Supplemental Material

This article contains the following supporting material:

• Supplemental Figures 1-3 -
  Supplementary Figure 1: Rescue of directional persistence by UNC-104 constructs. Analogous to the directionality plot in Fig 3., persistence of movement was measured and four representative worms for each construct are shown in these polar plots.
  
  Supplementary Figure 2: Phenotypes of worms expressing a large deletion in the stalk domain(Δ654-1339 and Δ878-1339), or PH domain point mutations (RR1479/80AA, K1522A, and K1534A).
  The movement of the transgenic worms (scale bar, 4.8 mm), agar tracks (scale bar, 0.13 mm), measurement of worm velocities, directional persistence, and analysis of synaptotagmin staining in the ventral nerve cord (scale bar, 10 μm) were performed as described in the legends to Fig. 2-4. Cell body staining is indicated by the arrowheads. While the deletion and substitution affect different domains in UNC-104, both constructs UNC-104Δ654-1339 and UNC-104Δ878-1339 show a similar partial rescue in coordinated movement but slow velocity (see Table 1). Surprisingly, the PH point mutant K1522A does not affect PI(4,5)P2 binding (Fig. 5), but UNC-104K1522A-expressing animals display slower velocities of movement, although the coordination of movement seen in the agar tracks and directional persistence of movement appears to be similar to animals expressing wt UNC-104.
  Immunofluorescence staining of synaptotagmin reveals a retention of synaptic vesicles in the cell body (arrow head), although the fluorescent signal in the ventral nerve cord remains high compared to unc-104 null (Fig. 4). UNC-104RR1479/80AA and UNC-104K1534A-expressing animals show wt type phenotype as expected from the PI(4,5)P2 binding assay (Fig. 5). See text for detailed description of phenotypes.
  
  Supplementary Figure 3: Quantitation of presynaptic vesicle localization in cultured neurons expressing PH domain mutants.
  Immunofluorescence images of cultured neurons as shown in Fig. 4 were used to quantify the relative localization of synaptotagmin in the soma and neurite. A region of interest was drawn around the cell body or the neurite, the average pixel intensity in these regions was measured, and the ratio of soma:neurite intensities is shown for neurons from unc-104(e1265), wt, UNC-104ΔPH, UNC-104KK1463/4AA, and UNC-104R1496A. Numbers indicate mean values with standard deviations from 5-9 neurons from 3 independent experiments.
  
  
• video1.mov (10 MB) - UNC-104::GFP movement in neuronal cell culture. Primary neuronal cell culture from unc-104 null worms rescued with UNC-104::GFP wt construct. This movie shows rapid movement of fluorescent particles in the neurite. Scale bar, 10 μm, frame rate 10x real time.
  

Prepared by: Journals Production Manager