Initiation of the clathrin complex formation requires the accumulation of phosphatidylinositol‑4,5‑bisphosphate (PIP2) and adaptor proteins, such as AP-2, at the pinching site [6, 7, 8]. In the case of clathrin-coated vesicles (CCV) formed at the trans-Golgi apparatus (TGA), AP-1 is essential [9, 10].
Growth of the clathrin coated pit requires BAR (Bin/Amphiphysin/Rvs) domain proteins [11, 12, 13] and reorganization of the actin network . The final scission step involves BAR domain proteins, dynamin and the dephosphorylation of PIP2. The latter step is suggested to function within a positive feedback loop, with regards to phosphatase activity [15, 16, 17]. The vesicles are then transported and sorted, based on receptor type or membrane composition , to the various destinations including the trans-Golgi network, endosomes and vacuoles.
amphiphysin and endophilin in mammals and Rvs161p and Rvs167p in yeast . The role of these BAR proteins is in membrane deformation, essentially promoting its tubulation. By binding to negatively charged membranes, a positive curvature is obtained that follows the concave topology of the protein's amphipathic α -helix dimer [28, 29]. F-BAR proteins, which belong to a sub-family of the BAR superfamily, possess a larger domain that is also concave in shape, yet shallower in its curvature. These proteins are proposed to generate vesicles with a larger radius compared to those proteins with possessing a BAR domain [30, 31]. In both cases the proteins may act as curvature sensors that reform the membrane into a shape to which they can readily bind . In the case of clathrin mediated endocytosis, the F-BAR proteins are believed to arrive at the site of clathrin-coated pit formation, before the BAR proteins, and as such may also be involved in nucleation of the CCP .
While the BAR domain proteins facilitate tubulation of the membrane, the adaptor proteins including AP-2 or those that possess the epsin N-terminal homology (ENTH) domain such as epsin , or the AP-180 N-terminal homology (ANTH) domains such as AP-180 continue to recruit the clathrin triskelion and other regulatory proteins required for the later stages of clathrin-coated vesicle (CCV) formation. Both the ENTH and ANTH domains are highly homologous and bind inositol phospholipids; especially PIP2 . Although both protein subclasses stimulate the formation of a clathrin triskelia network, only proteins possessing the ENTH domain influence membrane curvature, with the clathrin lattice produced by AP-180 stimulation having been shown to remain flat . This influence from the ENTH domains is believed to result from the formation of an additional α –helix ‘α0’ between the ENTH domain and the PIP molecule . It has been proposed that insertion of this domain between the lipid heads of the membrane bilayer may be sufficient to alter membrane curvature alone; however it may also be a synergistic response with clathrin assembly .
Dynamin is a non-classical GTPase, with a low affinity to GTP, but a high rate of GTP hydrolysis and GDP dissociation . It is suggested to detect the progression of invagination and therefore act as a sensor for the maturation of the CCP to a CCV . This is facilitated by its unique, non-classical GTPase properties, which best fit the function of dynamin to a two-step process of invagination: 1) the pre-collar rate limiting stage and 2) the post-collar fast assembly stage stimulated by GTP hydrolysis . The second stage is crucial for the scission of clathrin-coated vesicles. It has been proposed that dynamin controls the progress of CCV through a checkpoint system which may either lead to CCV maturation or abortion . To initiate abortion, dynamin will bind auxilin and Hsc70, which will in turn begin disassembly of the clathrin coat, a function normally carried out after scission of the mature vesicle . Similarly, progression through to maturation is governed by dynamin’s recruitment of various accessory proteins that monitor and control membrane curvature (amphiphysin/endophilin) or cargo (SNX9, grb2, TTP) .
As mentioned, various nucleation promotion factors including N-WASP, Arp2/3 and cortactin are also recruited to the endocytic site and promote actin polymerization during CCP maturation. These have been shown to co-localize with clathrin at the endocytic site [45, 46, 47] and are indicative of a role for actin cytoskeleton dyamics in clathrin-coated vesicle formation. Although this is well established in yeast cells [48, 49], the influence of the actin cytoskeleton on CCV maturation in mammalian cells is controversial.
In support of a correlation between actin cytoskeleton dynamics and CCV maturation, cortactin was shown to bind to dynamin-2 through its Src-homology 3 (SH3) domain [47, 50] and it was also reported that actin arrives at the endocytic site following a burst of dynamin recruitment . As dynamin is primarily active in the later stages of CCV maturation, just prior to and during scission, it was proposed that actin’s influence is particularly prominent in the later stages of clathrin-mediated endocytosis [51, 49].
Other studies however suggest the influence of actin cytoskeleton dynamics lies in scission of the vesicle, and not maturation or formation of the CCV. This was suggested when vesicle scission was reported to coincide with the peak level of Arp2/3 mediated actin polymerization  and when an 82% reduction in CCV scission was reported following latrunculin B treatment of Swiss 3T3 cells . Although This latter study failed to assess CCV formation, their results were supported by latrunculin A and jasplakinalide treatment of the same cell line. Here, an increase in the number of invaginated CCPs was detected indicating the drugs only inhibited CME at the scission stage .
It may be the case that an intact cytoskeleton at the endocytic site is not mandatory and its contribution to CCV formation and maturation will depend on the cell type and environment, as was suggested by a study that considered the importance of an intact actin cytoskeleton by inducing its depolymerization, or arresting its polymerization, whilst monitoring the process of clathrin-mediated endocytosis. . Alternative roles for the actin cytoskeleton were proposed when an increase in tubule growth was observed in COS-7 cells following treatment with Latrunculin B. It was concluded here that under normal circumstances, the actin cytoskeleton, along with dynamin, regulate tubule growth by maintaining membrane rigidity .
Normally PIP2 is protected from hydrolysis by the BDPs. With increasing membrane curvature however, the binding strength of the BDPs is weakened, exposing more membrane (and hence PIP2) to the phosphatases. This positive feedback loop promotes continued dephosphorylation of PIP2, and leads to further increases in membrane curvature and subsequently a narrowing of the endocytic bud neck .
Current evidence suggests that membrane curvature enhances phosphatase function by providing greater access to the lipid head groups for both increased binding and hydrolysis activity . This is indeed the case with phospholipase-c activity , and it has also been confirmed using cell cell-free assays with liposomes of varying sizes that Synj1 possesses an intrinsic preference for smaller vesicles with highly curved membranes .
Scission and dynamin accumulation is known to correspond to an abrupt decrease in PIP2 levels, and this has been attributed to the recruitment of Synj1 by the BAR domain proteins . Mathematical modeling demonstrates that the site of scission will be at the phase boundary where the amount of PIP2 decreases sharply . It has been postulated that this depletion of PIP2 results in a weakened hydrogen bond network in the upper part of the budding membrane whilst a strong hydrogen bond network remains in the lower part of the budding membrane. A subsequent imbalance in surface tension results . Also impacting membrane tension, and possibly contributing to vesicle scission, is the actin cytoskeleton. It has been shown that polymerization of the actin cytoskeleton is not crucial for vesicle scission in all cells; however, a high level of co-ordination was reported.
Although the exact mechanics of clathrin disassembly remain unclear, it has been established that for every clathrin triskelion removed from the vesicle coat, a single auxilin molecule is required. Although studies have shown that more auxilin can bind to the clathrin coat [68, 69], it is clear that only one is required for optimal disassembly . In contrast, three Hsc70 proteins are required to carry out the disassembly of a single clathrin triskelion and it has been shown in vitro that reducing the concentration of Hsc70:ATP stalls the reaction before its completion. Addition of more Hsc70:ATP allows disassembly to resume . Each of the three Hsc70 proteins is powered by the hydrolysis of ATP and therefore three ATP molecules are required for the disassembly of a single clathrin triskelion from the cage [71, 72].
In vitro biochemical studies suggest that the uncoating reaction is sensitive to pH, with efficient recruitment and binding of the chaperones occurring at pH6.0 and Hsc70 mediated dissocation of the clathrin triskelion occurring at pH7.0. At pH6.0 it was found that despite the ATPase activity of Hsc70 continuing, it was unable to disassemble the clathrin coat 
The rate of disassembly ranges substantially depending on the parameters of the experiments performed in its assessment. Measured in half-life (t½), studies using centrifugation-based experiments, which incorporate the adaptor proteins, report a slow disassembly of between 2 to 10 minutes [72, 74, 75]. Experiments that measure light scattering report much faster disassembly rates of approximately 10 seconds . These differences have been attributed to the stabilization each adaptor protein confers to the clathrin cage . Furthermore, additional in vitro experiments have shown that the time course of uncoating is non-linear and biphasic. Phase one involves a rapid burst of uncoating, followed by a second, slower phase of steady-state uncoating [76, 71]. A low ATP hydolysis rate is observed when ATP is bound in complex with Hsc70 and this has been suggested to govern the rate of uncoating following the initial burst of activity in phase one which subsequently limits the amount of disassembly that can follow .
Following dissociation of the clathrin triskelion from the clathrin cage, hsc70 remains bound to these clathrin triskelions, preventing their improper polymerization in the cytoplasm .