Journal: Life Science Alliance

Article Title: ADAP1 promotes invasive squamous cell carcinoma progression and predicts patient survival

doi: 10.26508/lsa.201900582

Figure Lengend Snippet: (A) Quantification of invaded cells through Matrigel-coated transwell. ADAP1-WT-expressing cells showed increased invasive capability compared with control and ADAP1-R49K-expressing cells. n = 3–4. *** P < 0.001. P value by unpaired t test is indicated. (B) RT-qPCR analysis of Arf6 mRNA in cells transduced with lentiviral Arf6 or scramble shRNAs. n = 3. (C) Quantification of cell invasion. The ADAP1-induced invasion was reduced by Arf6 knockdown. n = 4. *** P < 0.001. P value by unpaired t test is indicated. (D) Western blot analysis of cell lysates overexpressing ARF6-WT, ARF6-CA, and ADAP1. α-tubulin, loading control. (E) Quantification of invaded cells. ADAP1-induced cell invasion was reduced by ARF6-CA expression. n = 4. * P < 0.05. P value by unpaired t test is indicated. (F) RT-qPCR analysis of human ADAP1 ( hADAP1 ) RNA in SCC-61 cells transduced with lentiviral hADAP1 or scramble shRNAs. n = 3. (G) Western blot analysis of SCC-61 cell lysates transduced with scramble or hADAP1 shRNAs. (H) Quantification of invaded cells. Invasive activity of SCC-61 cells was reduced by hADAP1 knockdown. n = 3. Data are mean ± SD. *** P < 0.001, * P < 0.05. P value by unpaired t test is indicated. ns, not significant.

Article Snippet: For generating ARF6-expression lentiviral vectors, we obtained pcDNA3-Arf6 (WT) and Arf6 (Q67L) from Addgene (plasmid ID 79424 and 79425, respectively) and subcloned PCR-amplified Arf6 fragments into pLKO vector, which was followed by sequence confirmation.

Techniques: Expressing, Control, Quantitative RT-PCR, Transduction, Knockdown, Western Blot, Activity Assay

Journal: The EMBO Journal

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

doi: 10.15252/embj.2021107664

Figure Lengend Snippet: A, B Quantification of CTxB transport to the Golgi apparatus in either HeLa cells producing either mCherry, Arf6 T27N ‐mCherry, mCherry‐Rab8a T22N , or mCherry‐Rab6a′ T27N (A), or in BMMs producing either mCherry, Arf6 Q67L ‐mCherry, or Arf6 T27N ‐mCherry (B). Cells were transfected for 24 h (A) or transduced for 48 h (B) then incubated on ice with AlexaFluor488™‐Cholera Toxin subunit B (CTxB) for binding followed by a 20‐min (A) or 30‐min (B) incubation at 37°C to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti‐GM130 antibody). CTxB retrograde transport is expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker. Data are means ± SD from n = 3 to 4 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared with mCherry‐producing cells as determined by a one‐way ANOVA with Dunnett’s multiple comparisons test ( P < 0.05).

Article Snippet: Wild‐type, constitutively active, and dominant negative human Arf6, Arf6 Q67L , and Arf6 T27N were amplified from pcDNA3‐HA‐Arf6 (Addgene #10834), pcDNA3‐HA‐Arf6 Q67L (Addgene #10835), or pcDNA3‐HA‐Arf6 T27N (Addgene #10831) using primers WSU0433 and WSU0437 and cloned into either pEGFP‐N1 or pmCherry‐N1 (Clontech) using Eco RI and Kpn I restriction sites to generate pmEGFP‐N1‐Arf6, pEGFP‐N1‐Arf6 Q67L , pEGFP‐N1‐Arf6 T27N or pmCherry‐N1‐Arf6, pmCherry‐N1‐Arf6 Q67L , and pmCherry‐N1‐Arf6 T27N .

Techniques: Transfection, Incubation, Binding Assay, Staining, Marker

Journal: The EMBO Journal

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

doi: 10.15252/embj.2021107664

Figure Lengend Snippet: Representative confocal micrograph of HeLa cells transfected to produce either mCherry (red), GFP‐ACAP1 (green), and Arf6‐HA (blue; left hand panels) or mCherry‐BspF (red), GFP‐ACAP1 (green), and HA‐Arf6 (blue; right hand panels) and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 µm and 2 µm (insets). Representative Western blot analysis of co‐immunoprecipitations of myc‐ACAP1 and Arf6‐HA in the presence or absence of HA‐BspF. HeLa cells were transfected to produce Arf6‐HA and combinations of myc‐ACAP1 and HA‐BspF, or not, and myc‐ACAP1 was immunoprecipitated using anti‐myc‐conjugated magnetic beads. Input lysates (6% of post‐nuclear supernatants) and co‐immunoprecipitates were separated by SDS–PAGE and probed for Arf6‐HA, HA‐BspF and myc‐ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference ( P = 0.0017, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Quantification of Arf6 activity (GTP‐Arf6) in HeLa cells transfected to produce either mCherry and Arf6‐HA or mCherry‐BspF and Arf6‐HA by G‐LISA. Data are means ± SD of n = 3 independent experiments, normalized to mCherry‐producing controls. The asterisk indicates a statistically significant difference ( P = 0.0026, unpaired Student’s t ‐test) between BspF‐producing and control conditions. Bacterial replication in BMMs transduced to either produce GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 4 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Bacterial replication in BMMs transduced to either produce GFP, GFP‐ACAP1, or GFP‐ACAP1 R448Q and infected with either wild‐type (2308), Δ bspF , or complemented ∆ bspF (Δ bspF::bspF ) bacteria for 24 h. Data are means ± SD of n = 3 independent experiments, in which at least 100 cells were analyzed per experiment. Gray dots represent individual cells analyzed ( n > 300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences ( P < 0.05, two‐way ANOVA followed by Dunnett’s multiple comparisons test) between test and control conditions. Source data are available online for this figure.

Article Snippet: Wild‐type, constitutively active, and dominant negative human Arf6, Arf6 Q67L , and Arf6 T27N were amplified from pcDNA3‐HA‐Arf6 (Addgene #10834), pcDNA3‐HA‐Arf6 Q67L (Addgene #10835), or pcDNA3‐HA‐Arf6 T27N (Addgene #10831) using primers WSU0433 and WSU0437 and cloned into either pEGFP‐N1 or pmCherry‐N1 (Clontech) using Eco RI and Kpn I restriction sites to generate pmEGFP‐N1‐Arf6, pEGFP‐N1‐Arf6 Q67L , pEGFP‐N1‐Arf6 T27N or pmCherry‐N1‐Arf6, pmCherry‐N1‐Arf6 Q67L , and pmCherry‐N1‐Arf6 T27N .

Techniques: Transfection, Western Blot, Immunoprecipitation, Magnetic Beads, SDS Page, Control, Activity Assay, Infection, Bacteria

Journal: The EMBO Journal

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

doi: 10.15252/embj.2021107664

Figure Lengend Snippet: Representative confocal fluorescence micrographs of HeLa cells co‐transfected for 24 h to produce mCherry‐BspF and either Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). Localization of Arf6‐GFP, Arf6 Q67L ‐GFP, or Arf6 T27N ‐GFP to mCherry‐BspF‐labeled tubules was quantified in at least 300 individual cells per experiment. Data are means ± SD from n = 3 independent experiments.

Article Snippet: Wild‐type, constitutively active, and dominant negative human Arf6, Arf6 Q67L , and Arf6 T27N were amplified from pcDNA3‐HA‐Arf6 (Addgene #10834), pcDNA3‐HA‐Arf6 Q67L (Addgene #10835), or pcDNA3‐HA‐Arf6 T27N (Addgene #10831) using primers WSU0433 and WSU0437 and cloned into either pEGFP‐N1 or pmCherry‐N1 (Clontech) using Eco RI and Kpn I restriction sites to generate pmEGFP‐N1‐Arf6, pEGFP‐N1‐Arf6 Q67L , pEGFP‐N1‐Arf6 T27N or pmCherry‐N1‐Arf6, pmCherry‐N1‐Arf6 Q67L , and pmCherry‐N1‐Arf6 T27N .

Techniques: Fluorescence, Transfection, Labeling

Journal: The EMBO Journal

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

doi: 10.15252/embj.2021107664

Figure Lengend Snippet:

Article Snippet: Wild‐type, constitutively active, and dominant negative human Arf6, Arf6 Q67L , and Arf6 T27N were amplified from pcDNA3‐HA‐Arf6 (Addgene #10834), pcDNA3‐HA‐Arf6 Q67L (Addgene #10835), or pcDNA3‐HA‐Arf6 T27N (Addgene #10831) using primers WSU0433 and WSU0437 and cloned into either pEGFP‐N1 or pmCherry‐N1 (Clontech) using Eco RI and Kpn I restriction sites to generate pmEGFP‐N1‐Arf6, pEGFP‐N1‐Arf6 Q67L , pEGFP‐N1‐Arf6 T27N or pmCherry‐N1‐Arf6, pmCherry‐N1‐Arf6 Q67L , and pmCherry‐N1‐Arf6 T27N .

Techniques: Derivative Assay, Recombinant, Transduction, Sequencing, Software, cDNA Library Assay, Transformation Assay, Plasmid Preparation, Isolation, Activation Assay

Journal: The EMBO Journal

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

doi: 10.15252/embj.2021107664

Figure Lengend Snippet:

Article Snippet: Wild‐type, constitutively active, and dominant negative human Arf6, Arf6 Q67L , and Arf6 T27N were amplified from pcDNA3‐HA‐Arf6 (Addgene #10834), pcDNA3‐HA‐Arf6 Q67L (Addgene #10835), or pcDNA3‐HA‐Arf6 T27N (Addgene #10831) using primers WSU0433 and WSU0437 and cloned into either pEGFP‐N1 or pmCherry‐N1 (Clontech) using Eco RI and Kpn I restriction sites to generate pmEGFP‐N1‐Arf6, pEGFP‐N1‐Arf6 Q67L , pEGFP‐N1‐Arf6 T27N or pmCherry‐N1‐Arf6, pmCherry‐N1‐Arf6 Q67L , and pmCherry‐N1‐Arf6 T27N .

Techniques: Derivative Assay, Recombinant, Transduction, Sequencing, Software, cDNA Library Assay, Transformation Assay, Plasmid Preparation, Isolation, Activation Assay

Journal: The Journal of Neuroscience

Article Title: ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

doi: 10.1523/JNEUROSCI.1409-12.2012

Figure Lengend Snippet: Summary of ARF6-related constructs used for investigating the role of ARF6 in integrin-dependent neurite outgrowth and integrin transport, internalization and recycling

Article Snippet: Integrin α9 enhanced green fluorescent protein (EGFP)-N3 was obtained from Addgene (plasmid 13600), deposited by Dean Sheppard (University of California, San Francisco, CA), and has been described previously ( Eva et al., 2010 ). pXS-HA-ARF6 ( Radhakrishna et al., 1996 ), pcDNA3.1-ACAP1-myc ( Li et al., 2007b ), and pFLAG-CMV2-ACAP1-R448Q ( Jackson et al., 2000 ) were all gifts from Dr. Victor Hsu (Department of Medicine, Harvard Medical School, Boston, MA). pcDNA3-ARF6-Q67L and pcDNA3-ARF6-T27N ( Peters et al., 1995 ; Furman et al., 2002 ) were obtained from Addgene (plasmid 10835 and 10831, respectively) and deposited by Thomas Roberts/Richard Klausner (Harvard Medical School, Boston, MA). pFLAG-CMV6-EFA6 ( Brown et al., 2001 ) was a gift from Dr. Julie Donaldson (National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD).

Techniques: Construct, Dominant Negative Mutation, Mutagenesis, Activation Assay

Journal: The Journal of Neuroscience

Article Title: ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

doi: 10.1523/JNEUROSCI.1409-12.2012

Figure Lengend Snippet: ARF6 regulates β1 integrin localization in DRG axons and growth cones. Images are axons and growth cones of adult DRG neurons. ARF6 and β1 integrins partly colocalize in distinct regions (triangle) and vesicular structures (arrows) (A, maximum projection and single confocal section inset). Overlap is also present after expression of wt ARF6-HA in growth cone vesicles (B, arrows; inset is a single confocal section) and filopodia (B, dotted lines). Expression of dominant-negative ARF6 T27N leads to prominent β1 integrin localization at the growth cone periphery, where the two proteins colocalize (C, dotted lines). Colocalization is also evident in vesicles (C, arrows; inset is a single confocal section). Expression of constitutively active ARF6 Q67L results in accumulation of ARF6 in swollen intracellular structures within the growth cone, which also label for β1 integrin (D, circled), and colocalization is also apparent at vesicular structures of more usual size (D, arrows). Swollen vesicles containing ARF6 and β1 integrin were also present in DRG axons (E, inset is a single confocal section). Subcellular fractionation confirmed that ARF6 is enriched in the endosomal fraction (fraction 5) in maturely differentiated PC12 cells, as is β1 integrin and Rab11 (F). Scale bars: 10 μm. A–C and E are maximum projections with a single confocal slice magnified in the inset. D is a single confocal section.

Article Snippet: Integrin α9 enhanced green fluorescent protein (EGFP)-N3 was obtained from Addgene (plasmid 13600), deposited by Dean Sheppard (University of California, San Francisco, CA), and has been described previously ( Eva et al., 2010 ). pXS-HA-ARF6 ( Radhakrishna et al., 1996 ), pcDNA3.1-ACAP1-myc ( Li et al., 2007b ), and pFLAG-CMV2-ACAP1-R448Q ( Jackson et al., 2000 ) were all gifts from Dr. Victor Hsu (Department of Medicine, Harvard Medical School, Boston, MA). pcDNA3-ARF6-Q67L and pcDNA3-ARF6-T27N ( Peters et al., 1995 ; Furman et al., 2002 ) were obtained from Addgene (plasmid 10835 and 10831, respectively) and deposited by Thomas Roberts/Richard Klausner (Harvard Medical School, Boston, MA). pFLAG-CMV6-EFA6 ( Brown et al., 2001 ) was a gift from Dr. Julie Donaldson (National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD).

Techniques: Expressing, Dominant Negative Mutation, Fractionation

Journal: The Journal of Neuroscience

Article Title: ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

doi: 10.1523/JNEUROSCI.1409-12.2012

Figure Lengend Snippet: ARF6 vesicles are transported rapidly and contain β1 integrins. SNAP-tagged ARF6 (red) colocalizes with internalized β1 integrins (green) in PC12 cells live labeled with anti-β1–488, and subsequently fixed and imaged by confocal microscopy (A). Images shown are orthogonal views of a z-stack indicating vesicular colocalization, Scale bar, 10 μm. Spinning disk confocal microscopy was used to image maturely differentiated PC12 cells stably expressing ARF6-SNAP, and vesicle dynamics were quantified by tracking individual vesicles in neurites (B). Vesicles were tracked for their visible lifetime and the instantaneous velocity was calculated from the maximum distance moved by a vesicle between two frames (3 s). Maximum net movement over 10 frames is a measure of the furthest a vesicle traveled from its origin over 30 s. Anterograde and retrograde movements were included in the analysis. ARF6-SNAP movement is rapid and occurs over long distances. Differentiated PC12 cells stably expressing ARF6-SNAP were live labeled with anti-β1–488 and imaged by spinning disk confocal microscopy. ARF6-SNAP vesicles dual labeled for β1 integrin were observed moving anterogradely and retrogradely, and are shown here arriving at the growth cone (C). Arrows indicate a two-colored vesicle moving anterogradely. The rapid movement of this ARF6/β1 integrin-positive vesicle is indicated by the gentle slope displayed on a kymograph (D, arrows), which also displays a very slow moving structure (the very steep yellow line).

Article Snippet: Integrin α9 enhanced green fluorescent protein (EGFP)-N3 was obtained from Addgene (plasmid 13600), deposited by Dean Sheppard (University of California, San Francisco, CA), and has been described previously ( Eva et al., 2010 ). pXS-HA-ARF6 ( Radhakrishna et al., 1996 ), pcDNA3.1-ACAP1-myc ( Li et al., 2007b ), and pFLAG-CMV2-ACAP1-R448Q ( Jackson et al., 2000 ) were all gifts from Dr. Victor Hsu (Department of Medicine, Harvard Medical School, Boston, MA). pcDNA3-ARF6-Q67L and pcDNA3-ARF6-T27N ( Peters et al., 1995 ; Furman et al., 2002 ) were obtained from Addgene (plasmid 10835 and 10831, respectively) and deposited by Thomas Roberts/Richard Klausner (Harvard Medical School, Boston, MA). pFLAG-CMV6-EFA6 ( Brown et al., 2001 ) was a gift from Dr. Julie Donaldson (National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD).

Techniques: Labeling, Confocal Microscopy, Stable Transfection, Expressing

Journal: The Journal of Neuroscience

Article Title: ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

doi: 10.1523/JNEUROSCI.1409-12.2012

Figure Lengend Snippet: Manipulation of ARF6 regulates α9 integrin-dependent neurite outgrowth on tenascin. Differentiated PC12 cells stably expressing α9 integrin were trypsinized and electroporated with ARF6 regulatory proteins: ARF6 GEFs, either ARNO or EFA6, an ARF6 GAP, ACAP1, mutant ACAP1 R448Q lacking GAP activity, or wt ARF6 as indicated or mock transfected as a control. These were then grown on a substrate of tenascin for 2 d, in the presence or absence of an α9β1 blocking antibody (at a concentration that partly inhibits growth), and analyzed for maximum neurite length. Representative images are shown of each condition (A). Scale bar, 100 μm. Overexpression of ACAP1 increases α9-dependent outgrowth, which is inhibited by anti-α9β1, whereas ACAP1 R448Q expression inhibits outgrowth, which is inhibited further by anti-α9β1. Both EFA6 and ARNO inhibit α9-dependent outgrowth (ARNO expression results in the most marked effects) and outgrowth is inhibited further by addition of anti-α9β1. Expression of full-length ARF6 had no effect (B). Data were analyzed using ANOVA followed by post hoc analysis. In all cases, blocking with anti-α9β1 causes statistically significant changes (p < 0.001). Error bars indicate SEM.

Article Snippet: Integrin α9 enhanced green fluorescent protein (EGFP)-N3 was obtained from Addgene (plasmid 13600), deposited by Dean Sheppard (University of California, San Francisco, CA), and has been described previously ( Eva et al., 2010 ). pXS-HA-ARF6 ( Radhakrishna et al., 1996 ), pcDNA3.1-ACAP1-myc ( Li et al., 2007b ), and pFLAG-CMV2-ACAP1-R448Q ( Jackson et al., 2000 ) were all gifts from Dr. Victor Hsu (Department of Medicine, Harvard Medical School, Boston, MA). pcDNA3-ARF6-Q67L and pcDNA3-ARF6-T27N ( Peters et al., 1995 ; Furman et al., 2002 ) were obtained from Addgene (plasmid 10835 and 10831, respectively) and deposited by Thomas Roberts/Richard Klausner (Harvard Medical School, Boston, MA). pFLAG-CMV6-EFA6 ( Brown et al., 2001 ) was a gift from Dr. Julie Donaldson (National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD).

Techniques: Stable Transfection, Expressing, Mutagenesis, Activity Assay, Transfection, Blocking Assay, Concentration Assay, Over Expression

Journal: The Journal of Neuroscience

Article Title: ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

doi: 10.1523/JNEUROSCI.1409-12.2012

Figure Lengend Snippet: Manipulation of ARF6 affects axon growth in adult DRG neurons. Representative images of DRG neurons expressing ARF6 regulatory proteins as indicated (A). Expression of ACAP1 in adult DRG neurons increases axon growth after 2 d in standard culture conditions, while expression of GAP dead ACAP1 R448Q decreases axon growth. Expression of either EFA6 or ARNO (ARF6 GEFs) leads to a substantial reduction in DRG axon growth (B). Data were analyzed by ANOVA with post hoc analysis. p < 0.001 in all cases, compared with GFP-expressing controls. Scale bar, 100 μm.

Article Snippet: Integrin α9 enhanced green fluorescent protein (EGFP)-N3 was obtained from Addgene (plasmid 13600), deposited by Dean Sheppard (University of California, San Francisco, CA), and has been described previously ( Eva et al., 2010 ). pXS-HA-ARF6 ( Radhakrishna et al., 1996 ), pcDNA3.1-ACAP1-myc ( Li et al., 2007b ), and pFLAG-CMV2-ACAP1-R448Q ( Jackson et al., 2000 ) were all gifts from Dr. Victor Hsu (Department of Medicine, Harvard Medical School, Boston, MA). pcDNA3-ARF6-Q67L and pcDNA3-ARF6-T27N ( Peters et al., 1995 ; Furman et al., 2002 ) were obtained from Addgene (plasmid 10835 and 10831, respectively) and deposited by Thomas Roberts/Richard Klausner (Harvard Medical School, Boston, MA). pFLAG-CMV6-EFA6 ( Brown et al., 2001 ) was a gift from Dr. Julie Donaldson (National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD).

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

doi: 10.1523/JNEUROSCI.1409-12.2012

Figure Lengend Snippet: Manipulation of ARF6 activity regulates β1 integrin internalization and recycling. β1 integrin endocytosis and recycling were measured using a surface biotinylation protocol (described in detail in Materials and Methods) on maturely differentiated PC12 cells transfected with ARF6 regulatory proteins as indicated or mock transfected control cells. Integrin endocytosis was allowed to occur for 30 min, and internalized β1 integrin quantified by ELISA. Expression of the ARF6 GEFS ARNO and EFA6 increases β1 integrin internalization compared with mock transfected controls, while expression of GAP dead ACAP1 R448Q also increases internalization to a lesser extent. Expression of the ARF6 GAP ACAP1 has no effect (A). Integrin endocytosis was once again allowed to occur for 30 min, this time followed by removal of surface integrins, and subsequent incubation to allow recycling to occur for the time points indicated. ACAP1 expression resulted in a significant increase in the rate of β1 integrin recycling compared with controls (p < 0.05), while ACAP1 R448Q increased recycling to a lesser extent, although this did not achieve significance (p > 0.05). Expression of ARNO and EFA6 had no effect on the rate of recycling, but appeared to increase the rate of internalization of rapidly recycled integrins compared with controls, although again this was not significant (B). Transfected cells were lysed and examined by Western blot to check for expression of the transfected constructs (C). All extracts were run on the same blot, and then separated for antibody labeling, and reunited for simultaneous developing. D is a representative image of 12 d differentiated PC12 cells to highlight the neuronal morphology of the cells at the time of experimentation. Scale bar, 100 μm.

Article Snippet: Integrin α9 enhanced green fluorescent protein (EGFP)-N3 was obtained from Addgene (plasmid 13600), deposited by Dean Sheppard (University of California, San Francisco, CA), and has been described previously ( Eva et al., 2010 ). pXS-HA-ARF6 ( Radhakrishna et al., 1996 ), pcDNA3.1-ACAP1-myc ( Li et al., 2007b ), and pFLAG-CMV2-ACAP1-R448Q ( Jackson et al., 2000 ) were all gifts from Dr. Victor Hsu (Department of Medicine, Harvard Medical School, Boston, MA). pcDNA3-ARF6-Q67L and pcDNA3-ARF6-T27N ( Peters et al., 1995 ; Furman et al., 2002 ) were obtained from Addgene (plasmid 10835 and 10831, respectively) and deposited by Thomas Roberts/Richard Klausner (Harvard Medical School, Boston, MA). pFLAG-CMV6-EFA6 ( Brown et al., 2001 ) was a gift from Dr. Julie Donaldson (National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD).

Techniques: Activity Assay, Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Incubation, Western Blot, Construct, Antibody Labeling

Journal: The Journal of Neuroscience

Article Title: ARF6 Directs Axon Transport and Traffic of Integrins and Regulates Axon Growth in Adult DRG Neurons

doi: 10.1523/JNEUROSCI.1409-12.2012

Figure Lengend Snippet: ARF6 manipulation regulates the direction of α9 integrin axonal transport. Spinning disk confocal microscopy was used to image adult DRG neurons expressing α9 integrin-GFP, either alone or cotransfected with epitope-tagged ARF6 regulatory proteins. Cotransfection was confirmed after imaging by fixing and antibody labeling. One hundred percent of cotransfected neurons expressing α9-GFP also expressed their cotransfected partners. Dissociated adult DRG cultures were transfected with α9-GFP alone, and α9-GFP with either ACAP1-myc, ACAP1 R448Q FLAG, ARNO-FLAG, or EFA6-FLAG (A). Images of α9-GFP in DRG axons were acquired for 5 min, capturing frames every 3 s. These were used to generate movies and kymographs (E). Vesicles were then tracked for their visible lifetime using MetaMorph software and analyzed to quantify direction and velocity. None of the constructs expressed altered the balance between the mobile and immobile fraction of α9-GFP vesicles (B); however, differences were seen between the proportion of anterograde and retrograde vesicles within the mobile fraction. Coexpression of ACAP1 led to an increase in the proportion of anterograde movement and a decrease in retrograde movement, while ACAP1 R448Q expression had the opposite effect. Expression of EFA6 and ARNO increased retrograde transport and decreased anterograde transport. No statistical changes were found for the bidirectional fraction (C; p > 0.05). Data were analyzed by ANOVA with post hoc analysis. Analysis of instantaneous and average velocity revealed only small differences between groups (D). Cumulative frequency distributions were plotted for anterograde (D, left), and retrograde (D, right) instantaneous and average velocity, and analyzed using a Kruskal–Wallis test for variance. While no statistical differences were found, there was a trend for ARNO expression leading to slower anterograde transport and faster retrograde transport compared with controls (D; ARNO, purple lines; control, light blue lines). Scale bars: A, 100 μm; E, 5 μm.

Article Snippet: Integrin α9 enhanced green fluorescent protein (EGFP)-N3 was obtained from Addgene (plasmid 13600), deposited by Dean Sheppard (University of California, San Francisco, CA), and has been described previously ( Eva et al., 2010 ). pXS-HA-ARF6 ( Radhakrishna et al., 1996 ), pcDNA3.1-ACAP1-myc ( Li et al., 2007b ), and pFLAG-CMV2-ACAP1-R448Q ( Jackson et al., 2000 ) were all gifts from Dr. Victor Hsu (Department of Medicine, Harvard Medical School, Boston, MA). pcDNA3-ARF6-Q67L and pcDNA3-ARF6-T27N ( Peters et al., 1995 ; Furman et al., 2002 ) were obtained from Addgene (plasmid 10835 and 10831, respectively) and deposited by Thomas Roberts/Richard Klausner (Harvard Medical School, Boston, MA). pFLAG-CMV6-EFA6 ( Brown et al., 2001 ) was a gift from Dr. Julie Donaldson (National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD).

Techniques: Confocal Microscopy, Expressing, Cotransfection, Imaging, Antibody Labeling, Transfection, Software, Construct