Dataset Information

Analysis of subcellular Importin b1 interactomes by BioID and BAR proximity labelling

ABSTRACT: Importin 1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in detection of importin complex cargos, and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable Biotinylation by Antibody Recognition (BAR) analysis of Importin 1 interactomes.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Spinal Cord Motor Neuron, Hek-293 Cell, Hela Cell

SUBMITTER: Juan Oses-Prieto

LAB HEAD: Mike Fainzilber

PROVIDER: PXD032728 | Pride | 2022-10-15

REPOSITORIES: Pride

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Publications

A New Monoclonal Antibody Enables BAR Analysis of Subcellular Importin β1 Interactomes.

Song Didi-Andreas DA Alber Stefanie S Doron-Mandel Ella E Schmid Vera V Albus Christin A CA Leitner Orith O Hamawi Hedva H Oses-Prieto Juan A JA Dezorella Nili N Burlingame Alma L AL Fainzilber Mike M Rishal Ida I

Molecular & cellular proteomics : MCP 20220928 11

Importin β1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in the detection of importin complex cargos and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable biotinylation by antibody recognition analysis of importin β1 interactomes. The monoclonal antibody rec ...[more]

PMID: 36180036

Similar Datasets

Project description:Introduction: The mechanisms governing synaptonuclear transport, and the transcriptional pathways that are at the core of neuronal disorders are poorly understood. The importin family of nuclear import factors has pivotal roles in such pathways, due to their involvement in intracellular transport from both synapse to soma and cytoplasm to nucleus. Mammals express six different isoforms of importin α, which bind an Importin β to form transport complexes for specific cargoes. Methods: We addressed the roles of importin α isoforms in mammalian CNS by evaluation of a systematic series of importin α knockout mice. A comprehensive battery of behavioral tests was used to investigate spontaneous and novelty-induced locomotion, basal ganglia function, motor coordination, neuromuscular integration, anxiety-related behaviors and memory. Subsequent analyses using acute-brain slice electrophysiology, RNA-seq, bioinformatics, imaging and pharmacology was used in order to delineate the contribution of genes and signaling pathways involved in the identified phenotype and screen for new therapeutic approaches. Results and Discussion: During the course of comprehensive behavioral profiling of importin α mouse mutants, we identified an importin α knockout (KO) mouse with significantly reduced anxiety levels. Electrophysiological recordings in acute hippocampal slices showed a reduced short-term and presynaptic plasticity (paired-pulse facilitation deficit, lower PTP), while LTP was not affected in these mutants. We then performed transcriptional profiling (RNA-seq) from hippocampal extracts of wild type versus mutant animals after exposure to an anxiogenic drug. Computational analysis revealed differences in the expression of genes and the involvement of transcription factors. Drugs that activate LXR or the Sphingosine 1 phosphate receptor have robust anxiolytic effects, while a Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse. Thus, importin α5 influences anxiety by regulating nuclear import in neurons.

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