Figure 3.

Motility behaviour of G₃Mcs1-bound secretory vesicles. (A) Domain organization of Mcs1 in U. maydis. The molecule contains an MMD and is therefore considered to be a member of the fungal-specific class 17 myosins. Its tail region contains a CHS domain that was shown to participate in formation of the extracellular cell wall (Treitschke et al, 2010). Myosin-17 motors are therefore also considered to be class V CHSs. (B) Localization of G₃Mcs1 (Mcs1) in yeast-like cells that co-express a syntaxin-like plasma membrane protein (Sso1) fused to mCherry. Note few G₃Mcs1 signals in the plasma membrane of the mother cell (arrowhead). Bar represents micrometers. (C) Motility of G₃Mcs1 (green) in a photo-bleached bud (bleach). G₃Mcs1 travels to the apex where it often rests for several seconds (image series). Cell edge is given in blue. Time is given in seconds; bar represents micrometers. See also Supplementary Movie S2. (D) Kymograph showing bi-directional motility of G₃Mcs1 in a bud that was photo-bleached (bleached). Signals often pause before they move back to the cell centre (left). Note that an anterograde signal splits in two after reaching the apical region (arrowhead). Time is given in seconds; distance is given in micrometers. The image was contrast inverted. (E) Image series showing pausing and subsequent insertion of a G₃Mcs1 signal (green) into the plasma membrane, labelled with the syntaxin-like Sso1 fused to mCherry (red). After long pausing, G₃Mcs1-bound vesicle gets in close proximity and eventually fuses with the plasma membrane (arrowheads). Time is given in seconds; bar represents micrometers. See also Supplementary Movie S3. (F) Bar chart showing the behaviour of Mcs1-carrying vesicles at the growing bud. Most vesicles reach the plasma membrane and turn around (cortical turning). Some signals are turning without contact with the plasma membrane (cytoplasmic turning). A minority gets inserted into the plasma membrane (membrane insertion). Total observation time is 1089.8 s. Sample size n is given. See also Supplementary Movie S4. (G) Image series showing recovery of G₃Mcs1 signals after photo-bleaching at the growth region (upper image series) and in the mother cell (lower image series). The plasma membrane is labelled by the syntaxin mCherry-Sso1 (red). Pre: prior to photo-bleaching, 0′: immediately after the bleach, 5′: 5 min after photo-bleaching. Bar represents micrometers. (H) Bar chart showing the recovery of G₃Mcs1 signals in the plasma membrane after local photo-bleaching. Statistical significance was tested using an unpaired t-test with Welch's correction. Double asterisks indicate statistical significance to control at P<0.01 and triple asterisks indicate significance to control at P<0.0001. All bars are given as mean±s.e.m., sample size n is indicated.