SUPPLEMENTARY FIGURE LEGENDS Supplemental FIG. 1. Localization of myosin Vb in cultured neurons varies with maturation stage. A and B, localization of myosin Vb in cultured hippocampal neurons. A, in DIV 7 neurons myosin Vb shows a somato-dendritic distribution with punctate staining in the dendritic shaft and enrichment in the perinuclear region (white arrowheads). B, in DIV 28 neurons, myosin Vb is enriched in the perinuclear region (white arrowheads), and is found in dendritic spines (black arrowheads). C, neurons were labeled with antibodies against myosin Vb and the presynaptic marker synaptophysin (Syn). C, in DIV 7 (left panels) and DIV 14 neurons (middle panels), myosin Vb was mainly absent from syn-positive sites (arrowheads). In DIV 28 neurons (right panels), myosin Vb was detected at sites positive for synaptophysin (arrowheads). D, distribution of GluR1 also varies with maturation stage. Double labeling of hippocampal neurons with GluR1 and Syn shows that GluR1 is weakly clustered and mainly lacking form synaptophysin-positive puncta at DIV 7 (left panels; arrowheads). At DIV 14, GluR1 is present in clusters that colocalize with synaptophysin (right panels; arrowheads). Scale bars, 10 µm (A and B); and 1 µm (C and D). Supplemental FIG. 2. Overexpression of mutant myosin Vb does not alter localization of Golgi and ER resident proteins. A and D, localization of endogenous Golgi resident protein GM130 and the endoplasmic reticulum (ER) marker calnexin in DIV 12 neurons. B, relative distribution of endogenous GluR1 and GM130. C and E, neurons (DIV 9) were transfected with the tail region of myosin Vb (MyoVb CT) fused to GFP and stained at DIV 12 for GluR1 and GM130, or calnexin. MyoVb CT expression resulted in enhanced GluR1 accumulation in a perinuclear compartment that did not colocalize with GM130 (C) (arrowheads). Also, MyoVb CT did not alter the distribution of GM130 (C) or calnexin (E), suggesting an intact Golgi apparatus and ER compartment exist in these cells. At least 20 cells per group were analyzed. Scale bar, 5 µm. 15
Supplemental FIG. 3. The effects of mutant myosin Vb on GluR1 trafficking is neuron-specific. A-H, COS-7 or PC12 cells were transfected with the tail region of myosin Vb (MyoVb CT) fused to GFP with or without GluR1. Cells were fixed 48 hours later and stained with Rab11 or GluR1 antibodies. A, C, E, and G show representative images of endogenous Rab11 (Rab endo) or transfected GluR1 distribution in COS-7 and PC12 cells in the absence of MyoVb CT. B and D, in both COS-7 cells (B) and PC12 cells (D), MyoVb CT expression induced Rab11 redistribution in a perinuclear compartment (arrowheads). F and H, MyoVb CT did not trigger perinuclear accumulation of GluR1 in either COS-7 (F) or PC12 cells (H) (arrowheads). Scale bars: 5 µm. Supplemental FIG. 4. Myosin Vb does not directly associate with GluR1. HEK-293 cells were transfected with either the full-length (GFP MyoVb FL) or the C-terminal tail domain of myosin Vb (GFP MyoVb CT) fused to GFP, and either with HA-tagged GluR1 or Flag-tagged BERP. 24 hours later, cells were harvested and GFP antibodies were used to immunoprecipitates GFP-fused myosin Vb constructs. Left panels in A and B show the expression of the different constructs in starting lysates. Right panels in A and B show the co-immunoprecipitates using GFP, HA, or FLAG antibodies. Both GFP MyoVb CT (A) and GFP MyoVb FL (B) co-immunoprecipitated BERP, but not GluR1 (n=2 independent experiments). Supplemental FIG. 5. Colocalization of endogenous myosin Vb and Rab11 in hippocampal neurons. Staining of myosin Vb and Rab11 shows a partial overlap in the perinuclear region and in dendrites. Enlarged boxed area is shown below. This analysis reveals two populations of myosin Vb- and Rab11- positive structures. White arrows point to examples of Rab11-positive structures surrounded by myosin Vb- positive puncta. Black arrows point to examples of overlapping Rab11- and myosin Vb positive puncta. Scale bars, 10 µm (full view image) and 1 µm (enlarged panels). Supplemental FIG. 6. Effects of myosin Vb mutant lacking Rab11 binding region on endogenous Rab11 distribution. COS-7 cells or neurons were transfected with the tail region of myosin Vb that lacks the Rab11 binding region (MyoVb CT Rab11) fused to GFP. Cells were fixed 24 to 72 hrs later, and stained for endogenous Rab11. A and C, representative images of endogenous Rab11 (Rab endo) distribution in untransfected COS-7 and neuronal cells. Expression of MyoVb CT Rab11 failed to redistribute endogenous Rab11 in COS-7 cells (B) (arrowheads) or in neurons (D). Scale bars: 5 µm. Supplemental FIG. 7. Expression of a mutant form of Rab11 alters myosin Vb and GluR1 distribution in neurons. A-E, cultured neurons (DIV 9) were transfected with a dominant-negative form of Rab11 (Rab11-S25N) or MyoVb CT. At DIV 13, neurons were stained for myosin Vb (MyoVb endo) and GluR1. A and B, representative images of untransfected cells stained for endogenous GluR1 (A) and myosin Vb (B). Expression of Rab11-S25N results in redistribution of myosin Vb (C) and GluR1 in the perinuclear region (D) (arrowheads). However the effect of Rab11-S25N on GluR1 in the perinuclear region was less dramatic when compared to MyoVb CT. (E) (arrowheads). Scale bar: 5 µm. 16
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