mouse monoclonal anti spectrin 3a9  (Developmental Studies Hybridoma Bank)

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 to D ) Left: Stitched SIM images showing the distributions of endogenous endocytic pits, clathrin (A), Cav1 (B), Flot1 (C), or EndoA2 (D) in WT neurons. Endocytic pits are shown in green, with compartment markers MAP2 (magenta) and neurofacsin (yellow). Scale bar, 10 μm. Right: Enlarged SIM images of the three boxed regions on the left, corresponding to soma, dendrite, and AIS compartments, respectively. Scale bar, 2 μm. ( E ) Boxplots showing the area fraction of endogenous endocytic pits in different compartments of WT neurons. ( F ) Left: SIM images of tau (magenta) and endogenous endocytic pits (green) in distal axons of WT neurons. Right: The same as on the left, but in βII-spectrin KD neurons. Scale bars, 2 μm. ( G ) Boxplots showing the area fraction of endogenous endocytic pits (green) in distal axons of WT and βII-spectrin KD neurons. ( H ) Left: SIM images of MAP2 (magenta) and endogenous endocytic pits (green) in dendrites of WT neurons. Right: The same as on the left, but in βII-spectrin KD neurons. Scale bars, 2 μm. ( I ) Boxplots showing the area fraction of endogenous endocytic pits in dendrites of WT and βII-spectrin KD neurons.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques:

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 spatial distributions of clathrin, Cav1, Flot1, and EndoA2 endocytic pits, relative to periodic MPS lattice in axons. ( B ) Schematic illustrating two distinct types of endocytic pits based on their spatial positioning relative to periodic βII-spectrin lattice in axons. Class I pits do not overlap with MPS lattice, whereas class II do. The MPS was visualized by immunostaining with antibodies targeting the C terminus of βII-spectrin, which mark the centers of spectrin tetramers. ( C ) The same as in (B) but showing spatial relationships with the periodic adducin lattice in axons. The MPS was visualized by immunostaining with antibodies targeting the adducin, which mark the terminal ends of spectrin tetramers. ( D ) Left: Dual-color STORM images of βII-spectrin (magenta) and endogenous clathrin (green) in axons. Right: Magnified views of class I and class II CCPs in the boxed regions. Scale bars, 10 μm (left), 5 μm (middle), and 200 nm (right). ( E ) PCCs between βII-spectrin and endogenous clathrin under experimental and randomized conditions. ( F ) Left: Averaged dual-color STORM images of βII-spectrin (magenta) and endogenous clathrin (green), generated by aligning individual STORM images to the centers of CCPs. Right: Radial intensity profiles of the averaged images shown on the left. Scale bar, 100 nm. ( G to I ) The same as in (D) to (F) but for βII-spectrin (magenta) and exogenously expressed Cav1 (green). ( J to L ) The same as in (D) to (F) but for adducin (magenta) and exogenously expressed Flot1 (green). ( M to O ) The same as in (D) to (F) but for adducin (magenta) and exogenously expressed EndoA2 (green). ( P ) Percentages of class I and class II pits for endogenous clathrin, exogenously expressed Cav1, exogenously expressed Flot1 and exogenously expressed EndoA2 in axons.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Immunostaining, Generated

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 spatial distribution of clathrin, Cav1, Flot1, and EndoA2 endocytic pits, relative to periodic MPS lattice in dendrites. ( B ) Schematic illustrating two distinct types of endocytic pits based on their spatial positioning relative to periodic βIII-spectrin or adducin lattice in dendrites. Class I pits do not overlap with MPS lattice, whereas class II pits do. The MPS was visualized by immunostaining with antibodies targeting the N terminus of βIII-spectrin or adducin, which both mark terminal ends of spectrin tetramers. ( C ) Left: Dual-color STORM images of βIII-spectrin (magenta) and endogenous clathrin (green) in dendrites. Right: Magnified views of class I and class II CCPs in the boxed regions. Scale bars, 10 μm (left), 5 μm (middle), 200 nm (right). ( D ) PCCs between βIII-spectrin and endogenous clathrin under experimental and randomized conditions. ( E ) Left: Averaged dual-color STROM images of βIII-spectrin (magenta) and endogenous clathrin (green), generated by aligning individual STORM images to the centers of CCPs. Right: Radial intensity profile of averaged images shown on the left. Scale bar, 100 nm. ( F to H ) The same as in (C) to (E) but for adducin (magenta) and exogenously expressed Cav1 (green). ( I to K ) The same as in (C) to (E) but for adducin (magenta) and exogenously expressed Flot1 (green). ( L to N ) The same as in (C) to (E) but for adducin (magenta) and exogenously expressed EndoA2 (green). ( O ) Percentages of class I and class II endocytic pits for endogenous clathrin, exogenously expressed Cav1, exogenously expressed Flot1, and exogenously expressed EndoA2 in dendrites.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Immunostaining, Generated

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 ) Confocal fluorescence images of MAP2 (magenta) and internalized CF568-transferrin (green) in somatodendritic region of WT or βII-spectrin KD neurons treated with CF568-transferrin for 2, 10, and 20 min. Scale bars, 10 μm. ( B ) Time course of CF568-transferrin endocytosis in somatodendritic regions of WT and βII-spectrin KD neurons, quantified by the area fraction of transferrin-positive endosomes. Solid lines represent single-exponential fits to the data. ( C ) Confocal fluorescence images of MAP2 (gray), internalized HA-mGluR5a (green), and endogenous Cav1 (magenta) in somatodendritic region of WT or βII-spectrin KD neurons overexpressing HA-mGluR5a and treated with anti-HA antibody for 5, 10, and 20 min. Scale bars, 10 μm. ( D ) Time course of Cav1-mediated HA-mGluR5a endocytosis in the somatodendritic regions of WT and βII-spectrin KD neurons, quantified by the area fraction of Cav1-positive HA-mGluR5a endosomes. Solid lines represent single-exponential fits to the data. ( E ) SIM images of internalized NCAM1 (green) and endogenous EndoA2 (magenta) in axonal (top) and somatodendritic (bottom) regions of WT or βII-spectrin KD neurons treated with anti-NCAM1 antibody for 30 min. Scale bars, 2 μm. ( F ) Boxplots of EndoA2-mediated NCAM1 endocytosis in axonal and somatodendritic regions of WT and βII-spectrin KD neurons, quantified by the area proportion of EndoA2-positive NCAM1 endosomes.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Fluorescence

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 ligand-induced ERK activation via three major endocytic pathways: CME of TfR, LRME of HA-mGluR5a, and FEME of NCAM1. ( B ) Top: Epi-fluorescence images showing pERK immunostaining in neurons without ligand treatment, neurons treated with CF568-transferrin, and neurons overexpressing HA-mGluR5a treated with anti-HA antibody. Bottom: The same as the top but with neurons pretreated with dyngo-4a before ligand treatment. Scale bars, 25 μm. ( C ) Time course of ERK activation in neurons under the same conditions as in (B). ( D ) 3D STORM images of immunostained βIII-spectrin in dendrites of neurons under various treatments. First column: neurons pretreated with dimethyl sulfoxide (DMSO), dyngo-4a, U0126, MDL, or VAD. Second column: neurons pretreated with the same inhibitors followed by CF568-transferrin treatment. Third column: neurons overexpressing HA-mGluR5a pretreated with the same inhibitors followed by the anti-HA antibody treatment. Fourth column: neurons pretreated with the same inhibitors followed by anti-NCAM1 antibody treatment. Scale bars, 1 μm. Color scale bar represents the z -coordinate information. ( E ) Averaged 1D autocorrelation amplitudes of βIII-spectrin, calculated for the same conditions as in (D). ( F ) SIM images of MAP2 (magenta) and internalized CF568-transferrin (green) in neurons pretreated with DMSO, MDL, or VAD followed by CF568-transferrin treatment. Scale bars, 2 μm. ( G ) Boxplots of transferrin-positive endosome area fractions. ( H ) Confocal fluorescence images of MAP2 (magenta) and internalized HA-mGluR5a (green) in neurons overexpressing HA-mGluR5a pretreated with DMSO, MDL, or VAD followed by anti-HA antibody treatment. Scale bars, 10 μm. ( I ) Boxplots of HA-mGluR5a endosome area fractions. ( J ) Schematic summarizing the proposed positive feedback mechanism: Receptor endocytosis via CME, LRME, or FEME activates ERK signaling, which triggers calpain- and caspase-mediated MPS degradation; MPS disruption in turn facilitates further endocytosis, establishing a positive feedback loop.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Activation Assay, Fluorescence, Immunostaining, Disruption

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: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Fluorescence, Activation Assay

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 to D ) Left: Stitched SIM images showing the distributions of endogenous endocytic pits, clathrin (A), Cav1 (B), Flot1 (C), or EndoA2 (D) in WT neurons. Endocytic pits are shown in green, with compartment markers MAP2 (magenta) and neurofacsin (yellow). Scale bar, 10 μm. Right: Enlarged SIM images of the three boxed regions on the left, corresponding to soma, dendrite, and AIS compartments, respectively. Scale bar, 2 μm. ( E ) Boxplots showing the area fraction of endogenous endocytic pits in different compartments of WT neurons. ( F ) Left: SIM images of tau (magenta) and endogenous endocytic pits (green) in distal axons of WT neurons. Right: The same as on the left, but in βII-spectrin KD neurons. Scale bars, 2 μm. ( G ) Boxplots showing the area fraction of endogenous endocytic pits (green) in distal axons of WT and βII-spectrin KD neurons. ( H ) Left: SIM images of MAP2 (magenta) and endogenous endocytic pits (green) in dendrites of WT neurons. Right: The same as on the left, but in βII-spectrin KD neurons. Scale bars, 2 μm. ( I ) Boxplots showing the area fraction of endogenous endocytic pits in dendrites of WT and βII-spectrin KD neurons.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques:

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 spatial distributions of clathrin, Cav1, Flot1, and EndoA2 endocytic pits, relative to periodic MPS lattice in axons. ( B ) Schematic illustrating two distinct types of endocytic pits based on their spatial positioning relative to periodic βII-spectrin lattice in axons. Class I pits do not overlap with MPS lattice, whereas class II do. The MPS was visualized by immunostaining with antibodies targeting the C terminus of βII-spectrin, which mark the centers of spectrin tetramers. ( C ) The same as in (B) but showing spatial relationships with the periodic adducin lattice in axons. The MPS was visualized by immunostaining with antibodies targeting the adducin, which mark the terminal ends of spectrin tetramers. ( D ) Left: Dual-color STORM images of βII-spectrin (magenta) and endogenous clathrin (green) in axons. Right: Magnified views of class I and class II CCPs in the boxed regions. Scale bars, 10 μm (left), 5 μm (middle), and 200 nm (right). ( E ) PCCs between βII-spectrin and endogenous clathrin under experimental and randomized conditions. ( F ) Left: Averaged dual-color STORM images of βII-spectrin (magenta) and endogenous clathrin (green), generated by aligning individual STORM images to the centers of CCPs. Right: Radial intensity profiles of the averaged images shown on the left. Scale bar, 100 nm. ( G to I ) The same as in (D) to (F) but for βII-spectrin (magenta) and exogenously expressed Cav1 (green). ( J to L ) The same as in (D) to (F) but for adducin (magenta) and exogenously expressed Flot1 (green). ( M to O ) The same as in (D) to (F) but for adducin (magenta) and exogenously expressed EndoA2 (green). ( P ) Percentages of class I and class II pits for endogenous clathrin, exogenously expressed Cav1, exogenously expressed Flot1 and exogenously expressed EndoA2 in axons.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Immunostaining, Generated

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 spatial distribution of clathrin, Cav1, Flot1, and EndoA2 endocytic pits, relative to periodic MPS lattice in dendrites. ( B ) Schematic illustrating two distinct types of endocytic pits based on their spatial positioning relative to periodic βIII-spectrin or adducin lattice in dendrites. Class I pits do not overlap with MPS lattice, whereas class II pits do. The MPS was visualized by immunostaining with antibodies targeting the N terminus of βIII-spectrin or adducin, which both mark terminal ends of spectrin tetramers. ( C ) Left: Dual-color STORM images of βIII-spectrin (magenta) and endogenous clathrin (green) in dendrites. Right: Magnified views of class I and class II CCPs in the boxed regions. Scale bars, 10 μm (left), 5 μm (middle), 200 nm (right). ( D ) PCCs between βIII-spectrin and endogenous clathrin under experimental and randomized conditions. ( E ) Left: Averaged dual-color STROM images of βIII-spectrin (magenta) and endogenous clathrin (green), generated by aligning individual STORM images to the centers of CCPs. Right: Radial intensity profile of averaged images shown on the left. Scale bar, 100 nm. ( F to H ) The same as in (C) to (E) but for adducin (magenta) and exogenously expressed Cav1 (green). ( I to K ) The same as in (C) to (E) but for adducin (magenta) and exogenously expressed Flot1 (green). ( L to N ) The same as in (C) to (E) but for adducin (magenta) and exogenously expressed EndoA2 (green). ( O ) Percentages of class I and class II endocytic pits for endogenous clathrin, exogenously expressed Cav1, exogenously expressed Flot1, and exogenously expressed EndoA2 in dendrites.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Immunostaining, Generated

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 ) Confocal fluorescence images of MAP2 (magenta) and internalized CF568-transferrin (green) in somatodendritic region of WT or βII-spectrin KD neurons treated with CF568-transferrin for 2, 10, and 20 min. Scale bars, 10 μm. ( B ) Time course of CF568-transferrin endocytosis in somatodendritic regions of WT and βII-spectrin KD neurons, quantified by the area fraction of transferrin-positive endosomes. Solid lines represent single-exponential fits to the data. ( C ) Confocal fluorescence images of MAP2 (gray), internalized HA-mGluR5a (green), and endogenous Cav1 (magenta) in somatodendritic region of WT or βII-spectrin KD neurons overexpressing HA-mGluR5a and treated with anti-HA antibody for 5, 10, and 20 min. Scale bars, 10 μm. ( D ) Time course of Cav1-mediated HA-mGluR5a endocytosis in the somatodendritic regions of WT and βII-spectrin KD neurons, quantified by the area fraction of Cav1-positive HA-mGluR5a endosomes. Solid lines represent single-exponential fits to the data. ( E ) SIM images of internalized NCAM1 (green) and endogenous EndoA2 (magenta) in axonal (top) and somatodendritic (bottom) regions of WT or βII-spectrin KD neurons treated with anti-NCAM1 antibody for 30 min. Scale bars, 2 μm. ( F ) Boxplots of EndoA2-mediated NCAM1 endocytosis in axonal and somatodendritic regions of WT and βII-spectrin KD neurons, quantified by the area proportion of EndoA2-positive NCAM1 endosomes.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Fluorescence

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 ligand-induced ERK activation via three major endocytic pathways: CME of TfR, LRME of HA-mGluR5a, and FEME of NCAM1. ( B ) Top: Epi-fluorescence images showing pERK immunostaining in neurons without ligand treatment, neurons treated with CF568-transferrin, and neurons overexpressing HA-mGluR5a treated with anti-HA antibody. Bottom: The same as the top but with neurons pretreated with dyngo-4a before ligand treatment. Scale bars, 25 μm. ( C ) Time course of ERK activation in neurons under the same conditions as in (B). ( D ) 3D STORM images of immunostained βIII-spectrin in dendrites of neurons under various treatments. First column: neurons pretreated with dimethyl sulfoxide (DMSO), dyngo-4a, U0126, MDL, or VAD. Second column: neurons pretreated with the same inhibitors followed by CF568-transferrin treatment. Third column: neurons overexpressing HA-mGluR5a pretreated with the same inhibitors followed by the anti-HA antibody treatment. Fourth column: neurons pretreated with the same inhibitors followed by anti-NCAM1 antibody treatment. Scale bars, 1 μm. Color scale bar represents the z -coordinate information. ( E ) Averaged 1D autocorrelation amplitudes of βIII-spectrin, calculated for the same conditions as in (D). ( F ) SIM images of MAP2 (magenta) and internalized CF568-transferrin (green) in neurons pretreated with DMSO, MDL, or VAD followed by CF568-transferrin treatment. Scale bars, 2 μm. ( G ) Boxplots of transferrin-positive endosome area fractions. ( H ) Confocal fluorescence images of MAP2 (magenta) and internalized HA-mGluR5a (green) in neurons overexpressing HA-mGluR5a pretreated with DMSO, MDL, or VAD followed by anti-HA antibody treatment. Scale bars, 10 μm. ( I ) Boxplots of HA-mGluR5a endosome area fractions. ( J ) Schematic summarizing the proposed positive feedback mechanism: Receptor endocytosis via CME, LRME, or FEME activates ERK signaling, which triggers calpain- and caspase-mediated MPS degradation; MPS disruption in turn facilitates further endocytosis, establishing a positive feedback loop.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Activation Assay, Fluorescence, Immunostaining, Disruption

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: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for IF (Synaptic Systems, 314004), mouse anti-tau antibody 1:500 dilution for IF (BD Biosciences, 556319), guinea pig anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 dilution for IF (Synaptic Systems, 188002), chicken anti-neurofascin antibody (R&D system, AF3235), rabbit anti-CHC antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-Cav1 antibody 1:400 dilution for IF (Cell Signaling Technology, 3238S), mouse anti-Flot1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-365704), mouse anti–αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz Biotechnology, sc-515592), mouse anti-βII spectrin antibody 1:200 dilution for IF (BD Biosciences, 612563), rabbit anti-adducin antibody 1:500 dilution for IF (Abcam, ab51130), chicken anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A10262), rabbit anti-GFP antibody 1:500 dilution for IF (Thermo Fisher Scientific, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz Biotechnology, sc-515737), mouse anti-HA antibody 1:200 dilution for HA-mGluR5a internalization and IF (Thermo Fisher Scientific, 26183), rat anti-NCAM1 (CD56) antibody 1:40 dilution for NCAM1 internalization and IF (Cedarlane, CL10008AP), rat anti-TfR (CD71) antibody 1:500 dilution for IF (Bio-Rad, MCA1033GA), goat anti–LDL receptor (LDLR) antibody 1:100 dilution for IF (Thermo Fisher Scientific, PA5-46987), rabbit anti–phospho-ERK antibody 1:300 dilution (Cell Signaling Technology, 4370S), mouse anti-Aβ42 antibody 1:200 dilution for IF (BioLegend, 805501), and rabbit anti–cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Fluorescence, Activation Assay