# Bilayer synthesis?

If we want to design a bilayer from Myristic acid (14 carbon fatty acid). The average bond length between C-C is 1.5 A. What will be average thickness of the membrane?

Edited to include the OP attempt that was posted in a comment:

There are 13 C-C bonds in Myristic acid, 13*1.5=19.5A. 1A=o.1nm… so 19.5A=1.95nm, so the average thickness would be 1.95nm. As membrane is bilayer it will be 3.9nm

Lewis & Engelman (1983) Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles. J. Mol. Biol. 166: 211 - 217.

Table 1 and Figure 3 have the information that you need. For C14:0 the thickness of the hydrocarbon bilayer is given as 23 Å. Just in case this is homework, I'll leave you to convert that to nm.

Just to follow up on the comments, as hinted at by Shigeta, if you wanted to calculate an estimated bilayer thickness you would have to take into account the C-C-C bond angle which for tetrahedral carbon is 109.46°.

Thus the distance between two C atoms in an extended C chain is 2 x 1.5 x sin(109.46/2) = 2.45 Å

For C14:0 the length is then 6.5 x 2.45 = 15.9 Å (from methyl C to carbonyl C).

Therefore, calculated bilayer thickness = 31.8 Å plus any distance between the two C chains in the centre of the bilayer. This is still a lot bigger than the reported 23 Å.

Clearly the bilayer does not consist of extended hydrocarbon chains. The chains are presumably partially collapsed (on average) and may also interdigitate to some extent.

## Artificial bilayer tissue-like materials

Soft responsive materials that mimic and extend the function of biological tissues could revolutionize biomanufacturing, computing, robotics, sensing, and separations. In this project, we aim to mimic the reconfigurability and adaptability of biological tissues using a modular system of dynamic, self-assembling components. This will enable the design, synthesis, and assembly of a new class of multifunctional soft materials. These materials will form the platform for research into a number of emerging and established scientific concepts including neuromorphic computing, catalysis in flow chemistry, biomimetic separations, interspecies electron transfer, cell-free synthetic biology, and soft robotics. Specifically, we propose to develop a droplet interface bilayer (DIB) platform that integrates membrane forming molecules (amphiphilic block copolymers, lipids, and peptoids), membrane transporters (membrane proteins and artificial supramolecular channels), whole cells (engineered and native), and nanomaterials (quantum dots, 2d materials, responsive polymers) to form individually-addressable molecularly-gated compartments within a polymerizable matrix. DIB leverages the assembly of nanoscale biomimetic membranes (bilayers) between lipid or amphiphilic block copolymer coated aqueous droplets immersed in a hydrophobic medium, such as a polymerizable oil or organogel. The DIB offers several specific advantages for creating compartmentalized biomimetic materials: First, the bilayer that forms between droplets enables reconstitution of stimuli-responsive biomolecules, including membrane proteins, membrane active peptides and artificial channels, which can be used to control transport between compartments. Second, the DIB method uniquely enables the patterned assembly of 2- and 3-dimensional droplet networks that mimic the hierarchical organization and membrane-separation of cells in living tissues and which can exhibit collective functionality. The research proposed during the seed project will focus on a specific application and demonstration of the DIB platform through development of a novel selective separation/reactive “living” membrane that can remove a commonly found aqueous pollutant - nitrate. Nitrate is responsible for eutrophication of water bodies such as lakes and the Gulf of Mexico leading to large algal blooms and eutrophication. We propose to implement three strategies to demonstrate the versatility of the DIB platform for nitrate capture and degradation using enzymes and microbes that can potentially be used as deployable materials for degrading nitrate.

The Center for Dynamics and Control of Materials (CDCM) is supported by the National Science Foundation under NSF Award Number DMR-1720595. Additional support is provided by The University of Texas at Austin.

## The Endoplasmic Reticulum

The endoplasmic reticulum is an organelle that is responsible for the synthesis of lipids and the modification of proteins.

### Learning Objectives

Describe the structure of the endoplasmic reticulum and its role in synthesis and metabolism

### Key Takeaways

#### Key Points

• If the endoplasmic reticulum (ER) has ribosomes attached to it, it is called rough ER if it does not, then it is called smooth ER.
• The proteins made by the rough endoplasmic reticulum are for use outside of the cell.
• Functions of the smooth endoplasmic reticulum include synthesis of carbohydrates, lipids, and steroid hormones detoxification of medications and poisons and storage of calcium ions.

#### Key Terms

• lumen: The cavity or channel within a tube or tubular organ.
• reticulum: A network

### The Endoplasmic Reticulum

The endoplasmic reticulum (ER) is a series of interconnected membranous sacs and tubules that collectively modifies proteins and synthesizes lipids. However, these two functions are performed in separate areas of the ER: the rough ER and the smooth ER. The hollow portion of the ER tubules is called the lumen or cisternal space. The membrane of the ER, which is a phospholipid bilayer embedded with proteins, is continuous with the nuclear envelope.

### Rough ER

Rough Endoplasmic Reticulum: This transmission electron micrograph shows the rough endoplasmic reticulum and other organelles in a pancreatic cell.

The rough endoplasmic reticulum (RER) is so named because the ribosomes attached to its cytoplasmic surface give it a studded appearance when viewed through an electron microscope. Ribosomes transfer their newly synthesized proteins into the lumen of the RER where they undergo structural modifications, such as folding or the acquisition of side chains. These modified proteins will be incorporated into cellular membranes—the membrane of the ER or those of other organelles —or secreted from the cell (such as protein hormones, enzymes ). The RER also makes phospholipids for cellular membranes. If the phospholipids or modified proteins are not destined to stay in the RER, they will reach their destinations via transport vesicles that bud from the RER’s membrane. Since the RER is engaged in modifying proteins (such as enzymes, for example) that will be secreted from the cell, the RER is abundant in cells that secrete proteins. This is the case with cells of the liver, for example.

### Smooth ER

The smooth endoplasmic reticulum (SER) is continuous with the RER but has few or no ribosomes on its cytoplasmic surface. Functions of the SER include synthesis of carbohydrates, lipids, and steroid hormones detoxification of medications and poisons and storage of calcium ions. In muscle cells, a specialized SER called the sarcoplasmic reticulum is responsible for storage of the calcium ions that are needed to trigger the coordinated contractions of the muscle cells.

## Contents

When phospholipids are exposed to water, they self-assemble into a two-layered sheet with the hydrophobic tails pointing toward the center of the sheet. This arrangement results in two “leaflets” that are each a single molecular layer. The center of this bilayer contains almost no water and excludes molecules like sugars or salts that dissolve in water. The assembly process is driven by interactions between hydrophobic molecules (also called the hydrophobic effect). An increase in interactions between hydrophobic molecules (causing clustering of hydrophobic regions) allows water molecules to bond more freely with each other, increasing the entropy of the system. This complex process includes non-covalent interactions such as van der Waals forces, electrostatic and hydrogen bonds.

### Cross section analysis Edit

The lipid bilayer is very thin compared to its lateral dimensions. If a typical mammalian cell (diameter

10 micrometers) were magnified to the size of a watermelon (

1 ft/30 cm), the lipid bilayer making up the plasma membrane would be about as thick as a piece of office paper. Despite being only a few nanometers thick, the bilayer is composed of several distinct chemical regions across its cross-section. These regions and their interactions with the surrounding water have been characterized over the past several decades with x-ray reflectometry, [4] neutron scattering [5] and nuclear magnetic resonance techniques.

The first region on either side of the bilayer is the hydrophilic headgroup. This portion of the membrane is completely hydrated and is typically around 0.8-0.9 nm thick. In phospholipid bilayers the phosphate group is located within this hydrated region, approximately 0.5 nm outside the hydrophobic core. [6] In some cases, the hydrated region can extend much further, for instance in lipids with a large protein or long sugar chain grafted to the head. One common example of such a modification in nature is the lipopolysaccharide coat on a bacterial outer membrane, [7] which helps retain a water layer around the bacterium to prevent dehydration.

Next to the hydrated region is an intermediate region that is only partially hydrated. This boundary layer is approximately 0.3 nm thick. Within this short distance, the water concentration drops from 2M on the headgroup side to nearly zero on the tail (core) side. [8] [9] The hydrophobic core of the bilayer is typically 3-4 nm thick, but this value varies with chain length and chemistry. [4] [10] Core thickness also varies significantly with temperature, in particular near a phase transition. [11]

### Asymmetry Edit

In many naturally occurring bilayers, the compositions of the inner and outer membrane leaflets are different. In human red blood cells, the inner (cytoplasmic) leaflet is composed mostly of phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol and its phosphorylated derivatives. By contrast, the outer (extracellular) leaflet is based on phosphatidylcholine, sphingomyelin and a variety of glycolipids. [12] [13] In some cases, this asymmetry is based on where the lipids are made in the cell and reflects their initial orientation. [14] The biological functions of lipid asymmetry are imperfectly understood, although it is clear that it is used in several different situations. For example, when a cell undergoes apoptosis, the phosphatidylserine — normally localised to the cytoplasmic leaflet — is transferred to the outer surface: There, it is recognised by a macrophage that then actively scavenges the dying cell.

Lipid asymmetry arises, at least in part, from the fact that most phospholipids are synthesised and initially inserted into the inner monolayer: those that constitute the outer monolayer are then transported from the inner monolayer by a class of enzymes called flippases. [15] [16] Other lipids, such as sphingomyelin, appear to be synthesised at the external leaflet. Flippases are members of a larger family of lipid transport molecules that also includes floppases, which transfer lipids in the opposite direction, and scramblases, which randomize lipid distribution across lipid bilayers (as in apoptotic cells). In any case, once lipid asymmetry is established, it does not normally dissipate quickly because spontaneous flip-flop of lipids between leaflets is extremely slow. [17]

It is possible to mimic this asymmetry in the laboratory in model bilayer systems. Certain types of very small artificial vesicle will automatically make themselves slightly asymmetric, although the mechanism by which this asymmetry is generated is very different from that in cells. [18] By utilizing two different monolayers in Langmuir-Blodgett deposition [19] or a combination of Langmuir-Blodgett and vesicle rupture deposition [20] it is also possible to synthesize an asymmetric planar bilayer. This asymmetry may be lost over time as lipids in supported bilayers can be prone to flip-flop. [21]

### Phases and phase transitions Edit

At a given temperature a lipid bilayer can exist in either a liquid or a gel (solid) phase. All lipids have a characteristic temperature at which they transition (melt) from the gel to liquid phase. In both phases the lipid molecules are prevented from flip-flopping across the bilayer, but in liquid phase bilayers a given lipid will exchange locations with its neighbor millions of times a second. This random walk exchange allows lipid to diffuse and thus wander across the surface of the membrane. [22] Unlike liquid phase bilayers, the lipids in a gel phase bilayer have less mobility.

The phase behavior of lipid bilayers is determined largely by the strength of the attractive Van der Waals interactions between adjacent lipid molecules. Longer-tailed lipids have more area over which to interact, increasing the strength of this interaction and, as a consequence, decreasing the lipid mobility. Thus, at a given temperature, a short-tailed lipid will be more fluid than an otherwise identical long-tailed lipid. [10] Transition temperature can also be affected by the degree of unsaturation of the lipid tails. An unsaturated double bond can produce a kink in the alkane chain, disrupting the lipid packing. This disruption creates extra free space within the bilayer that allows additional flexibility in the adjacent chains. [10] An example of this effect can be noted in everyday life as butter, which has a large percentage saturated fats, is solid at room temperature while vegetable oil, which is mostly unsaturated, is liquid.

Most natural membranes are a complex mixture of different lipid molecules. If some of the components are liquid at a given temperature while others are in the gel phase, the two phases can coexist in spatially separated regions, rather like an iceberg floating in the ocean. This phase separation plays a critical role in biochemical phenomena because membrane components such as proteins can partition into one or the other phase [23] and thus be locally concentrated or activated. One particularly important component of many mixed phase systems is cholesterol, which modulates bilayer permeability, mechanical strength, and biochemical interactions.

### Surface chemistry Edit

While lipid tails primarily modulate bilayer phase behavior, it is the headgroup that determines the bilayer surface chemistry. Most natural bilayers are composed primarily of phospholipids, but sphingolipids and sterols such as cholesterol are also important components. [24] Of the phospholipids, the most common headgroup is phosphatidylcholine (PC), accounting for about half the phospholipids in most mammalian cells. [25] PC is a zwitterionic headgroup, as it has a negative charge on the phosphate group and a positive charge on the amine but, because these local charges balance, no net charge.

Other headgroups are also present to varying degrees and can include phosphatidylserine (PS) phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). These alternate headgroups often confer specific biological functionality that is highly context-dependent. For instance, PS presence on the extracellular membrane face of erythrocytes is a marker of cell apoptosis, [26] whereas PS in growth plate vesicles is necessary for the nucleation of hydroxyapatite crystals and subsequent bone mineralization. [27] [28] Unlike PC, some of the other headgroups carry a net charge, which can alter the electrostatic interactions of small molecules with the bilayer. [29]

### Containment and separation Edit

The primary role of the lipid bilayer in biology is to separate aqueous compartments from their surroundings. Without some form of barrier delineating “self” from “non-self”, it is difficult to even define the concept of an organism or of life. This barrier takes the form of a lipid bilayer in all known life forms except for a few species of archaea that utilize a specially adapted lipid monolayer. [7] It has even been proposed that the very first form of life may have been a simple lipid vesicle with virtually its sole biosynthetic capability being the production of more phospholipids. [30] The partitioning ability of the lipid bilayer is based on the fact that hydrophilic molecules cannot easily cross the hydrophobic bilayer core, as discussed in Transport across the bilayer below. The nucleus, mitochondria and chloroplasts have two lipid bilayers, while other sub-cellular structures are surrounded by a single lipid bilayer (such as the plasma membrane, endoplasmic reticula, Golgi apparatus and lysosomes). See Organelle. [31]

Prokaryotes have only one lipid bilayer - the cell membrane (also known as the plasma membrane). Many prokaryotes also have a cell wall, but the cell wall is composed of proteins or long chain carbohydrates, not lipids. In contrast, eukaryotes have a range of organelles including the nucleus, mitochondria, lysosomes and endoplasmic reticulum. All of these sub-cellular compartments are surrounded by one or more lipid bilayers and, together, typically comprise the majority of the bilayer area present in the cell. In liver hepatocytes for example, the plasma membrane accounts for only two percent of the total bilayer area of the cell, whereas the endoplasmic reticulum contains more than fifty percent and the mitochondria a further thirty percent. [32]

### Signaling Edit

Probably the most familiar form of cellular signaling is synaptic transmission, whereby a nerve impulse that has reached the end of one neuron is conveyed to an adjacent neuron via the release of neurotransmitters. This transmission is made possible by the action of synaptic vesicles loaded with the neurotransmitters to be released. These vesicles fuse with the cell membrane at the pre-synaptic terminal and release its contents to the exterior of the cell. The contents then diffuse across the synapse to the post-synaptic terminal.

Lipid bilayers are also involved in signal transduction through their role as the home of integral membrane proteins. This is an extremely broad and important class of biomolecule. It is estimated that up to a third of the human proteome are membrane proteins. [33] Some of these proteins are linked to the exterior of the cell membrane. An example of this is the CD59 protein, which identifies cells as “self” and thus inhibits their destruction by the immune system. The HIV virus evades the immune system in part by grafting these proteins from the host membrane onto its own surface. [32] Alternatively, some membrane proteins penetrate all the way through the bilayer and serve to relay individual signal events from the outside to the inside of the cell. The most common class of this type of protein is the G protein-coupled receptor (GPCR). GPCRs are responsible for much of the cell's ability to sense its surroundings and, because of this important role, approximately 40% of all modern drugs are targeted at GPCRs. [34]

In addition to protein- and solution-mediated processes, it is also possible for lipid bilayers to participate directly in signaling. A classic example of this is phosphatidylserine-triggered phagocytosis. Normally, phosphatidylserine is asymmetrically distributed in the cell membrane and is present only on the interior side. During programmed cell death a protein called a scramblase equilibrates this distribution, displaying phosphatidylserine on the extracellular bilayer face. The presence of phosphatidylserine then triggers phagocytosis to remove the dead or dying cell.

The lipid bilayer is a very difficult structure to study because it is so thin and fragile. In spite of these limitations dozens of techniques have been developed over the last seventy years to allow investigations of its structure and function.

### Electrical measurements Edit

Electrical measurements are a straightforward way to characterize an important function of a bilayer: its ability to segregate and prevent the flow of ions in solution. By applying a voltage across the bilayer and measuring the resulting current, the resistance of the bilayer is determined. This resistance is typically quite high (10 8 Ohm-cm 2 or more) [35] since the hydrophobic core is impermeable to charged species. The presence of even a few nanometer-scale holes results in a dramatic increase in current. [36] The sensitivity of this system is such that even the activity of single ion channels can be resolved. [37]

### Fluorescence microscopy Edit

Electrical measurements do not provide an actual picture like imaging with a microscope can. Lipid bilayers cannot be seen in a traditional microscope because they are too thin. In order to see bilayers, researchers often use fluorescence microscopy. A sample is excited with one wavelength of light and observed in a different wavelength, so that only fluorescent molecules with a matching excitation and emission profile will be seen. Natural lipid bilayers are not fluorescent, so a dye is used that attaches to the desired molecules in the bilayer. Resolution is usually limited to a few hundred nanometers, much smaller than a typical cell but much larger than the thickness of a lipid bilayer.

### Electron microscopy Edit

Electron microscopy offers a higher resolution image. In an electron microscope, a beam of focused electrons interacts with the sample rather than a beam of light as in traditional microscopy. In conjunction with rapid freezing techniques, electron microscopy has also been used to study the mechanisms of inter- and intracellular transport, for instance in demonstrating that exocytotic vesicles are the means of chemical release at synapses. [38]

### Nuclear magnetic resonance spectroscopy Edit

31 P-NMR(nuclear magnetic resonance) spectroscopy is widely used for studies of phospholipid bilayers and biological membranes in native conditions. The analysis [39] of 31 P-NMR spectra of lipids could provide a wide range of information about lipid bilayer packing, phase transitions (gel phase, physiological liquid crystal phase, ripple phases, non bilayer phases), lipid head group orientation/dynamics, and elastic properties of pure lipid bilayer and as a result of binding of proteins and other biomolecules.

### Atomic force microscopy Edit

A new method to study lipid bilayers is Atomic force microscopy (AFM). Rather than using a beam of light or particles, a very small sharpened tip scans the surface by making physical contact with the bilayer and moving across it, like a record player needle. AFM is a promising technique because it has the potential to image with nanometer resolution at room temperature and even under water or physiological buffer, conditions necessary for natural bilayer behavior. Utilizing this capability, AFM has been used to examine dynamic bilayer behavior including the formation of transmembrane pores (holes) [40] and phase transitions in supported bilayers. [41] Another advantage is that AFM does not require fluorescent or isotopic labeling of the lipids, since the probe tip interacts mechanically with the bilayer surface. Because of this, the same scan can image both lipids and associated proteins, sometimes even with single-molecule resolution. [40] [42] AFM can also probe the mechanical nature of lipid bilayers. [43]

### Dual polarisation interferometry Edit

Lipid bilayers exhibit high levels of birefringence where the refractive index in the plane of the bilayer differs from that perpendicular by as much as 0.1 refractive index units. This has been used to characterise the degree of order and disruption in bilayers using dual polarisation interferometry to understand mechanisms of protein interaction.

### Quantum chemical calculations Edit

Lipid bilayers are complicated molecular systems with many degrees of freedom. Thus, atomistic simulation of membrane and in particular ab initio calculations of its properties is difficult and computationally expensive. Quantum chemical calculations has recently been successfully performed to estimate dipole and quadrupole moments of lipid membranes. [44]

### Passive diffusion Edit

Most polar molecules have low solubility in the hydrocarbon core of a lipid bilayer and, as a consequence, have low permeability coefficients across the bilayer. This effect is particularly pronounced for charged species, which have even lower permeability coefficients than neutral polar molecules. [45] Anions typically have a higher rate of diffusion through bilayers than cations. [46] [47] Compared to ions, water molecules actually have a relatively large permeability through the bilayer, as evidenced by osmotic swelling. When a cell or vesicle with a high interior salt concentration is placed in a solution with a low salt concentration it will swell and eventually burst. Such a result would not be observed unless water was able to pass through the bilayer with relative ease. The anomalously large permeability of water through bilayers is still not completely understood and continues to be the subject of active debate. [48] Small uncharged apolar molecules diffuse through lipid bilayers many orders of magnitude faster than ions or water. This applies both to fats and organic solvents like chloroform and ether. Regardless of their polar character larger molecules diffuse more slowly across lipid bilayers than small molecules. [49]

### Ion pumps and channels Edit

Two special classes of protein deal with the ionic gradients found across cellular and sub-cellular membranes in nature- ion channels and ion pumps. Both pumps and channels are integral membrane proteins that pass through the bilayer, but their roles are quite different. Ion pumps are the proteins that build and maintain the chemical gradients by utilizing an external energy source to move ions against the concentration gradient to an area of higher chemical potential. The energy source can be ATP, as is the case for the Na + -K + ATPase. Alternatively, the energy source can be another chemical gradient already in place, as in the Ca 2+ /Na + antiporter. It is through the action of ion pumps that cells are able to regulate pH via the pumping of protons.

In contrast to ion pumps, ion channels do not build chemical gradients but rather dissipate them in order to perform work or send a signal. Probably the most familiar and best studied example is the voltage-gated Na + channel, which allows conduction of an action potential along neurons. All ion pumps have some sort of trigger or “gating” mechanism. In the previous example it was electrical bias, but other channels can be activated by binding a molecular agonist or through a conformational change in another nearby protein. [50]

### Endocytosis and exocytosis Edit

Some molecules or particles are too large or too hydrophilic to pass through a lipid bilayer. Other molecules could pass through the bilayer but must be transported rapidly in such large numbers that channel-type transport is impractical. In both cases, these types of cargo can be moved across the cell membrane through fusion or budding of vesicles. When a vesicle is produced inside the cell and fuses with the plasma membrane to release its contents into the extracellular space, this process is known as exocytosis. In the reverse process, a region of the cell membrane will dimple inwards and eventually pinch off, enclosing a portion of the extracellular fluid to transport it into the cell. Endocytosis and exocytosis rely on very different molecular machinery to function, but the two processes are intimately linked and could not work without each other. The primary mechanism of this interdependence is the large amount of lipid material involved. [51] In a typical cell, an area of bilayer equivalent to the entire plasma membrane will travel through the endocytosis/exocytosis cycle in about half an hour. [52] If these two processes were not balancing each other, the cell would either balloon outward to an unmanageable size or completely deplete its plasma membrane within a short time.

Exocytosis in prokaryotes: Membrane vesicular exocytosis, popularly known as membrane vesicle trafficking, a Nobel prize-winning (year, 2013) process, is traditionally regarded as a prerogative of eukaryotic cells. [53] This myth was however broken with the revelation that nanovesicles, popularly known as bacterial outer membrane vesicles, released by gram-negative microbes, translocate bacterial signal molecules to host or target cells [54] to carry out multiple processes in favour of the secreting microbe e.g., in host cell invasion [55] and microbe-environment interactions, in general. [56]

### Electroporation Edit

Electroporation is the rapid increase in bilayer permeability induced by the application of a large artificial electric field across the membrane. Experimentally, electroporation is used to introduce hydrophilic molecules into cells. It is a particularly useful technique for large highly charged molecules such as DNA, which would never passively diffuse across the hydrophobic bilayer core. [57] Because of this, electroporation is one of the key methods of transfection as well as bacterial transformation. It has even been proposed that electroporation resulting from lightning strikes could be a mechanism of natural horizontal gene transfer. [58]

This increase in permeability primarily affects transport of ions and other hydrated species, indicating that the mechanism is the creation of nm-scale water-filled holes in the membrane. Although electroporation and dielectric breakdown both result from application of an electric field, the mechanisms involved are fundamentally different. In dielectric breakdown the barrier material is ionized, creating a conductive pathway. The material alteration is thus chemical in nature. In contrast, during electroporation the lipid molecules are not chemically altered but simply shift position, opening up a pore that acts as the conductive pathway through the bilayer as it is filled with water.

Lipid bilayers are large enough structures to have some of the mechanical properties of liquids or solids. The area compression modulus Ka, bending modulus Kb, and edge energy Λ , can be used to describe them. Solid lipid bilayers also have a shear modulus, but like any liquid, the shear modulus is zero for fluid bilayers. These mechanical properties affect how the membrane functions. Ka and Kb affect the ability of proteins and small molecules to insert into the bilayer, [59] [60] and bilayer mechanical properties have been shown to alter the function of mechanically activated ion channels. [61] Bilayer mechanical properties also govern what types of stress a cell can withstand without tearing. Although lipid bilayers can easily bend, most cannot stretch more than a few percent before rupturing. [62]

As discussed in the Structure and organization section, the hydrophobic attraction of lipid tails in water is the primary force holding lipid bilayers together. Thus, the elastic modulus of the bilayer is primarily determined by how much extra area is exposed to water when the lipid molecules are stretched apart. [63] It is not surprising given this understanding of the forces involved that studies have shown that Ka varies strongly with osmotic pressure [64] but only weakly with tail length and unsaturation. [10] Because the forces involved are so small, it is difficult to experimentally determine Ka. Most techniques require sophisticated microscopy and very sensitive measurement equipment. [43] [65]

In contrast to Ka, which is a measure of how much energy is needed to stretch the bilayer, Kb is a measure of how much energy is needed to bend or flex the bilayer. Formally, bending modulus is defined as the energy required to deform a membrane from its intrinsic curvature to some other curvature. Intrinsic curvature is defined by the ratio of the diameter of the head group to that of the tail group. For two-tailed PC lipids, this ratio is nearly one so the intrinsic curvature is nearly zero. If a particular lipid has too large a deviation from zero intrinsic curvature it will not form a bilayer and will instead form other phases such as micelles or inverted micelles. Addition of small hydrophilic molecules like sucrose into mixed lipid lamellar liposomes made from galactolipid-rich thylakoid membranes destabilises bilayers into micellar phase. [66] Typically, Kb is not measured experimentally but rather is calculated from measurements of Ka and bilayer thickness, since the three parameters are related.

Fusion is the process by which two lipid bilayers merge, resulting in one connected structure. If this fusion proceeds completely through both leaflets of both bilayers, a water-filled bridge is formed and the solutions contained by the bilayers can mix. Alternatively, if only one leaflet from each bilayer is involved in the fusion process, the bilayers are said to be hemifused. Fusion is involved in many cellular processes, in particular in eukaryotes, since the eukaryotic cell is extensively sub-divided by lipid bilayer membranes. Exocytosis, fertilization of an egg by sperm activation, and transport of waste products to the lysozome are a few of the many eukaryotic processes that rely on some form of fusion. Even the entry of pathogens can be governed by fusion, as many bilayer-coated viruses have dedicated fusion proteins to gain entry into the host cell.

There are four fundamental steps in the fusion process. [25] First, the involved membranes must aggregate, approaching each other to within several nanometers. Second, the two bilayers must come into very close contact (within a few angstroms). To achieve this close contact, the two surfaces must become at least partially dehydrated, as the bound surface water normally present causes bilayers to strongly repel. The presence of ions, in particular divalent cations like magnesium and calcium, strongly affects this step. [69] [70] One of the critical roles of calcium in the body is regulating membrane fusion. Third, a destabilization must form at one point between the two bilayers, locally distorting their structures. The exact nature of this distortion is not known. One theory is that a highly curved "stalk" must form between the two bilayers. [71] Proponents of this theory believe that it explains why phosphatidylethanolamine, a highly curved lipid, promotes fusion. [72] Finally, in the last step of fusion, this point defect grows and the components of the two bilayers mix and diffuse away from the site of contact.

The situation is further complicated when considering fusion in vivo since biological fusion is almost always regulated by the action of membrane-associated proteins. The first of these proteins to be studied were the viral fusion proteins, which allow an enveloped virus to insert its genetic material into the host cell (enveloped viruses are those surrounded by a lipid bilayer some others have only a protein coat). Eukaryotic cells also use fusion proteins, the best-studied of which are the SNAREs. SNARE proteins are used to direct all vesicular intracellular trafficking. Despite years of study, much is still unknown about the function of this protein class. In fact, there is still an active debate regarding whether SNAREs are linked to early docking or participate later in the fusion process by facilitating hemifusion. [74]

In studies of molecular and cellular biology it is often desirable to artificially induce fusion. The addition of polyethylene glycol (PEG) causes fusion without significant aggregation or biochemical disruption. This procedure is now used extensively, for example by fusing B-cells with myeloma cells. [75] The resulting “hybridoma” from this combination expresses a desired antibody as determined by the B-cell involved, but is immortalized due to the melanoma component. Fusion can also be artificially induced through electroporation in a process known as electrofusion. It is believed that this phenomenon results from the energetically active edges formed during electroporation, which can act as the local defect point to nucleate stalk growth between two bilayers. [76]

Lipid bilayers can be created artificially in the lab to allow researchers to perform experiments that cannot be done with natural bilayers. They can also be used in the field of Synthetic Biology, to define the boundaries of artificial cells. These synthetic systems are called model lipid bilayers. There are many different types of model bilayers, each having experimental advantages and disadvantages. They can be made with either synthetic or natural lipids. Among the most common model systems are:

To date, the most successful commercial application of lipid bilayers has been the use of liposomes for drug delivery, especially for cancer treatment. (Note- the term “liposome” is in essence synonymous with “vesicle” except that vesicle is a general term for the structure whereas liposome refers to only artificial not natural vesicles) The basic idea of liposomal drug delivery is that the drug is encapsulated in solution inside the liposome then injected into the patient. These drug-loaded liposomes travel through the system until they bind at the target site and rupture, releasing the drug. In theory, liposomes should make an ideal drug delivery system since they can isolate nearly any hydrophilic drug, can be grafted with molecules to target specific tissues and can be relatively non-toxic since the body possesses biochemical pathways for degrading lipids. [77]

The first generation of drug delivery liposomes had a simple lipid composition and suffered from several limitations. Circulation in the bloodstream was extremely limited due to both renal clearing and phagocytosis. Refinement of the lipid composition to tune fluidity, surface charge density, and surface hydration resulted in vesicles that adsorb fewer proteins from serum and thus are less readily recognized by the immune system. [78] The most significant advance in this area was the grafting of polyethylene glycol (PEG) onto the liposome surface to produce “stealth” vesicles, which circulate over long times without immune or renal clearing. [79]

The first stealth liposomes were passively targeted at tumor tissues. Because tumors induce rapid and uncontrolled angiogenesis they are especially “leaky” and allow liposomes to exit the bloodstream at a much higher rate than normal tissue would. [80] More recently [ when? ] work has been undertaken to graft antibodies or other molecular markers onto the liposome surface in the hope of actively binding them to a specific cell or tissue type. [81] Some examples of this approach are already in clinical trials. [82]

Another potential application of lipid bilayers is the field of biosensors. Since the lipid bilayer is the barrier between the interior and exterior of the cell, it is also the site of extensive signal transduction. Researchers over the years have tried to harness this potential to develop a bilayer-based device for clinical diagnosis or bioterrorism detection. Progress has been slow in this area and, although a few companies have developed automated lipid-based detection systems, they are still targeted at the research community. These include Biacore (now GE Healthcare Life Sciences), which offers a disposable chip for utilizing lipid bilayers in studies of binding kinetics [83] and Nanion Inc., which has developed an automated patch clamping system. [84] Other, more exotic applications are also being pursued such as the use of lipid bilayer membrane pores for DNA sequencing by Oxford Nanolabs. To date, this technology has not proven commercially viable.

A supported lipid bilayer (SLB) as described above has achieved commercial success as a screening technique to measure the permeability of drugs. This parallel artificial membrane permeability assay PAMPA technique measures the permeability across specifically formulated lipid cocktail(s) found to be highly correlated with Caco-2 cultures, [85] [86] the gastrointestinal tract, [87] blood–brain barrier [88] and skin. [89]

By the early twentieth century scientists had come to believe that cells are surrounded by a thin oil-like barrier, [90] but the structural nature of this membrane was not known. Two experiments in 1925 laid the groundwork to fill in this gap. By measuring the capacitance of erythrocyte solutions, Hugo Fricke determined that the cell membrane was 3.3 nm thick. [91]

Although the results of this experiment were accurate, Fricke misinterpreted the data to mean that the cell membrane is a single molecular layer. Prof. Dr. Evert Gorter [92] (1881–1954) and F. Grendel of Leiden University approached the problem from a different perspective, spreading the erythrocyte lipids as a monolayer on a Langmuir-Blodgett trough. When they compared the area of the monolayer to the surface area of the cells, they found a ratio of two to one. [93] Later analyses showed several errors and incorrect assumptions with this experiment but, serendipitously, these errors canceled out and from this flawed data Gorter and Grendel drew the correct conclusion- that the cell membrane is a lipid bilayer. [25]

This theory was confirmed through the use of electron microscopy in the late 1950s. Although he did not publish the first electron microscopy study of lipid bilayers [94] J. David Robertson was the first to assert that the two dark electron-dense bands were the headgroups and associated proteins of two apposed lipid monolayers. [95] [96] In this body of work, Robertson put forward the concept of the “unit membrane.” This was the first time the bilayer structure had been universally assigned to all cell membranes as well as organelle membranes.

Around the same time, the development of model membranes confirmed that the lipid bilayer is a stable structure that can exist independent of proteins. By “painting” a solution of lipid in organic solvent across an aperture, Mueller and Rudin were able to create an artificial bilayer and determine that this exhibited lateral fluidity, high electrical resistance and self-healing in response to puncture, [97] all of which are properties of a natural cell membrane. A few years later, Alec Bangham showed that bilayers, in the form of lipid vesicles, could also be formed simply by exposing a dried lipid sample to water. [98] This was an important advance, since it demonstrated that lipid bilayers form spontaneously via self assembly and do not require a patterned support structure.

In 1977, a totally synthetic bilayer membrane was prepared by Kunitake and Okahata, from a single organic compound, didodecyldimethylammonium bromide. [99] It clearly shows that the bilayer membrane was assembled by the van der Waals interaction.

## Synthesis of Membrane Proteins and the "Signal Hypothesis"

All of a cell’s proteins are synthesized by ribosomes, including, of course, those proteins that are destined for inclusion in the plasma membrane.

Cytoplasmic ri­bosomes in eukaryotic cells occur in two states: (1) “attached” (ribosomes associated with the mem­branes of the endoplasmic reticulum) and (2) “free” (ribosomes freely dispersed in the cytosol). Both attached and free ribosomes are be­lieved to contribute proteins to the plasma membrane.

Synthesis of Membrane Proteins and the “Signal Hypothesis”:

Principally as a result of the work of G. Blobel, D. D. Sabatini, C. M. Redman, C. Milstein, J. E. Rothman, J. Lenard, and H. F. Lodish, the mechanism that routes newly synthesized proteins to their proper des­tinations in the cell has gradually unfolded. A major contribution to this end has been the confirmation of the signal hypothesis proposed in the early 1970s by Blobel and Sabatini.

According to this hypothesis (Fig. 15-19), proteins that are to be either (a) secreted from the cell, (b) dispatched to lysosomes, or (c) incor­porated into the plasma membrane or membranes of the endoplasmic reticulum are encoded by mRNA molecules that contain a special nucleotide sequence called a “signal.”

The signal segment encodes a chain of about 16 to 26 amino acids that appears at or near the beginning of the polypeptide chain. Near its N- terminus, the signal sequence contains polar, basic residues, whereas the central domain is apolar. When a ribosome attaches to the mRNA in the cytosol and begins to translate the message, the signal sequence or signal peptide emerging from the ribosome is rec­ognized by a ribonucleoprotein complex in the cytosol called the signal recognition particle (i.e., SRP).

SRP, which consists of a 7 S cytoplasmic RNA mole­cule and six polypeptides, binds to the signal se­quence, bringing about a temporary halt to protein synthesis by that ribosome. Synthesis is resumed only if the SRP-ribosome complex attaches to the endo­plasmic reticulum ribosomes synthesizing polypep­tides that lack a signal sequence do not interact with SRP and do not attach to the endoplasmic reticulum.

The amino acid sequences of a number of signal pep­tides have now been determined. Although they con­tain a specific distribution of hydrophobic and charged residues, no primary sequence homologies appear to exist. Consequently, it is be­lieved that SRP must recognize certain features con­tained in the signal peptide’s secondary and tertiary structure.

SRP-ribosome complexes attach to the endoplas­mic reticulum at specific sites occupied by SRP recep­tors (also called docking proteins). Once “docking” is completed, the SRP-ribosome-docking protein inter­action is replaced by a functional ribosome- membrane junction and the synthesis of the polypep­tide encoded by the mRNA is resumed. SRP returns to the cytosol where it can participate in another round of signal recognition and docking (i.e., the “SRP cycle” of Fig. 15-19a).

When protein synthesis is resumed by the docked ribosome, the elongating polypeptide chain passes through the ER membrane into the intracisternal space. This process, termed “translocation,” is presumed to involve active partici­pation of elements of the membrane. Translocation of the growing polypeptide into the intracisternal space is depicted in Figure 15-19 as taking place through a pore-like opening in the membrane solely to indicate that the membrane’s permeability barrier is tran­siently altered.

However, it is not known with cer­tainty whether translocation through the membrane takes place through such a proteinaceous water-filled channel or directly through the lipid bilayer. In most instances, the signal sequence is eventually cleaved from the remainder of the growing polypeptide by an extrinsic enzyme called signal peptidase attached to the lumenal surface of the ER.

If the protein being synthesized is destined for secretion from the cell, completion of synthesis is followed by the protein’s re­lease from the ribosome into the intracisternal space. The ribosome then detaches from the membrane and the mRNA and may participate in another round of protein synthesis. At the same time, the permeability barrier of the ER is restored.

Proteins discharged into the ER cisternae in this manner are ultimately con­veyed to the Golgi apparatus for chemical modifica­tion prior to secretion. In the case of pro­teins destined to be regular constituents of the endoplasmic reticulum or the plasma membrane, translocation into the intracisternal space is aborted before synthesis is finished so that the polypeptide is left anchored in the membrane (Fig. 15-19b). The in­formation halting the translocation is likely contained in the polypeptide itself and is recognized by the translocation apparatus.

For peripheral membrane proteins facing the exte­rior of the cell or the lumenal phase of the ER, the sig­nal sequence is followed by synthesis of the hydro- philic portion of the polypeptide. If an integral membrane protein is being synthesized, the hydro- philic portion is followed by a hydrophobic segment that remains anchored in the lipid bilayer. For pro­teins that span the membrane, synthesis is completed with the production of a final hydrophilic segment that faces the cytosol.

By comparing parts a and b of Figure 15-19, it is seen that the major distinction between the synthesis of secretory proteins and membrane proteins is that secretory proteins are released into the lumenal phase of the ER, whereas membrane proteins remain an­chored in the ER.

Addition of sugars to presumptive plasma membrane glycoproteins may occur soon after the hydrophilic portions of the molecules enter the ER cisternae. The membrane glycoprotein then migrates from the ER to the Golgi apparatus. Although the mechanism for this transfer is still uncertain, it is be­lieved to take place either by dispatchment of small vesicles from the ER, which then migrate to and fuse with the Golgi membranes, or by lat­eral “flow” along the ER membranes to the Golgi. Glycosylation of the membrane proteins is completed in the Golgi apparatus.

The Golgi apparatus dis­patches completed plasma membrane glycoproteins as small vesicles that migrate to and fuse with the plasma membrane. The overall process is summarized in Figure 15-20, which also shows that the intracellular/extracellular orientation of the mem­brane protein is maintained throughout its passage from the ER to the plasma membrane.

Some integral proteins have hydrophilic parts that face the interior of the cell and some peripheral proteins are associated only with the mem­brane’s cytoplasmic face. Although a similar mecha­nism is not precluded for the synthesis of these mem­brane proteins, they could be synthesized by free ribosomes.

Following release of these proteins in the cytosol they may diffuse to the plasma membrane. In­tegral proteins would be spontaneously inserted into the membrane by a hydrophobic segment (Fig. 15- 20), whereas peripheral proteins would attach to the membrane through polar interactions. Peripheral pro­teins reaching the plasma membrane in this manner could not pass through the membrane to the exterior surface because they could not traverse the hydropho­bic membrane core.

Prokaryotic cells do not contain an endoplasmic reticulum. Secretory proteins and new plasma membrane proteins are synthesized by ri­bosomes that attach to the inner surface of the plasma membrane. As in eukaryotic cells, ribosome attach­ment follows synthesis of a signal peptide encoded in the protein’s mRNA. The protein is then dispatched through the cell membrane and into the extracellular space.

Synthesis of Membrane Lipids:

In eukaryotic cells, phospholipid synthesis is associ­ated with the endoplasmic reticulum, whereas in pro­karyotic cells lipid synthesis is a property of the cyto­plasmic half of the plasma membrane. It is therefore likely that lipid synthesis in eukaryotic cells takes place in the cytoplasmic half of the ER membranes.

Following synthesis, the phospholipid becomes at least temporarily part of the interior monolayer but may ei­ther be enzymatically translocated to the outer layer or flip-flop between the two layers. In view of the fact that outer monolayer lipids are derived from the inner monolayer, an absolute asymmetry is precluded.

Because in prokaryotic cells lipid synthesis occurs in the plasma membrane, incorporation into the mem­brane’s structure is direct. In eukaryotic cells, how­ever, presumptive plasma membrane lipid must make its way from the ER to the plasma membrane.

This is believed to occur by one or both of two processes. Newly synthesized lipids inserted into ER membranes may make their way to the plasma membrane by the same mechanism that translocates ER membrane proteins (Fig. 15-20) that is both membrane proteins and lipids pass from the ER to the Golgi apparatus and are later dispatched to the plasma membrane via small vesicles.

The cytosol of eukaryotic cells contains a number of phospholipid transport proteins that function to transfer phospholipid molecules from one cellular membrane to another. These transport pro­teins might also play a major role in mediating the passage of phospholipids from the membranes of the ER to the plasma membrane.

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## New rapid synthesis developed for bilayer graphene and high-performance transistors

This is concept art of the crystal structure (top view) of AB-stacked bilayer graphene. Credit: Peter Allen, UCSB

Researchers at University of California, Santa Barbara, in collaboration with Rice University, have recently demonstrated a rapid synthesis technique for large-area Bernal (or AB) stacked bilayer graphene films that can open up new pathways for digital electronics and transparent conductor applications.

The invention also includes the first demonstration of a bilayer graphene double-gate field-effect transistor (FET), showing record ON/OFF transistor switching ratio and carrier mobility that could drive future ultra-low power and low-cost electronics.

Graphene is the thinnest known (

0.5 nanometer per layer) 2-dimensional atomic crystal. It has attracted wide interest due to its promising electrical and thermal properties and potential applications in electronics and photonics. However, many of those applications are significantly restricted by the zero band gap of graphene that results in leaky transistors not suitable for digital electronics.

"In addition to its atomically smooth surfaces, a considerable band gap of up to

0.25 eV can be opened up in bilayer graphene by creating a potential difference between the two layers, and thereby breaking the inherent symmetry, if the two layers can be aligned along a certain (Bernal or AB) orientation" explained Kaustav Banerjee, professor of electrical and computer engineering and Director of the Nanoelectronics Research Lab at UCSB. "The dual-gated transistors were specifically designed to allow such potential difference to be established between the layers through one of the gates, while the second gate modulated the carriers in the channel," he added. Banerjee's research team also includes UCSB researchers Wei Liu, Stephan Kraemer, Deblina Sarkar, Hong Li and Professor Pulickel Ajayan of Rice University. Their study was recently published in Chemistry of Materials.

This is concept art of a schematic view of an AB-stacked graphene film synthesized by UC Santa Barbara researchers using a stoichiometry engineered bifunctional alloy catalyst. Credit: Peter Allen, UCSB

The graphene films were grown in a deterministic manner using an engineered bifunctional (Cu:Ni) alloy surface at a relatively low temperature of 920 °C. Large-area (> 3 inch x 3 inch) Bernal (or AB) stacked bilayer graphene growth was demonstrated within few minutes and with nearly 100% area coverage. The bilayer graphene films exhibited electron mobility as high as 3450 cm2/(V*s), which is comparable to that of exfoliated bilayer graphene, thereby confirming very high-quality. The quality of grown graphene was further corroborated by demonstration of high-performance FETs with record ON/OFF ratio that is a key requirement in low-power digital electronics.

"Achieving surface catalytic graphene growth mode and precise control of the surface carbon concentration were key factors for the favorable growth kinetics for AB stacked bilayer graphene," explained Wei Liu, a post-doctoral researcher in Banerjee's group and a co-author of the article. In 2011, Banerjee's group demonstrated a large-area monolayer graphene synthesis method using a copper substrate as catalyst.

Bilayer graphene is close to monolayer graphene in terms of the film thickness with a hexagonal atomic structure and can be derived from its layered bulk form (graphite) in which adjacent layers are held together by relatively weak van der Waals forces. "However, apart from its band gap tunability, bilayer graphene has some key advantages over monolayer graphene. It has higher density of states and suffers much less from interface effects, which are beneficial for improving the current carrying capability," Liu continued.

"This demonstration is very impressive and should have far-reaching implications for the entire 2D materials community," commented Professor Ali Javey, of University of California, Berkeley and a Co-Director of the Bay Area Photovoltaic Consortium (BAPVC).

## Peroxisomes

Peroxisomes are small, round organelles enclosed by single membranes. They carry out oxidation reactions that break down fatty acids and amino acids. They also detoxify many poisons that may enter the body. Alcohol is detoxified by peroxisomes in liver cells. A byproduct of these oxidation reactions is hydrogen peroxide, H2O2, which is contained within the peroxisomes to prevent the chemical from causing damage to cellular components outside of the organelle. Hydrogen peroxide is safely broken down by peroxisomal enzymes into water and oxygen.

## Endoplasmic Reticulum Function

The ER plays a number of roles within the cell, from protein synthesis and lipid metabolism to detoxification of the cell. Cisternae, each of the small folds of the endoplasmic reticulum, are commonly associated with lipid metabolism. This creates the plasma membrane of the cell, as well as additional endoplasmic reticulum and organelles. They also appear to be important in maintaining the Ca 2+ balance within the cell and in the interaction of the ER with mitochondria. This interaction also influences the aerobic status of the cell.

Although ER sheets and tubules appear to have distinct functions, there isn’t a perfect delineation of roles. For instance, in mammals tubules and sheets can interconvert, making the cells adaptable to various conditions. The relationship between structure and function in the ER has not been completely elucidated.

### Protein Synthesis and Folding

Protein synthesis occurs in the rough endoplasmic reticulum. Although translation for all proteins begins in the cytoplasm, some are moved into the ER in order to be folded and sorted for different destinations. Proteins that are translocated into the ER during translation are often destined for secretion. Initially, these proteins are folded within the ER and then moved into the Golgi apparatus where they can be dispatched towards other organelles.

For instance, the hydrolytic enzymes in the lysosome are generated in this manner. Alternately, these proteins could be secreted from the cell. This is the origin of the enzymes of the digestive tract. The third potential role for proteins translated in the ER is to remain within the endomembrane system itself. This is particularly true for chaperone proteins that assist in the folding of other proteins. The genes encoding these proteins are upregulated when the cell is under stress from unfolded proteins.

### Lipid Synthesis

The smooth endoplasmic reticulum plays an important role in cholesterol and phospholipid biosynthesis. Therefore, this section of the ER is important not only for the generation and maintenance of the plasma membrane but of the extensive endomembrane system of the ER itself.

The SER is enriched in enzymes involved in sterol and steroid biosynthetic pathways and is also necessary for the synthesis of steroid hormones. Therefore the SER is extremely prominent in the cells of the adrenal gland that secrete five different groups of steroid hormones that influence the metabolism of the entire body. The synthesis of these hormones also involves enzymes within the mitochondria, further underscoring the relationship between these two organelles.

### Calcium Store

The SER is an important site for the storage and release of calcium in the cell. A modified form of the SER called sarcoplasmic reticulum forms an extensive network in contractile cells such as muscle fibers. Calcium ions are also involved in the regulation of metabolism in the cell and can change cytoskeletal dynamics.

The extensive nature of the ER network allows it to interact with the plasma membrane and use Ca 2+ for signal transduction and modulation of nuclear activity. In association with mitochondria, the ER can also use its calcium stores to induce apoptosis in response to stress.

## What Is the Purpose of a Phospholipid Bilayer?

The phospholipid bilayer's function is to maintain a barrier between the cell and its external environment and to store and transport a variety of proteins that are essential to the cell's function. It controls what enters and exits the cell.

The phospholipid bilayer serves as the main barrier between the cell's internal components and its extracellular environment, which consists mainly of the cytoplasm. By doing this, it controls and maintains a balance of molecules that are present in the cell, such as proteins and ions. Several proteins exist in the bilayer that help to control what enters and exits the cell. These are often referred to as transmembrane or transporter proteins. The cell alters the gene expression of these proteins to create more or less in response to its need to transport items across the membrane.

While the uptake of molecules into the cell is one major function, the bilayer also serves to prevent certain molecules from entering that may be harmful to the cell. Molecules housed on the surface of the bilayer have several other functions in addition to transport. For example, they may serve as communicating molecules, signaling messages between the internal part of the cell and external molecules such as proteins.