Binding of Vac14 to neuronal nitric oxide synthase: Characterisation of a new internal PDZ-recognition motif

AbstractPDZ domains mediate protein interactions primarily through either classical recognition of carboxyl-terminal motifs or PDZ/PDZ domain associations. Several studies have also described internal modes of PDZ recognition, most of which depend on β-finger structures. Here, we describe a novel interaction between the PDZ domain of nNOS and Vac14, the activator of the PtdIns(3)P 5-kinase PIKfyve. Binding assays using various Vac14 deletion constructs revealed a β-finger independent interaction that is based on a novel internal motif. Mutational analyses reveal essential residues within the motif allowing us to define a new type of PDZ domain interaction

ENTH/ANTH proteins and clathrin-mediated membrane budding

The epsin N-terminal homology (ENTH) domain is an evolutionarily conserved protein module found primarily in proteins that participate in clathrin-mediated endocytosis. Structural analyses and ligand-binding studies have shown that a set of proteins previously designated as harboring an ENTH domain in fact contain a highly similar, yet unique module referred to as an AP180 N-terminal homology (ANTH) domain. ENTH and ANTH (E/ANTH) domains bind both inositol phospholipids and proteins and contribute to the nucleation and formation of clathrin coats on membranes. ENTH domains also function in the development of membrane curvature through lipid remodeling during the formation of clathrin-coated vesicles. E/ANTH-bearing proteins have recently been shown to function with adaptor protein-1 and GGA adaptors at the trans-Golgi network, which suggests that E/ANTH domains are universal components of the machinery for clathrin-mediated membrane budding

Endophilin regulates JNK activation through its interaction with the germinal center kinase-like kinase

The endophilin family of proteins function in clathrin-mediated endocytosis. Here, we have identified and cloned the rat germinal center kinase-like kinase (rGLK), a member of the GCK (germinal center kinase) family of e-Jun N-terminal kinase (JNK) activating enzymes, as a novel endophilin I-binding partner. The interaction occurs both in vitro and in cells and is mediated by the Src homology 3 domain of endophilin I and a region of rGLK containing the endophilin consensus-binding sequence PPRPPPPR. Overlay analysis of rat brain extracts demonstrates that endophilin I is a major Src homology 3 domain-binding partner for rGLK. Overexpression of full-length endophilin I activates rGLK-mediated JNK activation, whereas N- and C-terminal fragments of endophilin I block JNK activation. Thus, endophilin I appears to have a novel function in JNK activation

Synaptojanin Forms Two Separate Complexes in the Nerve Terminal: INTERACTIONS WITH ENDOPHILIN AND AMPHIPHYSIN

Endophilin is a recently discovered src homology 3 domain-containing protein that is a major in vitro binding partner for synaptojanin. To further characterize endophilin, we generated an antipeptide antibody. Endophilin is enriched in the brain, and immunofluorescence analysis reveals a high concentration of the protein in synaptic terminals, where it colocalizes with synaptojanin. In vitro binding assays demonstrate that endophilin binds through its src homology 3 domain to synaptojanin, and immunoprecipitation analysis with the antiendophilin antibody reveals that endophilin is stably associated with synaptojanin in the nerve terminal. Immunoprecipitation with an antibody against amphiphysin I and II, which interact through their src homology 3 domains with dynamin and synaptojanin at sites distinct from those for endophilin, reveals a second stable complex, which includes dynamin and synaptojanin but excludes endophilin. These data demonstrate that synaptojanin is present in two separate complexes in the nerve terminal and support an important role for endophilin in the regulation of synaptojanin function

The identification of high-performing antibodies for Coiled-coil-helix-coiled-coil-helix domain containing protein 10 (CHCHD10) for use in Western Blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved, 1 approved with reservations]

CHCHD10 is a mitochondrial protein, implicated in the regulation of mitochondrial morphology and cristae structure, as well as the maintenance of mitochondrial DNA integrity. Recently discovered to be associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in its mutant form, the scientific community would benefit from the availability of validated anti-CHCHD10 antibodies. In this study, we characterized four CHCHD10 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. As this study highlights high-performing antibodies for CHCHD10, we encourage readers to use it as a guide to select the most appropriate antibody for their specific needs

Enthoprotin: a novel clathrin-associated protein identified through subcellular proteomics

Despite numerous advances in the identification of the molecular machinery for clathrin-mediated budding at the plasma membrane, the mechanistic details of this process remain incomplete. Moreover, relatively little is known regarding the regulation of clathrin-mediated budding at other membrane systems. To address these issues, we have utilized the powerful new approach of subcellular proteomics to identify novel proteins present on highly enriched clathrin-coated vesicles (CCVs). Among the ten novel proteins identified is the rat homologue of a predicted gene product from human, mouse, and Drosophila genomics projects, which we named enthoprotin. Enthoprotin is highly enriched on CCVs isolated from rat brain and liver extracts. In cells, enthoprotin demonstrates a punctate staining pattern that is concentrated in a perinuclear compartment where it colocalizes with clathrin and the clathrin adaptor protein (AP)1. Enthoprotin interacts with the clathrin adaptors AP1 and with Golgi-localized, γ-ear–containing, Arf-binding protein 2. Through its COOH-terminal domain, enthoprotin binds to the terminal domain of the clathrin heavy chain and stimulates clathrin assembly. These data suggest a role for enthoprotin in clathrin-mediated budding on internal membranes. Our study reveals the utility of proteomics in the identification of novel vesicle trafficking proteins

Connecdenn, a novel DENN domain-containing protein of neuronal clathrin-coated vesicles functioning in synaptic vesicle endocytosis

Clathrin-coated vesicles (CCVs) are responsible for the endocytosis of multiple cargo, including synaptic vesicle membranes. We now describe a new CCV protein, termed connecdenn, that contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a poorly characterized protein module found in multiple proteins of unrelated function and a C-terminal peptide motif domain harboring three distinct motifs for binding the α-ear of the clathrin adaptor protein 2 (AP-2). Connecdenn coimmunoprecipitates and partially colocalizes with AP-2, and nuclear magnetic resonance and peptide competition studies reveal that all three α-ear-binding motifs contribute to AP-2 interactions. In addition, connecdenn contains multiple Src homology 3 (SH3) domain-binding motifs and coimmunoprecipitates with the synaptic SH3 domain proteins intersectin and endophilin A1. Interestingly, connecdenn is enriched on neuronal CCVs and is present in the presynaptic compartment of neurons. Moreover, connecdenn has a uniquely stable association with CCV membranes because it resists extraction with Tris and high-salt buffers, unlike most other CCV proteins, but it is not detected on purified synaptic vesicles. Together, these observations suggest that connecdenn functions on the endocytic limb of the synaptic vesicle cycle. Accordingly, disruption of connecdenn interactions with its binding partners through overexpression of the C-terminal peptide motif domain or knock down of connecdenn through lentiviral delivery of small hairpin RNA both lead to defects in synaptic vesicle endocytosis in cultured hippocampal neurons. Thus, we identified connecdenn as a component of the endocytic machinery functioning in synaptic vesicle endocytosis, providing the first evidence of a role for a DENN domain-containing protein in endocytosis

The identification of high-performing antibodies for RNA-binding protein FUS for use in Western Blot, immunoprecipitation, and immunofluorescence [version 2; peer review: 2 approved]

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community.  In this study, we characterized ten FUS commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs

Predictors of self-perceived cultural responsiveness in entry-level physiotherapy students in Australia and Aotearoa New Zealand

Background: Ensuring physiotherapy students are well prepared to work safely and effectively in culturally diverse societies upon graduation is vital. Therefore, determining whether physiotherapy programs are effectively developing the cultural responsiveness of students is essential. This study aimed to evaluate the level of self-perceived cultural responsiveness of entry level physiotherapy students during their training, and explore the factors that might be associated with these levels. Methods: A cross sectional study of physiotherapy students from nine universities across Australia and Aotearoa New Zealand was conducted using an online self-administered questionnaire containing three parts: The Cultural Competence Assessment tool, Altemeyer’s Dogmatism scale, and the Marlowe-Crowne social desirability scale- short form. Demographic data relating to university, program, and level of study were also collected. Data was analysed using one-way ANOVA, t-tests and multiple regression analysis. Results: A total of 817 (19% response rate) students participated in this study. Overall, students had a moderate level of self-perceived cultural responsiveness (Mean (SD) = 5.15 (0.67)). Fewer number of weeks of clinical placement attended, lower levels of dogmatism, and greater social desirability were related to greater self-perceived cultural responsiveness. Additionally, fourth year undergraduate students perceived themselves to be less culturally responsive than first and second year students (p < 0.05). Conclusions: These results provide educators with knowledge about the level of self-perceived cultural responsiveness in physiotherapy students, and the factors that may need to be assessed and addressed to support the development of culturally responsive practice

The changing landscape : ecosystem responses to urbanization and pollution across climatic and societal gradients

Author Posting. © Ecological Society of America, 2008. This article is posted here by permission of Ecological Society of America for personal use, not for redistribution. The definitive version was published in Frontiers in Ecology and the Environment 6 (2008): 264–272, doi:10.1890/070147.Urbanization, an important driver of climate change and pollution, alters both biotic and abiotic ecosystem properties within, surrounding, and even at great distances from urban areas. As a result, research challenges and environmental problems must be tackled at local, regional, and global scales. Ecosystem responses to land change are complex and interacting, occurring on all spatial and temporal scales as a consequence of connectivity of resources, energy, and information among social, physical, and biological systems. We propose six hypotheses about local to continental effects of urbanization and pollution, and an operational research approach to test them. This approach focuses on analysis of “megapolitan” areas that have emerged across North America, but also includes diverse wildland-to-urban gradients and spatially continuous coverage of land change. Concerted and coordinated monitoring of land change and accompanying ecosystem responses, coupled with simulation models, will permit robust forecasts of how land change and human settlement patterns will alter ecosystem services and resource utilization across the North American continent. This, in turn, can be applied globally.We thank the NSF LTER program for its support