High abundance of ArfGAP1 found in the mossy fibers in hilus of the dentate gyrus region of the mouse brain.

The Arf GTPase-activating protein ArfGAP1 and its brain-specific isoform ArfGAP1B play an important role in neurotransmission. Here we analyzed the distribution of ArfGAP1 in the mouse brain. We found high levels of ArfGAP1 in the mouse dentate gyrus where it displayed especially elevated level in the polymorph layer (hilus). Importantly, the ArfGAP1 signal follows the pathway of the granular cell axons so-called mossy fibers which extend from the dentate gyrus to CA3 via stratum lucidum and partially stratum oriens. Additionally, we identified differential expression of ArfGAP1 in the isocortex. Thus, staining with anti-ArfGAP1 antibodies allows distinction between cortical cell layers 1, 2, 3 and 5 from 4 and 6. Taken together, our data suggest that ArfGAP1 can be used as a specific marker of the dentate mossy fibers and as for visualization of cortical layers in immunohistochemical studies

ArfGAP1 protein localization in mouse dentate gyrus and cerebelum.

<p>A, B. coronal view of the DG, poDG–polymorph layer (hilus) of the DG, grDG–granular cell layer of the DG, sr–stratum radiatum, slu–stratum lucidum, so–sorstratum oriens. CA1, CA2 and CA3 –Cornu Ammonis of the hippocampus. C, D. Coronal view of the cerebellum, ANcr1 and ANcr2 –ansiform cruciform lobule 1 and 2, arb–arbor vitae; ArfGAP1 (green), DAPI stain (blue). B,D–control experiments with the omission of primary antibody and staining only with secondary antibody. Magnification 10X. The scale bars represent 210 μm.</p

ArfGAP1 immunoreactivity in the hippocampus.

<p>A. Coronal plane. B. schematic view of the mossy fibers projections. C. Sagittal plane. SP-MF–suprapyramidal bundle and IIP-MF–intra- and infrapyramidal bundles of the mossy fibers projections. C. Sagittal plane. poDG–polymorph layer (hilus) of the DG, grDG–granular cell layer of the DG, sr–stratum radiatum, slu–stratum lucidum, so–sorstratum oriens. CA1, CA2 and CA3 –Cornu Ammonis of the hippocampus. The scale bars represent 200 μm.</p

Western blot detection of total ArfGAP1 and ArfGAP1^B in adult mouse brain regions.

<p>A. Data were normalized to GAPDH protein levels for each region. DG–dentate gyrus, Cer–cerebellum, OB–olfactory bulb, IsoCtx–isocortex, TH–thalamus. Data expressed as the mean ± SEM, n = 3. B. Linear regression analysis of the pairs of data obtained using the two antibodies from each tissue in each of the 3 experiments.</p

ArfGAP1 is localized in the mossy fibers of the mouse DG.

<p>A, B. Double labeling of ArfGAP1(green) and ZnT3 (red). Yellow staining indicates colocalization of ArfGAP1 and ZnT3. C, D. Double labeling of ArfGAP1(green) and GFAP (red). F, E. Omission of the primary antibody and staining only with secondary antibody. Abbreviations: CA1, CA2 and CA3 –Cornu Ammonis of the hippocampus. Coronal DG (dentate gyrus) view of ventral (A, C, F) and dorsal (B, D, E) hippocampus. TH–thalamus. mf–mossy fibers. Magnification 10X.The scale bars represent 210 μm.</p

Discrete Determinants in ArfGAP2/3 Conferring Golgi Localization and Regulation by the COPI Coat

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein–protein and protein–lipid interactions to promote GTP hydrolysis in Arf1-GTP