017, 8, 1163-1169. 38. Naik, A. R., Kulkarni, S. P., Lewis, K. T., Taatjes, D. J., Jena, B. P. Functional Reconstitution of the Insulin-Secreting Porosome Complex in Live Cells. Endocrinology 2016, 157, 54-60. 39. Bogan, J. S., Xu, Y., Hao, M. Cholesterol accumulation increases insulin granule size and impairs membrane trafficking. Traffic (Copenhagen, Denmark) 2012, 13, 14661480. 40. Wang, S., Lee, J.-S., Bishop, N., Jeremic, A., Cho, W.J., Chen, X., Mao, G.Z., Taatjes, D.J., Jena, B.P. 3D organization and function of the cell: Golgi budding and vesicle biogenesis to docking at the porosome complex. Histochem. Cell Biol. 2012, 137, 703-718.


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


29 “TOC Graphic”


Self-Assembly and Biogenesis of the Cellular Membrane are Dictated

Recommend Documents