Clathrin assembly lymphoid myeloid leukemia protein (CALM) is a clathrin assembly protein with a domain structure similar to the neuron-specific assembly protein AP180. We have previously found that CALM is expressed in neurons and present in synapses. We now report that CALM has a neuron-related function: it facilitates the endocytosis of the synaptic vesicle protein VAMP2 from the plasma membrane. Overexpression of CALM leads to the reduction of cell surface VAMP2, whereas knockdown of CALM by RNA interference results in the accumulation of surface VAMP2. The AP180 N-terminal homology (ANTH) domain of CALM is required for its effect on VAMP2 trafficking, and the ANTH domain itself acts as a dominant-negative mutant. Thus, our results reveal a role for CALM in directing VAMP2 trafficking during endocytosis. Neurotransmitter release is crucial to neuron function. In the presynaptic terminal, neurotransmitter release begins with the fusion of synaptic vesicles (SV) to the presynaptic plasma membrane (1). SV fusion is mediated by the SNARE proteins, which include the VAMP2 (also known as synap-tobrevin 2) on the vesicle and syntaxin 1 and SNAP25 on the plasma membrane (2,3). A recent quantitative analysis of SV constituents reveals a startling abundance of VAMP2 on SV, twice that of the next most abundant SV protein-synaptophysin (4). This finding emphasizes the importance of VAMP2 for SV. A fundamental question concerning the function of SV is how the neuron precisely constitutes and effectively preserves the vesicle components. Following SV fusion and subsequent exocytosis to release neurotransmitters, the retrieval of SV components that have dispersed to the presynaptic plasma membrane is thought to be accomplished primarily by clathrin-mediated endocytosis (5-7). Live cell image studies of hippocampal neurons show that surface VAMP2, most of which originates from SV, does not confine itself to the fusion site; instead, the protein diffuses along the axonal membrane even beyond the synapse (8,9). Although there is no doubt that endocytosis plays an indispensable role in the retrieval of SV components, it is unclear exactly how the SV acquires and maintains the individual SV-associated proteins-including VAMP2-through repeated cycles of exocytosis and endocytosis. Studies of Caenorhabditis elegans (10,11) and Drosophila (12) mutants have associated the loss of functional assembly protein (AP)180 with the misplacement of VAMP2 from its usual location on SV to the plasma membrane. AP180, a clathrin assembly protein, has been well characterized for its function in promoting the assembly of clathrin-coated vesicles at the plasma membrane (13,14). The membrane accumulation of VAMP2 in the AP180 mutants suggests that the assembly protein AP180, at least in Caenorhabditis elegans and Drosophila, has a specialized role in recruiting and directing VAMP2 from the plasma membrane to SV during endocytosis. Clathrin assembly lymphoid myeloid leukemia protein (CALM) is a clathrin assembly protein that structurally resembles A...

Dermatofibrosarcoma protuberans (DFSP) is a slow growing, low- to intermediate-grade dermal soft-tissue tumor. It has a high local recurrence rate but low metastatic potential. It is characterized by a uniform spindle cell arrangement, classically with a storiform pattern and CD34 immunoreactivity. The histomorphology and immunophenotype overlap with a broad range of other neoplasms. The standard treatment is complete surgical excision. The surgical procedures include wide local excision (WLE) with tumor free margins, Mohs micrographic surgery (MMS) and amputation. Unresectable DFSPs are treated with radiation therapy and/or targeted therapy. DFSP has characteristic t(17; 22) (q22; q13), resulting in a COL1A1- PDGFB fusion transcripts in more than 90% of DFSPs. Molecular detection of the gene rearrangement or fusion transcripts is helpful for the diagnosis of patients with atypical morphology and for screening candidates for targeted therapy with tyrosine kinase inhibitors. The aims of the present review are to update the clinical presentation, tumorigenesis and histopathology of DFSP and its variants for diagnosis and differential diagnosis from other benign and malignant tumors, to compare the advantages and drawbacks of WLE and MMS, to propose the baseline for selecting surgical procedure based on tumor’s location, size, stage and relationship with surrounding soft tissue and bone structures, and to provide a biologic rationale for the systemic therapy. We further propose a modified clinical staging system of DFSP and a surveillance program for the patients after surgical excision.

Emerging data suggest that, much like epithelial cells, the polarized growth of neurons requires both the secretory and endocytic pathways. The clathrin assembly proteins AP180 and CALM (clathrin assembly lymphoid myeloid protein) are known to be involved in clathrin-mediated endocytosis, but their roles in mammalian neurons and, in particular, in developmental processes before synaptogenesis are unknown. Here we provide evidence that AP180 and CALM play critical roles in establishing the polarity and controlling the growth of axons and dendrites in embryonic hippocampal neurons. Knockdown of AP180 primarily impairs axonal development, whereas reducing CALM levels results in dendritic dystrophy. Conversely, neurons that overexpress AP180 or CALM generate multiple axons. Ultrastructural analysis shows that CALM affiliates with a wider range of intracellular trafficking organelles than does AP180. Functional analysis shows that endocytosis is reduced in both AP180-deficient and CALM-deficient neurons. Additionally, CALMdeficient neurons show disrupted secretory transport. Our data demonstrate previously unknown functions for AP180 and CALM in intracellular trafficking that are essential in the growth of neurons.

No abstract

Clathrin assembly proteins AP180 and CALM regulate the assembly of clathrin-coated vesicles (CCVs), which mediate diverse intracellular trafficking processes, including synaptic vesicle (SV) recycling at the synapse. Although studies using several invertebrate model systems have indicated a role for AP180 in SV recycling, less is known about AP180’s or CALM’s function in the synapse of mammalian neurons. In this study, we examined synapses of rat hippocampal neurons in which the level of AP180 or CALM had been reduced by RNA interference (RNAi). Using light microscopy, we visualized synaptic puncta in these AP180- or CALM-reduced neurons by co-expressing Synaptophysin::EGFP (Syp::EGFP). We found that neurons with reduced AP180 or reduced CALM had smaller Syp::EGFP-illuminated puncta. Using electron microscopy, we further examined the ultrastructure of the AP180- or CALM-reduced presynaptic terminals. We found that SVs became variably enlarged in both the AP180-reduced and CALM-reduced presynaptic terminals. Lower AP180 and CALM also reduced the density of SVs and the size of SV clusters. Our findings demonstrate that in the presynaptic terminals of hippocampal neurons, AP180 and CALM have a similar role in regulating synaptic vesicles. This overlapping activity may be necessary for high-precision and high-efficacy SV formation during endocytosis.

The overproduction and extracellular buildup of amyloid-β peptide (Aβ) is a critical step in the etiology of Alzheimer's disease. Recent data suggest that intracellular trafficking is of central importance in the production of Aβ. Here we use a neuronal cell line to examine two structurally similar clathrin assembly proteins, AP180 and CALM. We show that RNA interference-mediated knockdown of AP180 reduces the generation of Aβ 1-40 and Aβ 1-42, whereas CALM knockdown has no effect on Aβ generation. Thus AP180 is among the traffic controllers that oversee and regulate amyloid precursor protein processing pathways. Our results also suggest that AP180 and CALM, while similar in their domain structures and biochemical properties, are in fact dedicated to separate trafficking pathways in neurons.

No abstract

Background: We previously described planar areal differences in adult mouse visual, somatosensory, and neocortex that collectively discriminated C57BL/6J and DBA/2J inbred strain identity. Here we use a novel application of established methods of two-dimensional geometric morphometrics to examine shape differences in the cortical area maps of these inbred strains.