Clathrin‐Mediated Endocytosis at the Synaptic Terminal: Bridging the Gap Between Physiology and Molecules

It has long been known that the maintenance of fast communication between neurons requires that presynaptic terminals recycle the small vesicles from which neurotransmitter is released. But the mechanisms that retrieve vesicles from the cell surface are still not understood. Although we have a wealth of information about the molecular details of endocytosis in non‐neuronal cells, it is clear that endocytosis at the synapse is faster and regulated in distinct ways. A satisfying understanding of these processes will require molecular events to be manipulated while observing endocytosis in living synapses. Here, we review recent work that seeks to bridge the gap between physiology and molecules to unravel the endocytic machinery operating at the synaptic terminal.     

Ikarugamycin: A Natural Product Inhibitor of Clathrin‐Mediated Endocytosis

Ikarugamycin (IKA) is a previously discovered antibiotic, which has been shown to inhibit the uptake of oxidized low‐density lipoproteins in macrophages. Furthermore, several groups have previously used IKA to inhibit clathrin‐mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA has yet to be performed. Consequently, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We show that IKA has an IC₅₀ of 2.7 μm in H1299 cells and acutely inhibits CME, but not other endocytic pathways, in a panel of cell lines. Although long‐term incubation with IKA has cytotoxic effects, the short‐term inhibitory effects on CME are reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking.      

Nonmuscle Myosin II Is a Critical Regulator of Clathrin‐Mediated Endocytosis

Variable requirements for actin during clathrin‐mediated endocytosis (CME) may be related to regional or cellular differences in membrane tension. To compensate, local regulation of force generation may be needed to facilitate membrane curving and vesicle budding. Force generation is assumed to occur primarily through actin polymerization. Here we examine the role of myosin II using loss of function experiments. Our results indicate that myosin II acts on cortical actin scaffolds primarily in the plane of the plasma membrane (bottom arrow) to generate changes that are critical for enhancing CME progression.      

Sorting of Clathrin‐Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin‐Mediated Endocytosis

Clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE) co‐exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6‐associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6‐GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6‐GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin‐coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6‐GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.      

Endocytosis of Ubiquitylation‐Deficient EGFR Mutants via Clathrin‐Coated Pits is Mediated by Ubiquitylation

Signaling by epidermal growth factor receptor (EGFR) is controlled by endocytosis. However, mechanisms of EGFR endocytosis remain poorly understood. Here, we found that the EGFR mutant lacking known ubiquitylation, acetylation and clathrin adaptor AP‐2‐binding sites (21KRΔAP2) was internalized at relatively high rates via the clathrin‐dependent pathway in human duodenal adenocarcinoma HuTu‐80 cells. RNA interference analysis revealed that this residual internalization is strongly inhibited by depletion of Grb2 and the E2 ubiquitin‐conjugating enzyme UbcH5b/c, and partially affected by depletion of the E3 ubiquitin ligase Cbl and ubiquitin‐binding adaptors, indicating that an ubiquitylation process is involved. Several new ubiquitin conjugation sites were identified by mass spectrometry in the 21KRΔAP2 mutant, suggesting that cryptic ubiquitylation may mediate endocytosis of this mutant. Total internal reflection fluorescence microscopy imaging of HuTu‐80 cells transfected with labeled ubiquitin adaptor epsin1 demonstrated that the ubiquitylation‐deficient EGFR mutant was endocytosed through a limited population of epsin‐enriched clathrin‐coated pits (CCPs), although with a prolonged CCP lifetime. Native EGFR was recruited with the same efficiency into CCPs containing either AP‐2 or epsin1 that were tagged with fluorescent proteins by genome editing of MDA‐MD‐231 cells. We propose that two redundant mechanisms, ubiquitylation and interaction with AP‐2, contribute to EGFR endocytosis via CCPs in a stochastic fashion.      

Polarised Clathrin‐Mediated Endocytosis of EGFR During Chemotactic Invasion

Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin‐mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA‐MB‐231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin‐mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin‐mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin‐mediated endocytosis to directed cell motility.      

Disruption of Clathrin‐Mediated Trafficking Causes Centrosome Overduplication and Senescence

The Hsc70 cochaperone, G cyclin‐associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin‐dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin‐mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin‐dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication.     

Synaptic and vesicular co-localization of the glutamate transporters VGLUT1 and VGLUT2 in the mouse hippocampus

Vesicular glutamate transporters (VGLUTs) are essential to glutamatergic synapses and determine the glutamatergic phenotype of neurones. The three known VGLUT isoforms display nearly identical uptake characteristics, but the associated expression domains in the adult rodent brain are largely segregated. Indeed, indirect evidence obtained in young VGLUT1-deficient mice indicated that in cells that co-express VGLUT1 and VGLUT2, the transporters may be targeted to different synaptic vesicles, which may populate different types of synapses formed by the same neurone. Direct evidence for a systematic segregation of VGLUT1 and VGLUT2 to distinct synapses and vesicles is lacking, and the mechanisms that may convey this segregation are not known. We show here that VGLUT1 and VGLUT2 are co-localized in many layers of the young hippocampus. Strikingly, VGLUT2 co-localizes with VGLUT1 in the mossy fibers at early stages. Furthermore, we show that a fraction of VGLUT1 and VGLUT2 is carried by the same vesicles at these stages. Hence, hippocampal neurones co-expressing VGLUT1 and VGLUT2 do not appear to sort them to separate vesicle pools. As the number of transporter molecules per vesicle affects quantal size, the developmental window where VGLUT1 and VGLUT2 are co-expressed may allow for greater plasticity in the control of quantal release.     

Choline Acetyltransferase: Regulation and Coupling with Protein Kinase and Vesicular Acetylcholine Transporter on Synaptic Vesicles

Both the membrane-bound choline acetyltransferase (MChAT) and soluble ChAT (SChAT) were found to be activated by ATP-mediated protein phosphorylation. ATP activation of MChAT but not SChAT was found to depend on the integrity of proton gradient of synaptic vesicles because conditions disrupting the proton gradient also abolished the activation of MChAT by ATP. Among the kinases studied, Ca2+/calmodulin kinase II is most effective in activation of MChAT. Transport of ACh into synaptic vesicles by vesicular acetylcholine transporter (VAChT) is also proton gradient-dependent; therefore we proposed that there is a functional coupling between ACh synthesis and its packaging into synaptic vesicles. This notion is supported by the following findings: first, the newly synthesized [3H]-ACh from [3H]-choline was taken up much more efficiently than the pre-existing ACh; second, ATP-activation of MChAT was abolished when VAChT was inhibited by the specific inhibitor vesamicol; third, the activity of ChAT was found to be markedly increased when neurons are under depolarizing conditions.     

The Sybtraps: Control of Synaptobrevin Traffic by Synaptophysin, α‐Synuclein and AP‐180

Synaptobrevin II (sybII) is a key fusogenic molecule on synaptic vesicles (SVs) therefore the active maintenance of both its conformation and location in sufficient numbers on this organelle is critical in both mediating and sustaining neurotransmitter release. Recently three proteins have been identified having key roles in the presentation, trafficking and retrieval of sybII during the fusion and endocytosis of SVs. The nerve terminal protein α‐synuclein catalyses sybII entry into SNARE complexes, whereas the monomeric adaptor protein AP‐180 is required for sybII retrieval during SV endocytosis. Overarching these events is the tetraspan SV protein synaptophysin, which is a major sybII interaction partner on the SV. This review will evaluate recent studies to propose working models for the control of sybII traffic by synaptophysin and other Sybtraps (syb II tra fficking p artners ) and suggest how dysfunction in sybII traffic may contribute to human disease.      

Characterization of a single-copy Arabidopsis gene encoding a protein showing limited similarity to the N-terminus of the mammalian clathrin-assembly protein AP180.

The Arabidopsis 194 gene encoding a protein containing sequence similarity to an N-terminal region of the clathrin-assembly protein AP180 has been identified in a 4.9-kb region of genomic DNA upstream of the gene encoding the high mobility group protein HMG-I/Y. The gene consists of 12 exons and 11 introns, identified by comparison with partial cDNAs and using the NetPlantGene programme, and encodes a protein of 584 amino acid residues. The C-terminal region of the protein contains 8 tandem repeats of a 17-amino-acid-residue sequence. Southern blot analysis of genomic DNA from Columbia and Landsberg ecotypes of Arabidopsis indicates the presence of a single copy of the 194 gene. The 194 gene is expressed in all organs of Arabidopsis including roots, stems, leaves, flowers, and developing siliques, as determined by northern blot analysis.     

α-Synuclein facilitates endocytosis by elevating the steady-state levels of phosphatidylinositol 4,5-bisphosphate

α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson''s disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we investigate the associations of α-Syn with the acidic phosphoinositides (PIPs), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P₂). Our results show that α-Syn colocalizes with PIP₂ and the phosphorylated active form of the clathrin adaptor protein 2 (AP2) at clathrin-coated pits. Using endocytosis of transferrin as an indicator for clathrin-mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI(4,5)P₂ levels on the plasma membrane. In accord with their effects on PI(4,5)P₂ levels, the PD associated A30P, E46K, and A53T mutations in α-Syn further enhance CME in neuronal and nonneuronal cells. However, lysine to glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, which interfere with phospholipid binding, are ineffective in enhancing CME. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by the α-Syn mutations and associates with their effects on PI(4,5)P₂ levels, however, with the exception of the A30P mutation. This study provides evidence for a critical involvement of PIPs in α-Syn–mediated membrane trafficking.