app695  (Addgene inc)

Journal: Science Advances

Article Title: Membrane-associated periodic skeleton regulates major forms of endocytosis in neurons through a signaling-driven positive feedback loop

doi: 10.1126/sciadv.aeb0803

Figure Lengend Snippet: ( A ) Schematic illustrating the sequential cleavage of APP695 by β-secretase and γ-secretase to produce Aβ42. ( B ) Schematic illustrating the structure of SEP-APP. ( C ) Left: Epi-fluorescence images of pERK in neurons overexpressing SEP-APP without ligand treatment. Middle: The same as the left but treated with GFP nanobody. Right: The same as the middle but with dyngo-4a preincubation before GFP nanobody treatment. Scale bars, 25 μm. ( D ) Time course of ERK activation in neurons under the same conditions as in (C). ( E ) 3D STORM images of immunostained βIII-spectrin in dendrites of neurons pretreated with DMSO, dyngo-4a, U0126, MDL, or VAD followed by GFP nanobody treatment. Scale bars, 1 μm. ( F ) Averaged 1D autocorrelation amplitude of βIII-spectrin, calculated for the same conditions as in (E). ( G ) Confocal fluorescence images of CTB (magenta) and internalized SEP-APP (green) in neurons pretreated with DMSO, MDL, or VAD followed by GFP nanobody treatment. Scale bars, 10 μm. ( H ) Boxplots of SEP-APP endosome area fractions. ( I ) Left: Confocal fluorescence images of MAP2 (magenta) and intracellular Aβ42 (green) in WT neurons, neurons overexpressing APPwt, and neurons overexpressing APPswe. Right: The same as the left but in βII-spectrin KD neurons. Scale bars, 10 μm. ( J ) Boxplots of intracellular Aβ42 area fractions in somatodendritic regions of neurons. ( K ) Left, SIM images of MAP2 (magenta) and cleaved caspase-3 (green) in WT neurons, neurons overexpressing APPwt, and neurons overexpressing APPswe. Right: The same as the left but in βII-spectrin KD neurons. Scale bars, 2 μm. ( L ) Boxplots of cleaved caspase-3 area fractions in dendrites of neurons. ( M ) Schematic illustrating APP endocytosis triggers downstream ERK signaling, leading to MPS degradation through caspase- and calpain-mediated spectrin cleavage. This degradation further accelerates APP endocytosis, promoting intracellular Aβ42 accumulation and caspase-3 activation.

Article Snippet: Coding sequences for CAV1, FLOT1, SH3GL1 (synthesized by Twist Bioscience), APP695 (Addgene, plasmid #114193), and Basp1 (GenoCopoeia, plasmid # EX-Mm10271-M98) were fused to a C-terminal GFP via a flexible polypeptide linker and cloned into the FUGW vector, generating GFP-tagged constructs for Cav1, Flot1, EndoA2, APPwt, and Basp1.

Techniques: Fluorescence, Activation Assay

Journal: bioRxiv

Article Title: Macropinocytosis of amyloid precursor protein requires the adaptor protein Fe65 and the recruitment and activity of Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641070

Figure Lengend Snippet: N2a cells transfected with APP695 and LAMP1-mCh (red) and incubated with fluorescently-tagged A) N-terminal anti-APP (APP), B) Aβ-region targeting 6E10 antibodies, or C) anti-NCAM antibodies (green) on ice for 20 minutes, then immediately fixed or allowed to incubate at 37°C for 15 minutes. Colocalization was assessed between crosslinked APP and LAMP1 (white pixels). D) Quantification of the mean % of APP colocalized with LAMP1 from three replicate experiments (n=3; 10 images per replicate), with significance calculated by a one-way ANOVA with Tukey’s test. E) N2a cells transfected with APP695 and LAMP1-mCh (red) that were treated with DMSO, 20μM Pitstop2 or 10μM EIPA. APP was then bound/crosslinked by antibody and imaged after 15 minutes. Colocalization was assessed between crosslinked APP and LAMP1 (white pixels). F) Quantification of the mean % of APP colocalized with LAMP1 (n=3; 10 images per replicate), with significance calculated by a one-way ANOVA with Tukey’s test. Data is presented as mean ± SEM. * p<0.05; Scale bar = 5μm .

Article Snippet: APP695-AENATA (pCAX-APP-AENATA) was a gift from Dennis Selkoe and Tracy Young-Pearse (Addgene plasmid #30144).

Techniques: Transfection, Incubation

Journal: bioRxiv

Article Title: Macropinocytosis of amyloid precursor protein requires the adaptor protein Fe65 and the recruitment and activity of Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641070

Figure Lengend Snippet: A) N2a cells transfected with Fe65-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Cells were incubated with fluorescent anti-APP or anti-NCAM antibodies (red) on ice, then immediately fixed on ice as a baseline (00:00) or incubated for 30 seconds (00:30), 2 minutes (02:00), 5 minutes (5:00), and 10 minutes (10:00). Colocalization between signals is indicated by white pixels. B/C) Quantification of colocalization data (n=3; 15 images per replicate) is represented as the difference between the colocalization at each timepoint to the colocalization at baseline (mean % colocalized at XX:XX – mean % colocalized at 00:00). * denotes significant difference to baseline within condition; # denotes significant difference between groups at the indicated timepoint. D) N2a cells transfected with Arf6-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Experiment above was repeated with Arf6-EGFP expressing cells. Colocalization between signals is indicated by white pixels. E/F) Quantification of colocalization data (n=3; 15 images per replicate). Data representation and analysis from G/H) was repeated for N2a cells expressing Arf6-GFP. * denotes significant difference to baseline within condition; # denotes significant difference between groups at the indicated timepoint. Significance was measured by a two-way ANOVA with a Tukey’s test using a single pooled variance. Data is presented as mean ± SEM. */# p<0.05; Scale bar = 5μm .

Article Snippet: APP695-AENATA (pCAX-APP-AENATA) was a gift from Dennis Selkoe and Tracy Young-Pearse (Addgene plasmid #30144).

Techniques: Transfection, Incubation, Expressing

Journal: bioRxiv

Article Title: Macropinocytosis of amyloid precursor protein requires the adaptor protein Fe65 and the recruitment and activity of Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641070

Figure Lengend Snippet: A) N2a cells transfected with Rac1-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Cells were incubated with tagged anti-APP or anti-NCAM antibodies (red) on ice and then were either immediately fixed on ice as a baseline (00:00), or incubated for 30 seconds (00:30), 2 minutes (02:00), 5 minutes (5:00), and 10 minutes (10:00) at 37°C 5% CO2 prior to fixation. Colocalization between signals is indicated by white pixels. B/C) Quantification of colocalization data (n=3; 15 images per replicate) is represented as the difference between the colocalization at each timepoint to the colocalization at baseline (mean % colocalized at XX:XX – mean % colocalized at 00:00). * denotes significant difference to baseline within condition; # denotes significant difference between groups at the indicated timepoint. D) N2a cells transfected with Cdc42-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Experiment from A) was repeated with Cdc42-EGFP expressing cells. Colocalization between signals is indicated by white pixels. E/F) Quantification of colocalization data (n=3; 15 images per replicate). Data representation and analysis from B/C) was repeated for N2a cells expressing Cdc42-EGFP. * denotes significant difference to baseline within condition; # denotes significant difference between groups at the indicated timepoint. G) N2a cells transfected with RhoA-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Experiment from A) was repeated with RhoA-EGFP expressing cells. Colocalization between signals is indicated by white pixels. H/I) Quantification of colocalization data (n=3; 15 images per replicate). Data representation and analysis from B/C) was repeated for N2a cells expressing RhoA-EGFP. * denotes significant difference to baseline within condition; # denotes significant difference between groups at the indicated timepoint. Significance was measured by a two-way ANOVA with a Tukey’s test using a single pooled variance. Data is presented as mean ± SEM. */# p<0.05; Scale bar = 5μm .

Article Snippet: APP695-AENATA (pCAX-APP-AENATA) was a gift from Dennis Selkoe and Tracy Young-Pearse (Addgene plasmid #30144).

Techniques: Transfection, Incubation, Expressing

Journal: bioRxiv

Article Title: Macropinocytosis of amyloid precursor protein requires the adaptor protein Fe65 and the recruitment and activity of Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641070

Figure Lengend Snippet: A) N2a cells transfected with Fe65-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Cells were treated with 0.1% DMSO, 5μM NAV-2729, 10μM EHT 1864, 10μM ML 141, and 35μM Rhosin. After treatment, cells were incubated with tagged anti-APP antibodies (red) on ice and then fixed following an incubation for 30 seconds. Colocalization was assessed between Fe65 and antibody bound/crosslinked APP or PLC8PH (white pixels). B) Quantification of the mean % of Fe65 colocalized with APP (left) or PLC8PH (right) from three replicate experiments (n=3; 15 images per replicate). C) N2a cells transfected with Arf6-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Cells were treated with 0.1% DMSO, 5μM NAV-2729, 10μM EHT 1864, 10μM ML 141, and 35μM Rhosin. After treatment, cells were incubated with tagged anti-APP antibodies (red) on ice and then fixed following a 30 second incubation. Colocalization was assessed between Arf6 and bound/crosslinked APP or PLC8PH (white pixels). D) Quantification of the mean % of Arf6 colocalized with APP (left) or PLC8PH (right) from three replicate experiments (n=3; 15 images per replicate). Significant changes in the percentage colocalized between treatments were assessed by a one-way ANOVA with Tukey’s test . Data is presented as mean ± SEM. *p<0.05; Scale bar = 5μm .

Article Snippet: APP695-AENATA (pCAX-APP-AENATA) was a gift from Dennis Selkoe and Tracy Young-Pearse (Addgene plasmid #30144).

Techniques: Transfection, Incubation

Journal: bioRxiv

Article Title: Macropinocytosis of amyloid precursor protein requires the adaptor protein Fe65 and the recruitment and activity of Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641070

Figure Lengend Snippet: A) N2a cells transfected with Rac1-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Cells were treated with 0.1% DMSO, 5μM NAV-2729, 10μM EHT 1864, 10μM ML 141, and 35μM Rhosin. After treatment, cells were incubated with tagged anti-APP antibodies (red) on ice and then fixed following a 30 second incubation. Colocalization was assessed between Rac1 and bound/crosslinked APP or PLC8PH (white pixels). B) Quantification of the mean % of Rac1 colocalized with APP (left) or PLC8PH (right) from three replicate experiments (n=3; 15 images per replicate). C) N2a cells transfected with Cdc42-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Cells were treated as described in A), then incubated with tagged anti-APP antibodies (red) on ice and fixed following a 30 second incubation. Colocalization was assessed between Cdc42 and bound/crosslinked APP or PLC8PH (white pixels). D) Quantification of the mean % of Cdc42 colocalized with APP (left) or PLC8PH (right) from three replicate experiments (n=3; 15 images per replicate). E) N2a cells transfected with RhoA-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Cells were treated as described in A), then incubated with tagged anti-APP antibodies (red) on ice and then fixed following an incubation for 30 seconds. Colocalization was assessed between RhoA and bound/crosslinked APP or PLC8PH (white pixels). F) Quantification of the mean % of RhoA colocalized with APP (left) or PLC8PH (right) from three replicate experiments (n=3; 15 images per replicate). Significant changes in the percentage colocalized between treatments were assessed by a one-way ANOVA with Tukey’s test . Data is presented as mean ± SEM. *p<0.05; Scale bar = 5μm .

Article Snippet: APP695-AENATA (pCAX-APP-AENATA) was a gift from Dennis Selkoe and Tracy Young-Pearse (Addgene plasmid #30144).

Techniques: Transfection, Incubation