MyoVb is Concentrated in Dendritic Spines and Traffics with REs
(A) Schematic diagram of MyoVb and its association with REs via Rab11/Rab11-FIP2.
(B) Cultured hippocampal neurons (DIV19) were fixed and immunolabeled for endogenous MyoVb and PSD-95 (left). Recycling endosomes (REs) were labeled by Alexa 647-transferrin uptake (Alexa-Tf, middle) or TfR-mCherry expression (TfR-mCh, right), followed by MyoVb staining. Colocalization is indicated by yellow arrows. Red arrows indicate REs in dendritic shafts that are not associated with MyoVb. Dashed white lines indicate the dendritic outline determined by a GFP cell fill. Scale bar, 2 μm.
(C-D) Hippocampal neurons expressing TfR-mCh and GFP-MyoVb FL (C) or GFP-MyoVb ΔC (D) and were imaged over time. Red arrows in (C) indicate the coordinated movement of GFP-MyoVb FL and TfR-mCh into and out of the spine head in two examples (C1, C2). Green arrows in (D) indicate the lack of correlated movement between GFP-MyoVb ΔC and TfR-mCh. Time is indicated in min:sec. Scale bars, 2 μm. See Movies S1-S3.
(E) Means ± SEM of the correlation coefficient between GFP-MyoVb FL or GFP-MyoVb-ΔC and TfR-mCh over time. n = 31 spines from 3 neurons for each; p<0.001 for all time points.

Binding of MyoVb to Rab11-FIP2 is Induced by a Ca²⁺-Dependent Conformational Switch
(A) Ca²⁺ stimulates the actin-activated ATPase activity of purified MyoVb. Data represent means ± SD; n = 4. The calculated V_max and K_actin are 1.17 ± 0.21 s^-1 motor^-1 and 12.58 ± 6.81 μM in the absence of Ca²⁺, and 12.07 ± 1.21 s^-1motor^-1 and 7.11 ± 1.78 μM in the presence of 100 μM Ca²⁺.
(B) Ultracentrifugation sedimentation analysis of MyoVb under Ca²⁺-free or 100 μM Ca²⁺conditions. The sedimentation coefficient of MyoVb decreased from 14.34 S in the absence of Ca²⁺ to 10.82 S in the presence of 100 μM Ca²⁺.
(C) Model diagram indicating the proposed Ca²⁺-regulated conformational switch and cargo binding by MyoVb. Top panel, wildtype MyoVb is folded into an inactive structure at low Ca²⁺ levels. High Ca²⁺ switches MyoVb to an extended structure that binds to Rab11/Rab11-FIP2, resulting in membrane recruitment. Middle panel, a mutant of MyoVb lacking 15 amino acids required for Rab11-FIP2 binding (MyoVb-ΔRBD) is incapable of binding to REs at low or high Ca²⁺ levels. Bottom panel, deleting part of the coiled-coil region (MyoVb-CCtr) generates a constitutively unfolded MyoVb mutant that binds REs independent of Ca²⁺.
(D) Schematic diagram of MyoVb illustrating its domain architecture. Brackets indicate MyoVb domains that interact with other proteins. CaM, calmodulin. Numbers indicate amino acids.
(E) Brain lysates were subjected to immunoprecipitation (IP) with rabbit Rab11 or MyoVb antibodies and immunoblot (IB) analysis with the indicated antibodies. Pre-immune rabbit serum was used as a negative control.
(F) Co-IP was performed on lysates of 293T cells expressing V5-tagged MyoVb and GFP-tagged Rab11-FIP2 in different concentrations of free Ca²⁺. ms, control mouse serum.
(G) Data represent means ± SEM of the normalized fraction of MyoVb bound to GFP-Rab11-FIP2 at various levels of free Ca²⁺. *p<0.05 relative to 0 mM Ca²⁺.
(H) Co-IP was performed on lysates from 293T cells expressing the indicated constructs at 0 or 1 mM free Ca²⁺. A representative blot from three independent experiments is shown.

MyoVb Mediates RE Translocation into Spines Following LTP-Inducing Stimuli
(A-C) Time lapse images of TfR-mCh and GFP-MyoVb-WT (A), ΔRBD (B), or CCtr (C) in a dendritic spine. Two time points before and a six minute time lapse after glycine (black arrow) are shown. Red arrows indicate co-transport of GFP-MyoVb WT or CCtr and REs following glycine stimulation. Green arrows indicate the lack of correlated movement between GFP-MyoVb ΔRBD and RE. Time is indicated in min:sec. See Movies S6-S8. Scale bar, 2 μm.
(D-F) Cumulative probability plots of GFP-MyoVb WT (D), ΔRBD (E), and CCtr (F) fluorescence intensity on REs before and 2.5 min after glycine. The ΔRBD mutant blocks, while the CCtr mutant occludes the glycine-induced association of MyoVb with REs.
(G) Data indicate means ± SEM of the fold increase in TfR-mCh fluorescence intensity 10-15 min after glycine in spines initially lacking REs. **p<0.001; t-test.

Acute Blockade of LTP by Chemical Genetic Inhibition of MyoVb
(A) Schematic diagram illustrating the chemical genetic inhibition strategy using a transgenic mouse line expressing V5-tagged MyoVb-Y119G. See text for details.
(B) Acute inhibition of MyoVb motility disrupts LTP in hippocampal slices. Hippocampal slices were acutely prepared from MyoVb-WT (WT) or MyoVb-Y119G transgenic (Tg) mice, and AMPA receptor-mediated EPSCs were measured as illustrated in Figure S9A. Left panel, LTP was induced using high-frequency stimulation (arrow). PE-ADP or water was included in the recording pipette solution. n = 7, 7 and 12 animals for WT PE-ADP, Tg water and Tg PE-ADP respectively. Error bars indicate SEM. Right panel, representative EPSC traces (averages of six consecutive EPSCs) were evoked from CA1 pyramidal cells in each condition at the times indicated (1 and 2, corresponding to time points in the left panel graph). Scale: 200 pA, 10 msec.
(C) Schematic model for spine mobilization of AMPA receptor-containing recycling endosomes by Ca²⁺-regulated MyoVb during LTP. See text for details.

MyoVb is Recruited to REs that Move Into Spines Upon LTP Stimuli
(A) Hippocampal neurons expressing TfR-GFP (red) to label REs and mCherry as a cell fill were stained for MyoVb (green) either before (Unstim) or after glycine stimulation. White arrows indicate REs co-labeled with MyoVb. Scale bar, 2 μm.
(B) Means ± SEM of the fraction of MyoVb associated with REs before or after a glycine stimulus. * p<0.01 relative to control, t-test.
(C) Hippocampal neurons expressing GFP-MyoVb and TfR-mCh were imaged before and after glycine. Times are indicated in min:sec. Red arrows indicate the movement of GFP-MyoVb and REs into spines in two examples (C1, C2). Scale bars, 2 μm. See Movies S5-S6.
(D) Quantitative analysis of normalized GFP-MyoVb and TfR-mCh intensity in the spines shown in (C) over time. Black lines indicate periods of glycine application and double arrow-headed lines correspond to the time lapse after glycine shown in (C).
(E) Cumulative probability plot of the average intensity of GFP-MyoVb on individual REs before and 5 min after glycine.
(F) Quantitative analysis of GFP-MyoVb and RE movement into spines. The intensity at each data point is the average intensity across a population of spines from the corresponding time periods normalized by the averaged intensity 0-5 min before glycine (Gly) stimulation. *p<0.01 and #p<0.01 relative to GFP-MyoVb and TfR-mCh before glycine respectively.
(G) GFP-MyoVb spine translocation events (events/min/100 μm of dendrite) were measured after glycine (Gly) with or without APV, and normalized to basal conditions. Data represent means ± SEM. **p < 0.001 relative to Gly.
(H) Cumulative probability plot of the average intensity of GFP-MyoVb on individual REs before (Basal) and 5 min after stimulation (5 μM glutamate, 200 μM glycine, 3 min) in the presence (H1) or absence (H2, 0 Ca²⁺) of 2 mM extracellular Ca²⁺.

MyoVb Constitutively Traffics REs in Spines by Interacting with Rab11-FIP2
(A) Hippocampal neurons expressing GFP-MyoVb WT, ΔRBD, or CCtr were stained for endogenous Rab11-FIP2. The dendritic outline was determined by a mCherry cell fill. Colocalization is indicated by white arrows. Scale bar, 2 μm.
(B) Pixel-by-pixel fluorescence intensity correlations between GFP-MyoVb-WT, ΔRBD, or CCtr with endogenous Rab11-FIP2. Correlation coefficients (CC) are indicated. AFU, arbitrary fluorescence units.
(C) Hippocampal neurons expressing TfR-mCh, GFP, and V5-tagged MyoVb WT, ΔRBD or CCtr were stained for V5. The dendritic outline was determined by GFP. Scale bar, 2 μm.
(D) Quantitative analysis of the normalized fraction of MyoVb associated with REs. Data represent means ± SEM. **p <0.001 relative to WT; t-test.
(E) Quantitative analysis of RE mobility in spines over time. Neurons expressing TfR-mCh and GFP-MyoVb WT or mutants were imaged for 5 min at 37°C. The normalized TfR-mCh fluorescence (F/F₀) in 30 dendritic spines for each construct was plotted over time.
(F) Coefficient of variance (CV) of the normalized intensity of TfR-mCh in spines over a 5 min time lapse. Data represent means ± SEM of the CV. *p<0.01 relative to WT; t-test.

MyoVb is Required for LTP-Induced Exocytosis, AMPA Receptor Insertion, and Spine Growth
(A) Hippocampal neurons expressing MyoVb shRNA were transfected with TfR-SEP either alone (MyoVb RNAi) or together with RNAi-resistant MyoVb-WT (Rescue). Exocytosis from REs was monitored by TfR-SEP fluorescence before and 15 min after glycine. Fluorescence intensity is indicated as a pseudocolor scale. White and red arrows indicated spines with increased and unchanged/decreased TfR-SEP signal after glycine stimulation, respectively. Scale bars, 2 μm and 1 μm. See Movies S9-S11.
(B) Quantitative analysis of surface appearing TfR-SEP in spines. Data represent means ± SEM of the fractional increase in fluorescence intensity (ΔF/F₀). The black bar indicates the period of glycine application. Vec, vector control; Scram, scrambled shRNA.
(C) Data presented as in (B) for neurons expressing MyoVb shRNA along with RNAi-resistant versions of MyoVb-WT, MyoVb-CCtr, or MyoVb-ΔRBD. Note the ordinate scale difference between the graphs in (B) and (C).
(D) Hippocampal neurons expressing MyoVb shRNA were transfected with SEP-GluR1 either alone (MyoVb RNAi) or together with RNAi-resistant MyoVb-WT (Rescue). Stimulus-induced AMPA receptor insertion was monitored by increased SEP-GluR1 fluorescence before and 15 min after glycine stimulation. Yellow and red arrows indicate spines with increased and decreased SEP-GluR1 after glycine respectively. Yellow boxes indicated corresponding magnified regions. Scale bars, 2 μm.
(E) Quantitative analysis of SEP-GluR1 in spines after glycine stimulation. Data represent means ± SEM of the normalized SEP-GluR1 fluorescence intensity in spines (F/F₀). SEP-GluR1 F/F₀ in spines: Vec, 1.87 ± 0.10; Scram, 1.93 ± 0.13; RNAi, 1.07 ± 0.03; WT, 1.97 ± 0.22; CCtr, 1.99 ± 0.14; ΔRBD, 1.22 ± 0.05; Vec + APV (APV), 0.85 ± 0.02. n = 703, 900, 337, 661, 945, and 497 spines from 14, 14, 12, 16, 21, 14 and 10 cells for Vec, RNAi, Scram, WT, CCtr, ΔRBD and APV, respectively; **p<0.001 relative to Vec or Scram; t-test.
(F) Depleting endogenous MyoVb inhibits stimulus-induced spine growth. Yellow and red arrows indicate dendritic protrusions that appeared and disappear after glycine respectively. Scale bars, 2 μm.
(G) Quantitative analysis of spine formation following LTP stimulation. New protrusions per 100 μm of dendrite: Vec, 10.9 ± 2.4; Scram, 10.8 ± 2.1; RNAi, -4.0 ± 2.6; Rescue, 8.9 ± 2.2; Vec + APV, 2.2 ± 1.9. n = 12, 15, 27, 11 and 8 cells for Vec, Scram, RNAi, Rescue, and Vec + APV, respectively. **p <0.001, *p<0.05 relative to Vec or Scram; t-test.
(H) Quantitative analysis of spine growth following LTP stimulation. Normalized spine size S/S₀: Vec, 1.52 ± 0.07; Scram, 1.81 ± 0.15; RNAi, 0.88 ± 0.04; Rescue, 1.91 ± 0.17; Vec + APV, 0.84 ± 0.05. n = 104, 74, 241, 132, and 105 spines from 6, 9, 14, 8 and 5 cells for Vec, Scram, RNAi, Rescue and Vec + APV, respectively. **p <0.001 relative to Vec or Scram; t-test.