Effect of Clathrin Assembly Lymphoid Myeloid Leukemia Protein Depletion on Clathrin Coat Formation

Department of Cell Biology, Center of Anatomy, Hannover Medical School, Carl-Neuberg Str. 1, D-30625 Hannover, Germany.
Traffic (Impact Factor: 4.35). 01/2006; 6(12):1225-34. DOI: 10.1111/j.1600-0854.2005.00355.x
Source: PubMed


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.

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Available from: Ernst Joachim Ungewickell, Dec 04, 2014
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    • "Instead, our data are most consistent with a model according to which clathrin/AP180 depending on the frequency of firing reform Syb2-containing SVs either directly from the plasma membrane via conventional CME or from ELVs following clathrin-independent membrane retrieval (Kononenko and Haucke, 2015; Kononenko et al., 2014), possibly involving ultrafast (Watanabe et al., 2013b; Watanabe et al., 2014) or other forms of bulk endocytosis (Cheung and Cousin, 2013). Genetic manipulation of endocytic proteins in this model would lead to alterations in SV size and shape due to defects in membrane deformation and/or coat assembly as reported for several endocytic protein mutants (Dittman and Ryan, 2009; Ferguson et al., 2007; Milosevic et al., 2011; Nonet et al., 1999; Saheki and De Camilli, 2012; Zhang et al., 1998), acute perturbation of AP180 function in squid axons (Morgan et al., 1999), and endocytic vesicles in non-neuronal cells depleted of the clathrin-associated AP180 paralog CALM (Meyerholz et al., 2005; Miller et al., 2015). Neurons from AP180 À/À mice suffer from accelerated synaptic rundown, indicative of faster depletion of the releasable vesicle pool in response to repeated stimulation (see Figure 3F). "
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    ABSTRACT: Neurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis. Here we demonstrate that loss of the Syb2-specific endocytic adaptor AP180 causes a moderate activity-dependent reduction of vesicular Syb2 levels, defects in SV reformation, and a corresponding impairment of neurotransmission that lead to excitatory/inhibitory imbalance, epileptic seizures, and premature death. Further reduction of Syb2 levels in AP180(-/-)/Syb2(+/-) mice results in perinatal lethality, whereas Syb2(+/-) mice partially phenocopy loss of AP180, indicating that reduced vesicular Syb2 levels underlie the observed defects in neurotransmission. Thus, a large vesicular Syb2 pool maintained by AP180 is crucial to sustain efficient neurotransmission and SV reformation.
    Neuron 09/2015; DOI:10.1016/j.neuron.2015.08.034 · 15.05 Impact Factor
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    • "CALM encodes a 652 aa protein with multiple domains such as AP180 N-terminal homology (ANTH) domain, DPF motif, NPF motif, and type I and II clathrin-binding sequences (CBS I and II), of which expression is ubiquitously observed in various organs [12]–[18]. Knockdown of CALM by RNA interference leads to the formation of larger and more irregular, clathrin-coated vesicles in HeLa cells, indicating that CALM is required for proper formation of clathrin-coated vesicles [19]. CALM was originally isolated as a part of the fusion gene CALM/AF10, which results from the chromosomal translocation t(10;11) (p13;q14) [20]. "
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    ABSTRACT: CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage-Sca-1+KIT+ (LSK) cells decreased in the fetal liver of CALM-/- mice. Also, colony forming activity was impaired in CALM-/- LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM-/- LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM-/- murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM-/- MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM-/- MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM-/- MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM-/- MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM-/- KIT+ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy.
    PLoS ONE 10/2014; 9(10):e109441. DOI:10.1371/journal.pone.0109441 · 3.23 Impact Factor
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    • "Since a critical AP-2 binding DPF peptide motif is encoded by exon 13, the loss of exon 13 is expected to reduce AP-2 binding [22]. Loss of this DPF motif may be compensated by the DIF and/or FESVF motifs encoded within exons 12 and 14, respectively [22]. Isoforms lacking exons 13 and 14 were also detected that would lack both the DPF and FESVF motifs and would be expected to have particularly low AP-2 binding. "
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    ABSTRACT: Novel Alzheimer's disease (AD) risk factors have been identified by genome-wide association studies. Elucidating the mechanism underlying these factors is critical to the validation process and, by identifying rate-limiting steps in AD risk, may yield novel therapeutic targets. Here, we evaluated the association between the AD-associated polymorphism rs3851179 near PICALM, which encodes a clathrin-coated pit accessory protein. Immunostaining established that PICALM is expressed predominately in microvessels in human brain. Consistent with this finding, PICALM mRNA expression correlated with expression of the endothelial genes vWF and CD31. Additionally, we found that PICALM expression was modestly increased with the rs3851179A AD-protective allele. Analysis of PICALM isoforms found several isoforms lacking exons encoding elements previously identified as critical to PICALM function. Increased expression of the common isoform lacking exon 13 was also associated with the rs3851179A protective allele; this association was not apparent when this isoform was compared with total PICALM expression, indicating that the SNP is associated with total PICALM expression and not this isoform per se. Interestingly, PICALM lacking exons 2-4 was not associated with rs3851179 but was associated with rs592297, which is located in exon 5. Thus, our primary findings are that multiple PICALM isoforms are expressed in the human brain, that PICALM is robustly expressed in microvessels, and that expression of total PICALM is modestly correlated with the AD-associated SNP rs3851179. We interpret these results as suggesting that increased PICALM expression in the microvasculature may reduce AD risk.
    PLoS ONE 03/2014; 9(3):e91242. DOI:10.1371/journal.pone.0091242 · 3.23 Impact Factor
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