Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins.

Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.

Delivery of negatively charged liposomes into the atherosclerotic plaque of apolipoprotein E-deficient mouse aortic tissue.

Liposomes have been used to diagnose and treat cancer and, to a lesser extent, cardiovascular disease. We previously showed the uptake of anionic liposomes into the atheromas of Watanabe heritable hyperlipidemic rabbits within lipid pools. However, the cellular distribution of anionic liposomes in atherosclerotic plaque remains undescribed. In addition, how anionic liposomes are absorbed into atherosclerotic plaque is unclear. We investigated the uptake and distribution of anionic liposomes in atherosclerotic plaque in aortic tissues from apolipoprotein E-deficient (ApoE(-/-)) mice. To facilitate the tracking of liposomes, we used liposomes containing fluorescently labeled non-silencing small interfering RNA. Confocal microscopy analysis showed the uptake of anionic liposomes into atherosclerotic plaque and colocalization with macrophages. Transmission electron microscopy analysis revealed anionic liposomal accumulation in macrophages. To investigate how anionic liposomes cross the local endothelial barrier, we examined the role of clathrin-mediated endocytosis in human coronary artery endothelial cells (HCAECs) treated with or without the inflammatory cytokine tumor necrosis factor (TNF)-α. Pretreatment with amantadine, an inhibitor of clathrin-mediated endocytosis, significantly decreased liposomal uptake in HCAECs treated with or without TNF-α by 77% and 46%, respectively. Immunoblot analysis showed that endogenous clathrin expression was significantly increased in HCAECs stimulated with TNF-α but was inhibited by amantadine. These studies indicated that clathrin-mediated endocytosis is partly responsible for the uptake of liposomes by endothelial cells. Our results suggest that anionic liposomes target macrophage-rich areas of vulnerable plaque in ApoE(-)(/)(-) mice; this finding may lead to the development of novel diagnostic and therapeutic strategies for treating vulnerable plaque in humans.

Association of adaptor protein TRIP8b with clathrin.

TPR-containing Rab8b-interacting protein (TRIP8b) is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, hyperpolarization-activated cyclic nucleotide-regulated channel (HCN) channels and G protein-coupled receptor calcium-independent receptor of α-latrotoxin. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro-binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat.

Effect of electromagnetic field on endocytosis of cationic solid lipid nanoparticles by human brain-microvascular endothelial cells.

This study investigates the electromagnetic field (EMF)-regulated transport of cationic solid lipid nanoparticles (CSLNs) across human brain-microvascular endothelial cells (HBMECs). The positive charge of CSLNs was from dioctadecyldimethyl ammonium bromide and stearylamine, and radiofrequency EMF was applied to HBMECs for promoting uptake of CSLNs. Immunochemical staining revealed that the expression of clathrin on the membrane of HBMECs enhanced during vesicular endocytosis of CSLNs. However, CSLNs and EMF slightly affected the expression of P-glycoprotein on the membrane of HBMECs. An exposure to EMF yielded negligible increase in the permeability of free saquinavir (SQV) across the HBMEC monolayer. Nevertheless, the permeability of SQV across the HBMEC monolayer increased about 17-fold when SQV was entrapped in CSLNs. Moreover, the permeability of SQV across the HBMEC monolayer increased about 22-fold by applying the CSLN encapsulation and EMF exposure. CSLNs and EMF could produce synergistic effect on improving the brain-targeting delivery.

Exposure to ELF magnetic fields modulate redox related protein expression in mouse macrophages.

The interaction of extremely low frequency (ELF) magnetic fields (MF) with cells can induce alterations in various cell physiological processes. Here, we present evidence that exposure of mouse macrophages to 50 Hz, 1.0 mT MF lead to immune cell activation seen as increased production of reactive oxygen species (ROS), and also to modulation on the expression level of important proteins acting in redox regulatory processes and thus explaining the noted changes in ROS levels seen after exposure. The MF exposure caused slight and transient decreases after short term exposures (2h or less) of clathrin, adaptin, PI3-kinase, protein kinase B (PKB) and PP2A, whereas longer exposures had no effect. The levels of the NAD(P)H oxidase subunit gp91phox oscillated between increased and normal levels compared to controls. The stress proteins Hsp70 and Hsp110 exhibited increased levels at certain time points, but not generally. The effects of MF on protein levels are different from the effects exerted by 12-O-tetradecanolyphobol-13-acetate (TPA) or LPS, although all three factors cause increases in ROS release. This suggests that ELF MF interacts with other cellular constituents than these chemicals, although induced pathways at least partially converge.

AT1 receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through a microtubule-dependent endocytic pathway.

Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via AT1 (AT1a) receptor-mediated endocytosis, but the underlying cellular mechanisms remain poorly understood. The present study tested the hypothesis that the microtubule- rather than the clathrin-dependent endocytic pathway regulates AT1-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of AT1 receptors, clathrin light (LC) and heavy chain (HC) proteins, and type 1 microtubule-associated proteins (MAPs; MAP-1A and MAP-1B) in PT cells were knocked down by their respective small interfering (si) RNAs before AT1-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. AT1 siRNAs inhibited AT1 expression and blocked ANG II-induced NHE-3 expression in PT cells, as expected (P < 0.01). Clathrin LC or HC siRNAs knocked down their respective proteins by approximately 90% with a peak response at 24 h, and blocked the clathrin-dependent uptake of Alexa Fluor 594-transferrin (P < 0.01). However, neither LC nor HC siRNAs inhibited AT1-mediated uptake of FITC-ANG II or affected ANG II-induced NHE-3 expression. MAP-1A or MAP-1B siRNAs markedly knocked down MAP-1A or MAP-1B proteins in a time-dependent manner with peak inhibitions at 48 h (>76.8%, P < 0.01). MAP protein knockdown resulted in approximately 52% decreases in AT1-mediated FITC-ANG II uptake and approximately 66% decreases in ANG II-induced NHE-3 expression (P < 0.01). These effects were associated with threefold decreases in ANG II-induced MAP kinases ERK 1/2 activation (P < 0.01), but not with altered AT1 expression or clathrin-dependent transferrin uptake. Both losartan and AT1a receptor deletion in mouse PT cells completely abolished the effects of MAP-1A knockdown on ANG II-induced NHE-3 expression and activation of MAP kinases ERK1/2. Our findings suggest that the alternative microtubule-dependent endocytic pathway, rather than the canonical clathrin-dependent pathway, plays an important role in AT1 (AT1a)-mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells.

Enhanced synaptic vesicle traffic in hippocampus of phenytoin-resistant kindled rats.

AIM: Intractable epilepsy is characterized of seizure resistance to the anti-epileptic drugs. The underlying mechanisms are still elusive. Alterations of synaptic vesicle traffic may be one of the candidate mechanisms. METHODS: Phenytoin-resistant and phenytoin-non resistant epileptic rats were selected in the amygdala kindled adult male Wistar rats. Synaptotagmin-I and clathrin were determined by cDNA microarry analysis and Western blotting in the hippocampus of phenytoin-resistant and phenytoin-nonresistant kindled rats, which were associated with the exocytosis and endocytosis of the synaptic vesicle traffic. RESULTS: Microarry analysis showed both synaptotagmin-I and clathrin mRNA were up-regulated at least 3.06 fold accompanied with their correspondent proteins increased by 52.3 +/- 6.4 % and 76.7 +/- 12.4 % respectively in the hippocampus of phenytoin-resistant rats as compared with those in phenytoin-nonresistant rats. There were no significant differences in plasma phenytoin concentrations between the two groups. CONCLUSIONS: The increased expressions of synaptotagmin-I and clathrin in the hippocampus of phenytoin-resistant kindled rats play a role in the development of intractable epilepsy.

Rhythmic cycle of clathrin-coated pit formation at the trans-golgi network in human MDA-MB-435 cells.

We studied the in vivo dynamics of enhanced green fluorescent protein-tagged clathrin light chain a (GFP-CLCa) at the trans-Golgi network (TGN) in MDA-MB-435 cells. The intensity of fluorescence signals of GFP-CLCa periodically increased and decreased at the TGN approximately every 100 s. This suggests that the formation of clathrin-coated pits occurs synchronously and periodically at the TGN.

Effect of clathrin assembly lymphoid myeloid leukemia protein depletion on clathrin coat formation.

The endocytic accessory clathrin assembly lymphoid myeloid leukemia protein (CALM) is the ubiquitously expressed homolog of the neuron-specific protein AP180 that has been implicated in the retrieval of synaptic vesicle. Here, we show that CALM associates with the alpha-appendage domain of the AP2 adaptor via the three peptide motifs 420DPF, 375DIF and 489FESVF and to a lesser extent with the amino-terminal domain of the clathrin heavy chain. Reducing clathrin levels by RNA interference did not significantly affect CALM localization, but depletion of AP2 weakens its association with the plasma membrane. In cells, where CALM levels were reduced by RNA interference, AP2 and clathrin remained organized in somewhat enlarged bright fluorescent puncta. Electron microscopy showed that the depletion of CALM drastically affected the clathrin lattice structure. Round-coated buds, which are the predominant features in control cells, were replaced by irregularly shaped buds and long clathrin-coated tubules. Moreover, we noted an increase in the number of very small cages that formed on flat lattices. Furthermore, we noticed a redistribution of endosomal markers and AP1 in cells that were CALM depleted. Taken together, our findings indicate a critical role for CALM in the regulation and orderly progression of coated bud formation at the plasma membrane.

Role of the endocytic machinery in the sorting of lysosome-associated membrane proteins.

The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin-and to a lesser extent the other AP complexes-are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

Skeletal muscle expression of clathrin and mannose 6-phosphate receptor in experimental chloroquine-induced myopathy.

Previous studies suggest that the muscle fiber lysosome system plays a central role in the increased formation of autophagosomes and autolysosomes that occurs in the context of chloroquine-induced myopathy. The goal of this study was to characterize the contribution of receptor-mediated intracellular transport, particularly the endosomal pathway, to the abnormal accumulation of vacuoles in experimental chloroquine myopathy. Expression of the mannose 6-phosphate receptor (M6PR) and clathrin were analyzed in innervated and denervated rat soleus muscles after treatment with either saline or chloroquine. Accumulation of vacuoles was observed only in chloroquine-treated denervated muscles. Further, clathrin immunostaining and M6PR messenger ribonucleic acid (mRNA) were significantly increased in denervated soleus muscle from saline- and chloroquine-treated rats compared to contralateral, innervated muscles. However, there was no difference in clathrin levels when comparing saline- and chloroquine-treated denervated muscles. These data suggest that chloroquine activates the transport of newly synthesized lysosomal enzymes from the secretory pathway via the trans-Golgi network of the Golgi apparatus (an endosomal pathway) as well as autophagosome formation (an autophagic process) in skeletal muscles. Vacuoles may subsequently accumulate secondary to abnormal formation or turnover of autolysosomes at or after fusion of autophagosomes with early endosomes.

Clathrin self-assembly involves coordinated weak interactions favorable for cellular regulation.

The clathrin triskelion self-assembles into a polyhedral coat surrounding membrane vesicles that sort receptor cargo to the endocytic pathway. A triskelion comprises three clathrin heavy chains joined at their C-termini, extending into proximal and distal leg segments ending in a globular N-terminal domain. In the clathrin coat, leg segments entwine into parallel and anti-parallel interactions. Here we define the contributions of segmental interactions to the clathrin assembly reaction and measure the strength of their interactions. Proximal and distal leg segments were found to lack sufficient affinity to form stable homo- or heterodimers under assembly conditions. However, chimeric constructs of proximal or distal leg segments, trimerized by replacement of the clathrin trimerization domain with that of the invariant chain protein, were able to self-assemble in reversible reactions. Thus clathrin assembly occurs because weak leg segment affinities are coordinated through trimerization, sharing a dependence on multiple weak interactions with other biopolymers. Such polymerization is sensitive to small environmental changes and is therefore compatible with cellular regulation of assembly, disassembly and curvature during formation of clathrin-coated vesicles.

Dynamics and regulation of clathrin coats at specialized endocytic zones of dendrites and spines.

Endocytosis is a fundamental mechanism by which neurons control intercellular signaling, nutrient uptake, and synaptic transmission. This process is carried out by the assembly of clathrin coats and the budding of clathrin-coated vesicles from the neuronal plasma membrane. Here, we demonstrate that in young neurons, clathrin assembly and disassembly occur rapidly, locally, and repeatedly at "hot spots" throughout dendrites and at the tips of dendritic filopodia. In contrast, clathrin coats in mature dendrites reside in stable, long-lasting zones at sites of endocytosis, where clathrin undergoes continuous exchange with local cytosolic pools. In dendritic spines, endocytic zones lie lateral to the postsynaptic density (PSD) where they develop and persist independent of synaptic activity, akin to the PSD itself. These results reveal the presence of a novel specialization dedicated to endocytosis near the postsynaptic membrane.

Controlled elimination of clathrin heavy-chain expression in DT40 lymphocytes.

We exploited the high rate of homologous recombination shown by the chicken B cell line DT40 to inactivate the endogenous alleles for clathrin heavy chain and replace them with human clathrin complementary DNA under the control of a tetracycline-regulatable promoter. Clathrin repression perturbed the activities of Akt-mediated and mitogen-activated protein kinase-mediated signaling pathways and induced apoptosis; this finding suggests that in DT40 cells clathrin helps to maintain the integrity of antiapoptotic survival pathways. We also describe a variant cell line in which these signaling pathways were unaffected by clathrin down-regulation. This variant cell line did not undergo apoptosis in the absence of clathrin and was used to examine the effects of clathrin depletion on membrane-trafficking pathways. Receptor-mediated and fluid-phase endocytosis were both substantially inhibited, and transferrin-receptor recycling was modestly inhibited. Surprisingly, clathrin removal did not affect the morphology or biochemical composition of lysosomes.

Distribution of dynamins in testis and their possible relation to spermatogenesis.

Dynamin 2 and dynamin 3 are highly expressed in testis. However, their functions in the tissue remain unclear. Considering that dynamin 1, neuron-specific isoform of dynamin, plays a pivotal role in endocytosis, functions of dynamin 2 and dynamin 3 in testis must be essential. Cellular expression and subcellular localization of dynamin 2 and dynamin 3 in testis were investigated. Dynamin 2 and dynamin 3 were highly expressed in germ cells and Sertoli cells, constituents of seminiferous tubules. By immunofluorescence it was revealed that dynamin 2 colocalizes with clathrin both at the plasmamembrane and at Golgi in a cell line of Sertoli cells. Immunoreactivity for dynamin 3, on the other hand, appeared as finer puncta, which did not colocalize with clathrin, suggesting that these two dynamins have distinct functions in Sertoli cells. In the klotho deficient mouse testis, which demonstrates disorder in spermatogenesis, expression of dynamin 2 and dynamin 3 was drastically reduced indicating possible association of these proteins with spermatogenesis.

High-resolution localization of clathrin assembly protein AP180 in the presynaptic terminals of mammalian neurons.

Synaptic vesicles (SVs) assemble at the presynaptic compartment through a clathrin-dependent mechanism that involves one or more assembly proteins (APs). The assembly protein AP180 is especially efficient at facilitating clathrin cage formation, but its precise ultrastructural localization in neurons is unknown. Using immunoelectron microscopy, we demonstrate the presynaptic localization of AP180 in axon terminals of rat cerebellar neurons. In contrast, the assembly protein AP2 was associated with both the presynaptic plasma membrane and the cytosolic side of the membrane at postsynaptic and extrasynaptic sites. Furthermore, ultrastructural analysis of primate retina showed that AP180 immunoreactivity was preferentially and highly enriched at ribbon synapses, where glutamate is released tonically at high levels and rapid vesicle turnover is essential. To maintain functional synaptic transmission, neurotransmitter-filled SVs must be readily available, and this requires proper reassembly of new vesicles. The expression of AP180, in addition to AP-2, in the clathrin-mediated endocytic pathway might add another level of control to SV reformation for efficient assembly of clathrin, effectively controlling the size of assembled vesicles and faithfully recovering SV-specific components.

AP180 and AP-2 interact directly in a complex that cooperatively assembles clathrin.

Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics. AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623-680 of AP180. The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex. These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo.

UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles.

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.

An arf1Delta synthetic lethal screen identifies a new clathrin heavy chain conditional allele that perturbs vacuolar protein transport in Saccharomyces cerevisiae.

ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1Delta allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1Delta). Most of the swa mutants exhibit phenotypes comparable to arf1Delta mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3' end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y.

Clathrin self-assembly is regulated by three light-chain residues controlling the formation of critical salt bridges.

Clathrin self-assembly into a polyhedral lattice mediates membrane protein sorting during endocytosis and organelle biogenesis. Lattice formation occurs spontaneously in vitro at low pH and, intracellularly, is triggered by adaptors at physiological pH. To begin to understand the cellular regulation of clathrin polymerization, we analyzed molecular interactions during the spontaneous assembly of recombinant hub fragments of the clathrin heavy chain, which bind clathrin light-chain subunits and mimic the self-assembly of intact clathrin. Reconstitution of hubs using deletion and substitution mutants of the light-chain subunits revealed that the pH dependence of clathrin self-assembly is controlled by only three acidic residues in the clathrin light-chain subunits. Salt inhibition of hub assembly identified two classes of salt bridges which are involved and deletion analysis mapped the clathrin heavy-chain regions participating in their formation. These combined observations indicated that the negatively charged regulatory residues, identified in the light-chain subunits, inhibit the formation of high-affinity salt bridges which would otherwise induce clathrin heavy chains to assemble at physiological pH. In the presence of light chains, clathrin self-assembly depends on salt bridges that form only at low pH, but is exquisitely sensitive to regulation. We propose that cellular clathrin assembly is controlled via the simple biochemical mechanism of reversing the inhibitory effect of the light-chain regulatory sequence, thereby promoting high-affinity salt bridge formation.