Disassembly Of Clathrin Coats Research Articles

Temporal Vestibular Deficits in synaptojanin 1 (synj1) Mutants.

The lipid phosphatase synaptojanin 1 (synj1) is required for the disassembly of clathrin coats on endocytic compartments. In neurons such activity is necessary for the recycling of endocytosed membrane into synaptic vesicles. Mutations in zebrafish synj1 have been shown to disrupt the activity of ribbon synapses in sensory hair cells. After prolonged mechanical stimulation of hair cells, both phase locking of afferent nerve activity and the recovery of spontaneous release of synaptic vesicles are diminished in synj1 mutants. Presumably as a behavioral consequence of these synaptic deficits, synj1 mutants are unable to maintain an upright posture. To probe vestibular function with respect to postural control in synj1 mutants, we developed a method for assessing the vestibulospinal reflex (VSR) in larvae. We elicited the VSR by rotating the head and recorded tail movements. As expected, the VSR is completely absent in pcdh15a and lhfpl5a mutants that lack inner ear function. Conversely, lhfpl5b mutants, which have a selective loss of function of the lateral line organ, have normal VSRs, suggesting that the hair cells of this organ do not contribute to this reflex. In contrast to mechanotransduction mutants, the synj1 mutant produces normal tail movements during the initial cycles of rotation of the head. Both the amplitude and temporal aspects of the response are unchanged. However, after several rotations, the VSR in synj1 mutants was strongly diminished or absent. Mutant synj1 larvae are able to recover, but the time required for the reappearance of the VSR after prolonged stimulation is dramatically increased in synj1 mutants. Collectively, the data demonstrate a behavioral correlate of the synaptic defects caused by the loss of synj1 function. Our results suggest that defects in synaptic vesicle recycling give rise to fatigue of ribbons synapses and possibly other synapses of the VS circuit, leading to the loss of postural control.

Proteomic Analysis of Restored Insulin Production and Trafficking in Obese Diabetic Mouse Pancreatic Islets Following Euglycemia.

For the treatment of patients with prediabetes or diabetes, clinical evidence has emerged that β-cell function can be restored by glucose-lowering therapeutic strategies. However, little is known about the molecular mechanisms underlying this functional adaptive behavior of the pancreatic β-cell. This study examines the dynamic changes in protein expression and phosphorylation state associated with (pro)insulin production and secretory pathway function mediated by euglycemia to induce β-cell rest in obese/diabetic db/db islet β-cells. Unbiased quantitative profiling of the protein expression and phosphorylation events that occur upon β-cell adaption during the transition from hyperglycemia to euglycemia was assessed in isolated pancreatic islets from obese diabetic db/db and wild-type (WT) mice using quantitative proteomics and phosphoproteomics together with bioinformatics analysis. Dynamic changes in the expression and phosphorylation of proteins associated with pancreatic β-cell (pro)insulin production and complementary regulated-secretory pathway regulation were observed in obese diabetic db/db islets in a hyperglycemic environment, relative to WT mouse islets in a normal euglycemic environment, that resolved when isolated db/db islets were exposed to euglycemia for 12 h in vitro. By similarly treating WT islets in parallel, the effects of tissue culture could be mostly eliminated and only those changes associated with resolution by euglycemia were assessed. Among such regulated protein phosphorylation-dependent signaling events were those associated with COPII-coated vesicle-dependent ER exit, ER-to-Golgi trafficking, clathrin-coat disassembly, and a particular association for the luminal Golgi protein kinase, FAM20C, in control of distal secretory pathway trafficking, sorting, and granule biogenesis. Protein expression and especially phosphorylation play key roles in the regulation of (pro)insulin production, correlative secretory pathway trafficking, and the restoration of β-cell secretory capacity in the adaptive functional β-cell response to metabolic demand, especially that mediated by glucose.

Danger-associated peptide signaling in Arabidopsis requires clathrin

The Arabidopsis thaliana endogenous elicitor peptides (AtPeps) are released into the apoplast after cellular damage caused by pathogens or wounding to induce innate immunity by direct binding to the membrane-localized leucine-rich repeat receptor kinases, PEP RECEPTOR1 (PEPR1) and PEPR2. Although the PEPR-mediated signaling components and responses have been studied extensively, the contributions of the subcellular localization and dynamics of the active PEPRs remain largely unknown. We used live-cell imaging of the fluorescently labeled and bioactive pep1 to visualize the intracellular behavior of the PEPRs in the Arabidopsis root meristem. We found that AtPep1 decorated the plasma membrane (PM) in a receptor-dependent manner and cointernalized with PEPRs. Trafficking of the AtPep1-PEPR1 complexes to the vacuole required neither the trans-Golgi network/early endosome (TGN/EE)-localized vacuolar H(+)-ATPase activity nor the function of the brefeldin A-sensitive ADP-ribosylation factor-guanine exchange factors (ARF-GEFs). In addition, AtPep1 and different TGN/EE markers colocalized only rarely, implying that the intracellular route of this receptor-ligand pair is largely independent of the TGN/EE. Inducible overexpression of the Arabidopsis clathrin coat disassembly factor, Auxilin2, which inhibits clathrin-mediated endocytosis (CME), impaired the AtPep1-PEPR1 internalization and compromised AtPep1-mediated responses. Our results show that clathrin function at the PM is required to induce plant defense responses, likely through CME of cell surface-located signaling components.

Clathrin-coat disassembly illuminates the mechanisms of Hsp70 force generation.

Hsp70s use ATP hydrolysis to disrupt protein:protein associations or move macromolecules. One example is Hsc70-mediated disassembly of clathrin coats that form on vesicles during endocytosis. We exploit the exceptional features of these coats to test three models—Brownian ratchet, power-stroke and entropic pulling—proposed to explain how Hsp70s transform their substrates. Our data rule out the ratchet and power-stroke models, and instead support a collision pressure mechanism whereby collisions between clathrin coat walls and Hsc70s drive coats apart. Collision pressure is the complement to the pulling force described in the entropic pulling model. We also find that self-association can augment collision pressure to allow disassembly of clathrin lattices predicted to resist disassembly. These results illuminate how Hsp70s generate the forces that transform their substrates.

Clathrin Coat Disassembly by the Yeast Hsc70/Ssa1p and Auxilin/Swa2p Proteins Observed by Single-particle Burst Analysis Spectroscopy

The role of clathrin-coated vesicles in receptor-mediated endocytosis is conserved among eukaryotes, and many of the proteins required for clathrin coat assembly and disassembly have orthologs in yeast and mammals. In yeast, dozens of proteins have been identified as regulators of the multistep reaction required for endocytosis, including those that regulate disassembly of the clathrin coat. In mammalian systems, clathrin coat disassembly has been reconstituted using neuronal clathrin baskets mixed with the purified chaperone ATPase 70-kDa heat shock cognate (Hsc70), plus a clathrin-specific co-chaperone, such as the synaptic protein auxilin. Yet, despite previous characterization of the yeast Hsc70 ortholog, Ssa1p, and the auxilin-like ortholog, Swa2p, testing mechanistic models for disassembly of nonneuronal clathrin coats has been limited by the absence of a functional reconstitution assay. Here we use single-particle burst analysis spectroscopy, in combination with fluorescence correlation spectroscopy, to follow the population dynamics of fluorescently tagged yeast clathrin baskets in the presence of purified Ssa1p and Swa2p. An advantage of this combined approach for mechanistic studies is the ability to measure, as a function of time, changes in the number and size of objects from a starting population to the reaction products. Our results indicate that Ssa1p and Swa2p cooperatively disassemble yeast clathrin baskets into fragments larger than the individual triskelia, suggesting that disassembly of clathrin-coated vesicles may proceed through a partially uncoated intermediate.

Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating

Heat shock cognate protein 70 (Hsc70) supports remodeling of protein complexes -- for example, disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by electron cryomicroscopy, in the vicinity of each occupied site and that accumulation of local strains destabilises the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes.

FKBP36 Forms Complexes with Clathrin and Hsp72 in Spermatocytes

The testes-specific peptidyl-prolyl cis/ trans isomerase FKBP36 plays a crucial role in male meiosis. Here we show that the catalytic domain of FKBP36 binds to clathrin heavy chain (CHC) of clathrin. Despite wild-type FKBP36 not displaying PPIase activity, the introduction of the R81L substitution resulted in catalysis of prolyl isomerization, which is comparable to the regulated activity of FKBP38. Furthermore, the TPR domain of FKBP36 interacts with Hsp72. In fact, FKBP36 preferentially binds to Hsp72 among the members of the Hsp70 family and is thus the first TPR-containing protein which discriminates between Hsp70 proteins. The clathrin-FKBP36-Hsp72 complexes resulting from both identified interactions are bound to the matrices of clathrin-coated vesicles in spermatocytes, which indicates a possible role of FKBP36 and Hsp72 in the disassembly of clathrin coats.

The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis

BackgroundLigand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux), a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood.ResultsTo understand the role of auxilin in Notch ligand endocytosis, we have analyzed several point mutations affecting specific domains of dAux. In agreement with previous work, analysis using these stronger dAux alleles shows that dAux is required for several Notch-dependent processes, and its function during Notch signaling is required in the signaling cells. In support of the genetic evidences, the level of Delta appears elevated in dAux deficient cells, suggesting that the endocytosis of Notch ligand is disrupted. Deletion analysis shows that the clathrin-binding motif and the J-domain, when over-expressed, are sufficient for rescuing dAux phenotypes, implying that the recruitment of Hsc70 to clathrin is a critical role for dAux. However, surface labeling experiment shows that, in dAux mutant cells, Delta accumulates at the cell surface. In dAux mutant cells, clathrin appears to form large aggregates, although Delta is not enriched in these aberrant clathrin-positive structures.ConclusionOur data suggest that dAux mutations inhibit Notch ligand internalization at an early step during clathrin-mediated endocytosis, before the disassembly of clathrin-coated vesicles. Further, the inhibition of ligand endocytosis in dAux mutant cells possibly occurs due to depletion of cytosolic pools of clathrin via the formation of clathrin aggregates. Together, our observations argue that ligand endocytosis is critical for Notch signaling and auxilin participates in Notch signaling by facilitating ligand internalization.

A Doubleheader in Endocytosis

Synaptojanin1 degrades the signaling lipid phosphatidylinositol-4,5-bisphosphate and facilitates compensatory endocytosis, clathrin-coat disassembly, and vesicle reavailability at active synapses. In this issue of Neuron, Mani et al. provide new information about the separate roles of synaptojanin's two phosphatase domains and its interactions with endophilin in regulating these important aspects of the vesicle cycle.

Drosophila melanogaster auxilinregulates the internalization of Delta to control activity of the Notch signaling pathway

We have isolated mutations in the Drosophila melanogaster homologue of auxilin, a J-domain–containing protein known to cooperate with Hsc70 in the disassembly of clathrin coats from clathrin-coated vesicles in vitro. Consistent with this biochemical role, animals with reduced auxilin function exhibit genetic interactions with Hsc70 and clathrin. Interestingly, the auxilin mutations interact specifically with Notch and disrupt several Notch-mediated processes. Genetic evidence places auxilin function in the signal-sending cells, upstream of Notch receptor activation, suggesting that the relevant cargo for this auxilin-mediated endocytosis is the Notch ligand Delta. Indeed, the localization of Delta protein is disrupted in auxilin mutant tissues. Thus, our data suggest that auxilin is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal-sending cells needs to proceed past coat disassembly to activate Notch.

Novel functions of clathrin light chains: clathrin heavy chain trimerization is defective in light chain-deficient yeast.

Clathrin is a major coat protein involved in sorting and retention of proteins at the late Golgi and in endocytosis from the cell surface. The clathrin triskelion contains three heavy chains, which provide the structural backbone of the clathrin lattice and three light chains, which are thought to regulate the formation or disassembly of clathrin coats. To better understand the function of the clathrin light chain, we characterized yeast strains carrying a disruption of the clathrin light chain gene (CLC1). Light chain-deficient cells showed phenotypes similar to those displayed by yeast that have a disruption in the clathrin heavy chain gene (CHC1). In clc1-delta cells, the steady state level of the clathrin heavy chain was reduced to 20%-25% of wild-type levels and most of the heavy chain was not trimerized. If CHC1 was overexpressed in clc1-delta cells, heavy chain trimers were detected and several clc1-delta phenotypes were partially rescued. These results indicate that the light chain is important for heavy chain trimerization and the heavy chain still has some function in the absence of the light chain. In yeast, deletion of CHC1 is lethal in strains carrying the scd1-i allele, while strains carrying the scd1-v allele can survive without the heavy chain. In previous studies we isolated several multicopy suppressors of inviability of chc1-delta scd1-i cells. Surprisingly, one of these suppressors, SCD4, is identical to CLC1. Overexpression of CLC1 in viable chc1-delta scd1-v strains rescued some but not all of the phenotypes displayed by these cells. In the absence of the heavy chain, the light chain was not found in a high molecular mass complex, but still associated with membranes. These results suggest that the light chain can function independently of the clathrin heavy chain in yeast.