Figure 5.
MIM localizes to the site of fusion and pore formation. (A) Confocal intensity-projection of a representative SG expressing Glue-DsRed (Costantino et al., 2008; green; expressed under the endogenous sgs3 promoter), and a functional MIM-Emerald (magenta; Fig. S5 B), showing at increased magnification the dynamic localization of MIM to cytoplasmic and apical clusters. Yellow squares mark the magnified areas. Top right: Dynamic MIM cytoplasmic clusters merging (black dashed lines) and splitting (white dashed lines; also in Video 8). Bottom right: A representative LSV (SRRF intensity-projection) during exocytosis, showing MIM localization to the sites of fusion and pore formation (dashed circle) and remaining throughout secretion (also in Video 9). Time mm:ss; relative to fusion (of bottom right LSV). Scale bars = 20 µm (left), 10 µm (top right), 1 µm (bottom right). (B) Correlative confocal and FIB-SEM showing MIM-Emerald fluorescence associated with the membrane of a putative fusion site. Top left: Confocal intensity-projection showing the fluorescence from a representative SG after resin embedding. Yellow squares mark the targeted region for FIB-SEM imaging. Bottom left: Overlay of the transformed fluorescence microscopy (FM) image onto a resliced XZ plane of the FIB-SEM stack. Correlation precision can be evaluated from the correspondence between the Glue-DsRed (green) and the LSVs in FIB-SEM. Marked area (yellow rectangle) shows MIM-Emerald appearing as a ring surrounding an LSV. Right: FIB-SEM XY view of the marked area in the XZ view, showing MIM localization to the putative fusion site on the plasma membrane (PM). Plane shown in the XZ view is marked by a dashed line. Scale bars = 20 µm (left), 1 µm (middle and right). UAS expression in A and B was driven by c135-GAL4.

MIM localizes to the site of fusion and pore formation. (A) Confocal intensity-projection of a representative SG expressing Glue-DsRed (Costantino et al., 2008; green; expressed under the endogenous sgs3 promoter), and a functional MIM-Emerald (magenta; Fig. S5 B), showing at increased magnification the dynamic localization of MIM to cytoplasmic and apical clusters. Yellow squares mark the magnified areas. Top right: Dynamic MIM cytoplasmic clusters merging (black dashed lines) and splitting (white dashed lines; also in Video 8). Bottom right: A representative LSV (SRRF intensity-projection) during exocytosis, showing MIM localization to the sites of fusion and pore formation (dashed circle) and remaining throughout secretion (also in Video 9). Time mm:ss; relative to fusion (of bottom right LSV). Scale bars = 20 µm (left), 10 µm (top right), 1 µm (bottom right). (B) Correlative confocal and FIB-SEM showing MIM-Emerald fluorescence associated with the membrane of a putative fusion site. Top left: Confocal intensity-projection showing the fluorescence from a representative SG after resin embedding. Yellow squares mark the targeted region for FIB-SEM imaging. Bottom left: Overlay of the transformed fluorescence microscopy (FM) image onto a resliced XZ plane of the FIB-SEM stack. Correlation precision can be evaluated from the correspondence between the Glue-DsRed (green) and the LSVs in FIB-SEM. Marked area (yellow rectangle) shows MIM-Emerald appearing as a ring surrounding an LSV. Right: FIB-SEM XY view of the marked area in the XZ view, showing MIM localization to the putative fusion site on the plasma membrane (PM). Plane shown in the XZ view is marked by a dashed line. Scale bars = 20 µm (left), 1 µm (middle and right). UAS expression in A and B was driven by c135-GAL4.

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