Journal: The Journal of Cell Biology

Article Title: KIF13A drives AMPA receptor synaptic delivery for long-term potentiation via endosomal remodeling

doi: 10.1083/jcb.202003183

Figure Lengend Snippet: Activity-dependent transport of AMPARs is mediated by KIF13A. (A) Left: Representative images of hippocampal neuronal cultures after 7 d in vitro, infected either with a control (top row) or the shKIF13A-expressing lentivirus (bottom row; red channel) for 7 d. Cultures were fixed and stained for surface GluA1 (nonpermeabilizing conditions, magenta) and total GluA1 (after permeabilization, cyan). Scale bars, 20 µm. Right: Quantifications of fluorescence intensity, measured separately in the somatic and dendritic compartments for both surface (magenta) and total (cyan) GluA1 channels. Plots show mean fluorescence intensity for the individual experiments and mean ± SEM for each pool of receptors in the different compartments. n = 32 for both control neurons and shKIF13A-expressing neurons from six independent experiments. (B) Left: Representative images for dissociated hippocampal neurons infected with lentivirus, as described for A. Immunocytochemistry was performed after baseline or 15 min of cLTP induction. Scale bars, 2.5 µm. Dashed lines outline neuron morphology (from mask of the mCherry channel). Arrowheads indicate individual spines used for the quantification of both surface (left) and total (right) GluA1 immunostaining. Plots on the right show cumulative probability distributions of fluorescence intensity from individual spines for each condition described above. Gray traces represent spines from neurons infected with a control lentivirus, and red traces show spines from neurons infected with a lentivirus expressing shKIF13A. Dashed lines represent baseline values, while continuous lines represent values after cLTP induction. P values compare cLTP versus baseline condition (black for control and red for shKIF13A), according to the Kolmogorov–Smirnov test (left graph, surface GluA1) or control versus shKIF13A in baseline condition (right graph, total GluA1). n = 1,229 spines (24 neurons) for baseline conditions and n = 1,183 spines (26 neurons) for cLTP conditions, from control neurons. n = 750 spines (25 neurons) for baseline conditions and n = 608 spines (24 neurons) for cLTP conditions from shKIF13A-expressing neurons, analyzed from three independent experiments. (C) Quantifications of fluorescence intensity in the adjacent dendritic shaft from each condition described in B. Plots show mean fluorescence intensity after cLTP normalized to mean baseline intensity for each condition for the individual experiments and mean ± SEM for each pool of receptors (surface GluA1, magenta; total GluA1, cyan). n = 26 control neurons and n = 24 shKIF13A-expressing neurons from three independent experiments. (D) Left: Representative images of spine morphology from mCherry channel of the experiments described in B. Scale bars, 2.5 µm. Right, cumulative probability distribution of fluorescence intensity at spines over the value at the adjacent dendrite from the different conditions described for B. Dashed lines represent baseline values, while continuous lines represent values after cLTP induction. P values compare cLTP versus baseline condition (black for control and red for shKIF13A) according to the Kolmogorov–Smirnov test.

Article Snippet: Antibodies for immunoprecipitation were anti-GFP (4 µg/ml), anti-GluA1 (10 µg/ml; rabbit; AB1504; Merck Millipore), or anti–centaurin-α1 (5 µg/ml).

Techniques: Activity Assay, In Vitro, Infection, Expressing, Staining, Fluorescence, Immunocytochemistry, Immunostaining

Journal: The Journal of Cell Biology

Article Title: KIF13A drives AMPA receptor synaptic delivery for long-term potentiation via endosomal remodeling

doi: 10.1083/jcb.202003183

Figure Lengend Snippet: Protein interactions between GluA1, KIF13A and centaurin-α1 upon LTP induction. (A) GFP-immunoprecipitation from protein extracts of 7‒10 DIV organotypic hippocampal slices expressing GFP-KIF13A-ST protein or GFP as control. Extracts were obtained from slices maintained at basal conditions (aCSF) or from slices undergoing cLTP induction for different times (7.5 min, 15 min) or from slices recovered for 15 min following cLTP (Recovery), as indicated. Coimmunoprecipitated proteins (“Bound”) and input fractions were analyzed by Western blot using antibodies for GluA1 and centaurin-α1. IP, immunoprecipitation. Left, representative Western blots. Right, quantification of the amount of coprecipitated GluA1 normalized to baseline (dashed line). Dark symbols represent mean ± SEM; gray symbols represent individual values from each experiment. n = 5, 4, 5, and 4 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively. (B) Left: Similar to A, with the immunoprecipitation of endogenous GluA1. Nonimmune rabbit IgGs were used as immunoprecipitation control. GluA1, centaurin-α1, and KIF13A were detected by Western blot. Right: Quantification of the coprecipitated amount of KIF13A (green circles) and centaurin-α1 (purple circles). P values (P = 0.009 and P = 0.003) represent significant difference from baseline (Friedman test). n = 4, 4, 4, and 3 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of KIF13A coimmunoprecipitation. n = 5, 5, 5, and 4 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of centaurin-α1 coimmunoprecipitation. (C) Left: Similar to B, with the immunoprecipitation of endogenous centaurin-α1 and Western blot detection of endogenous KIF13A, GluA1, and centaurin-α1. Nonimmune goat IgGs were used as immunoprecipitation control. Right: Quantifications of the coprecipitated KIF13A (green circles) and GluA1 (black circles). P value represents significant difference from baseline (P = 0.009, Friedman test). n = 4, 4, 4, and 3 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of KIF13A coimmunoprecipitation. n = 5, 5, 5, and 4 experiments for baseline, 7.5-min cLTP, 15-min cLTP, and recovery, respectively, in the case of GluA1 coimmunoprecipitation.

Article Snippet: Antibodies for immunoprecipitation were anti-GFP (4 µg/ml), anti-GluA1 (10 µg/ml; rabbit; AB1504; Merck Millipore), or anti–centaurin-α1 (5 µg/ml).

Techniques: Immunoprecipitation, Expressing, Western Blot

Journal: The Journal of Cell Biology

Article Title: KIF13A drives AMPA receptor synaptic delivery for long-term potentiation via endosomal remodeling

doi: 10.1083/jcb.202003183

Figure Lengend Snippet: Immunoprecipitation of GFP-KIF13A-ST and effect of phosphatase inhibitors on centaurin-α1 coprecipitation. (A) GFP-immunoprecipitation from protein extracts of 7–10 DIV organotypic hippocampal slices expressing GFP-KIF13A-ST protein, or GFP as control, as described for of the main text. The immunoprecipitation of the recombinant protein was detected by Western blot with an anti-GFP antibody. GFP-KIF13A-ST appears as multiple bands, probably due to proteolytic degradation. Arrowhead indicates full-length recombinant protein; asterisks indicate light and heavy chains of the IgG used for immunoprecipitation. (B) Representative Western blots from experiments similar to of the main text but in the presence (+inhibitors) or absence (−inhibitors) of phosphatase inhibitors in the homogenization buffer. Nonimmune rabbit IgGs were used as immunoprecipitation control. GluA1, centaurin-α1, and pGluA1 (S845) were detected by Western blot. (C) Quantification of the amount of pGluA1 (S845) immunoprecipitated in both conditions (black circles, with phosphatase inhibitors; red squares, without phosphatase inhibitors). Darker lines represent the mean ± SEM, while lighter lines show individual values for each experiment. n = 4 experiments. (D) Quantification of the amount of centaurin-α1 protein coprecipitated in both conditions (black circles, with phosphatase inhibitors; red squares, without phosphatase inhibitors) and normalized to baseline coprecipitated protein. Darker lines represent the mean ± SEM, while lighter lines show individual values for each experiment. n = 4 experiments.

Article Snippet: Antibodies for immunoprecipitation were anti-GFP (4 µg/ml), anti-GluA1 (10 µg/ml; rabbit; AB1504; Merck Millipore), or anti–centaurin-α1 (5 µg/ml).

Techniques: Immunoprecipitation, Expressing, Recombinant, Western Blot, Homogenization