This essay summarizes the structure and function of membranes and the proteins within them, and describes their role in trafficking and transport, and their involvement in health and disease. Techniques for studying membranes are also discussed. Biological membranes consist of a double sheet known as a bilayer of lipid molecules. This structure is generally referred to as the phospholipid bilayer. In addition to the various types of lipids that occur in biological membranes, membrane proteins and sugars are also key components of the structure.
Membrane proteins play a vital role in biological membranes, as they help to maintain the structural integrity, organization and flow of material through membranes. Sugars are found on one side of the bilayer only, and are attached by covalent bonds to some lipids and proteins. Three types of lipid are found in biological membranes, namely phospholipids, glycolipids and sterols.
Phospholipids consist of two fatty acid chains linked to glycerol and a phosphate group. Phospholipids containing glycerol are referred to as glycerophospholipids. An example of a glycerophospholipid that is commonly found in biological membranes is phosphatidylcholine PC Figure 1 a , which has a choline molecule attached to the phosphate group. Serine and ethanolamine can replace the choline in this position, and these lipids are called phosphatidylserine PS and phosphatidylethanolamine PE , respectively.
Phospholipids can also be sphingophospholipids based on sphingosine , such as sphingomyelin. Glycolipids can contain either glycerol or sphingosine, and always have a sugar such as glucose in place of the phosphate head found in phospholipids Figure 1 b. Sterols are absent from most bacterial membranes, but are an important component of animal typically cholesterol and plant mainly stigmasterol membranes.
Cholesterol has a quite different structure to that of the phospholipids and glycolipids. The sugars attached to lipids and proteins can act as markers due to the structural diversity of sugar chains. For example, antigens composed of sugar chains on the surface of red blood cells determine an individual's blood group. These antigens are recognized by antibodies to cause an immune response, which is why matching blood groups must be used in blood transfusions.
Other carbohydrate markers are present in disease e. All membrane lipids are amphipathic—that is, they contain both a hydrophilic water-loving region and a hydrophobic water-hating region. Thus the most favourable environment for the hydrophilic head is an aqueous one, whereas the hydrophobic tail is more stable in a lipid environment. The amphipathic nature of membrane lipids means that they naturally form bilayers in which the hydrophilic heads point outward towards the aqueous environment and the hydrophobic tails point inward towards each other Figure 2 a.
When placed in water, membrane lipids will spontaneously form liposomes, which are spheres formed of a bilayer with water inside and outside, resembling a tiny cell Figure 2 b. This is the most favourable configuration for these lipids, as it means that all of the hydrophilic heads are in contact with water and all of the hydrophobic tails are in a lipid environment. Spontaneous formation of bilayers by membrane lipids. The hydrophilic heads pink circles will always face the aqueous environment in bilayers a and liposomes b. The hydrophobic tails will face inward away from the water.
Early experiments by E. Grendel in were the first to demonstrate that biological membranes are bilayers. These researchers extracted the lipids from red blood cells and found that they occupied a space that was twice the surface area of the cell. Red blood cells contain no internal membranes, so they deduced that the plasma membrane must be composed of two layers of lipids. The fluid mosaic model proposed by Jonathan Singer and Garth Nicolson in describes the dynamic and fluid nature of biological membranes.
Lipids and proteins can diffuse laterally through the membrane. Phospholipids can diffuse relatively quickly in the leaflet of the bilayer in which they are located. A phospholipid can travel around the perimeter of a red blood cell in around 12 s, or move the length of a bacterial cell within 1 s. Phospholipids can also spin around on their head-to-tail axis, and their lipid tails are very flexible.
These different types of movements create a dynamic, fluid membrane which surrounds cells and organelles. Membrane proteins can also move laterally in the bilayer, but their rates of movement vary and are generally slower than those of lipids. In some cases, membrane proteins are held in particular areas of the membrane in order to polarize the cell and enable different ends of the cell to have different functions.
One example of this is the attachment of a glycosyl-phosphatidylinositol GPI anchor to proteins to target them to the apical membrane of epithelial cells and exclude them from the basolateral membrane. Fluorescence photobleaching is one experimental method that is used by scientists to demonstrate visually the motility of proteins and lipids in a bilayer Figure 3. A lipid or membrane protein located on the surface of a cell is tagged with a fluorescent marker such as green fluorescent protein GFP. A beam of laser light is then focused on to a small area of the cell surface using a fluorescence microscope in order to bleach the fluorescent tags in this area so that they no longer emit a fluorescence signal.
This small area of membrane is observed over time and gradually the fluorescence increases again, indicating that other tagged proteins or lipids are diffusing into this region from elsewhere in the membrane. This demonstrates that the lipid bilayer surrounding cells is fluid in nature and allows lateral diffusion of both lipids and membrane proteins. Cells expressing a GFP-labelled protein in the endoplasmic reticulum were subjected to photobleaching. Adapted from Figure 1b from Lippincott-Schwartz, J. This is due to the energetic barrier encountered when forcing the hydrophilic head in the case of lipids or hydrophilic regions in the case of proteins through the hydrophobic environment of the inside of the membrane.
This near absence of vertical movement allows the inner and outer leaflets of the bilayer to maintain different lipid compositions, and enables membrane proteins to be inserted in the correct orientation for them to function. However, some enzymes facilitate the process of lipid flip-flop from one leaflet to another. These flippases, or phospholipid translocators, use ATP to move lipids across the bilayer to the other leaflet.
In eukaryotic cells, flippases are located in various organelles, including the endoplasmic reticulum ER , where they flip-flop newly synthesized lipids. Biological membranes are formed by adding to a pre-existing membrane. In prokaryotes this occurs on the inner leaflet of the plasma membrane, facing the cytoplasm. Lipids then leave the ER and travel through the secretory pathway for distribution to various subcellular compartments or the plasma membrane.
In eukaryotic cells, enzymes that span the ER catalyse the formation of membrane lipids. In the cytoplasmic leaflet of the ER membrane, two fatty acids are bound, one by one, to glycerol phosphate from the cytoplasm. This newly formed diacylglycerol phosphate is anchored in the ER membrane by its fatty acid chains.
The phosphate is then replaced by the head group e. Flippases in the ER membrane can then move some of these newly formed lipids to the luminal side of the ER membrane. Similarly, flippases in prokaryotes can transfer new lipids from the inner leaflet of the plasma membrane to the outer leaflet. These flippases are responsible for adjusting the lipid composition of each layer of the membrane. In eukaryotes, lipids must then be distributed to the various intracellular membranes. The traffic of vesicles between organelles in combination with signals that direct particular lipids to specific locations is required to create the correct lipid composition in all of the cellular membranes Figure 4.
The Golgi then sends lipids in vesicles to various destinations, including the plasma membrane and lysosomes. Lipids and proteins are internalized from the plasma membrane into endosomes. Organelles, such as mitochondria, acquire lipids from the ER by a different mechanism. Water-soluble proteins called phospholipid-exchange proteins remove phospholipids from the ER membrane and deposit them in the membranes of the appropriate organelles.
The main compartments of eukaryotic cells are shown. Arrows indicate movement of lipid vesicles between them, with colours at the tail end indicating origin and those at the head end indicating destination. The inner and outer leaflets of bilayers differ in their lipid composition. In mammalian cells, the outer leaflet of the plasma membrane contains predominantly PC and sphingomyelin, whereas PS and PE are found on the inner leaflet. During programmed cell death apoptosis , PS is no longer restricted to the inner leaflet of the plasma membrane.
It is exposed on the outer leaflet by the action of an enzyme called scramblase which is a type of flippase enzyme. PS is negatively charged, unlike PC, which has no net charge. The movement of PS into the outer leaflet therefore changes the charge of the plasma membrane as viewed from the outside of the cell.
This change in surface charge labels the apoptotic cell for phagocytosis by phagocytic cells such as macrophages. Lipid composition also varies between the organelles within eukaryotic cells. Cholesterol is synthesized in the ER, but the ER membrane has a relatively low cholesterol content, as much of the cholesterol is transported to other cellular membranes. The prevalence of cholesterol in membranes increases through the secretory pathway, with more in the Golgi than in the ER the trans -Golgi network is richer in cholesterol than the cis -Golgi , and most in the plasma membrane.
This increase in cholesterol through the secretory pathway results in slightly thicker membranes in the late Golgi and plasma membrane compared with the ER, and is thought to be a contributing factor to protein sorting through the pathway, as membrane proteins in the plasma membrane generally have longer hydrophobic transmembrane domains than membrane proteins that reside in the ER. Membrane proteins are the nanomachines that enable membranes to send and receive messages and to transport molecules into and out of cells and compartments.
Without membrane proteins the phospholipid membrane would present an impenetrable barrier and cells would be unable to communicate with their neighbours, transport nutrients into the cell or waste products out of it, or respond to external stimuli. Both unicellular and multicellular organisms need membrane proteins in order to live. The membrane proteins that are present in a particular membrane determine the substances to which it will be permeable and what signal molecules it can recognize.
In eukaryotic cells, the synthesis of membrane proteins destined for the plasma membrane, ER or any other membrane-bound compartment begins on cytosolic ribosomes. After a short segment of protein has been synthesized, the ribosome, mRNA and nascent protein chain associate with the ER, where the rest of the protein is made and simultaneously inserted into the membrane. We now know that there is an N-terminal signal sequence within membrane proteins.
These signal sequences are not identical but share a common motif, namely a hydrophobic stretch of 20—30 amino acids, a basic region at the N-terminus and a polar domain at the C-terminus of the signal.
Structure and organization of membranes
These N-terminal signal sequences are recognized by the signal recognition particle SRP , which has binding sites for the signal sequence, ribosome and the SRP receptor which is embedded in the ER membrane. Upon binding the SRP, the ribosome pauses protein synthesis. The translocon is a protein pore through which membrane protein chains can be threaded into the membrane. It has a laterally opening gate to allow newly synthesized proteins into the ER membrane.
Once the ribosome is at the translocon, the SRP dissociates and protein synthesis resumes. This process is referred to as co-translational targeting, and the main events are summarized in Figure 5. The key steps of ER targeting are summarized. Each component is labelled and the ER membrane is represented by double blue lines. The signal sequence shown in black becomes the first transmembrane domain of the protein in this example.
Co-translational targeting is the dominant mechanism for protein delivery to the ER in higher eukaryotes, whereas yeast and prokaryotes favour post-translational targeting, whereby proteins are delivered to the ER after completion of synthesis. Post-translational targeting also occurs in higher eukaryotes, often when a membrane protein is so small that the signal sequence does not emerge until the whole protein has been synthesized. Membrane-spanning proteins are diverse in structure and function. In addition, there are other non-spanning proteins which associate with the bilayer, often using a hydrophobic anchor.
A vital class of membrane proteins are those involved in active or passive transport of materials across the cell membrane or other subcellular membranes surrounding organelles. For a cell or an organism to survive, it is crucial that the right substances enter cells e. Molecules can cross biological membranes in several different ways depending on their concentration on either side of the membrane, their size and their charge.
Some molecules, including water, can simply diffuse through the membrane without assistance. However, large molecules or charged molecules cannot cross membranes by simple diffusion. Charged molecules such as ions can move through channels passively, down electrochemical gradients. This requires channel proteins but no energy input. Passive transport can also be mediated by carrier proteins that carry specific molecules such as amino acids down concentration gradients, again without any requirement for energy. Active transport moves species against concentration gradients and requires energy, which is obtained from ATP, from light, or from the downhill movement of a second type of molecule or ion within the same transporter Figure 6.
The different types of membrane proteins involved in passive and active transport are shown. Passive transport is the movement of molecules across biological membranes down concentration gradients. This type of transport does not require energy. Channels form water-filled pores and thus create a hydrophilic path that enables ions to travel through the hydrophobic membrane.
These channels allow downhill movement of ions, down an electrochemical gradient. Both the size and charge of the channel pore determine its selectivity. Different channels have pores of different diameters to allow the selection of ions on the basis of size. The amino acids that line the pore will be hydrophilic, and their charge will determine whether positive or negative ions travel through it. Channels are not always open. They can be gated by ligands which bind to some part of the protein, either by a change in membrane potential voltage gated or by mechanical stress mechanosensitive.
The nicotinic acetylcholine receptor is an example of a ligand-gated ion channel which opens upon binding the neurotransmitter acetylcholine Figure 7. The nicotinic acetylcholine receptor is a pentameric membrane protein composed of five subunits arranged in a ring, with a pore through the centre.
In the closed state, the pore is blocked by large hydrophobic amino acid side chains which rotate out of the way upon acetylcholine binding to make way for smaller hydrophilic side chains, allowing the passage of ions through the pore. This creates a net movement of positive charges into the cell, resulting in a change in membrane potential. Acetylcholine released by motor neurons at the neuromuscular junction travels across the synapse and binds to nicotinic acetylcholine receptors in the plasma membrane of the muscle cells, causing membrane depolarization.
The pentameric structure of the receptor is shown, with the pore region P indicated. Transmembrane helices M1—M4 are labelled in each subunit. The bilayer is shown in orange. Reproduced from Berridge, M. Carrier proteins are the other class of membrane proteins, apart from channels, which can facilitate passive transport of substances down concentration gradients. Carrier proteins transport molecules much more slowly than channels, as a number of conformational changes in the carrier are required for the transport of the solute across the membrane.
A molecule such as a sugar binds to the carrier protein on one side of the membrane where it is present at a high concentration. Upon binding, the carrier changes conformation so that the sugar molecule then faces towards the opposite side of the membrane. The concentration of sugar on this side is lower, so dissociation occurs and the sugar is released. This is downhill movement and, although slower than movement through channels, it requires no energy.
A heritable change in the CFTR gene which results in a single amino acid deletion in the protein causes cystic fibrosis. This is a serious illness in which thick mucus accumulates in the lungs, causing a significantly lower than average life expectancy in patients who have the disease. Unimpaired ion transport is vital for our survival and health, and conditions such as cystic fibrosis highlight the need for research into these types of proteins.
The transport of molecules across a membrane against a concentration gradient requires energy, and is referred to as active transport. Calcium ions signal many events, including muscle contraction, neurotransmitter release and cellular motility. There are many P-type ATPases, and they are conserved in evolution across many species.
We have now obtained three-dimensional structures of SERCA in a number of conformational states, which allow scientists to visualize the transport process. The three cytoplasmic domains, phosphorylation P , nucleotide binding N and actuator A are labelled. Secondary active transport requires an ion electrochemical gradient to drive the uphill transport of another solute. The downhill movement of one species drives the uphill movement of the other. This can be symport in which both types of molecule or ion travel across the membrane in the same direction or antiport in which the two species travel in opposite directions , as shown in Figure 9.
In order to understand more fully the mechanisms of action of membrane proteins such as the transporters described here, we can determine their three-dimensional protein structures. As a result of huge advances in structural biology in the last 50 years, we now have access to many thousands of protein structures in online databases. This enables researchers to visualize the structure of their protein of interest, and thus gain insight into its mechanism.
The structure of whale myoglobin was solved in using X-ray crystallography, earning John C. This was the first protein structure to be solved using this technique, and since then thousands of proteins have been solved using this method. X-ray crystallography works by firing a beam of X-rays at a crystalline structure and measuring the diffraction of the X-rays after they have passed through the structure of interest.
This generates an electron density map, showing where different atoms in the structure are located. For regular crystalline solids such as salts this is relatively straightforward, but for large irregular molecules such as proteins it can present many technical challenges. Before a protein is subjected to X-ray beams, it must first be purified and crystallized.
In nature, proteins exist in the busy milieu of a cell, surrounded by thousands of other types of proteins, as well as lipids and other molecules. A common method of obtaining enough of the protein of interest involves expressing the relevant gene in a system such as bacteria. The gene is tagged with a small protein tag which can be used to isolate the protein of interest. Bacterial systems allow large amounts of protein to be produced cheaply and quickly. However, if the protein of interest is from a species that is only distantly related to that in which it is normally expressed e.
International Journal of Cell Biology
In addition, the expression of membrane proteins that make pores or channels can kill the host organism. A pure protein sample is then crystallized by allowing water to evaporate away, in exactly the same way as a solution of salt will form crystals naturally when left to dry. Optimum conditions for this must be determined, and crystallization conditions are not always straightforward, as they differ from one protein to another.
For soluble proteins such as myoglobin this is easier than for insoluble membrane proteins. Membrane proteins have lipid-soluble domains that will not dissolve in an aqueous medium. This significantly decreases the ease with which membrane protein structures can be solved using X-ray diffraction. However, there are ways in which scientists can overcome this difficulty.
Generally, membrane proteins are removed from the membrane in which they were made and placed in an environment of lipids and detergents for crystallization. Sometimes the lipids associated with the protein are apparent in the crystal structure. The number of solved crystal structures of proteins is constantly growing as technology improves and expertise is shared among scientists to help to optimize conditions for crystal production. The Protein Data Bank PDB is an online archive of protein structures which can be freely accessed by scientists worldwide.
The number of membrane protein structures in the PDB is increasing rapidly with the refinement of crystallization techniques Figure The increase in the number of solved crystal structures of membrane proteins is shown from , when the first such structure was solved. Adapted from White, S. Nature , — Nuclear magnetic resonance NMR spectroscopy is another valuable technique for elucidating membrane protein structure. Molecules are placed in a magnetic field and the resonance properties of different atomic nuclei are measured, which gives an indication of where in a particular molecule different atoms are located.
Generally, NMR is limited to smaller proteins, below around 35 kDa in size. It also offers the potential to visualize proteins in a more physiologically relevant environment e. Another advantage of NMR is that it does not require the protein to be locked in a crystal lattice—a structure which can distort the natural shape of the protein.
Electron microscopy can also be used to study membrane protein structure. By freezing membrane proteins in their natural lipid environments, it is possible to investigate their structure using high-resolution electron microscopy. This provides a snapshot of the naturally occurring conformation of individual proteins in the bilayer. The lipids that surround membrane proteins in biological membranes play an important role in the activity of these proteins.
As was mentioned earlier, some membrane protein crystal structures include lipids bound to the outside surface of the transmembrane domains of the proteins. It is thought that these lipids bind tightly to the protein, and have a long-lived interaction with the transmembrane region. In other cases, lipids are thought to interact briefly with membrane proteins, rapidly moving away and being replaced by other membrane lipids.
The activity of membrane proteins is considered to be dependent to some extent on the lipids that surround them in the membrane. These types of interaction can be studied by placing a purified form of the protein of interest in an artificial bilayer and measuring its activity. By altering the types of lipid present in the artificial bilayer, deductions can be made about the lipids that the protein requires in order to be active. Fluorescence spectroscopy and electron spin resonance are two techniques that are used to measure how strongly membrane proteins interact with specific lipids around them.
Molecular dynamics simulations use computer algorithms to work through theoretical problems. These simulated experiments are useful for investigating interactions between membrane proteins and lipids, as in real membranes these interactions are often so fleeting that they are very difficult to measure. Molecular dynamics simulations have predicted that in the case of the nicotinic acetylcholine receptor, the negatively charged lipid, phosphatidic acid, is required for activity.
These simulations have also shown that cholesterol stabilizes the receptor and that the phosphatidic acid forms a shell around the protein which is more long-lasting than the interactions with other membrane lipids. Although molecular dynamics simulations are extremely useful, they are limited by the assumptions and approximations on which they are based.
As in many areas of biology, a combination of experimental and computational research is required if real progress is to be made in understanding the complexity of biological membranes. Inside the plasma membrane that surrounds eukaryotic cells lie many other membranes which define the intracellular compartments, or organelles.
Each of these organelles has distinct functions and contains specific complements of proteins adapted for these roles. With the exception of a few proteins that are coded for by the mitochondrial genome, synthesis of all of the proteins that are required in these organelles begins on ribosomes in the cytoplasm, and therefore the proteins must be directed to the correct destination.
We have seen earlier how this is achieved with membrane proteins, and most organelles have some kind of signal sequence that can be recognized by various receptors and which ensures that the protein arrives at the correct organelle. Besides the specific protein complement of each organelle, the lipid make-up of the bilayers surrounding organelles varies. Lipids are synthesized in the ER, and flippases move lipid molecules between leaflets of the bilayer. For organelles in the secretory pathway and the plasma membrane, lipid transport into these compartments is mediated by vesicular membrane traffic through the pathway.
The cholesterol concentration in membranes increases from the ER through the Golgi to the plasma membrane. Cholesterol makes membranes thicker and more rigid, so the low levels of cholesterol in the ER membrane render it thin and facilitate the insertion of newly synthesized membrane and secretory proteins. PC becomes relatively less abundant through this pathway, with more found in the ER than at the plasma membrane. PS and PE are found throughout the secretory pathway in the cytosolic leaflet of the membranes.
This differential lipid composition through the secretory pathway is achieved by targeting specific lipids into transport vesicles. Proteins included in these vesicles act as labels and direct the lipids to the right compartment. Forward-moving anterograde vesicles destined for the plasma membrane are rich in cholesterol. Lipids also move backwards through the secretory pathway, from the plasma membrane towards the ER. This is known as retrograde traffic. Retrograde vesicles from the Golgi are enriched in lipids such as PC, which are concentrated in the ER.
The lipid composition of the mitochondria is very different from that of the secretory pathway compartments. Mitochondrial membranes are much richer in PE and cardiolipin than is the ER. Cardiolipin is synthesized in the mitochondria and is predominantly confined to this organelle. As membrane proteins have evolved along with their organelles and surrounding lipids, it follows that different lipid compositions are required in different organelles for the optimum activity of the proteins within their membranes.
The activity of this carrier protein is dependent on the presence of cardiolipin, which is relatively abundant in mitochondrial membranes. The targeting of newly synthesized membrane and secretory proteins to the ER has already been briefly discussed. However, there are many different destinations within the cell to which a protein can be sent, and sometimes proteins are located in more than one of these.
The signals and protein machinery that are required to target proteins to the correct compartment are many and various, and much of the detail of the exact mechanisms involved has yet to be clarified.
Traffic through the secretory pathway is by vesicular transport in both anterograde and retrograde directions. Proteins and lipids can be included and excluded from vesicles by various means in order to selectively determine which molecules move forward or backward through the pathway.
Biological membranes | Essays in Biochemistry
Vesicles are coated with proteins that determine their destination. Nesprin-1 is a giant tail-anchored nuclear envelope protein composed of an N-terminal F-actin binding domain, a long linker region formed by multiple spectrin repeats and a C-terminal transmembrane domain. Based on this structure, it connects the nucleus to the actin cytoskeleton. Earlier reports had shown that Nesprin-1 binds to nuclear envelope proteins emerin and lamin through C-terminal spectrin repeats.
These repeats can also self-associate. We focus on the N-terminal Nesprin-1 sequences and show that they interact with Nesprin-3, a further member of the Nesprin family, which connects the nucleus to the intermediate filament network. We show that upon ectopic expression of Nesprin-3 in COS7 cells, which are nearly devoid of Nesprin-3 in vitro, vimentin filaments are recruited to the nucleus and provide evidence for an F-actin interaction of Nesprin-3 in vitro. We propose that Nesprins through interactions amongst themselves and amongst the various Nesprins form a network around the nucleus and connect the nucleus to several cytoskeletal networks of the cell.
The nuclear envelope is a barrier separating the nucleus from the cytoplasm. It consists of two lipid bilayers, the outer nuclear membrane ONM which is continuous with the endoplasmic reticulum ER and the inner nuclear membrane INM. The INM is intimately linked with the nuclear lamina, a network of intermediate filament proteins, the lamins, and lamina-associated proteins.
In addition to its barrier function the NE provides a link to the cytoskeleton through which the shape of the nucleus and its position in the cell is maintained. Important players in this scenario are the Nesprins [ 1 ]. Nesprins nuclear envelope spectrin repeat proteins comprise a large family of spectrin repeat SR containing type II transmembrane proteins localizing to both nuclear membranes with evolutionarily conserved orthologs in lower organisms including S. To date, four proteins belonging to the Nesprin family have been identified in mammals, each encoded by a different gene that gives rise to multiple isoforms.
Nesprin-3 harbors an N-terminal binding site for plectin, a large cytolinker which can interact with intermediate filaments, microtubules and actin filaments, and a C-terminal transmembrane region [ 13 , 14 ]. Nesprin-4 binds to kinesin-1 and is involved in microtubule-dependent nuclear positioning [ 15 ]. Nesprins are also essential components of the LINC complex linker of nucleoskeleton and cytoskeleton that traverses the NE to connect the nuclear interior with the cytoskeleton in the cytoplasm.
Several biologically important functions have been attributed to the LINC complex including nuclear anchorage, nuclear migration, anchoring the MTOC to the nucleus, ciliogenesis, and regulation of chromosome dynamics [ 19 — 21 ]. Here we focus on the N-terminal region of Nesprin The spectrin repeat is an ancient fold and has already been found in proteins in the amoebozoan lineage [ 28 ]. Structurally, the spectrin repeat is distinguished from other three-helix domains via its characteristic length, its left-handed twist, and localization of the termini to the distal ends of the domain. They also serve as a platform for cytoskeletal and signal transduction proteins [ 29 ].
Self-association through spectrin repeats has been shown for a C-terminal Nesprin-1 isoform where two molecules interact with each other through distinct C-terminal spectrin repeats to form an antiparallel dimer [ 26 , 32 ]. Furthermore, the mechanical properties of spectrin repeats make them ideal candidates as components in structures that are exposed to great mechanical stress, such as the cell cortex, the muscle sarcomere, and stress fibers.
We carried out an analysis of sequences contained in the N-terminal isoform Nesprin previously Enaptin [ 11 ] and investigated possible interactions. We detected interactions between spectrin repeats and an interaction of Nesprin-1 with Nesprin Nesprin-1 also binds to Nesprin-3 enhancing the network and furthermore connecting the nucleus through this interaction to the intermediate filament network.
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- Cytoskeletal Interactions at the Nuclear Envelope Mediated by Nesprins?
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A polypeptide encompassing residues — could not be expressed to detectable levels. The following antibodies were used: The cells were imaged using a confocal laser scanning microscope. The purification of GST and the fusion proteins and pull down assays were performed as described elsewhere [ 37 ]. The blotting times varied from 48 to 72 hours depending on the size of the proteins to be detected. Equal loading was assessed by Ponceau S staining of the blots. Signals were detected using appropriate horseradish peroxidase coupled secondary antibodies followed by enhanced chemiluminescence.
The assay was performed with pH values between 6. Supernatant and pellet fractions were separated and resuspended in 5x SDS sample buffer. Actin was isolated from D. Nesprin-1 has several isoforms that perform varied functions in different cell types. We used monoclonal antibody K that was generated against a recombinant polypeptide harbouring the last two spectrin repeats aa — of mouse Nesprin to study Nesprin-1 isoform expression Figure 1 a. This region corresponds to the C-terminus of mouse CPG2 and does not show homology to Nesprin-2 or -3 making the antibody specific for Nesprin Thus the data indicate the presence of N-terminal Nesprin-1 polypeptides of varying size Figure 1 b.
We cannot exclude the possibility that the smaller forms are breakdown products of the larger protein. Spectrin repeat-containing proteins can form higher-order structures by virtue of interaction among their spectrin repeats; they are also platforms for interaction with other proteins [ 29 ]. Like Nesprin-1, Nesprin-3 also dimerizes as shown by coimmunoprecipitation experiments. Furthermore, it coprecipitates with the ABD of plectin [ 13 ].
To determine whether N-terminal sequences of Nesprin-1 can interact with themselves we used bacterially produced GST-fusion proteins encompassing several spectrin repeats of Nesprin aa —, —, and —; Figure 2 a to pull down the corresponding GFP-tagged proteins from COS7 cells. All fusion proteins had the ability to pull down their GFP-tagged counterparts, albeit to differing degrees whereas GST alone did not Figure 2 b. By pull down experiments we next investigated the possibility of an association between Nesprin-1 and Nesprin-3 through their spectrin repeats.
GST served as negative control for Nesprin-3 binding. Our data suggest that Nesprin-1 associates with Nesprin-3 through several spectrin repeats. We performed yeast-two hybrid analysis to obtain additional support for the association between Nesprin-1 and No interaction was detected in the negative controls Figure 3 b.
In general, the yeast-two hybrid assay is a first screen for the identification of novel binding partners which then needs to be confirmed by independent methods. In our case we first carried out two sets of independent experiments and only then used the yeast two-hybrid assay which appears to have given a false-positive reaction. Based on the findings that the ABD of Nesprin-1 can bind to F-actin with high affinity and can also bundle actin filaments [ 11 ] we next asked the question whether the interaction between Nesprin-1 and Nesprin-3 interferes with the F-actin binding activity of Nesprin-1 and performed competitive F-actin co-sedimentation assays in the presence of Nesprin-3 polypeptides.
We therefore carried out all further experiments at a pH below 7. Instead, the protein was also observed in the F-actin pellet Figure 4 a. The latter proposal is not unprecedented. GST NE3 SR1,2,3 co-precipitated with F-actin in spin down assays whereas in the absence of F-actin the majority of the protein stayed in the supernatant under polymerizing conditions Figure 4 a. In these assays SR1,2 did bind to F-actin. Upon addition of actin the majority of the Nesprin-3 peptide pelleted with F-actin.
We conclude that NE SR1,2 has the ability to interact with F-actin whereas for SR1 and SR2 we cannot exclude the possibility that a successful F-actin interaction is prevented by inappropriate folding of the polypeptides.