Journal: Scientific Reports

Article Title: Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter

doi: 10.1038/s41598-017-05637-x

Figure Lengend Snippet: Preferential binding of JHC1-64 to wt DAT and its R60A mutant in filopodia and cell edges of HEK293 cells. ( A ) Chemical sstructure of JHC1-064 . ( B ) Cells transiently expressing wt YFP-HA-DAT, and R60A or W63A mutants of YFP-HA-DAT were incubated with 100 nM JHC1-64 at RT for 30 min. Live-cell imaging was performed through 515 nm (YFP, green ) and 561 nm (JHC1-64, red ) filter channels. Maximal projections of 5 z-planes are shown. Arrows point to examples of peripheral filopodia. Scale bars, 10μm. ( C ) YFP-HA-DAT or the R60A mutant were treated with 10-200 nM JHC1-64 for 30 min at RT. The ratio of JHC1-64 and YFP fluorescence intensities (designated JHC/YFP) was quantitated for whole cells as described in ‘Methods’. Bars represent mean values (±SEM; n = 3). ( D ) Cells were treated with 100 nM JHC1-64 as in ( B ), and the time dependence of the JHC/YFP ratio was determined by time-lapse imaging of YFP and JHC1-64. Mean values are presented (±SEM, n = 3). ( E ) Examples of the segmentation masks generated as described in “Methods” for quantification of the mean fluorescence intensities of peripheral filopodia ( left ) and non-filopodial membrane (flat membrane, FM; right ) regions from YFP images obtained in experiments represented in ( B ). ( F ) Cells expressing YFP-HA-DAT or R60A were imaged after incubation with 100 nM JHC1-64 for 30 min at RT. The JHC/YFP ratio in filopodia and FM masks was quantitated in individual cells. Results are shown as mean values of JHC/YFP ratios relative to the ratios in FM mask in each individual cell (±SEM, n = 10). *p < 0.05, **p < 0.01.

Article Snippet: To generate R60A mutation in the template of YFP-DAT (no HA tag, Addgene plasmid # 90228) , the 1622 bp DAT sequence (begins with bp 242 in DAT cDNA) in YFP-HA-R60A was replaced by the corresponding sequence from the YFP-DAT construct using PflMI (5’ end) and SmaI (3’ end) restriction sites.

Techniques: Binding Assay, Mutagenesis, Expressing, Incubation, Live Cell Imaging, Fluorescence, Imaging, Generated, Membrane

Journal: Scientific Reports

Article Title: Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter

doi: 10.1038/s41598-017-05637-x

Figure Lengend Snippet: Binding of JHC1-64 increases the concentration of R60A in filopodia of HEK293 cells. ( A ) Cells transiently expressing YFP-HA-DAT or the R60A mutant were incubated with 100 nM JHC1-64 for 30 min at RT. Live-cell imaging was performed through 515 nm (YFP, green ) and 561 nm (JHC1-64, red ) filter channels. Maximal z-projections of 5 consecutive x-y-confocal planes are shown. Scale bars, 10μm. ( B ) Insets represent high magnification images of the regions marked by the white rectangle in ( A ). Arrows point to representative filopodia. ( C ) The Filopodia/FM ratios of mean intensities of the YFP fluorescence were calculated as described in the “Methods”. Results are shown as mean values (±SEM, n = 10). **p < 0.01.

Article Snippet: To generate R60A mutation in the template of YFP-DAT (no HA tag, Addgene plasmid # 90228) , the 1622 bp DAT sequence (begins with bp 242 in DAT cDNA) in YFP-HA-R60A was replaced by the corresponding sequence from the YFP-DAT construct using PflMI (5’ end) and SmaI (3’ end) restriction sites.

Techniques: Binding Assay, Concentration Assay, Expressing, Mutagenesis, Incubation, Live Cell Imaging, Fluorescence

Journal: Scientific Reports

Article Title: Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter

doi: 10.1038/s41598-017-05637-x

Figure Lengend Snippet: Binding of cocaine increases the concentration of R60A (but not W63A) in the filopodia of HEK293 cells. ( A ) Cells transiently expressing wt YFP-HA-DAT, or its R60A or W63A mutants were incubated with 10 μM cocaine for 30 min at RT. Live-cell imaging was performed through the 515 nm (YFP, green ) filter channel. Maximal z-projections of 5 consecutive x-y-confocal planes are shown. ( B ) Insets represent high magnification images of the regions marked by the white rectangle in ( A ). Arrows point to representative filopodia. ( C ) The filopodia/FM ratios of mean intensities of the YFP fluorescence were calculated as described in “Methods”. Results are shown as mean values (±SEM, n = 10). ***p < 0.001.

Article Snippet: To generate R60A mutation in the template of YFP-DAT (no HA tag, Addgene plasmid # 90228) , the 1622 bp DAT sequence (begins with bp 242 in DAT cDNA) in YFP-HA-R60A was replaced by the corresponding sequence from the YFP-DAT construct using PflMI (5’ end) and SmaI (3’ end) restriction sites.

Techniques: Binding Assay, Concentration Assay, Expressing, Incubation, Live Cell Imaging, Fluorescence

Journal: Scientific Reports

Article Title: Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter

doi: 10.1038/s41598-017-05637-x

Figure Lengend Snippet: Binding of cocaine results in the enrichment of the R60A mutant relative to co-expressed wt DAT in the filopodia of HEK293 cells. ( A ) HEK293 cells co-expressing RFP-HA-DAT with YFP-HA-DAT, YFP-HA-DAT-R60A or YFP-HA-DAT-W63A were imaged through 515 nm (YFP, green ) and 561 nm (RFP, red ) filter channels before and after cocaine treatment (30 min at RT). Maximal z-projections of 5 consecutive x-y-confocal planes are shown. Scale bars, 10μm. ( B ) Insets represent high magnification images of the regions marked by the white rectangle in ( A ). Arrows point to examples of peripheral filopodia. ( C–E ) The Filopodia/FM ratios of YFP and RFP fluorescence intensities in cells co-expressing RFP-HA-DAT with YFP-HA-R60A ( C ), YFP-HA-W63A ( D ) or wt YFP-HA-DAT ( E ) were calculated in experiments exemplified in ( A,B ) as described in “Methods”. Results are shown as mean ± SEM, n = 10. ( F ) The YFP/RFP ratio in filopodia was calculated in experiments represented in ( A,B ). Results are shown as mean ± SEM, n = 10. *p < 0.05, ***p < 0.001. *p < 0.05, ***p < 0.001.

Article Snippet: To generate R60A mutation in the template of YFP-DAT (no HA tag, Addgene plasmid # 90228) , the 1622 bp DAT sequence (begins with bp 242 in DAT cDNA) in YFP-HA-R60A was replaced by the corresponding sequence from the YFP-DAT construct using PflMI (5’ end) and SmaI (3’ end) restriction sites.

Techniques: Binding Assay, Mutagenesis, Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter

doi: 10.1038/s41598-017-05637-x

Figure Lengend Snippet: The IC interaction network that regulates the opening/closure of the IC vestibule and the transition between OF and IF states, shows distinctive dynamics in wt DAT and mutants W63A and R60A. ( A ) In wt DAT, the salt-bridge R60-D436 and close interactions between W63 and the F332 and Y335 stabilize substrate/sodium-binding site in the OF state. ( B ) In the mutant W63A, substitution of W63 by alanine breaks the stabilizing network of molecular interaction; F76 side chain dihedral angle χ 1 rotates from ± 180° to −60°, leading to the opening of the IC vestibule. ( C ) Substitution of R60 by alanine breaks the salt-bridge R60-D436. Yet, alternative IC salt bridges form, such as R260-E61, which stably maintain the closed state of the IC vestibule. Residues at positions 60 and 63 are displayed in VDW format, as well as D436. In all diagrams, residues within 3 Å of W63 or W63A are shown in licorice format. Hydrated EC regions are indicated by green shaded areas.

Article Snippet: To generate R60A mutation in the template of YFP-DAT (no HA tag, Addgene plasmid # 90228) , the 1622 bp DAT sequence (begins with bp 242 in DAT cDNA) in YFP-HA-R60A was replaced by the corresponding sequence from the YFP-DAT construct using PflMI (5’ end) and SmaI (3’ end) restriction sites.

Techniques: Binding Assay, Mutagenesis, Stable Transfection

Journal: Scientific Reports

Article Title: Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter

doi: 10.1038/s41598-017-05637-x

Figure Lengend Snippet: Cocaine and its analog bind and stabilize the outward-facing (OF) state to the wt DAT and the R60A mutant. ( A ) Cocaine binds near the S1 site of R60A in the OF state of the mutant, and closely interacts with D79, R85, Y156, F320 and D476. ( B,C ) Cocaine binds near the S1 ( B ) and S2 ( C ) sites of wt DAT in the OF state. ( D ) JHC1-64 binds near the S2 site of R60A and inserts toward the S1 site. (E,F) JHC1-64 binds to the vicinity of the S1 ( E ) and S2 ( F ) sites of wt DAT. Cyan, blue, dark red, light red , and yellow spheres represent the respective carbon, nitrogen, oxygen, chloride, and sulfur atoms of JHC1-64.

Article Snippet: To generate R60A mutation in the template of YFP-DAT (no HA tag, Addgene plasmid # 90228) , the 1622 bp DAT sequence (begins with bp 242 in DAT cDNA) in YFP-HA-R60A was replaced by the corresponding sequence from the YFP-DAT construct using PflMI (5’ end) and SmaI (3’ end) restriction sites.

Techniques: Mutagenesis

Journal: Scientific Reports

Article Title: Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter

doi: 10.1038/s41598-017-05637-x

Figure Lengend Snippet: Binding of zinc increases the concentration of R60A in filopodia of HEK293 cells. ( A) Cells transiently expressing YFP-DAT or the R60A mutant of YFP-DAT were incubated with 10 μM zinc chloride for 30 min at RT. Live-cell imaging was performed through the 515 nm (YFP, green ) filter channel. Maximal z-projections of 5 consecutive x-y-confocal planes are shown. Scale bars, 10 μm. ( B ) Insets represent high magnification images of the regions marked by the white rectangle in ( A ). Arrows point to examples of peripheral filopodia. ( C ) The filopodia/FM ratios of YFP fluorescence intensities were quantitated in experiments exemplified in ( A , B ). Results are shown as mean ± SEM, n = 10. *p < 0.05.

Article Snippet: To generate R60A mutation in the template of YFP-DAT (no HA tag, Addgene plasmid # 90228) , the 1622 bp DAT sequence (begins with bp 242 in DAT cDNA) in YFP-HA-R60A was replaced by the corresponding sequence from the YFP-DAT construct using PflMI (5’ end) and SmaI (3’ end) restriction sites.

Techniques: Binding Assay, Concentration Assay, Expressing, Mutagenesis, Incubation, Live Cell Imaging, Fluorescence

Journal: Scientific Reports

Article Title: Targeting of dopamine transporter to filopodia requires an outward-facing conformation of the transporter

doi: 10.1038/s41598-017-05637-x

Figure Lengend Snippet: AMPH treatment reduces the concentration of wt DAT in the filopodia of HEK293 cells. ( A ) Cells transiently expressing YFP-HA-DAT were treated with AMPH or vehicle (control) at 37°C for 1 hr. Cells were then incubated with 100 nM JHC1-64 for 10 min. Images were acquired through 515 nm (YFP, green) and 561 nm (JHC1-64, red) filter channels. The ratio of JHC1-64 and YFP fluorescence intensities (JHC/YFP) was calculated for individual cells. Results are shown as mean ± SEM, n = 5. ****p < 0.0001. ( B ) Cells transiently expressing YFP-HA-DAT were incubated with 10 μM AMPH or vehicle in KRH at 37 °C for 30 min. Live-cell imaging was performed through 515 nm (YFP, green) filter channel. Maximal z-projections of 5 consecutive x-y-confocal planes are shown. Scale bars, 10 μm. ( C ) Insets represent high magnification images of the regions marked by the white rectangle in ( B ). Arrows point on examples of peripheral filopodia. ( D ) The filopodia/FM ratios of YFP fluorescence intensities were calculated in experiments exemplified in ( B , C ). Results are shown as mean ± SEM, n = 8. **p < 0.01.

Article Snippet: To generate R60A mutation in the template of YFP-DAT (no HA tag, Addgene plasmid # 90228) , the 1622 bp DAT sequence (begins with bp 242 in DAT cDNA) in YFP-HA-R60A was replaced by the corresponding sequence from the YFP-DAT construct using PflMI (5’ end) and SmaI (3’ end) restriction sites.

Techniques: Concentration Assay, Expressing, Control, Incubation, Fluorescence, Live Cell Imaging

Journal: The Journal of Biological Chemistry

Article Title: Formyl Peptide Receptors from Immune and Vomeronasal System Exhibit Distinct Agonist Properties *

doi: 10.1074/jbc.M112.375774

Figure Lengend Snippet: Concentration-response curves of two mouse immune Fprs (mFpr1 and mFpr2) for selected agonists. A , our analysis revealed several subtype-specific agonists. Temporin A amide is selective for mFpr2 and has an EC 50 of 690 n m . T20, β-amyloid 16–22 and V3gp120 HIV (BK-130) are selective for mFpr1 with an EC 50 of 200 n m , 4,100 n m, and 4,500 n m , respectively. NDI-6I prefers mFpr1 with an EC 50 of 0.6 n m but can also activate mFpr2 with an EC 50 of 57 n m . B , mFpr1 and mFpr2 but not mFpr-rs1 are activated by DMSO at concentrations above 0.1%. First unspecific effects of DMSO are observed above 2%. The DMSO activation is dose-dependent with an EC 50 of 97 m m for mFpr1 and 111 m m for mFpr2. These experiments are based on at least five independent transfections. C , analysis of Ca 2+ signals evoked by DMSO (2%). These signals depend on the presence of the G protein α-subunit Gα 16 . Bars show mean responses of duplicates for two independent transfections. D , cross-desensitization of DMSO (2%) evoked Ca 2+ responses by fMLF (30 μ m ). Bars show mean responses of triplicates for three independent transfections. Error bars , S.D. **, p ≤ 0.01; ***, p ≤ 0.001.

Article Snippet: Temporin A amide, T20, ADP715 HIV-I, V3gp120 HIV (JR-FL), V3gp120 HIV (BK-130), μPAR 84-95, and β-amyloid 16–22 were purchased from Annaspec/MoBiTec. fMLF and DMSO were obtained from Sigma.

Techniques: Concentration Assay, Activation Assay, Transfection