Clathrin, Endocytosis

A synaptic vesicle cycle. The number of synaptic vesicles in a single synapse in the brain varies from fewer than 100 to several hundred. In specialized synapses there may be thousands. However, at any moment only a fraction of the total are in the "active zone," often aligned along the presynaptic membrane (Fig. 30-20A) or in specialized ribbons such as those in Fig. 30-10B. The vesicles are normally reused repeatedly, undergoing a cycle of filling with neurotransmitter, translocation to the active zone, ATP-dependent priming, exocytosis with release of the neurotransmitter into the synaptic cleft, coating with clathrin, endocytosis, and acidification as outlined in Fig. 30-20B.554-557 The entire cycle may be completed within 40-60 s to avoid depletion of active vesicles.558 559 A key event in the cycle is the arrival of an action potential at the presynaptic neuron end. 

Specialized regions of internalization from the plasma membrane, coated with a polyhedral lattice of the protein clathrin. It is in these regions that the first step of the process of endocytosis takes place, with the formation of clathrin-coated endocytic vesicles. 

A process in which a substance gains entry into a cell. Endocytic mechanisms are crucial for a variety of cellular functions such as the uptake of nutrients, regulation of cell surface expression of receptors, maintenance of cell polarity, and more. Receptor-mediated endocytosis via clathrin-coated pits is the most studied endocytic process, which is important for regulation of the time and magnitude of signals generated by a variety of cell-surface receptors. 

Biochemical characterization of clathrin-coated vesicles revealed that their major coat components are clathrin and various types of adaptor complexes. Clathrin assembles in triskelions that consist of three heavy chains of approximately 190 kDa and three light chains of 30 40 kDa. Four types of adaptor complexes have been identified to date, AP-1, AP-2, AP-3 and AP-4 (AP for adaptor protein). Whereas AP-1, AP-3 and AP-4 mediate sorting events at the TGN and/or endosomes, AP-2 is involved in endocytosis at the plasma membrane. Each adaptor complex is a hetero-tetrameric protein complex, and the term adaptin was extended to all subunits of these complexes. One complex is composed of two large adaptins (one each of y/a/S/s and [31-4, respectively, 90-130 kDa), one medium adaptin (pi -4, <50 kDa), and one small adaptin (ol-4, <20 kDa). In contrast to AP-1, AP-2 and AP-3, which interact directly with clathrin and are part of the clathrin-coated vesicles, AP-4 seems to be involved in budding of a certain type of non-clathrin-coated vesicles at the TGN. 

NHE5. The distribution of this isoform is distinct, being in neuronal-rich areas of the central nervous system. Low levels have also been found in testis, spleen and skeletal muscle. Like the preceding isoforms, NHE5 is found in the plasma membrane and is internalised by clathrin-associated endocytosis into recycling endosomes. The normal role of NHE5 is unknown but its malfunction is speculated to contribute to the development of neurodegenerative disease. 

Pelkmans L, Fava E, Grabner H, Hannus M, Habermann B, Krausz E, Zerial M (2005) Genomewide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 436 78-86... 

LDL (apo B-lOO, E) receptors occur on the cell surface in pits that are coated on the cytosolic side of the cell membrane with a protein called clathrin. The glycoprotein receptor spans the membrane, the B-lOO binding region being at the exposed amino terminal end. After binding, LDL is taken up intact by endocytosis. The apoprotein and cholesteryl ester are then hydrolyzed in the lysosomes, and cholesterol is translocated into the cell. The receptors are recycled to the cell surface. This influx of cholesterol inhibits in a coordinated manner HMG-CoA synthase, HMG-CoA reductase, and, therefore, cholesterol synthesis stimulates ACAT activ-... 

Figure 41 -15. Two types of endocytosis. An endocytotic vesicle (V) forms as a result of invagination of a portion of the plasma membrane. Fluid-phase endocytosis (A) is random and nondirected. Receptor-mediated endocytosis (B) is selective and occurs in coated pits (CP) lined with the protein clathrin (the fuzzy material). Targeting is provided by receptors (black symbols) specific for a variety of molecules. This results in the formation of a coated vesicle (CV).

Most proteins that are synthesized on membrane-bound polyribosomes and are destined for the Golgi apparatus or plasma membrane reach these sites inside transport vesicles. The precise mechanisms by which proteins synthesized in the rough ER are inserted into these vesicles are not known. Those involved in transport from the ER to the Golgi apparatus and vice versa—and from the Golgi to the plasma membrane— are mainly clathrin-free, unlike the coated vesicles involved in endocytosis (see discussions of the LDL receptor in Chapters 25 and 26). For the sake of clarity, the non-clathrin-coated vesicles will be referred to in... 

Since endocytosis ofLDH was confirmed by TEM images (Figure 13.9), forthe next step, its specific endocytic pathway for membrane entry was determined by immunofluorescence and confocal microscopy. Cells were incubated with LDH-FITC, fixed with 3.7% freshly made formaldehyde, and then stained with either anti-clathrin antibody or anti-caveolin-1 antibody both conjugated to the red fluorescent dye Texas Red (TR). The confocal microscopic images showed that green fluorescent... 

LDH-FITC is well overlapped with red fluorescent clathrin-TR, but not with caveolin-1-TR (Figure 13.10). This is dear evidence that clathrin-mediated endocytosis is the prindpal mechanism for the cellular internalization of LDH particles. Caveolae-mediated endocytosis, if any, seems not to be responsible for LDH uptake. 

Fig. 13.11 Effects of clathrin-mediated endocytosis inhibitors on the internalization of LDH-FITC. Cellular uptake (%) of LDH was calculated by comparison with that in the absence of inhibitor (100%).

The 80 kDa glycoprotein transferrin (Tf) is responsible for intracellular iron transport via the transferrin receptor (TfR), using a clathrin-dependent endocytosis process. Tumor tissues frequently overexpress the TfR. While natural Tf recycles to... 

Rejman J, Oberle V, Zuhom IS, Hoekstra D (2004) Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J 377 159-169... 

Rejman J, Bragonzi A, Conese M (2005) Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol Ther 12 468 174... 

The bulk of pinocytosis in the nervous system is mediated by clathrin-mediated endocytosis (CME) [55] and this is the best-characterized pathway. More detail about clathrin-mediated pathways will be given when receptor-mediated endocytosis and the synaptic vesicle cycle pathways are considered. Pinocytosis through CME is responsible for uptake of essential nutrients such as cholesterol bound to low density lipoprotein (LDL) and transferring, but also plays a role in regulating the levels of membrane pumps and channels in neurons. Finally, CME is critical for normal synaptic vesicle recycling. 

The constitutive pathway has not been studied as intensively as regulated secretion [54]. In particular, relatively little is known about targeting and regulatory mechanisms for these transport vesicles. Clathrin seems not to be directly involved in the constitutive secretory pathway. Antibodies that disrupt clathrin assembly in vitro inhibit endocytosis, but constitutive exocytosis is not affected [63]. 

Retrieval of membrane components in the secretory pathway through receptor-mediated endocytosis (RME) is a clathrin-coat-dependent process [5]. The clathrin coat provides stability to the vesicle core and allows uptake of specific membrane proteins for reuse or degradation. RME shows a remarkable degree of specificity, allowing cells to internalize with astonishing efficiency only those selected molecules independent of their extracellular concentration. 

Extracellular ligands (hormones, neurotrophins, carrier protein, adhesion molecules, small molecules, etc.) will bind to specific transmembrane receptors. This binding of specific ligand induces the concentration of the receptor in coated pits and internalization via clathrin-coated vesicles. One of the best studied and characterized examples of RME is the internalization of cholesterol by mammalian cells [69]. In the nervous system, there are a plethora of different membrane receptors that bind extracellular molecules, including neurotrophins, hormones and other cell modulators, being the best studied examples. This type of clathrin-mediated endocytosis is an amazingly efficient process, capable of concentrating... 

As noted above, synaptic vesicles are not typically generated at the level of the TGN. Instead, they are assembled from endocytosed material retrieved from the synaptic plasma membrane. Synaptic vesicle and plasma membrane lipids and proteins are synthesized in the endoplasmic reticulum and modified in the Golgi apparatus, where they are then packaged in secretory vesicles. These synaptic precursors are delivered to the plasma membrane from the cell body by the constitutive secretory pathway. Synaptic vesicle proteins must be retrieved by clathrin-mediated synaptic vesicle endocytosis, a variant of RME with some neuron-specific components. Once the vesicle sheds its clathrin coat, the uncoated vesicle fuses with a... 

AP2 and clathrin AP2 is a protein complex that binds to a specific receptor on synaptic vesicles and plasma membranes to trigger assembly of clathrin for endocytosis. 

In the classic model of synaptic vesicle recycling in nerve terminals, synaptic vesicles fuse completely with the plasma membrane and the integrated vesicle proteins move away from the active zone to adjacent membrane regions (Fig. 9-9A). In these regions, clathrin-mediated synaptic vesicle endocytosis takes place rapidly after neurotransmitter release (within seconds) [64]. The process starts with the formation of a clathrin-coated pit that invaginates toward the interior of the cell and pinches off to form a clathrin-coated vesicle [83]. Coated vesicles are transient organelles that rapidly shed their coats in an ATP/chaperone dependent process. Once uncoated, the recycled vesicle fuses with a local EE for reconstitution as a synaptic vesicle. Subsequently, the recycled synaptic vesicle is filled with neurotransmitter and it returns to the release site ready for use. This may be the normal pathway when neurotransmitter release rates are modest. Clathrin/ EE-based pathways become essential when synaptic proteins have been incorporated into the presynaptic plasma membrane. 

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