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Pallidin is a novel interacting protein for cytohesin-2 and regulates the early endosomal pathway and dendritic formation in neurons.

Journal of neurochemistry | 2018

Cytohesin-2 is a member of the guanine nucleotide exchange factors for ADP ribosylation factor 1 (Arf1) and Arf6, which are small GTPases that regulate membrane traffic and actin dynamics. In this study, we first demonstrated that cytohesin-2 localized to the plasma membrane and vesicles in various subcellular compartment in hippocampal neurons by immunoelectron microscopy. Next, to understand the molecular network of cytohesin-2 in neurons, we conducted yeast two-hybrid screening of brain cDNA libraries using cytohesin-2 as bait and isolated pallidin, a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) involved in endosomal trafficking. Pallidin interacted specifically with cytohesin-2 among cytohesin family members. Glutathione S-transferase pull-down and immunoprecipitation assays further confirmed the formation of a protein complex between cytohesin-2 and pallidin. Immunofluorescence demonstrated that cytohesin-2 and pallidin partially colocalized in various subsets of endosomes immunopositive for EEA1, syntaxin 12, and LAMP2 in hippocampal neurons. Knockdown of pallidin or cytohesin-2 reduced cytoplasmic EEA1-positive early endosomes. Furthermore, knockdown of pallidin increased the total dendritic length of cultured hippocampal neurons, which was rescued by co-expression of wild-type pallidin but not a mutant lacking the ability to interact with cytohesin-2. In contrast, knockdown of cytohesin-2 had the opposite effect on total dendritic length. The present results suggested that the interaction between pallidin and cytohesin-2 may participate in various neuronal functions such as endosomal trafficking and dendritic formation in hippocampal neurons. Cover Image for this issue: doi: 10.1111/jnc.14197.

Pubmed ID: 30151872 RIS Download

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Anti-α-Tubulin antibody (antibody)

RRID:AB_477593

This monoclonal targets α-Tubulin

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MAP2 (microtubule-associated protein-2) antibody (antibody)

RRID:AB_2571557

This polyclonal targets mouse MAP2, 927-1104 aa (NM008632)

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Iba1 antibody (antibody)

RRID:AB_2224402

This polyclonal targets AIF1

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LAMP-2 (C-20) (antibody)

RRID:AB_2134754

This polyclonal targets LAMP2

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EEA1 (antibody)

RRID:AB_397830

This monoclonal targets EEA1

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Anti-HA-tag (antibody)

RRID:AB_591844

This polyclonal targets HA-tag

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PLDN antibody (antibody)

RRID:AB_2164174

This polyclonal targets PLDN

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GFAP (glial fibrillary acidic protein) antibody (antibody)

RRID:AB_2571708

This polyclonal targets mouse GFAP, 335-400 aa (AF332061)

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either: ICR or MF1 (organism)

RRID:MGI:5653323

Strain Type: Not Specified This is a legacy resource.

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C57BL/6J (organism)

RRID:IMSR_JAX:000664

Mus musculus with name C57BL/6J from IMSR.

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HeLa (cell line)

RRID:CVCL_0030

Cell line HeLa is a Cancer cell line with a species of origin Homo sapiens

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GFAP (glial fibrillary acidic protein) antibody (antibody)

RRID:AB_2571708

This polyclonal targets mouse GFAP, 335-400 aa (AF332061)

View all literature mentions

C57BL/6J (organism)

RRID:IMSR_JAX:000664

Mus musculus with name C57BL/6J from IMSR.

View all literature mentions

either: ICR or MF1 (organism)

RRID:MGI:5653323

Strain Type: Not Specified This is a legacy resource.

View all literature mentions

Anti-α-Tubulin antibody (antibody)

RRID:AB_477593

This monoclonal targets α-Tubulin

View all literature mentions

Anti-HA-tag (antibody)

RRID:AB_591844

This polyclonal targets HA-tag

View all literature mentions

Iba1 antibody (antibody)

RRID:AB_2224402

This polyclonal targets AIF1

View all literature mentions

LAMP-2 (C-20) (antibody)

RRID:AB_2134754

This polyclonal targets LAMP2

View all literature mentions

EEA1 (antibody)

RRID:AB_397830

This monoclonal targets EEA1

View all literature mentions

MAP2 (microtubule-associated protein-2) antibody (antibody)

RRID:AB_2571557

This polyclonal targets mouse MAP2, 927-1104 aa (NM008632)

View all literature mentions

PLDN antibody (antibody)

RRID:AB_2164174

This polyclonal targets PLDN

View all literature mentions

HeLa (cell line)

RRID:CVCL_0030

Cell line HeLa is a Cancer cell line with a species of origin Homo sapiens

View all literature mentions