Here is my paper on Clathrin

Tommy Kelsey
FYS: Harnessing Life’s Molecular Machines
Paper 3

Clathrin

Molecular machines are the basic mechanical models that drive some of the most important physiological functions. The abilities of the eye to see and the muscle to flex are both physiological responses to the work of millions of molecular machines. Molecular machines are simply molecular compounds that are designed to carry out various forms of mechanical movement. For example, the actin in the muscle fibers are pulled closer together as a result of shape change in the myosin fibers of the muscle. With the understanding of these molecular machines that run the biological processes of organisms, enormous advances can be made in the fight against viruses such as the JC and simian viruses. Each cell has thousands of molecular machines that are responsible for many different tasks, such as DNA replication, particle transport, and intracellular communication. Clathrin is a molecular machine that is important for allowing foreign substances to enter the cell.
Clathrin is a protein that is responsible for creating coated pits and vesicles that are necessary for bringing substances into and out of the cell and bypassing the cellular membrane. This process, called endocytosis, is responsible for transporting molecules, such as large polar proteins, past the hydrophobic cellular membrane. Clathrin assists this process by forming a coated pit on the inner surface of the plasma membrane. This vesicle then buds off and forms a relatively large vesicle on the inner part of the cell membrane. This vesicle brings with is not only the wanted substance but also a small volume of extracellular fluid. These coated pits and vesicles were first observed by Thomas Roth and Keith Porter in 1964.
Clathrin molecules are brought together on the portion of the cell membrane that is to become a vesicle by adaptor molecules. An example of this is the creation of a synaptic vesicle in the neuron using the protein AP180. This protein promotes polymerization of clathrin and also recruits them to one destination. Once the clathrin molecules are brought together on a specific part of the membrane, they begin to form a soccer ball shaped bubble with flat hexagons and pentagons as the borders. To understand how this structure is built by this molecular machine it is important to understand the structure of clathrin.
http://www.rcsb.org/
Figure 1
The clathrin has a triskelion structure with three long and skinny arms. Triskelion simply means that this machine has three main arms branching off its main central body (Figure 1). These arms come into play later on when forming the bubble necessary for transportation. At the end of each of the legs, is a foot that is the main component in binding to the adaptor proteins. These legs are flexible enough to form a soccer ball like sphere with flat hexagons and pentagons as the border. This hollow sphere is then able to encase the desired package and bring it into the cell. The full capsule ends up being much larger than the proteins being carried which is the reason why extra cellular fluid is also brought into the cell. If clathrin molecules are isolated and incubated at the correct temperature, they would spontaneously assemble into the spherical soccer ball shape.
As shown in Figure 1, the legs of clathrin consist of a series of 10 alpha helix repeats joined by hairpin turns, seven of which make up one leg of the protein. These segments are formally known as clathrin heavy-chain repeats. Each repeat consists of two faces of helices that is involved in the interaction of other helix faces of another clathrin protein. This may be the way in which the clathrin molecules are able to attach to each other to make the spherical vesicle. This can be supported by the finding of similar helices faces in other proteins that are involved in the formation of vacuoles.
Endocytosis is a process that allows for the uptake of external materials by cells and utilizes receptors to prevent uptake of unwanted materials. A specific receptor protein is embedded in the membrane of the cell and binds to the extracellular molecule, also known as the ligand. The receptor’s chemical structure allows it to “recognize” the specific macromolecule which prevents the internalization of unwanted foreign substances. After the receptor binds the recognized macromolecule, that portion of the cellular membrane undergoes endocytosis. Clathrin molecules are dispersed evenly on the internal side of the plasma membrane. Accessory proteins are responsible for clathrin recruitment. As clathrin converge near the receptor protein and the desired macromolecule, the inner portion of the membrane is drawn in as well, as shown in Figure 2. As more and more clathrin are drawn inward, a vesicle is formed with the bonded clathrin molecules that outer shell and that portion of the membrane is the inner part of the vesicle. The receptor proteins and their binded “cargo” are on the inner part of the vesicle.
Figure 2
http://www.cytochemistry.net/Cell-biology/recend.htm#clathrin

Accessory proteins, such as AP 180 and epsin, are responsible for the recruitment polymerization of the clathrin molecules, and membrane bending. Different accessory proteins are responsible for determining how many clathrin molecules are to be brought together to form the vesicle. Essentially, the accessory proteins are involved in determining the size of the vesicle. This is due to the size of the “cargo” that the vesicle is transporting. Adaptor proteins, AP 2 being the most well known, link the membrane cargo to the clathrin coat. Once the cargo is loaded, another protein called dynamin enacts scission of the clathrin and separates the vesicle from the membrane. Once the vesicle breaks off the membrane, the clathrin molecules disperse and continue to carry out the same process. The clathrin are then recycled and used again in the same process later.
Although this process is used to transport necessary products through the cell membrane into the cell, viruses and other harmful molecules can be transported into the cell via endocytosis. An example of this is JC virus which is a demylinating disease that occurs in immunosupressed patients, such as aids and cancer patients. Experiments have shown that these virions entered the cell through vesicles that form during routine endocytosis. Another virus known as simian virus 40 (SV 40) showed similar internalization kinetics as the JC virus. However, when agents were added that suppressed clathrin dependent endocytosis had no effect on SV 40 but inhibited the internalization of the JC virus. It is clear that clathrin dependent endocytosis had a major effect on the JC virus’s ability to enter cells.
Another experiment studied the effects of inhibiting clathrin dependent endocytosis using anti-clathrin antibodies and the effects on the Semliki Forest virus. After introducing an anti-clathrin inhibitor, clathrin cumulated in the cytoplasm of the cell rather than near the cellular membrane. In effect, the number of coated pits and vesicles formed in the membrane were reduced significantly. It had been previously proven that Semliki Forest virus entered the cell by clathrin dependent endocytosis. Since the number of vesicles and coated pits drastically decreased, the Semliki Forest virus could not enter the cell through its usual route. The uptake of the virus by the cell decreased by 40-50% which is a very significant difference. Again, it can be concluded that inhibiting the clathrin dependent endocytosis using anti-clathrin antibodies had a significant effect on viral uptake (Figure 3). Another experiment, using different methods to inhibit budding and formation of clathrin vesicles, showed that FV, Sindbis virus, and human rhinovirus depended on the clathrin endocytosis pathway for cell entry.
Figure 3
www.britannica.com/ebc/art-698

From each of these experiments it is shown that some viruses can be prevented, or at least minimized by the utilization of clathrin endocytosis. In each case it was evident that the viruses entered the cell through the vesicles formed by the clathrin molecule. Once endocytosis was subdued using anti-clathrin antibodies and other budding inhibitors, the uptake of the viruses was significantly decreased. This phenomenon could be very important for future research in virus prevention.