Clathrin Assembly Lymphoid Myeloid Leukemia Research Articles

Using quantitative microscopy approaches to study the formation of clathrin-coated vesicles.

Clathrin-coated vesicles (CCVs) are endocytic carriers that mediate the internalization of ligands and their receptors from the plasma membrane. CCVs assembly requires the coordinated recruitment of many proteins that promote receptor sorting and membrane curvature formation. Clathrin triskelia together with the adaptors AP2 and clathrin assembly lymphoid myeloid leukemia protein (CALM) are the most represented components of CCVs. CALM supports the formation of membrane curvature that is essential for the growth of CCVs. Membrane curvature can be reached only by overcoming local plasma membrane tension that, together with the availability of CCV components, determines the type of clathrin structures that forms over the plasma membrane. We hypothesized that CALM recruitment could support the formation of CCVs even under mechanically stringent conditions, such as increased membrane tension. To investigate this, we used knock-in gene editing to obtain cells expressing fluorescently tagged CALM at endogenous levels and spinning-disc and lattice light sheet live-cell microscopy calibrated to the intensity of a single molecule to quantitatively study CALM recruitment during the formation of CCVs in normal conditions, upon membrane tension increase, and/or CALM depletion. Our results demonstrate that CALM promotes CCV completion under membrane tension increase possibly by a mechanism based on protein-protein crowding at the neck of the CCV. We also observed that CALM is differentially expressed across cell types and that given the small footprint of CALM, there is sufficient room on the CCV surface to recruit CALM molecules as needed, depending on the local membrane tension and cellular availability. In conclusion, our data suggest that the competence in clathrin-mediated endocytosis differs across cells and tissues and explain the reason why CALM is essential during embryo development but dispensable in other cellular contexts.

CALM supports clathrin-coated vesicle completion upon membrane tension increase

The most represented components of clathrin-coated vesicles (CCVs) are clathrin triskelia and the adaptors clathrin assembly lymphoid myeloid leukemia protein (CALM) and the heterotetrameric complex AP2. Investigation of the dynamics of AP180-amino-terminal-homology (ANTH) recruitment during CCV formation has been hampered by CALM toxicity upon overexpression. We used knock-in gene editing to express a C-terminal-attached fluorescent version of CALM, while preserving its endogenous expression levels, and cutting-edge live-cell microscopy approaches to study CALM recruitment at forming CCVs. Our results demonstrate that CALM promotes vesicle completion upon membrane tension increase as a function of the amount of this adaptor present. Since the expression of adaptors, including CALM, differs among cells, our data support a model in which the efficiency of clathrin-mediated endocytosis is tissue specific and explain why CALM is essential during embryogenesis and red blood cell development.

MLL and CALM Are Fused to AF10 in Morphologically Distinct Subsets of Acute Leukemia With Translocation t(10;11): Both Rearrangements Are Associated With a Poor Prognosis

AbstractThe translocation t(10;11)(p13;q14) has been observed in acute lymphoblastic leukemia (ALL) as well as acute myeloid leukemia (AML). A recent study showed a MLL/AF10fusion in all cases of AML with t(10;11) and various breakpoints on chromosome 11 ranging from q13 to q23. We recently cloned CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene), the fusion partner of AF10 at 11q14 in the monocytic cell line U937. To further define the role of these genes in acute leukemias, 10 cases (9 AML and 1 ALL) with cytogenetically proven t(10;11)(p12-14;q13-21) and well-characterized morphology, immunophenotype, and clinical course were analyzed. Interphase fluorescence in situ hybridization (FISH) was performed with 2 YACs flanking the CALM region, a YAC contig of the MLLregion, and a YAC spanning the AF10 breakpoint. Rearrangement of at least one of these genes was detected in all cases with balanced t(10;11). In 4 cases, including 3 AML with immature morphology (1 AML-M0 and 2 AML-M1) and 1 ALL, the signals of the CALM YACS were separated in interphase cells, indicating a translocation breakpoint within the CALM region. MLL was rearranged in 3 AML with myelomonocytic differentiation (2 AML-M2 and 1 AML-M5), including 1 secondary AML. In all 3 cases, a characteristic immunophenotype was identified (CD4+, CD13−, CD33+, CD65s+).AF-10 was involved in 5 of 6 evaluable cases, including 1 case without detectable CALM or MLL rearrangement. In 2 complex translocations, none of the three genes was rearranged. All cases had a remarkably poor prognosis, with a mean survival of 9.6 ± 6.6 months. For the 7 AML cases that were uniformly treated according to the AMLCG86/92 protocols, disease-free and overall survival was significantly worse than for the overall study group (P = .03 and P = .01, respectively). We conclude that the t(10;11)(p13;q14) indicates CALM and MLL rearrangements in morphologically distinct subsets of acute leukemia and may be associated with a poor prognosis.

Chlorpromazine eliminates acute myeloid leukemia cells by perturbing subcellular localization of FLT3-ITD and KIT-D816V

Mutated receptor tyrosine kinases (MT-RTKs) such as internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3 ITD) and a point mutation KIT D816V are driver mutations for acute myeloid leukemia (AML). Clathrin assembly lymphoid myeloid leukemia protein (CALM) regulates intracellular transport of RTKs, however, the precise role for MT-RTKs remains elusive. We here show that CALM knock down leads to severely impaired FLT3 ITD- or KIT D814V-dependent cell growth compared to marginal influence on wild-type FLT3- or KIT-mediated cell growth. An antipsychotic drug chlorpromazine (CPZ) suppresses the growth of primary AML samples, and human CD34+CD38- AML cells including AML initiating cells with MT-RTKs in vitro and in vivo. Mechanistically, CPZ reduces CALM protein at post transcriptional level and perturbs the intracellular localization of MT-RTKs, thereby blocking their signaling. Our study presents a therapeutic strategy for AML with MT-RTKs by altering the intracellular localization of MT-RTKs using CPZ.

A Novel Sequence in AP180 and CALM Promotes Efficient Clathrin Binding and Assembly.

The clathrin heavy chain N-terminal domain interacts with endocytic adapter proteins via clathrin binding motifs to assemble clathrin triskelia into cages. However, the precise mechanism of clathrin assembly is not yet known. Clathrin assembly protein AP180 has more clathrin binding motifs than any other endocytic protein and has a major role in the assembly of the clathrin coat during synaptic vesicle biogenesis. We now demonstrate that some of the previously identified binding motifs in AP180 may be non-functional and that a non-conventional clathrin binding sequence has a major influence on AP180 function. The related protein, clathrin assembly lymphoid myeloid leukemia protein (CALM), has fewer clathrin binding motifs and functions ubiquitously in clathrin-mediated endocytosis. The C-terminal ~16 kDa sub-domain in AP180, which has relatively high similarity with CALM, was shown in earlier work to have an unexplained role in clathrin binding. We identified the specific sequences in this sub-domain that bind to clathrin. Evidence for a role for these sequences in promoting clathrin binding was examined using in vitro and ex vivo experiments that compared the clathrin binding ability of site mutants with the wild type sequence. A sequence conserved in both AP180 and CALM (LDSSLA[S/N]LVGNLGI) was found to be the major interaction site and mutation caused a deficit in clathrin assembly, which is the first example of a mutation having this effect. In contrast, single or double mutation of DL(L/F) motifs in full length AP180 had no significant effect on clathrin binding, despite higher clathrin affinity for isolated peptides containing these motifs. We conclude that the novel clathrin interaction sites identified here in CALM and AP180 have a major role in how these proteins interface with clathrin. This work advances the case that AP180 and CALM are required to use a combination of standard clathrin N-terminal domain binding motifs and the sequence identified here for optimal binding and assembling clathrin.

Synaptophysin 1 Clears Synaptobrevin 2 from the Presynaptic Active Zone to Prevent Short-Term Depression.

Release site clearance is an important process during synaptic vesicle (SV) recycling. However, little is known about its molecular mechanism. Here we identify self-assembly of exocytosed Synaptobrevin 2 (Syb2) and Synaptophysin 1 (Syp1) by homo- and hetero-oligomerization into clusters as key mechanisms mediating release site clearance for preventing cis-SNARE complex formation at the active zone (AZ). In hippocampal neurons from Syp1 knockout mice, neurons expressing a monomeric Syb2 mutant, or after acute block of the ATPase N-ethylmaleimide-sensitive factor (NSF), responsible for cis-SNARE complex disassembly, we found strong frequency-dependent short-term depression (STD), whereas retrieval of Syb2 by compensatory endocytosis was only affected weakly. Defects in Syb2 endocytosis were stimulus- and frequency-dependent, indicating that Syp1 is not essential for Syb2 retrieval, but for its efficient clearance upstream of endocytosis. Our findings identify an SV protein as a release site clearance factor.

The ∼ 16 kDa C-terminal sequence of clathrin assembly protein AP180 is essential for efficient clathrin binding.

Brain-specific AP180 is present in clathrin coats at equal concentration to the adapter complex, AP2, and assembles clathrin faster than any other protein in vitro. Both AP180 and its ubiquitously expressed homolog clathrin assembly lymphoid myeloid leukemia protein (CALM) control vesicle size and shape in clathrin mediated endocytosis. The clathrin assembly role of AP180 is mediated by a long disordered C-terminal assembly domain. Within this assembly domain, a central acidic clathrin and adapter binding (CLAP) sub-domain contains all of the known short binding motifs for clathrin and AP2. The role of the remaining ∼16 kDa C-terminal sequence has not been clear. We show that this sequence has a separate function in ensuring efficient binding of clathrin, based on in vitro binding and ex vivo transferrin uptake assays. Sequence alignment suggests the C-terminal sub-domain is conserved in CALM.

The Biochemical Properties and Functions of CALM and AP180 in Clathrin Mediated Endocytosis.

Clathrin-mediated endocytosis (CME) is a fundamental process for the regulated internalization of transmembrane cargo and ligands via the formation of vesicles using a clathrin coat. A vesicle coat is initially created at the plasma membrane by clathrin assembly into a lattice, while a specific cargo sorting process selects and concentrates proteins for inclusion in the new vesicle. Vesicles formed via CME traffic to different parts of the cell and fuse with target membranes to deliver cargo. Both clathrin assembly and cargo sorting functions are features of the two gene family consisting of assembly protein 180 kDa (AP180) and clathrin assembly lymphoid myeloid leukemia protein (CALM). In this review, we compare the primary structure and domain organization of CALM and AP180 and relate these properties to known functions and roles in CME and disease.

Nuclear export signal within CALM is necessary for CALM‐AF10‐induced leukemia

The CALM–AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies. Certain HOXA cluster genes and MEIS1 genes are upregulated in patients and mouse models that express CALM-AF10. Wild-type clathrin assembly lymphoid myeloid leukemia protein (CALM) primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia. To investigate the influence of localization on leukemogenesis involving CALM-AF10, we determined the nuclear export signal (NES) within CALM that is necessary and sufficient for cytoplasmic localization of CALM-AF10. Mutations in the NES eliminated the capacity of CALM-AF10 to immortalize murine bone-marrow cells in vitro and to promote development of acute myeloid leukemia in mouse models. Furthermore, a fusion of AF10 with the minimal NES can immortalize bone-marrow cells and induce leukemia in mice. These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm.

Adaptor complex AP2/PICALM, through interaction with LC3, targets Alzheimer's APP-CTF for terminal degradation via autophagy

The hallmarks of Alzheimer's disease (AD) are the aggregates of amyloid-β (Aβ) peptides and tau protein. Autophagy is a major cellular pathway leading to the removal of aggregated proteins. We have reported recently that autophagy was responsible for amyloid precursor protein cleaved C-terminal fragment (APP-CTF) degradation and amyloid β clearance in an Atg5-dependent manner. Here we aimed to elucidate the molecular mechanism by which autophagy mediates the degradation of APP-CTF and the clearance of amyloid β. Through affinity purification followed by mass spectrum analysis, we identified adaptor protein (AP) 2 together with phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) as binding proteins of microtubule-associated protein 1 light chain 3 (LC3). Further analysis showed that AP2 regulated the cellular levels of APP-CTF. Knockdown of AP2 reduced autophagy-mediated APP-CTF degradation. Immunoprecipitation and live imaging analysis demonstrated that AP2 and PICALM cross-link LC3 with APP-CTF. These data suggest that the AP-2/PICALM complex functions as an autophagic cargo receptor for the recognition and shipment of APP-CTF from the endocytic pathway to the LC3-marked autophagic degradation pathway. This molecular mechanism linking AP2/PICALM and AD is consistent with genetic evidence indicating a role for PICALM as a risk factor for AD.

Identification of GSX2 and AF10 as NUP98 partner genes in myeloid malignancies

Identification of GSX2 and AF10 as NUP98 partner genes in myeloid malignancies

Uncoupling the Functions of CALM in VAMP Sorting and Clathrin-Coated Pit Formation

CALM (clathrin assembly lymphoid myeloid leukemia protein) is a cargo-selective adaptor for the post-Golgi R-SNAREs VAMPs 2, 3, and 8, and it also regulates the size of clathrin-coated pits and vesicles at the plasma membrane. The present study has two objectives: to determine whether CALM can sort additional VAMPs, and to investigate whether VAMP sorting contributes to CALM-dependent vesicle size regulation. Using a flow cytometry-based endocytosis efficiency assay, we demonstrate that CALM is also able to sort VAMPs 4 and 7, even though they have sorting signals for other clathrin adaptors. CALM homologues are present in nearly every eukaryote, suggesting that the CALM family may have evolved as adaptors for retrieving all post-Golgi VAMPs from the plasma membrane. Using a knockdown/rescue system, we show that wild-type CALM restores normal VAMP sorting in CALM-depleted cells, but that two non-VAMP-binding mutants do not. However, when we assayed the effect of CALM depletion on coated pit morphology, using a fluorescence microscopy-based assay, we found that the two mutants were as effective as wild-type CALM. Thus, we can uncouple the sorting function of CALM from its structural role.

Iron Deprivation Impairs Proliferation of CALM-AF10 leukemia Cells in Vitro and in Vivo

Abstract 1386 Background:The CALM-AF10 fusion protein arises from the t(10;11) chromosomal translocation and is found in 8–10% of T-cell acute lymphoblastic leukemias and a smaller percentage of acute myeloid leukemias; these hematopoietic malignancies are associated with a poor clinical outcome. The precise contributions of the Clathrin Assembly Lymphoid Myeloid leukemia (CALM) protein to leukemogenesis remain uncertain. CALM plays a role in clathrin-dependent endocytosis, which mediates the entry of growth factor receptors and nutrients into cells and is essential for the internalization of iron-bound transferrin. We have previously shown that Calm-deficient (Calm−/−) mouse fibroblasts derived from fit1 mice are iron deficient, and are more sensitive to treatment with iron chelators. Objective:Since CALM-AF10 leukemia cells are haploinsufficient for CALM, we hypothesize that reduced levels of CALM in CALM-AF10 leukemia cells cause a relative iron deficiency, and result in enhanced sensitivity to iron deprivation. Design/Methods:Fibroblasts and fetal liver hematopoietic progenitors (HP) were derived from Calm−/−, Calm+/− or Calm+/+ E14 embryos. The proliferation rates of non-immortalized fibroblasts were compared in the presence and absence of supplemental iron (ferric ammonium citrate (FAC)) or treatment with an iron chelator (deferoxamine (DFO)). Surface transferrin receptor expression was quantified by flow cytometry. Primary HP cells were cultured in the presence or absence of DFO and viable cell numbers were determined. To examine the anti-leukemic effect of iron deprivation in vivo, C57BL/6J-Tyrc-2J mice were fed a low-iron diet, transplanted with CALM-AF10-transduced Calm+/− leukemia cells and leukemia latency was determined. Results:Heterozygous Calm+/− fibroblasts exhibit CALM protein levels that are intermediate between their wildtype (Calm+/+) and deficient (Calm−/−) counterparts. Calm+/− cells display a slower rate of proliferation in vitro (50% reduction of viable cells) compared to their wildtype fibroblasts, and this growth deficiency can be corrected by iron supplementation with 50 mM FAC. The presence of reduced intracellular iron levels in Calm+/− fibroblasts was manifested by increased transferrin receptor expression relative to wildtype cells. In vitro, Calm+/− HP cells showed greater sensitivity to iron chelation by DFO (5 mM) than Calm+/+ controls: the relative number of viable cells in the presence of DFO was 20% lower in Calm+/− compared to Calm+/− HP cells. To assess the impact of iron deprivation in vivo, bone marrow cells from Calm+/−CALM-AF10 leukemic mice were injected into secondary recipient mice preconditioned by having received a low-iron diet for 8 weeks (n=9). The onset of leukemia in transplanted mice maintained on the iron-deficient diet was considerably delayed (median survival 93 days versus 63 days, p=0.013) relative to age- and gender-matched control mice fed a normal diet (n=9). Conclusions:We have shown that reduced CALM expression impairs iron import and consequently limits the rate of cell proliferation. Both in vitro and in vivo results suggest that CALM haploinsufficient CALM-AF10 leukemias are particularly sensitive to iron deprivation. This raises the possibility that iron chelation may be a previously unappreciated treatment option for patients with aggressive CALM-rearranged leukemias. We are currently studying the impact of iron chelation in murine CALM-AF10 in vivo leukemia models. Disclosures:No relevant conflicts of interest to declare.

Abrogation of MLL–AF10 and CALM–AF10-mediated transformation through genetic inactivation or pharmacological inhibition of the H3K79 methyltransferase Dot1l

The t(10;11)(p12;q23) translocation and the t(10;11)(p12;q14) translocation, which encode the MLL (mixed lineage leukemia)-AF10 and CALM (clathrin assembly lymphoid myeloid leukemia)-AF10 fusion oncoproteins, respectively, are two recurrent chromosomal rearrangements observed in patients with acute myeloid leukemia and acute lymphoblastic leukemia. Here, we demonstrate that MLL-AF10 and CALM-AF10-mediated transformation is dependent on the H3K79 methyltransferase Dot1l using genetic and pharmacological approaches in mouse models. Targeted disruption of Dot1l using a conditional knockout mouse model abolished in vitro transformation of murine bone marrow cells and in vivo initiation and maintenance of MLL-AF10 or CALM-AF10 leukemia. The treatment of MLL-AF10 and CALM-AF10 transformed cells with EPZ004777, a specific small-molecule inhibitor of Dot1l, suppressed expression of leukemogenic genes such as Hoxa cluster genes and Meis1, and selectively impaired proliferation of MLL-AF10 and CALM-AF10 transformed cells. Pretreatment with EPZ004777 profoundly decreased the in vivo spleen-colony-forming ability of MLL-AF10 or CALM-AF10 transformed bone marrow cells. These results show that patients with leukemia-bearing chromosomal translocations that involve the AF10 gene may benefit from small-molecule therapeutics that inhibit H3K79 methylation.

Role of Phosphatidylinositol Clathrin Assembly Lymphoid-Myeloid Leukemia (PICALM) in Intracellular Amyloid Precursor Protein (APP) Processing and Amyloid Plaque Pathogenesis

One of the pathological hallmarks of Alzheimer disease is the accumulation of amyloid plaques in the extracellular space in the brain. Amyloid plaques are primarily composed of aggregated amyloid β peptide (Aβ), a proteolytic fragment of the transmembrane amyloid precursor protein (APP). For APP to be proteolytically cleaved into Aβ, it must be internalized into the cell and trafficked to endosomes where specific protease complexes can cleave APP. Several recent genome-wide association studies have reported that several single nucleotide polymorphisms (SNPs) in the phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) gene were significantly associated with Alzheimer disease, suggesting a role in APP endocytosis and Aβ generation. Here, we show that PICALM co-localizes with APP in intracellular vesicles of N2a-APP cells after endocytosis is initiated. PICALM knockdown resulted in reduced APP internalization and Aβ generation. Conversely, PICALM overexpression increased APP internalization and Aβ production. In vivo, PICALM was found to be expressed in neurons and co-localized with APP throughout the cortex and hippocampus in APP/PS1 mice. PICALM expression was altered using AAV8 gene transfer of PICALM shRNA or PICALM cDNA into the hippocampus of 6-month-old APP/PS1 mice. PICALM knockdown decreased soluble and insoluble Aβ levels and amyloid plaque load in the hippocampus. Conversely, PICALM overexpression increased Aβ levels and amyloid plaque load. These data indicate that PICALM, an adaptor protein involved in clathrin-mediated endocytosis, regulates APP internalization and subsequent Aβ generation. PICALM contributes to amyloid plaque load in brain likely via its effect on Aβ metabolism.

The Clathrin Assembly Protein PICALM Is Required for Erythroid Maturation and Transferrin Internalization in Mice

Phosphatidylinositol binding clathrin assembly protein (PICALM), also known as clathrin assembly lymphoid myeloid leukemia protein (CALM), was originally isolated as part of the fusion gene CALM/AF10, which results from the chromosomal translocation t(10;11)(p13;q14). CALM is sufficient to drive clathrin assembly in vitro on lipid monolayers and regulates clathrin-coated budding and the size and shape of the vesicles at the plasma membrane. However, the physiological role of CALM has yet to be elucidated. Here, the role of CALM in vivo was investigated using CALM-deficient mice. CALM-deficient mice exhibited retarded growth in utero and were dwarfed throughout their shortened life-spans. Moreover, CALM-deficient mice suffered from severe anemia, and the maturation and iron content in erythroid precursors were severely impaired. CALM-deficient erythroid cells and embryonic fibroblasts exhibited impaired clathrin-mediated endocytosis of transferrin. These results indicate that CALM is required for erythroid maturation and transferrin internalization in mice.

CALM Insufficiency Impairs Leukemia Cell Proliferation by Limiting Iron Uptake

Abstract 1478 Background:The CALM-AF10 fusion protein that arises from the t(10;11) chromosomal translocation is found in both acute myeloid leukemia and 8–10% of T-cell acute lymphoblastic leukemia and is associated with a poor clinical outcome. The interaction of AF10 with the DOT1L histone methyltransferase has been shown to be necessary for CALM-AF10 mediated transformation. However, the role of the Clathrin Assembly Lymphoid Myeloid leukemia (CALM) protein in leukemogenesis remains uncertain. CALM plays a role in clathrin-mediated endocytosis. This process mediates the entry of various growth factor receptors and nutrients into cells and is essential for the internalization of iron-bound transferrin. Objective:We hypothesize that loss of CALM function, as a result of CALM haploinsufficiency and/or a dominant negative CALM-AF10 effect, may contribute to transformation. Design/Methods:To study the effects of CALM deficiency, we used genetically modified fit1 mice that harbor a Calm gene mutation that results in a severely truncated and nonfunctional Calm protein. Fetal liver (FL) cells from Calm−/−, Calm+/− or CalmWT/WT E14 embryos were retrovirally transduced with MLL-ENL or CALM-AF10 and studied in vitro or transplanted into syngeneic mice. Interleukin-3 dependent cell lines were established from Calm−/− or CalmWT control FL cells immortalized by CALM-AF10 or MLL-ENL. Results:CALM-AF10 leukemias induced in Calm−/− or Calm+/− precursors were considerably delayed compared with those induced in CalmWT controls. Clathrin-mediated endocytosis of fluorescently labeled transferrin receptor (TfR) was impaired in Calm−/− cells. This was accompanied by a 2–4 fold increase in surface expression of TfR by flow cytometry and an increase in total TfR protein as measured by Western blot in Calm−/− cells. Calm -deficient cells also displayed lower levels of the iron storage protein ferritin, suggestive of an iron deficient state. Each of these features was reversed by re-expression of CALM via retroviral transduction of Calm−/− cells with a CALM expression vector. Paralleling the prolonged latency of Calm−/− leukemias, we found that Calm -deficient cells proliferated at a considerably slower rate than CALM -rescued cells. This cell proliferation defect could be rescued by supplementing the cells with iron (ferric ammonium citrate, 50 μM), indicating that reduced iron availability limits the expansion of Calm -deficient cells. Intriguingly, Calm−/− cells were significantly more sensitive to the growth inhibitory effect of iron chelation (deferoxamine, 5 μM) than CALM -rescued cells. Conclusions:These observations suggest that loss of CALM function impairs iron import and consequently limits the rate of cell proliferation. This raises the possibility that CALM haploinsufficiency present in CALM-AF10 leukemias might render cells particularly responsive to iron chelation therapy. Disclosures:No relevant conflicts of interest to declare.

FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

The t(10;11)(p13;q14) translocation results in the fusion of the CALM (clathrin assembly lymphoid myeloid leukemia protein) and AF10 genes. This translocation is observed in acute myeloblastic leukemia (AML M6), acute lymphoblastic leukemia (ALL) and malignant lymphoma. Using a yeast two-hybrid screen, the four and a half LIM domain protein 2 (FHL2) was identified as a CALM interacting protein. Recently, high expression of FHL2 in breast, gastric, colon, lung as well as in prostate cancer was shown to be associated with an adverse prognosis. The interaction between CALM and FHL2 was confirmed by glutathione S-transferase-pulldown assay and co-immunoprecipitation experiments. The FHL2 interaction domain of CALM was mapped to amino acids 294–335 of CALM. The transcriptional activation capacity of FHL2 was reduced by CALM, but not by CALM/AF10, which suggests that regulation of FHL2 by CALM might be disturbed in CALM/AF10-positive leukemia. Extremely high expression of FHL2 was seen in acute erythroid leukemia (AML M6). FHL2 was also highly expressed in chronic myeloid leukemia and in AML with complex aberrant karyotype. These results suggest that FHL2 may play an important role in leukemogenesis, especially in the case of AML M6.

SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses

Neurotransmission depends on the exo-endocytosis of synaptic vesicles at active zones. Synaptobrevin 2 [also known as vesicle-associated membrane protein 2 (VAMP2)], the most abundant synaptic vesicle protein and a major soluble NSF attachment protein receptor (SNARE) component, is required for fast calcium-triggered synaptic vesicle fusion. In contrast to the extensive knowledge about the mechanism of SNARE-mediated exocytosis, little is known about the endocytic sorting of synaptobrevin 2. Here we show that synaptobrevin 2 sorting involves determinants within its SNARE motif that are recognized by the ANTH domains of the endocytic adaptors AP180 and clathrin assembly lymphoid myeloid leukemia (CALM). Depletion of CALM or AP180 causes selective surface accumulation of synaptobrevin 2 but not vGLUT1 at the neuronal surface. Endocytic sorting of synaptobrevin 2 is mediated by direct interaction of the ANTH domain of the related endocytic adaptors CALM and AP180 with the N-terminal half of the SNARE motif centered around M46, as evidenced by NMR spectroscopy analysis and site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the ANTH domain proteins AP180 and CALM as cargo-specific adaptors for synaptobrevin endocytosis. Defective SNARE endocytosis may also underlie the association of CALM and AP180 with neurodevelopmental and cognitive defects or neurodegenerative disorders.

Clathrin assembly proteins AP180 and CALM in the embryonic rat brain

Clathrin-coated vesicles are known to play diverse and pivotal roles in cells. The proper formation of clathrin-coated vesicles is dependent on, and highly regulated by, a large number of clathrin assembly proteins. These assembly proteins likely determine the functional specificity of clathrin-coated vesicles, and together they control a multitude of intracellular trafficking pathways, including those involved in embryonic development. In this study, we focus on two closely related clathrin assembly proteins, AP180 and CALM (clathrin assembly lymphoid myeloid leukemia protein), in the developing embryonic rat brain. We find that AP180 begins to be expressed at embryonic day 14 (E14), but only in postmitotic cells that have acquired a neuronal fate. CALM, on the other hand, is expressed as early as E12, by both neural stem cells and postmitotic neurons. In vitro loss-of-function studies using RNA interference (RNAi) indicate that AP180 and CALM are dispensable for some aspects of embryonic neurogenesis but are required for the growth of postmitotic neurons. These results identify the developmental stage of AP180 and CALM expression and suggest that each protein has distinct functions in neural development.