What is the average size of a cell membrane

The extent of protrusion of proteins from the cell membrane is evident. The fraction of membrane surface occupied by proteins in this cross section depiction is similar to that actually found in cells. Courtesy of David Goodsell. Unsurprisingly, membrane proteins are roughly as thick as the membranes they occupy. Often these proteins also have regions which extend into the space on either side of the membrane.

This is evident in Figure 3 where some of the membrane associated proteins are shown to scale in cross section. Due to these extra constituents that also include lipopolysaccharides, the overall membrane width is variably reported to be anywhere between 4 and 10 nm.

The value of 4 nm is most representative of the membrane shaved off from its outer and inner protrusions. This value is quite constant across different organellar membranes as shown recently for rat hepatocyte via x-ray scattering where the ER, Golgi, basolateral and apical plasma membranes, were 3.

We conclude by noting that the cell membrane area is about half protein BNID and the biology and physics of the dynamics taking place there is still intensively studied and possibly holds the key to the action of many future drugs. What is the thickness of the cell membrane? Reader Mode Figure 1: An electron micrograph of an E. Purchase Draft Download About us. How big are the molecular machines of the central dogma?

The recently developed super resolution fluorescence microscopy, stochastic optical reconstruction microscopy STORM , has broken the diffraction barrier of light. It can resolve the fine structures and dynamic processes that cannot be achieved with conventional fluorescence microscopy [28].

The super resolution images of microtubules, mitochondria and clathrin-coated pits have been acquired, demonstrating that STORM is a powerful tool for cell imaging [30]. A The scheme of this work. Cells were cultured on cover slips A1 , A2 and A3. C The image of the ectoplasmic side of the cell membrane MDCK cells prepared by shearing open the cells on a cover slip. Scale bars: nm in B—F. We first studied the ectoplasmic surface of MDCK cells.

In order to verify the feature of the native cell membrane, we directly imaged the ectoplasmic surface of living cells. The roughness of membrane surface was only 1. Cultured cells were soft and elevated by several micrometers, making it difficult to achieve high resolution; therefore, we took advantage of two other strategies, specifically shearing open and centrifuging, to obtain quasi-native cell membranes.

Figure 1C showed the high resolution image of the cell membrane's ectoplasmic side prepared by shearing open the cells. The ectoplasmic surface was found to be rather smooth with a roughness root mean square RMS of 0. No indents or particles were visible. As shown in Figure 1D , the ectoplasmic side of the cell membrane obtained by the hypotonic-centrifugation procedure presented a roughness RMS of 1.

To know whether this phenomenon was common for other types of nucleated mammalian cells, we imaged the ectoplasmic side of two other human cancer cell lines derived from different organs, A cells from lung and HeLa cells from cervix, by the shearing open approach.

To test our technique, we previously decorated antibodies on the cell membranes, and the result showed that the resolution of AFM was high enough to distinguish protruding proteins from the surface of cell membranes [32]. Meanwhile, it should be noted that we imaged the native or quasi-native cell membranes unfixed , which allowed us to detect the original state of cell membranes. We have found that the fixation of the cell membrane e.

Taken together, these results demonstrate that the ectoplasmic side of native nucleated mammalian cell membranes is smooth without any obvious protrusion. It is well established that proteins, such as receptors and glycosyl phosphatidyl inositol-anchored proteins GPI-APs , are present on the ectoplasmic surface of the cell membrane. To verify the location and organization of these membrane proteins, we treated the ectoplasmic side of the cell membrane with proteinase K, which can digest most proteins above the lipid bilayers, and monitored the real-time changes with time-lapse AFM.

The surface of undigested ectoplasmic side of the cell membrane was consistently smooth without any pits or protrusions, as indicated in the magnified image and the corresponding section analysis Figure 2A, 2D and 2E.

After digestion with proteinase K, most proteins were removed, except some undigested or half-digested proteins, as indicated by the blue arrows in Figure 2B and 2C , at the ectoplasmic side of the cell membrane. The heights between the undigested proteins and the local pits, as indicated by the green arrows in Figure 2B , ranged from 1. The width of the pits varied from 30 nm to 80 nm, with the majority around A The AFM topographic image of the ectoplasmic side of the cell membrane.

J and K The depth and width distributions of the pits after proteinase K treatment, respectively. N The AFM topographic image of the ectoplasmic side of the cell membrane without treatment. The indent widths exhibit variability between 40 nm and nm, with the majority of indentations having an average width of around These results demonstrate that the ectoplasmic side of the cell membrane comprises a layer of dense proteins, e. To further verify the relationship between the dense protein layer and the lipid bilayer, the ectoplasmic side of the cell membrane was digested with a more specific enzyme, collagenase 3, which can specifically digest certain membrane receptors at the extracellular side [35].

Figure 2N shows the untreated ectoplasmic side of the cell membrane, and the magnified image from Figure 2N is shown in Figure 2T. After treatment with collagenase 3, a few round pits appeared in the membrane, as indicated by the green arrows in Figure 2O , which can be distinguished more clearly from the corresponding amplitude image Figure 2Q, 2R and the section analysis Figure 2U, 2W.

The depth of pits was about 4 nm, which was consistent with the height of the extracellular segment of transmembrane proteins, e. The widths of the pits were also extended by about 20 nm Figure 2V, 2X. These results further reveal that the proteins at the ectoplasmic side of the cell membrane form a dense protein layer with a thickness of about 4 nm and that it sits on top of the lipid bilayer.

Most of the membrane proteins, such as receptors, on the ectoplasmic surface of cells are glycosylated. These glycoproteins play important structural and functional roles in cellular activities, such as cell-cell recognition and adhesion [37]. In order to precisely localize the carbohydrates, we utilized a super resolution microscopy known as STORM. In each imaging cycle, only a fraction of fluorophores was activated Figure 3A 1 , making it possible to precisely localize their positions Figure 3A2.

Other fluorophores can be localized by repeating these cycles Figure 3A3 , and the overall images can be reconstructed according to the positions of these fluorophores Figure 3A4 [38].

C The size distribution of the mannose clusters. Mannose is one of the common carbohydrates on the membrane surface. As depicted in Figure 3B , mannose clusters on the ectoplasmic side of the cell membrane were plentiful and obvious.

Because carbohydrates are incorporated with proteins, the distribution of carbohydrate microdomains implies that functional glycoproteins, such as receptors and transporters, may form microdomains in membranes to fulfill their functions efficiently. We further digested the ectoplasmic side of the cell membranes by PNGase F that can cleave most saccharides from glycoproteins, and in situ observed the changes by AFM Figure S3.

Unlike the ectoplasmic side of human erythrocyte membranes [32] , after digestion by PNGase F, the ectoplasmic side of MDCK cell membranes exhibited no apparent pits or indents on the smooth surface.

This result indicates the absence of a dense layer of saccharides on the ectoplasmic surface of the cell membranes, consistent with the patchy distribution of carbohydrates on the membrane surface shown by STORM imaging.

The distribution of proteins at the cytoplasmic side of the cell membrane is another key aspect of cell membrane structure and function. Various types of proteins can be found at the cytoplasmic side of the cell membrane, such as the intracellular domains of receptors and transporters. To achieve high-resolution imaging of the cytoplasmic side of the cell membrane by AFM, the cells were sheared open by hypotonic buffer Figure 4A , followed by hypertonic salt treatment that removed the membrane skeletons and non-transmembrane proteins.

Since the transmembrane proteins were inserted in the lipid bilayer, they were not removed by hypertonic buffer, as expected [31]. Figure 4B displays the fluorescent image of the cytoplasmic side of the cell membrane, in which abundant actin filaments green are visible on the membrane surface red.

The cytoskeletons were disrupted by high-salt treatment Figure 4C. The AFM topographical images of the cytoplasmic side of membranes before and after treatment with high-salt buffer are shown in Figure 4D and 4E , respectively. Dense actin filaments are shown as strips Figure 4D and 4G , while no obvious cytoskeleton can be observed in Figure 4E.

The average height of the membranes was The cytoplasmic side of cell membranes was rather rough and covered with proteins, which can be seen more clearly in the magnified image Figure 4F. The roughness RMS of the cytoplasmic side of membranes was 3. The height of the proteins measured from top to bottom was Based on the similarity of heights of the ectoplasmic protein layer and lipid bilayer both at about 4 nm Figure 2 , the total height of the cell membrane was calculated to be about 20 nm, consistent with the real size measured from the whole cell membrane Figure 4E.

The width of the protein microdomains was The distribution of distances of the adjacent protein domains from border to border was about These results demonstrate that the cytoplasmic side consists of protein microdomains scattered in the lipid bilayer. A The scheme for preparing the cytoplasmic side of the cell membrane. B and C The fluorescent images of the cytoplasmic side of the cell membrane before and after incubation with high-salt buffer, respectively.

The red membrane patches represent the lipid bilayer labeled with DiI, and the green fibers represent the actin filaments labeled with phalloidin-FITC. D and E The AFM topographic images of the cytoplasmic side of the cell membrane before and after incubation with high-salt buffer, respectively. F The high magnification image of the cytoplasmic side of the cell membrane.

G—I Cross section analysis along the green line in D—F , respectively. To investigate the relationship between the protein microdomains and lipid bilayer, the cytoplasmic side of membranes was treated with trypsin that could digest most membrane protein domains at the cytoplasmic side.

The topographical image of the digested cytoplasmic side of the cell membrane showed that most of the proteins had been removed, thereby revealing the relative smoothness of local membrane patches Figure 5A, 5C. Some undigested proteins were right above the lipid bilayer, as shown by the bright dots. The height of single-layered, digested membrane patches was 8. Double layers of digested membranes with an average height of A AFM image of the cytoplasmic side of the cell membrane after digestion with trypsin for 1 h.

The single and double layers of membranes are indicated by green and pink arrows, respectively. B The membranes were treated in situ with 0. C and E The magnified images from A and B , respectively. D and F The cross section analysis along the green lines in C and E , respectively. I The magnified image of the green square area in H. Triton X has been widely used to destroy the lipid bilayer by interacting gently with the lipids. We then used 0.

As a result, the average height of the remaining membrane decreased to about 4. The proteins Figure 5B and 5E on the remaining membrane surface may consist of membrane-anchoring proteins, such as GPI proteins, while the pits in the left membrane implicated sites of the transmembrane proteins, such as receptors.

These results further confirm that the whole cell membrane consists of inner dispersed protein domains 12 nm , a lipid bilayer 4 nm , and an ectoplasmic layer of dense proteins 4 nm.

To directly clarify whether cholesterol-enriched domains, i. Figure 5G shows the cytoplasmic side of the cell membrane after digestion by proteinase K. The left membrane was smooth with a height of 8. The height of the left membrane patches remained the same about 8 nm, as shown in Table 1 , except for many pits. The magnified image of the green square area is shown in Figure 5I.

Two major depth distributions of the pits are evident: one is at 4. Taken together, our data indicate that the cholesterol-enriched domains may be the protein microdomains on the cytoplasmic side of cell membranes Figure 4F. Cholesterol-enriched domains in the cell membranes are proposed to perform various functions through embedded proteins [14].

We attempted to locate the functional proteins associated with cholesterol-enriched domains. Band 3 serves as an ion transporter and the anchoring sites for ankyrin, protein 4. Although several studies have reported on the relationship between band 3 and cholesterol-enriched domains, direct observation at high resolution has not thus far been achieved [41].

Here, band 3 was localized at the cytoplasmic side of membranes using the super-resolution fluorescence microscopy afforded by STORM. Figure 5L shows the fluorescence images of band 3 on the cytoplasmic side of the cell membrane. This undoubtedly indicates the presence of cholesterol-enriched domains on the cytoplasmic side of the cell membrane and demonstrates that band 3 was localized in cholesterol-enriched domains. In order to confirm the exposure of proteins on the cytoplasmic and ectoplasmic sides of cell membranes, single-molecule force spectroscopy was applied to detect the amino groups on the surface of the cell membrane.

The aldehyde group linked onto the AFM tip could bind the exposed amino groups of membrane proteins, and this interaction was recorded in AFM force curves. The typical force curves acquired at the cytoplasmic and ectoplasmic sides of cell membranes, out of thousands of force curves, were shown in Figure 6B and 6C , respectively. In Figure 6B , multiple force events were evident in these force curves, and the maximum unbinding forces could reach about pN at a loading rate of 0.

However, only two or three force events were evident in the force curves in Figure 6C , and the maximum unbinding forces were less than pN at a loading rate of The overall binding probabilities, i. These results reveal that a large quantity of exposed amino groups are present on the cytoplasmic side, while fewer amino groups are present on the ectoplasmic side of membranes, essentially because most proteins on the ectoplasmic side of the cell membrane are glycosylated and compacted, while considering that there is a denser protein layer in the ectoplasmic side than that in the cytoplasmic side.

A The scheme of AFM tip functionalized with aldehyde group. B and C The typical force curves acquired on the cytoplasmic and ectoplasmic side of the cell membrane, respectively. The black and red lines represent the approaching and withdrawn curves, respectively. Using a combination of single-molecule techniques, including AFM, SMFS and STORM to study the structure of nucleated cell membranes in-situ , we found that 1 proteins at the ectoplasmic side of membrane form a dense protein layer 4 nm on top of a lipid bilayer; 2 proteins aggregate to form islands evenly dispersed at the cytoplasmic side of the cell membrane with a height of 10—12 nm; 3 cholesterol-enriched domains exist in the cell membrane; 4 carbohydrates stay in microdomains at the ectoplasmic side; and 5 exposed amino groups are asymmetrically distributed on both sides.

Proteins are asymmetrically distributed on the cell membrane surface. The ectoplasmic side of the cell membrane consists of various types of proteins, such as extracellular segments of receptors and the GPI-APs, above the lipid bilayer. The proteins at the ectoplasmic side of the cell membrane form a dense protein layer showing a smooth feature Figure 7A with a height of about 4 nm Figure 7C. The Problem of Size. Why are cells so small? Cells are so small that you need a microscope to examine them.

To answer this question we have to understand that, in order to survive, cells must constantly interact with their surrounding environment. Gases and food molecules dissolved in water must be absorbed and waste products must be eliminated.