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Plasma Membrane Proteins and Cytoskeletal Attachment

Plasma Membrane Proteins and Cytoskeletal Attachment



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Regarding membrane protein functions, which of the following statements is CORRECT?

a. Membrane proteins are responsible for both cell to cell recognition and cell anchoring and are stabilised by linking through to the microtubulele cytoskeletal fibres.

b. A protein that is enzymatically active and membrane-bound will function significantly differently to a protein that is enzymatically active and cytosolic.

c. Membrane proteins are manufactured in the internal cavity (lumen) of the rough ER.

d. Damage to the smooth ER could alter the makeup of the molecules that are displayed at the cell surface.

Regarding membrane proteins, what is incorrect in the statement “Membrane proteins are responsible for both cell to cell recognition and cell anchoring, and are stabilized by linking through to microtubules cytoskeletal fibres"?

I believe this to be wrong due to it normally being Actin Filaments and Intermediate Filaments that attach to Integrin proteins in the cell surface membrane.


a. Membrane proteins are responsible for both cell to cell recognition and cell anchoring and are stabilised by linking through to the microtubulele cytoskeletal fibres.

Not entirely correct as you yourself pointed out that integrins attach to actin filaments.

b. A protein that is enzymatically active and membrane-bound will function significantly differently to a protein that is enzymatically active and cytosolic.

Possible but not necessary.

c. Membrane proteins are manufactured in the internal cavity (lumen) of the rough ER.

They are secreted in the ER and modified there too but not manufactured there.

d. Damage to the smooth ER could alter the makeup of the molecules that are displayed at the cell surface.

Seems correct because the statement itself does not claim certainty. It says that the makeup of cell surface molecules (which could be lipids or proteins) could change in response to SER damage.


Elucidating membrane structure and protein behavior using giant plasma membrane vesicles

The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan) (ii) two-photon microscopy of the same dye and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.


Proteins in Plasma Membranes

Proteins make up the second major component of plasma membranes. Integral proteins are, as their name suggests, integrated completely into the membrane structure. In fact, their hydrophobic membrane-spanning regions interact with the hydrophobic region of the the phospholipid bilayer. We refer to some specialized types of integral proteins as integrins. See image below.

The fluid mosaic model of the plasma membrane describes the plasma membrane as a fluid combination of phospholipids, cholesterol, and proteins. Carbohydrates attached to lipids (glycolipids) and to proteins (glycoproteins) extend from the outward-facing surface of the membrane. Image Attribution: OpenStax Biology

Single-pass integral membrane proteins usually have a hydrophobic transmembrane segment that consists of 20–25 amino acids. Some span only part of the membrane—associating with a single layer. On the other hand, others stretch from one side of the membrane to the other, and are exposed on either side.

Some complex proteins consist of up to 12 segments of a single protein. These proteins fold extensively and embed themselves in the membrane (see image below). This type of protein has a hydrophilic region or regions, and one or several mildly hydrophobic regions. This arrangement of regions of the protein tends to orient the protein alongside the phospholipids, with the hydrophobic region of the protein adjacent to the tails of the phospholipids and the hydrophilic region or regions of the protein protruding from the membrane and in contact with the cytosol or extracellular fluid.

Integral membranes proteins may have one or more alpha-helices that span the membrane (examples 1 and 2). Conversely, they may have beta-sheets that span the membrane (example 3). Image Attribution: “Foobar”/Wikimedia Commons

Peripheral Proteins

We can find peripheral proteins on the exterior and interior surfaces of membranes. In fact, they attach either to integral proteins or to phospholipids. Along with integral proteins, peripheral proteins may serve as enzymes. They may also serve as structural attachments for the fibers of the cytoskeleton. Furthermore, they may serve as part of the cell’s recognition sites. We sometimes refer to this as “cell-specific” proteins. The body recognizes its own proteins and attacks foreign proteins associated with invasive pathogens.


Cell Membrane Plasma Membrane Membrane Proteins Cell Biology Functions of Plasma

Cells, whether it is animal, plant or bacteria, have many different parts or organelles. Each organelle has a different and important function within the cell. If any of the parts fail then the cell ceases to function and it begins to die.

One of the most important parts of a cell is called the plasma membrane (or cell membrane). The cell membrane has many important functions including, cell to cell recognition, regulation of what goes into and out of the cell, and cell to cell communication. The unique chemistry of the plasma membrane allows it to perform these functions.

The plasma membrane is composed of a phospholipid bilayer that has proteins and carbohydrates embedded in it. A typical lipid or triglyceride is composed of three fatty acids chemically bonded to a glycerol molecule. Regular lipids are insoluble in water. An example of this insolubility can be seen in salad dressing such as Italian salad dressing in which the layers separate when allowed to settle. One layer is the water layer and the other the oil or lipid layer. The lipids found in the lipid bilayer of the plasma membrane have exchanged one of the fatty acid molecules for a phosphate molecule. This changes the chemistry of the lipid molecule giving it a polar or charged “head” and a “nonpolar” or uncharged “tail”.

The lipid bilayer is composed of two layers of these phospholipids with the heads pointing “out.” This means that one polar head is in contact with the exterior of the cell and one polar head is in contact with the interior of the cell. And both the exterior and interior environments are water based environments. The tails or the unpolar portions of the phospholipids are sequestered together away from the water environment. If you place phosopholipids into a water based environment, they will spontaneously form the bilayer described above.

Embedded in the plasma membrane are proteins which can have various functions including: being marker molecules, attachment proteins, transport proteins, receptor proteins or enzymes. This protein embedded lipid model is called the fluid mosaic model because the proteins are not stationary within the lipid membrane.

Marker molecule proteins help the cells help the cells recognize each other. For example, marker molecules allow the immune system to tell the difference between self-cells and invading cells or bodies. Attachment proteins can attach the cell to other cells or to other extracellular molecules. Transport proteins allow molecules that are too big or that have a charge to enter into the membrane since only small and uncharged molecules can get through the membrane without the aid of transport proteins. This is because of the hydrophilic and hydrophobic nature of the membrane. Receptor proteins function in cell to cell communication. They accept or recognize the incoming chemical signal from other cells. And the enzymes in the membrane function as any other enzymes in that they lower the activation energy for chemical reactions.

There can also be carbohydrate molecules attached to the protein or lipid molecules of the plasma membrane. These carbohydrates help in the cell recognition and cell to cell signaling functions. There are also cholesterol molecules in the membrane which determine the degree of fluidity of the membrane.

It is the above mentioned proteins, lipids and carbohydrates that give the membrane its chemical properties and therefore determine the function of the plasma membrane which is the regulation of what goes into and out of the cell.


What Do Membranes Do?

Cell membranes serve as barriers and gatekeepers. They are semi-permeable, which means that some molecules can diffuse across the lipid bilayer but others cannot. Small hydrophobic molecules and gases like oxygen and carbon dioxide cross membranes rapidly. Small polar molecules, such as water and ethanol, can also pass through membranes, but they do so more slowly. On the other hand, cell membranes restrict diffusion of highly charged molecules, such as ions, and large molecules, such as sugars and amino acids. The passage of these molecules relies on specific transport proteins embedded in the membrane.

Figure 3: Selective transportSpecialized proteins in the cell membrane regulate the concentration of specific molecules inside the cell.© 2010 Nature Education All rights reserved.

Membrane transport proteins are specific and selective for the molecules they move, and they often use energy to catalyze passage. Also, these proteins transport some nutrients against the concentration gradient, which requires additional energy. The ability to maintain concentration gradients and sometimes move materials against them is vital to cell health and maintenance. Thanks to membrane barriers and transport proteins, the cell can accumulate nutrients in higher concentrations than exist in the environment and, conversely, dispose of waste products (Figure 3).

Other transmembrane proteins have communication-related jobs. These proteins bind signals, such as hormones or immune mediators, to their extracellular portions. Binding causes a conformational change in the protein that transmits a signal to intracellular messenger molecules. Like transport proteins, receptor proteins are specific and selective for the molecules they bind (Figure 4).

Figure 4: Examples of the action of transmembrane proteinsTransporters carry a molecule (such as glucose) from one side of the plasma membrane to the other. Receptors can bind an extracellular molecule (triangle), and this activates an intracellular process. Enzymes in the membrane can do the same thing they do in the cytoplasm of a cell: transform a molecule into another form. Anchor proteins can physically link intracellular structures with extracellular structures.© 2010 Nature Education All rights reserved.

Peripheral membrane proteins are associated with the membrane but are not inserted into the bilayer. Rather, they are usually bound to other proteins in the membrane. Some peripheral proteins form a filamentous network just under the membrane that provides attachment sites for transmembrane proteins. Other peripheral proteins are secreted by the cell and form an extracellular matrix that functions in cell recognition.


CONCLUSION AND PERSPECTIVES

Recent studies have revealed that cell wall production relies on a close interaction between the plasma membrane and the underlying cytoskeleton in plants. The cell wall, plasma membrane and cytoskeleton nexus could be mediated by a single bridging protein or through several proteins that form a scaffolding structure or that may participate in signaling pathways. However, the roles of the already identified candidate proteins are not clarified and further studies in this field are needed. It may be anticipated that high-end proteomics will aid in the identification of additional proteins that link the cytoskeleton and the plasma membrane and that in vitro and in vivo work using high-end cell biology techniques will reveal their functions. These data should significantly advance our understanding for how the cytoskeleton is tethered to the plasma membrane and will likely reveal insights into differences in cytoskeletal arrangements compared to yeast and animal cells.


The behaviour of the plasma membrane during plasmolysis: a study by UV microscopy

A high resolution ultraviolet (UV) bright-field microscope was used to analyse the formation of Hechtian strands and the Hechtian reticulation that remain attached to the cell wall after plasmolysis and deplasmolysis of onion inner epidermal cells. In real time video images, UV microscopy allowed a detailed investigation of the dynamic behaviour of the plasma membrane during the processes of osmotic water loss and uptake. Furthermore, the role of cytoskeletal elements as possible linkers of the plasma membrane to the cell wall was probed by application of cytoskeletal drugs during plasmolysis. Microtubules were depolymerized in oryzalin, and latrunculin B was used to destabilize actin microfilaments. The results showed no visible changes in the formation of the Hechtian reticulation or strands. Plasmolysis forms appeared to be normal, indicating stong membrane-to-wall attachments independent of cytoskeletal elements. During re-expansion of the protoplast in deplasmolysis, the plasma membrane incorporated Hechtian strands and subprotoplasts, fused with the Hechtian reticulation and finally realigned at the cell wall.


Plasma Membrane Proteins and Cytoskeletal Attachment - Biology

Cytoskeletal proteins are proteins that make up the cytoskeleton, flagella or cilia of cells. Generally, cytoskeletal proteins are polymers, and include tubulin (the protein component of microtubules), actin (the component of microfilaments) and lamin (the component of intermediate filaments. The cytoskeleton is a dynamic 3-dimensional structure that fills the cytoplasm, and is present in both eukaryotic and prokaryotic cells. The cytoskeleton acts as both muscle and skeleton, and plays a role in cell protection, cell motility (migration), cytokinesis, intracellular transport, cell division and the organization of the organelles within the cell.

Cytoskeletal Connections

The elements of the cytoskeleton are not independent. Different filaments are connected to one another, and to the surface of the cell. Motor proteins, which not only connect to the cytoskeleton but drive up and down filaments, are a special case. Crawling cells need to attach to surfaces, tissues in multicellular organisms must be attached to one another, and it is obviously important to connect the cytoskeleton to sites of attachment. A number of proteins are responsible for connecting the actin cytoskeleton to the plasma membrane. The best characterized system is in red blood cells (erythrocytes), which need to maintain a large surface area for maximal gas exchange but remain flexible enough to travel through capillaries. The characteristic biconcave shape is generated using tension from a network of F-actin, attached to the membrane through large complexes of several proteins. Two proteins, band III and glycophorin, are integral membrane-spanning proteins, and join the complexes to the membrane itself. Actin is bound by a large protein called spectrin, which is in turn connected to band III through ankyrin and to glycophorin. The net result is a compact crosslinked array of protein connecting multiple actin filaments to the whole area of the membrane.

Figure 1. The cytoskeleton. Actin filaments are shown in red, microtubules in green, and the nuclei are in blue.

Cytoskeletal Proteins and disease

Since the cytoskeleton is central to the lives of cells, it is hardly surprising to find that it is highly relevant to modern medicine. Several pathogens (for example, the bacterium Shigella and Vaccinia virus) make use of the host cytoskeleton to gain entry into and to infect neighbouring cells. Other organisms (e.g. the death cap toadstool Amanita phalloides and the meadow saffron Colchicum) make toxins that interfere with particular aspects of cytoskeletal function. Conversely, an increasing number of medicines work by attacking the cytoskeleton of disease-causing cells, for example paclitaxel for cancer and fluconazole for yeast infections.

1. Fuchs E. The cytoskeleton and disease: genetic disorders of intermediate filaments. Annual Review of Genetics, 1996, 30: 197&ndash231.

2. Desai A. Microtubule polymerization dynamics. Annual Review of Cell and Development Biology, 1998,13: 83&ndash117.


Proteins involved in the attachment of actin to the plasma membrane

Proteins that may be involved in two types of actin-membrane association are discussed. The first set includes α-actinin, vinculin, fimbrin and a new cytoskeletal protein that are all concentrated in adhesion plaques, those regions of cultured fibroblasts where bundles of actin microfilaments terminate and where the plasma membrane comes close to the underlying substrate. The properties of non-muscle α-actinin suggest that it functions to cross-link actin filaments and thereby stabilize microfilament bundles rather than functioning in their attachment to the membrane. Fimbrin also appears to be involved in bundling of filaments rather than in attachment. In contrast, vinculin binds to the ends of actin filaments in vitro and is probably the best candidate for a role in the attachment of actin to membranes at the adhesion plaque. The discovery of a new protein, 215k, of unknown function, in the adhesion plaque suggests that many more proteins remain to be identified in this region. Attachment of actin filaments to other regions of the plasma membrane is also considered and a protein is described that seems to be a spectrin homologue in brain and other tissues. The brain protein resembles erythrocyte spectrin in its physical properties, in binding actin, in being associated with cell membranes and in crossreacting immunologically. We suggest that the brain protein and erythrocyte spectrin both belong to a family of related proteins (the spectrins) which function in the attachment of actin to membranes in many different cell types.


MATERIALS AND METHODS

Antibodies

Anti-TTLL4 antibody was raised in guinea pigs immunized with the glutathione S-transferase (GST)–fused C-terminal domain of TTLL4 (TTLL4 C′50AA). The polyclonal anti-Ttll4 antibody was purified using a column filled with the GST-tagged TTLL4 C-terminal domain (TTLL4 C′50AA). The fractionated anti-TTLL4 antibody was further precleared against TTLL4 KO tissue lysates. The mAb 4A8 for NAP1 was a kind gift from Yukio Ishimi (Ibaraki University, Mito, Japan). Other antibodies used in this study were as follows: polyglutamylation (mouse mAb GT335 AG-20B-0020-C100 AdipoGen, San Diego, CA) NAP1LI (rabbit polyclonal ab33076 Abcam, Cambridge, United Kingdom) glycophorin A (mouse mAb ab129024 Abcam) carbonic anhydrase1 (rabbit polyclonal ab86280 Abcam) tropomyosin (mouse monoclonal ab7785 Abcam) tropomodulin1 (mouse monoclonal ab119025 Abcam) α-adducin (rabbit polyclonal A303-713A Bethyl Laboratories, Montgomery, TX) β-adducin (rabbit polyclonal A303-741A Bethyl) Ter119 (Alexa Fluor 488 anti-mouse TER-119/erythroid cells antibody 116215 BioLegend, San Diego, CA) actin (rabbit polyclonal A2066 Sigma-Aldrich, St. Louis, MO) spectrin β-chain (rabbit polyclonal ABT185 Millipore, Billerica, MA), Alexa fluorophore–conjugated secondary antibodies for immunofluorescence (Invitrogen, Carlsbad, CA), and horseradish peroxidase–conjugated secondary antibodies for Western blot analysis (Jackson Immuno Research Laboratories, West Grove, PA).

All experiments and treatments in mice were approved by the Institutional Animal Care and Use Committee at the Hamamatsu University School of Medicine. Ttll4 allele-trapped mice were purchased from Trans Genic (Kobe, Japan) and mated with wild-type C57BL/6J mice for at least 10 generations. Ttll4/−, Ttll4+/−, and wild-type littermates were obtained from heterozygous mating. Mice 8–12 mo of age, regardless of the sex unless otherwise specified, were used for each experiment. The bone marrow and spleen were dissected from adult C57BL/6J wild-type and Ttll4−/− mice. The organs were homogenized in lysis buffer (50 mM Tris-Cl, pH 7.5, 1% Triton X-100). For the hemolytic anemia model, adult C57BL/6J and Ttll4−/− mice were injected with 20 mg/kg PHZ (Sigma-Aldrich 114715) every 24 h for two consecutive days. Mice were killed at 48 h after injection, and hematological parameters were analyzed.

Genotyping of progenies by PCR

Genomic DNA was prepared from 4- to 5-mm tail samples and then used for genotyping by PCR. Oligonucleotides used were 5′-TTCTGTAGCTGGGCTTATT-3′ as the forward primer for the wild-type allele, 5′-AATCCCATGGTCCCACAAA-3′ as the forward primer for the trap vector, and 5′-CGGTGAAACCTCGACACA-3′ as the reverse primer for both the wild-type and trapped alleles.

Reverse transcription PCR

Total RNA was extracted from the testis and muscle tissues. Next 1 µg of RNA per sample was reverse-transcribed into cDNA with RTace (Toyobo, Osaka, Japan) and oligoT15 primer. The resulting cDNA was amplified with primers specific for Ttll4: forward, 5′-TATC TCGGAACTGTGTGGATTTGA-3′, and reverse, 5′-GAATGACCTGAATGCCAATGC-3′ and AmpliTaq Gold.

Blood collection and hematology analyses

Whole blood (200 μl) of adult Ttll4-KO and wild-type C57BL/6J was collected either via cardiac puncture or from the tail vein into Eppendorf tubes containing 2 μl of 10% EDTA or heparinized capillary tubes. RBC counts and indices were measured by an automated hematology analyzer (Celltac α Nihon Kohden, Tokyo, Japan) calibrated for mouse blood. For Giemsa staining, whole-blood smears were air-dried, methanol-fixed, and stained with Giemsa. Peripheral blood counts were also determined by diluting RBCs in RBC diluting fluid (3% sodium citrate, 1% Formalin) and then by manual counting using a hemocytometer. Reticulocytes were counted manually after staining with new methylene blue (Sigma-Aldrich). Images were acquired with an Olympus BX51 microscope equipped with an Olympus DP-72 camera (Tokyo, Japan).

Osmotic fragility and deformability analysis

Osmotic fragility was measured by the method of Gilligan et al. (1999). Whole blood was washed three times in isotonic buffer, and final hematocrit was adjusted to 5%. Diluted RBCs in a volume of 10 µl were added to 290 µl of lysis buffer of appropriate salt concentration and incubated for 20 min at room temperature. The lysed RBC suspension was then centrifuged, and the absorbance of the supernatant was measured at 540 nm. For measuring deformability, 30 µl of whole blood was mixed with 4 ml of 3.5% polyvinylpyrrolidone solution, and the deformability index was recorded by increasing applied sheer stress from 0 to 50 dynes/cm 2 using a custom-built ektacytometer.

RBC purification and preparation

We used three different methods to purify RBCs. RBCs were pelleted at 600 × g for 10 min at 4°C. After removal of the plasma and top cell layer, including the buffy coat, the cells were washed six times in RBC wash buffer (5 mM ethylene glycol tetraacetic acid [EGTA], 0.25% NaCl, 5.0 mM Na2HPO4, pH 7.5). Second, for purification by FACS, whole blood was washed once with PBS, and then 10 µl of blood was resuspended in FACS buffer (PBS, 0.5% bovine serum albumin [BSA], 1 mM EGTA, pH 7.5). The cell suspension was stained with TER-119 Alexa 488–conjugated antibody and then washed with PBS. Stained RBCs were sorted on a BD FACSAria (BD Biosciences, Franklin Lakes, NJ) and gated on the basis of their side scatter signals, forward scatter signals, and positive staining with TER-119 Alexa-488. The data were analyzed using FlowJo-V10 software. Third, a discontinuous gradient of 75% and 76.9% Percoll (Sigma-Aldrich) was used to separate RBCs.

Preparation of RBC membranes

To fractionate the soluble and membrane components of RBCs, washed RBCs were lysed with hypotonic buffer (20 mM Tris-Cl, 1 mM EGTA, pH 7.5), and the supernatant was separated by centrifugation. Next the cell pellet was washed repeatedly to remove all cytoplasmic content, and the final pellet consisting of RBC membrane ghosts was lysed directly in SDS–PAGE sample buffer, NP-40 lysis buffer, or RIPA buffer. The Mg 2+ membrane ghosts and Triton-insoluble skeletons were prepared as described previously (Moyer et al., 2010). Protein concentrations were measured using the BCA assay (Pierce, Rockford, IL). To determine the binding affinity of glutamylated and nonglutamylated NAP1 with the RBC membrane, Mg 2+ membrane ghosts were treated with increasing concentrations (110, 210, and 310 mM) of KCl in a medium of lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 0.2% Triton X-100, 10% glycerol, pH 7.5) for 1.0 h and then centrifuged at 20,000 × g for 30 min at 4°C. After centrifugation, the supernatant was collected, and the remaining pellet was directly lysed in SDS–PAGE sample buffer.

Immunoprecipitation assay

To immunoprecipitate glutamylated proteins, mAb GT335 (Adipogen) was linked to protein G Sepharose beads (GE Healthcare, Little Chalfont, United Kingdom). For immunoprecipitation of NAP1LI, the anti-NAP1L1 antibody (Abcam) was linked to protein G Sepharose beads. The whole RBC lysate was added to the beads and incubated overnight at 4°C. Bound proteins were directly eluted in SDS–PAGE sample buffer.

Immunoblotting and immunofluorescence

SDS–PAGE was performed as described by Laemmli (1970). Fairbanks running buffer (Fairbanks et al., 1971) was used to separate ankyrin from the spectrins. Gels were either stained with Coomassie blue or transferred to polyvinylidene fluoride membranes (Millipore) for immunoblotting. The membranes were probed with various primary antibodies, followed by horseradish peroxidase–conjugated secondary antibodies. Blots were developed with enhanced chemiluminescence reagents (ECL GE Healthcare) and exposed in a Fuji Film Intelligent Dark Box (Tokyo, Japan), where the images were captured using Image Reader LAS-3000 software. For immunohistochemistry, RBC samples were washed three times with PBS (330 mOsm) containing 5 mM glucose, fixed for 20 min in 4% paraformaldehyde and 0.02% glutaraldehyde in PBS at room temperature, and rinsed three times in rinsing buffer (PBS containing 0.1 M glycine). Fixed cells were then permeabilized with PBS containing 0.1 M glycine and 0.1% Triton X-100 for 5 min at room temperature and again rinsed three times in PBS containing 0.1 M glycine. To ensure complete neutralization of the unreacted aldehydes, RBCs were then incubated in rinsing buffer at room temperature for 1.5 h. Nonspecific binding was blocked by incubation in blocking buffer (PBS containing 0.05 mM glycine, 0.2% BSA, and 5% goat serum) overnight at 4°C. After immunostaining, RBCs were allowed to attach to glass coverslips for 20 min in a humidified chamber and mounted on glass slides using Vectashield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA) and observed by confocal laser scanning microscopy (FluoView FV1000 Olympus).

Ultrastructure studies

For SEM, whole blood was fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and postfixed in 1% OsO4. Next the fixed cells were dehydrated, freeze-dried, and plasma-coated. The cells were photographed using a Hitachi S-4800 scanning electron microscope. For TEM of the RBC membrane skeleton, copper grids of 400 mesh (Nisshin-EM, Tokyo, Japan) were coated with 0.5% (wt/vol) Formvar in ethylene dichloride (Davison and Colquhoun, 1985) and then were carbon-coated and subjected to glow discharge (HDT-400 Hydrophilic Treatment Device JEOL Datum, Tokyo, Japan). Collected blood cells were washed with PBS containing Mg and EGTA (Pi/NaCl/Mg 150 mM NaCl, 5 mM NaH2PO4, 2 mM NaN3, 2 mM MgCl2, 1 mM EGTA, pH 5.5). Washed RBCs were then coated onto the grids as described by Byers and Branton (1985). Of note, the protocol by Byers and Branton (1985) is for spreading the RBC skeleton on the grid however, our results (Figure 3A) indicate that the RBC membranes were not spread and remained at native mouse RBC size. TEM images of RBC membranes were obtained using a JEOL JEM-1220 transmission electron microscope with an accelerating voltage of 80 kV and equipped with a Gatan-Bioscan camera, Model 792 (Pleasanton, CA).