Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: ( A-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: Same as Left, but in βII-spectrin knockdown (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: Same as 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 immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Knockdown

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: ( A ) Widefield epi-fluorescence images of βII-spectrin in neurons transduced with adenoviruses expressing either scrambled (control) shRNA or βII-spectrin shRNA. Scale bar: 25 µm. ( B ) Boxplots showing the normalized fluorescence intensity of βII-spectrin. ( C-F ) Stitched SIM images showing the distributions of endogenous endocytic pits, clathrin ( C ), Cav1( D ), Flot1 ( E ), or EndoA2 ( F ) in wild-type (WT) neurons. Endocytic pits are shown in green, tau, a distal axon marker, is shown in magenta. Scale bar: 10 µm. Bottom: Enlarged SIM images of the boxed regions on the top, corresponding to distal axon compartments. Scale bar: 2 µm. ( G) Left: SIM images of neurofascin (magenta) and endogenous endocytic pits (green; clathrin, Cav1, Flot1, or EndoA2) at the AIS of WT neurons. Right: Same as Left, but in βII-spectrin knockdown (KD) neurons. Scale bars: 2 µm. ( H ) Boxplots showing the area fraction of endogenous endocytic pits at the AIS of WT and βII-spectrin KD neurons. ( I) Left: SIM images of MAP2 (magenta) and endogenous endocytic pits (green) in dendrites of WT neurons (DIV 7). Right: Same as Left, but in βII-spectrin knockdown (KD) neurons (DIV 7). Scale bars: 2 µm. ( J ) Boxplots showing the area fraction of endogenous endocytic pits in dendrites of WT and βII-spectrin KD neurons (DIV 7). Boxplots show the median and boundaries (first and third quartile); Whiskers denote 1.5 times the interquartile range of the box. p -values calculated with two-sided unpaired Student’s t -test.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Fluorescence, Transduction, Expressing, Control, shRNA, Marker, Knockdown

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

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 ) Same as in (B) but showing spatial relationships with the periodic adducin lattice in axons. The MPS wa 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 clathrin-coated pits (CCPs) in the boxed regions. Scale bars: 10 µm (left), 5 µm (middle), 200 nm (right). ( E ) Pearson correlation coefficients 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-I ) Same as in ( D–F ), but for βII-spectrin (magenta) and exogenously expressed Cav1 (green). ( J-L ) Same as in ( D–F) , but for adducin (magenta) and exogenously expressed Flot1 (green). ( M-O ) Same as in ( D–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 immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Immunostaining, Generated

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: ( A ) Left: Representative dual-color STORM images of the MPS (magenta) and surface-localized endocytic pits (green) in axons. Endogenous clathrin and exogenously expressed Cav1 were co-stained with βII-spectrin, while exogenously expressed Flot1 and exogenously expressed EndoA2 were co-stained with adducin. The y/z view corresponds to the white-boxed region in the x/y view. Right: Same as Left, but for endocytic pits internalized within the axonal cytoplasm. Scale bars: 200 nm. (B ) Average percentage of surface-localized endocytic pits in axons. Quantification includes only endogenous clathrin, and both endogenous and exogenously expressed Cav1, Flot1, and EndoA2. ( C ) Boxplots showing the diameters of surface-localized and internalized endocytic pits in axons. Quantification includes the same categories as in ( B ). ( D ) Average percentage of surface-localized CCPs in axons and dendrites under DMSO (control) or dyngo-4a treatment. ( E) Boxplots showing the diameters of membrane-localized and internalized CPPs in axons and dendrites under DMSO or dyngo-4a treatment. ( F-H) Same as ( A-C) , but for dendrites. Data are presented as mean ± s.e.m. ( n = 3 biological replicates per condition). Boxplots show the median and boundaries (first and third quartile); Whiskers denote 1.5 times the interquartile range of the box. p -values calculated with two-sided unpaired Student’s t -test.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Staining, Control, Membrane

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: ( A ) Dual-color STORM images of βII-spectrin (magenta) and wheat germ agglutinin (WGA, green) in axons. Scale bars: 10 µm (left), 5 µm (middle), 200 nm (right). ( B ) Pearson correlation coefficient between βII-spectrin and WGA under experimental and randomized conditions. ( C, D ) Same as ( A, B ), but for βII-spectrin (magenta) and cholera toxin subunit B (CTB, green). ( E ) Left: Dual-color STORM images of βII-spectrin (magenta) and exogenously expressed Flot1 (green) in axons. Right: Magnified views of Class I and Class II Flot1-pits in the boxed regions. Scale bars: 10 µm (left), 5 µm (middle), 200 nm (right). ( F ) Pearson correlation coefficients between βII-spectrin and exogenously expressed Flot1 under experimental and randomized conditions. ( G ) Left: Averaged dual-color STORM images of βII-spectrin (magenta) and exogenously expressed Flot1 (green), generated by aligning individual STORM images to the centers of Flot1-pits. Right: Radial intensity profiles of the averaged images shown on th left. Scale bar: 100 nm. ( H-J ) Same as in ( E–G ), but for βII-spectrin (magenta) and exogenously expressed EndoA2 (green). ( K ) Percentages of Class I and Class II pits for exogenously expressed Flot1 and exogenously expressed EndoA2 in axons. Data are presented as mean ± s.e.m. ( n = 3 biological replicates per condition). Boxplots show the median and boundaries (first and third quartile); Whisker denote 1.5 times the interquartile range of the box. p -values calculated with two-sided paired Student’s t -test.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Generated, Whisker Assay

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: ( A ) Left: Dual-color STORM images of βII-spectrin (magenta) and endogenous Cav1 (green) in axons. Right: Magnified views of Class I and Class II Cav1-pits in the boxed regions. Scale bars: 10 µm (left), 5 µm (middle), 200 nm (right). ( B ) Pearson correlation coefficients between βII-spectrin and endogenous Cav1 under experimental and randomized conditions. ( C, D ) Same as ( A, B ), but for adducin (magenta) and endogenous Flot 1 (green). (E, F ) Same as ( A, B ), but for adducin (magenta) and endogenous EndoA2 (green). ( G) Percentages of Class I and Class II pits for endogenous Cav1, endogenous Flot1 and endogenous EndoA2 in axons. ( H ) Left: Dual-color STORM images of βIII-spectrin (magenta) and endogenous Cav1 (green) in dendrites. Right: Representative enlarged images of Class I and Class II Cav1-pits from the boxed regions. Scale bars: 10 µm (left), 5 µm (middle), 200 nm (right). ( I ) Pearson correlation coefficients between βIII-spectrin and endogenous Cav1 under experimental and randomized conditions. ( J, K ) Same as in ( H, I ), but for adducin (magenta) and endogenous Flot1 (green) in dendrites. ( L, M ) Same as in ( H, I ), but for adducin (magenta) and endogenous EndoA2 (green) in dendrites. ( N) Percentages of Class I and Class II pits for endogenous Cav1, endogenous Flot1 and endogenous EndoA2 in dendrites. Data are presented as mean ± s.e.m. ( n = 3 biological replicates per condition). Boxplots show the median and boundaries (first and third quartile); Whiskers denote 1.5 times the interquartile range of the box. p -values calculated with two-sided paired Student’s t -test.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques:

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

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 type 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 clathrin-coated pits (CCPs) in the boxed regions. Scale bars: 10 µm (left), 5 µm (middle), 200 nm (right). (D ) Pearson correlation coefficients 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-H ) Same as in ( C–E) , but for adducin (magenta) and exogenously expressed Cav1 (green). (I-K) Same as in ( C–E ), but for adducin (magenta) and exogenously expressed Flot1 (green). ( L-N ) Same as in ( C–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 immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Immunostaining, Generated

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: ( A ) Widefield epi-fluorescence images of clathrin heavy chain (CHC) in neurons transduced with adenoviruses expressing either scrambled (control) shRNA or CHC shRNA. Scale bars: 25 µm. ( B ) Boxplots showing the normalized fluorescence intensity of CHC. ( C ) Confocal fluorescence images of MAP2 (magenta) and internalized CF568-transferrin (green) in somatodendritic region of CHC KD neurons pretreated with DMSO followed by CF568-transferrin treatment for 40 minutes, and WT neurons pretreated with DMSO, dyngo-4a, nystatin or EIPA for 30 minutes, followed by CF568-transferrin treatment for 40 minutes. Scale bars: 10 µm. ( D ) Boxplots of CF568-transferrin endocytosis in somatodendritic regions of neurons, quantified by the area fraction of transferrin endosomes. ( E ) Dual-color SIM images of internalized CF568-transferin (green) and endocytic pits (magenta; clathrin, Cav1, Flot1 or EndoA2) in dendrites of WT neurons treated with CF568-transferrin for 30 minutes. Regions of strong colocalization are indicated by white arrows. Scale bars: 2 µm. ( F ) Pearson correlation coefficients between internalized CF568-transferrin and endocytic pits. ( G ) Widefield epi-fluorescence images of surface-localized transferrin receptor (TfR) in WT and βII-spectrin KD neurons without ligand stimulation. Surface-localized TfR was immunostained with anti-TfR antibody following cell fixation without Triton permeabilization. Scale bars: 25 µm. ( H) Boxplots showing the normalized fluorescence intensity of surface TfR. ( I ) Widefield epi-fluorescence images of CTB (magenta) and internalized Dil-LDL (green) in somatodendritic region of CHC KD neurons pretreated with DMSO, followed by treatment with Dil-LDL for 40 minutes, and WT neurons pretreated with DMSO, dyngo-4a, nystatin or EIPA for 30 minutes, followed by treatment with Dil-LDL for 40 minutes. Scale bars: 10 µm. ( J ) Boxplots of Dil-LDL endocytosis in somatodendritic regions of neurons, quantified by the area fraction of LDL endosomes. ( K ) Left: Widefield epi-fluorescence images of internalized Dil-LDL in somatodendritic regions of WT neurons treated with Dil-LDL for 10, 20 and 40 minutes. Right: Same as Left, but in βII-spectrin KD neurons. Scale bars: 10 µm. ( L ) Time course of LDL endocytosis in the somatodendritic regions of WT and βII-spectrin KD neurons, quantified by the area fraction of LDL endosomes. Solid lines represent single-exponential fits. Data are presented as mean ± s.e.m. ( M ) Widefield epi-fluorescence images of surface-localized LDLR in WT and βII-spectrin KD neurons without ligand stimulation. Surface-localized LDLR was immunostained with anti-LDLR antibody following cell fixation without Triton permeabilization. Scale bars: 25 µm. ( N ) Boxplots showing the normalized fluorescence intensity of surface LDLR. Boxplots show the median and boundaries (first and third quartile); Whiskers denote 1.5 times the interquartile range of the box. p -values calculated with two-sided unpaired Student’s t -test.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Fluorescence, Transduction, Expressing, Control, shRNA

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

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 minutes. Scale bars: 10 µm. ( B ) Time course of CF568-transferrin endocytosis in somatodendriti 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 somatodendriti region of WT or βII-spectrin KD neurons overexpressing HA-mGluR5a and treated with anti-HA antibody for 5, 10, and 20 minutes. Scale bars: 10 µm. ( D ) Time course of Cav1-mediated HA-mGluR5 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 th data. ( E ) SIM images of internalized NCAM1 (green) and endogenous endoA2 (magenta) in axonal (top) and somatodendritic region (bottom) of WT or βII-spectrin KD neurons treated with anti-NCAM1 antibody for 30 minutes. 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 immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Fluorescence

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: ( A ) Dual-color SIM images of internalized HA-mGluR5a (green) and endocytic pits (magenta; clathrin, Cav1, Flot1 or EndoA2) in dendrites of WT neurons overexpressing HA-mGluR5a treated with anti-HA antibody for 30 minutes. Regions of strong colocalization are indicated by white arrows. Scale bars: 2 µm. ( B ) Pearson correlation coefficients between internalized HA-mGluR5a and endocytic pits. ( C ) Confocal fluorescence images of MAP2 (magenta) and internalized HA-mGluR5a (green) in the somatodendriti regions of CHC KD neurons overexpressing HA-mGluR5a pretreated with DMSO, followed by treatment with anti-HA antibody for 40 minutes, and WT neurons overexpressing HA-mGluR5a pretreated with DMSO, dyngo-4a, nystatin or EIPA for 30 minutes, followed by treatment with anti-HA antibody for 40 minutes. Scale bars: 10 µm. ( D ) Boxplots of HA-mGluR5a endocytosis in somatodendritic regions of neurons, quantified by the area fraction of HA-mGluR5a endosomes. ( E ) Widefield epi-fluorescence images of surface-localized HA-mGluR5a in WT and βII-spectrin KD neurons without ligand stimulation. Surface-localized HA-mGluR5a was immunostained with anti-HA antibody following cell fixation without Triton permeabilization. Scale bars: 25 µm. ( F ) Boxplots showing the normalized fluorescence intensity of surface HA-mGluR5a. ( G ) Dual-color SIM images of internalized NCAM1 (green) and endocytic pits (magenta; clathrin, Cav1, Flot1 or EndoA2) in axons of WT neurons treated with anti-NCAM1 antibody for 30 minutes. Regions of strong colocalization are indicated by white arrows. Scale bars, 2 µm. ( H ) Pearson correlation coefficients between internalized NCAM1 and endocytic pits. ( I ) Confocal fluorescence images of surface NCAM1 and internalized NCAM1 in CHC KD neurons pretreated with DMSO, followed by treatment with anti-NCAM1 antibody for 40 minutes, and WT neurons pretreated with DMSO, dyngo-4a, nystatin or EIPA for 30 minutes, followed by treatment with anti-NCAM1 antibody for 40 minutes. Scale bars: 10 µm. ( J ) Boxplots of NCAM1 endocytosis in neurons, quantified by the area fraction of NCAM1 endosomes. ( K ) Widefield epi-fluorescence images of surface-localized NCAM1 in WT and βII-spectrin KD neurons without ligand stimulation. Surface-localized NCAM1 was immunostained with anti-NCAM1 antibody following cell fixation without Triton permeabilization. Scale bars: 15 µm. ( L ) Boxplots showing the normalized fluorescence intensity of surface NCAM1. Boxplots show the median and boundaries (first and third quartile); Whiskers denote 1.5 times the interquartile range of the box. p -values calculated with two-sided unpaired Student’s t -test.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Fluorescence

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: ( A ) Schematic illustrating ligand-induced ERK activation via three major endocytic pathways: CME of transferrin receptor, 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: 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 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 immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Activation Assay, Fluorescence, Immunostaining, Disruption

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: ( A ) Left: Immunoblot detection of αII-spectrin cleavage in WT neurons treated with anti-NCAM1 antibody or CF568-transferrin for 60 minutes. For inhibitor conditions, neurons were pretreated with MDL or VAD prior to ligand-induced endocytosis. Right: Same as Left, but for βII-spectrin cleavage. ( B ) Left: Immunoblot detection of αII-spectrin cleavage in neurons overexpressing HA-mGluR5a treated with anti-HA antibody for 60 minutes. For inhibitor conditions, neurons were pretreated with MDL or VAD prior to ligand-induced endocytosis. Right: Same as Left, but for βII-spectrin cleavage. ( C ) Left: immunoblot detection of αII-spectrin cleavage in neurons overexpressing SEP-APP treated with GFP nanobody for 60 minutes. For inhibitor conditions, neurons were pretreated with MDL or VAD prior to ligand-induced endocytosis. Right: Same as Left, but for βII-spectrin cleavage.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Western Blot

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: ( A ) Boxplots of the area fraction of endogenous and overexpressed endocytic pits in axonal and dendritic neuronal compartments, quantified from the STORM data shown in and , and Figs. S5. ( B ) Widefield epi-fluorescence images of MAP2 (magenta) and βII-spectrin (green) in WT neurons, and neurons overexpressing Basp1, Cav1, Flot1, and EndoA2. Scale bars, 25 µm. ( C ) Boxplots of normalized fluorescence intensity of βII-spectrin in axons and dendrites of neurons. ( D ) 3D STORM images of immunostained βII-spectrin in axons and dendrites of WT neurons, and neurons overexpressing Basp1, Cav1, Flot1, and EndoA2. Scale bars, 1 µm. Color scale bar represents the z-coordinate information. ( E ) Averaged 1D autocorrelation amplitude of βII-spectrin in axons and dendrites of neurons. Boxplots show the median and boundaries (first and third quartile); Whiskers denote 1.5 times the interquartile range of the box. p -values calculated with two-sided unpaired Student’s t -test.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Fluorescence

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: A β 42 accumulation. ( 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: Same as left, but treated with GFP nanobody. Right: Same as middle, but with dyngo-4a pre-incubation 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: Same as 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: Same as 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 immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Fluorescence, Incubation, Activation Assay

Journal: bioRxiv

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

doi: 10.64898/2025.12.12.693977

Figure Lengend Snippet: ( A ) Confocal fluorescence images of CTB (magenta) and internalized SEP-APP (green) in somatodendritic regions of CHC KD neuron overexpressing SEP-APP pretreated with DMSO, followed by GFP nanobody treatment for 40 minutes, and WT neurons overexpressing SEP-APP pretreated with DMSO, dyngo-4a, nystatin, or EIPA for 30 minutes, followed by GFP nanobody treatment for 40 minutes. Scale bars: 10 µm. ( B ) Boxplots of SEP-APP endocytosis in somatodendritic regions of neurons, quantified by the area fraction of SEP-APP endosomes. ( C ) Left: Confocal fluorescence images of CTB (magenta) and internalized SEP-APP in somatodendritic region of WT neurons overexpressing SEP-APP treated with GFP nanobody for 5, 10, and 20 minutes. Right: Same as Left, but in βII-spectrin KD neurons. Scale bars: 10 µm. ( D ) Time course of APP endocytosis in the somatodendritic regions of WT and βII-spectrin KD neurons, quantified by the area fraction of APP endosomes. Solid lines represent single-exponential fits to the data. Data are presented as mean ± s.e.m. ( E ) Widefield epi-fluorescence images of surface-localized SEP-APP in WT and βII-spectrin KD neurons without ligand stimulation. Surface-localized SEP-APP was immunostained with anti-GFP antibody following cell fixation without Triton permeabilization. Scale bars: 25 µm. ( F ) Boxplots showing the normalized fluorescence intensity of surface SEP-APP. Boxplots show the median and boundaries (first and third quartile); Whiskers denote 1.5 times the interquartile range of the box. p -values calculated with two-sided unpaired Student’s t -test. ( G ) Widefield epi-fluorescence images of total SEP-APP in WT and βII-spectrin KD neurons without ligand stimulation. Total SEP-APP was immunostained with anti-GFP antibody following cell fixation and Triton permeabilization. Scale bars: 25 µm. ( H ) Boxplots showing the normalized fluorescence intensity of total SEP-APP. Boxplots show th median and boundaries (first and third quartile); Whiskers denote 1.5 times the interquartile range of the box. p -values calculated with two-sided unpaired Student’s t -test.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-tau antibody 1:500 dilution for immunofluorescence (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-clathrin heavy chain antibody 1:500 dilution for IF (Abcam, ab21679), rabbit anti-caveolin-1 antibody 1:400 dilution for IF (Cell signaling, 3238S), mouse anti-flotillin-1 antibody 1:100 dilution for IF (BD Biosciences, 610820), mouse anti-endophilinA2 antibody 1:100 dilution for IF (Santa Cruz, sc-365704), mouse anti-αII-spectrin (EnCor Biotechnology, MCA-3D7), mouse anti-βII spectrin antibody 1:200 dilution for IF (Santa Cruz, 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, A11122), goat anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, sc-9660), mouse anti-βIII spectrin antibody 1:100 dilution for IF (Santa Cruz, 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-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), rabbit anti-cleaved caspase-3 (Asp175) antibody 1:400 dilution for IF (Cell Signaling Technology, 9661).

Techniques: Fluorescence

Journal: The Journal of Neuroscience

Article Title: Ankyrin-R Links Kv3.3 to the Spectrin Cytoskeleton and Is Required for Purkinje Neuron Survival

doi: 10.1523/jneurosci.1132-21.2021

Figure Lengend Snippet: Figure 7. AnkR-spectrin interactions in the cerebellum. A, Immunostaining of sagittal cerebellum for AnkR (polyclonal, red) and b 1 spectrin (monoclonal, green). Scale bars: 20mm. B, Immunostaining of sagittal cerebellum for AnkR (monoclonal, red) and b 3 spectrin (polyclonal, green). Scale bars: 20mm. C, Immunostaining of sagittal cerebellum for b 1 spectrin in three- month Ank1F/F and Ank1F/F;Nestin-Cre. Scale bar: 50 mm. D, Immunostaining of sagittal cerebellum for b 3 spectrin in three-month Ank1F/F and Ank1F/F;Nestin-Cre. Scale bar: 50mm. E, Quantification of b 1 spectrin fluorescence intensity in the granule cell layer (GCL) of three-month Ank1F/F (N = 3; 686 6.1 AU) and Ank1F/F;Nestin-Cre (N = 3; 61 6 3.8 AU). F, Quantification of b 1 spectrin fluorescence intensity at the Purkinje neuron soma by measuring the fluorescence intensity of a line across the PCL and dividing by length in three-month sagittal cerebellum of Ank1F/F (N = 3; 396 2.4 AU) and Ank1F/F;Nestin-Cre (N = 3; 38 6 1.6 AU). Error bars indicate mean 6 SEM. G, Quantification of b 3 spectrin fluorescence intensity in the molecular layer (ML) of three-month Ank1F/F (N = 3; 63 6 2.2 AU) and Ank1F/F;Nestin-Cre (N = 3; 666 3.1 AU). H, Quantification of b 3 spectrin fluorescence intensity at the Purkinje neuron soma by meas- uring the fluorescence intensity of a line across the PCL and dividing by length in three-month sagittal cerebellum of Ank1F/F (N = 3; 52 6 4.4 AU) and Ank1F/F;Nestin-Cre (N = 3; 406 3.9 AU). Error bars indicate mean 6 SEM. I, Immunoblots of b 3 spectrin, AnkR, and IgG immunoprecipitation (IP) reactions using antibodies against AnkR and b 3 spectrin.

Article Snippet: The primary antibodies used here include: mouse monoclonal antibodies against AnkR (IF: 1:250, UC Davis/NIH NeuroMab Facility catalog #75- 380, RRID:AB_2491109), b 1 spectrin (IF: 1:250, UC Davis/NIH NeuroMab Facility catalog #73-374, RRID:AB_2315814), AnkG (IF: 1:500, UC Davis/NIH NeuroMab Facility catalog #73-146, RRID:AB_ 10697718), Kv3.3 (IF: 1:500, Antibodies-Online catalog #ABIN572016, RRID:AB_10782137), Calbindin (IF: 1:250, UC Davis/NIH NeuroMab Facility catalog #L109/57, RRID:AB_2877197), and Flag-tag or DDDDK-tag (WB: 1:1000, MBL International catalog #M185-3L, RRID: AB_11123930), rabbit polyclonal antibodies against AnkR (IF: 1:500, WB: 1:1000, RRID:AB_2833096), Ank1 (IF: 1:500, WB: 1:1000, Thermo Fisher Scientific catalog #PA5-63372, RRID:AB_2638015), AnkB (IF: 1:250, Santa Cruz Biotechnology catalog #sc-28560, RRID:AB_2242828), b 3 spectrin (IF: 1:500, WB: 1:1000, Novus catalog #NB110-58346, RRID:AB_877723), Calbindin D-28k (IF: 1:500, Swant catalog #CB38, RRID:AB_10000340), Kv3.3 (IF: 1:500, WB: 1:1000, Alomone Labs catalog #APC-102, RRID:AB_2040170), and GFP (WB: 1:1000, Thermo Fisher Scientific, catalog #A-11122, RRID:AB_221569).

Techniques: Immunostaining, Fluorescence, Western Blot, Immunoprecipitation

Journal: Frontiers in Cellular Neuroscience

Article Title: Prevention of noise-induced hearing loss by calpain inhibitor MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathway

doi: 10.3389/fncel.2023.1199656

Figure Lengend Snippet: MDL-28170 treatment inhibits noise-induced α-fodrin activation in the cochlear tissues. (A) Representative immunoblots from whole cochlear homogenates revealed 240 kDa α-fodrin and the cleaved 150 kDa fragment 3 h after noise exposure. GAPDH (37 kDa) was used as the sample loading control. (B,C) Semi-quantifications of each band density show that the ratio of cleaved to full α-fodrin increased with noise exposure and MDL treatment prevented such an increase (B) , while the cleaved band density divided by GAPDH was not different among the three groups (C) . Data are presented as means + SD; n = 3 in each group with one mouse (two cochleae) per sample, * p < 0.05. (D) Representative images were taken from the basal turn of cochlear epithelia at the time point 3 h after noise exposure. MDL treatment alone showed similar immunolabeling intensity for cleaved α-fodrin (red) in OHCs as the DMSO-treated group. Noise exposure significantly increased cleaved α-fodrin in OHCs, whereas MDL treatment prevented such effects. Phalloidin (green) was a counterstain for visualization of OHCs. The enlarged OHCs allow for better visualization of the immunolabeling. Scale bar = 10 μm. (E) Confocal images used to compare fluorescence intensity by semi-quantification of immunolabeling for cleaved α-fodrin in OHCs were acquired under the same settings and the same processing enhancement for the captured images was used. The person analyzing images was blind to the treatment groups. It confirmed a significant increase after noise exposure, whereas MDL treatment prevented such effects. Data are presented as means + SD; the n is indicated in the bar for each condition with use of one cochlea per mouse. ** p < 0.01, **** p < 0.0001.

Article Snippet: The specimens were first permeabilized in 2% Triton X-100 solution and then blocked with 10% normal goat serum for 30 min each step at room temperature, followed by incubation with primary antibodies: polyclonal rabbit anti-Calpain I (Abcam, Cambridge, UK, #ab39170, 1:50), polyclonal rabbit anti-Calpain II (Abcam #ab39165, 1:50), cleaved α-fodrin (Cell Signaling Technology, Danvers, MA, USA, #2121, 1:50), monoclonal rabbit anti-PI3K, p85α (Millipore, Burlington, MA, USA, #04-403, 1:50), monoclonal rabbit anti-p-Akt (Ser473) (Cell Signaling Technology #4060, 1:50) at 4°C for overnights (24–48 h).

Techniques: Activation Assay, Western Blot, Control, Immunolabeling, Fluorescence