Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 Å, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1

Development of a \u3cem\u3eMen’s Health\u3c/em\u3e Course for First-Year Undergraduates Using Culturally Responsive Teaching Strategies

Purpose A novel first-year experience course was developed using culturally responsive teaching strategies at an undergraduate liberal arts college in the southeastern USA to promote health advocacy and to provide students with an overview of male health. The course focuses on the biological, sociocultural, economic and gender influences that shape men\u27s health beliefs and practices. It also emphasizes health disparities in the USA among Black/African American men compared to other racial groups and intervention strategies to improve health outcomes. Design/methodology/approach The lecture and laboratory components of the course were designed as a blended learning environment with a modified flipped class model. Culturally relevant strategies guided the course design with three focus domains: academic success, cultural competence and sociopolitical consciousness. A community engagement model and service-learning activities were also incorporated in the design. The authors used course grades to gauge learning and implemented a survey to assess students\u27 perception of the knowledge gained in three realms: men\u27s health, health sciences and physical sciences. Findings This report describes the course design, highlights the value of using culturally responsive teaching strategies and service-learning projects to encourage students\u27 active learning. Course activity examples are discussed with student responses. The authors found that students\u27 perception of their knowledge in men\u27s health, health sciences and physical sciences increased and the students performed well in the course. Originality/value This is one of few biology courses in the nation that intentionally focuses on the unique health challenges of Black men, while empowering college students to develop culturally competent strategies to improve their health outcomes. The findings suggest that the students learned the material and that their perceived knowledge on men\u27s health increased. The authors urge other academic institutions and healthcare providers to consider implementation of similar courses in an effort to enhance male health equity

MeCP2 Regulates the Synaptic Expression of a Dysbindin-BLOC-1 Network Component in Mouse Brain and Human Induced Pluripotent Stem Cell-Derived Neurons

Clinical, epidemiological, and genetic evidence suggest overlapping pathogenic mechanisms between autism spectrum disorder (ASD) and schizophrenia. We tested this hypothesis by asking if mutations in the ASD gene MECP2 which cause Rett syndrome affect the expression of genes encoding the schizophrenia risk factor dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1 (BLOC-1), and associated interacting proteins. We measured mRNA and protein levels of key components of a dysbindin interaction network by, quantitative real time PCR and quantitative immunohistochemistry in hippocampal samples of wild-type and Mecp2 mutant mice. In addition, we confirmed results by performing immunohistochemistry of normal human hippocampus and quantitative qRT-PCR of human inducible pluripotent stem cells (iPSCs)-derived human neurons from Rett syndrome patients. We defined the distribution of the BLOC-1 subunit pallidin in human and mouse hippocampus and contrasted this distribution with that of symptomatic Mecp2 mutant mice. Neurons from mutant mice and Rett syndrome patients displayed selectively reduced levels of pallidin transcript. Pallidin immunoreactivity decreased in the hippocampus of symptomatic Mecp2 mutant mice, a feature most prominent at asymmetric synapses as determined by immunoelectron microcopy. Pallidin immunoreactivity decreased concomitantly with reduced BDNF content in the hippocampus of Mecp2 mice. Similarly, BDNF content was reduced in the hippocampus of BLOC-1 deficient mice suggesting that genetic defects in BLOC-1 are upstream of the BDNF phenotype in Mecp2 deficient mice. Our results demonstrate that the ASD-related gene Mecp2 regulates the expression of components belonging to the dysbindin interactome and these molecular differences may contribute to synaptic phenotypes that characterize Mecp2 deficiencies and ASD.Fil: Larimore, Jennifer. Agnes Scott College; Estados UnidosFil: Ryder, Pearl V.. University of Emory; Estados UnidosFil: Kim, Kun Yong. University of Yale. School of Medicine; Estados UnidosFil: Ambrose, L. Alex. Agnes Scott College; Estados UnidosFil: Chapleau, Christopher. University Of Alabama; Estados UnidosFil: Calfa, Gaston Diego. University Of Alabama; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gross, Christina. University of Emory; Estados UnidosFil: Bassell, Gary J.. University of Emory; Estados UnidosFil: Pozzo Miller, Lucas. University Of Alabama; Estados UnidosFil: Smith, Yoland. University of Emory; Estados UnidosFil: Talbot, Konrad. The Pennsylvania State University; Estados UnidosFil: Park, In Hyun. University of Yale. School of Medicine; Estados UnidosFil: Faundez, Victor. University of Emory; Estados Unido

Chemical-genetic disruption of clathrin function spares adaptor complex 3-dependent endosome vesicle biogenesis

A role for clathrin in AP-3–dependent vesicle biogenesis has been inferred from biochemical interactions and colocalization between this adaptor and clathrin. The functionality of these molecular associations, however, is controversial. We comprehensively explore the role of clathrin in AP-3–dependent vesicle budding, using rapid chemical-genetic perturbation of clathrin function with a clathrin light chain–FKBP chimera oligomerizable by the drug AP20187. We find that AP-3 interacts and colocalizes with endogenous and recombinant FKBP chimeric clathrin polypeptides in PC12-cell endosomes. AP-3 displays, however, a divergent behavior from AP-1, AP-2, and clathrin chains. AP-3 cofractionates with clathrin-coated vesicle fractions isolated from PC12 cells even after clathrin function is acutely inhibited by AP20187. We predicted that AP20187 would inhibit AP-3 vesicle formation from endosomes after a brefeldin A block. AP-3 vesicle formation continued, however, after brefeldin A wash-out despite impairment of clathrin function by AP20187. These findings indicate that AP-3–clathrin association is dispensable for endosomal AP-3 vesicle budding and suggest that endosomal AP-3–clathrin interactions differ from those by which AP-1 and AP-2 adaptors productively engage clathrin in vesicle biogenesis

Antagonism between germ cell-less and Torso receptor regulates transcriptional quiescence underlying germline/soma distinction

Transcriptional quiescence, an evolutionarily conserved trait, distinguishes the embryonic primordial germ cells (PGCs) from their somatic neighbors. In Drosophila melanogaster, PGCs from embryos maternally compromised for germ cell-less (gcl) misexpress somatic genes, possibly resulting in PGC loss. Recent studies documented a requirement for Gcl during proteolytic degradation of the terminal patterning determinant, Torso receptor. Here we demonstrate that the somatic determinant of female fate, Sex-lethal (Sxl), is a biologically relevant transcriptional target of Gcl. Underscoring the significance of transcriptional silencing mediated by Gcl, ectopic expression of a degradation-resistant form of Torso (torsoDeg) can activate Sxl transcription in PGCs, whereas simultaneous loss of torso-like (tsl) reinstates the quiescent status of gcl PGCs. Intriguingly, like gcl mutants, embryos derived from mothers expressing torsoDeg in the germline display aberrant spreading of pole plasm RNAs, suggesting that mutual antagonism between Gcl and Torso ensures the controlled release of germ-plasm underlying the germline/soma distinction

<i>Mecp2</i> Deficiency Does Not Affect the Content of BLOC-1 Sensitive Markers in the Dentate Gyrus.

<p>Images depict indirect quantitative immunofluorescence microscopy of PI4KIIα and VAMP2 (A–B) or immunoperoxidase light microscopy of VAMP7 (C–D) or the AP-3 δ subunit (E). A–E italic letters represent mutant <i>Mecp2</i> and BLOC-1 <i>Bloc1s8^sdy/sdy</i>. F–H Box plots depict the quantitation of immunoreactivities of antigens presented in A–E and <i>A–E</i>. P values were obtained by Mann-Whitney U test, n = 4 independent stainings from 2 animals per genotype.</p

Quantitative Real Time PCR Determination of BLOC-1 Subunit Transcripts.

<p>BLOC-1 subunit transcripts from symptomatic adult (A) and P7 (B) mice hippocampi and cortices were analyzed by qRT-PCR. Box plot depicts relative mRNA content for wild type (<i>Mecp2^+/y</i>, Blue) and <i>Mecp2</i> mutant tissue (<i>Mecp2^tm1.1Jae/y</i>, Red). P values were obtained by Mann-Whitney U test. Number of animals tested is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065069#pone-0065069-t001" target="_blank">Table 1</a>.</p