Structural Biochemistry/Endocytosis

Endocytosis
Endocytosis is a process in which substances enter the cell without passing through the cell membrane, but by forming an intracellular vesicle. This process allows the entry of large polar molecules that are not able to get into the hydrophobic plasma membrane. Also, it plays a important role in regulation of intracellular signaling. The intracellular vesicle is formed by the plasma membrane surrounding the potential food molecule. In essence, the cell engulfs the whole molecule in order to ingest it. A part of the cell is "invaginated" and can forms a vesicle or endosome that contains the molecule ingested. The different type of molecule that is associated with endocytosis is given specific names such as phagocytosis. The opposite of endocytosis is exocytosis which is the expulsion of such molecules.

Endocytosis in animal cells:
 * Phagocytosis: a cell engulfs a particle by surrounding it with pseudopodia and infusing it into the food vacuole, which contains hydrolytic enzymes. Only occurs in specialized cells such as the amoeba. Known as cell eating and can be used as an immune system defense! The endosomes attributed to phagocytosis is so large that they are usually referred to as a vacuole or phagosome.
 * Pinocytosis: the cell produces droplets of extracellular fluid into tiny vesicles that also fuse with lysosome containing enzymes, which breaks down the particles. Known as cell drinking. The amount of liquids entering are usually very small. Almost all cells undergo pinocytosis and they do so continuously.
 * Receptor-mediated endocytosis: membrane embedded proteins with specific receptor site are exposed to the extracellular fluid in order to bind with specific ligands. Then, vesicles are formed containing the ligand molecules. The material is then ingested and liberated from the vesicle.



Clathrin-Mediated Endocytosis (CME)
In the mechanism of clathrin-mediated endocytosis, triskelion structure clathrin perform self-assembly to form a regular lattice. At the mean time, adaptor proteins such as epsin, SNX9 then bind to the membrane receptor protein to form CCV (clathrin-coated vesicle). Disassembly immediately begins after the formation of the vesicle neck, and it is carried out by Hsc70 and its cofactor auxilin. The uncoated CCV can undergo further reaction in the cell.


 * 1) REDIRECT

Clathrin-Independent Endocytosis (CIE)
Clathrin-Independent Endocytosis

Since many cells don't have the required cytoplasm sequences to turn into clarthin-coated vesicles, there is a significant clathrin-independent endocytosis. There are several different CIE mechanisms.

1) Caveolar Endocytosis: This is used for the uptake of glycophingolipids and some viruses. It involves the caveolin coat and dynamin.

2) This is used for uptake of toxins from bacteria,GPI anchored proteins, and fluid-phase markers. This mode depends on actin, CDC42, ADP-ribosylation factor but is independent of dynamin.

3) This is used for uptake of integral membrane proteins that do not have the adaptor protein recognition sequences. It is independent of dynamin but is dependent on ARE6 GTPase.

All these forms of Clathrin-independent endocytosis require free cholesterol. Clathrin-independent endocytosis is also used prominently by proteins and lipids that are in sphingolipid-rich lipid raft membranes.

After the uptake of molecules, they are snet to distinct vesicles and are transferred to the early endosome. In the early endosome, there is a mixing of clathrin-independent and clathrin-dependent endocytosis cargo and then finally, recycling happens.

Membrane Curvature Stabilization
Endocytic membranes require high membrane curvature, but membranes were mostly found to be sheet-like structure. However, in recent study some small G proteins were observed to be capable of membrane curvature generation. In the process that CCP(clathrin-coated pit) is developed to form CCV (clathrin-coated vesicle), an amphipathic helix is introduced into lipid monolayer by epsin. By doing so, the helix remains at the glycerol backbone of the lipid,  and thus make the phospholipid moieties bent. Membrane curvature generation can be induced by the splay of the phospholipid. Also, cytoskeleton is important to membrane curvature regulation by promoting dynamin's fission ability.

r== Endocytic Recycling == Recycling of old endocytosis material is necessary when endocytic uptake occurs in order to maintain the shape and size of the cell and the plasma membrane. Recycling contributes to many processes such as nutrient intake, movement of cells, cytokinesis, and intracellular signaling. Recycling of materials depends on whether endocytosis was Clathrin dependent or Clathrin independent.

Early Endosome

The early endosome receives and sorts material that comes from CIE and CDE. Since the lumen of the early endosome is acidic, confirmational changes in proteins for ligand release from receptor occur. To enter the fast recycling pathways the following must happen: 1) membrane proteins and lipids must be separated from luminal content 2) generation of membrane tubules. Otherwise, the material can enter endocytic recycling compartment where recycling endosomes emerge.

Rapid Recycling Route

The fast recycling route is used for the transport of TFR and glycosphingolipids. A few studies have shown, RAB4 is important for recycling these materials. However, RAB4 inhibits rapid recycling and increases slow recycling. It seems that small interfering RNA might knock down RAB4 and increase rapid recycling. Recent evidence suggests that RAB5 might be the regulator of rapid recycling by localizing to the plasma membrane and to early endosomes.

Slow Recycling Route

This route is used for transporting cargo proteins from early endosome to ERC and then to the plasma membrane. In many cells, ERC is localized and is near the Golgi complex and the microtubule organizing center. However, in polar cells, the early endosome extends tubules that become the ERC. This model of transformation has been supported by live imaging studies.

ERC traffic

One of the reasons that endocytosis material is moved from early endosome to ERC might be to make sure that the material does not enter degradative compartments. In mammalian cells, sorting nexin 4 connects the early endosome and ERC. If nexin 4 is not present, TFR would be sorted to late endosome where it would be degraded.

ERC to the Plasma Membrane

Early explanations of recycling included ARF6 associated tubular endosomes that extend from ERC and carry material to the plasma membrane. The tubular endosomes would align with the microtubules and recycling would depend on both microtubules and actin. ARF6 would activate Phospholipase D2 (PLD2) which is present on tubular recycling endosome. PLD2 products (phosphatidic acid and diacylglycerol) take part in the recycling. Phosphatidic acid promotes membrane fission and might also cause the release of recycling carriers. Meanwhile, diacylglycerol promotes membrane fission and fusion. Thus, it might promote the fusion of the carriers with the plasma membrane again.

ARF6 also activates PtdIns (4)P5K enzyme which generates PtdIns(4,5)-bisphosphate. PtdIns(4,5)-bisphosphate is present on cell surface and on tubular endosome. PtdIns(4,5)-bisphosphate is also responsible for recruiting proteins to the plasma membrane which result in cell spreading, cell migrating, and wound healing.

Evidence of ARF6 ivolvement in CIE sorting and recycling:

1) Recycling of syndecan 1 and FGFR require PtIns(4,5)P2 (which is produced by ARF6 activation of PtdIns(4)P5K) and synthenin. When mutant synthenin is introduced which cannot bind to PtIns(4,5)P2, recycling of synedcan 1 and FGFR does not occur. This shows that without ARF6, synthenin cannot alone do the job of recycling and impairs cell spreading. 2) A cytoplasmic acidic cluster that is present on an inwardly rectifying potassium channel, Kir 3.4 binds to ARF6 GEF. This leads to activation of ARF6. Furthermore, there is an increase of Kir 3.4 on the plasma membrane suggesting that recycling has taken place and the material has been moved to the plasma membrane again.

ARF6 Inhibition

Extracellular signal regulated kinase (ERK) inhibits AFR6 activation. This inhibition causes a buildup of CIE tubular recycling endosomes which stops recycling. Other signaling molecules such as Ras, Rac, and Src proteins on these tubules might also change AFR6 activity and stop recycling.

Regulators of Recycling

1) ERC can be detected by the presence of RAB11 and other proteins. Since slow recycling occurs at the ERC, manipulating RAB11 could stop recycling and change the place of ERC in the cell. 2) RAB8 seems to be important in the early endosome to ERC transport and may also interact with ARF6. Thus manipulation of this protein could lead to inhibition of ARF6 and thus recycling. 3) ALIX was found to be a RME-1-binding protein that is needed for recylling TFR. Thus, destabilizing of ALIX could regulate recycling.