Figure 1.
Targeting vector design for rab30-Gal4, rab40-Gal4, rabX5-Gal4 and rabX6-Gal4.
20–22 kb genomic regions (black bars) were recombineered from bacterial artificial chromosomes (BACs) into attB-P[acman]-KO [17], [22]. Regions of a few kb are shown at higher resolution to reveal the structures of rab loci within these genomic regions. Sequences between red arrows were replaces with a Gal4 knock-in cassette [17]. For expression analyses, transgenic flies with the targeting vectors inserted in the same landing site were used.
Table 1.
Similarities of rab-Gal4 expression patterns based on expression patterns.
Table 2.
Similarities of Rab GTPases based on subcellular localization features.
Figure 2.
Cellular Expression Analysis of rab30-Gal4, rab40-Gal4, rabX5-gal4 and rabX6-Gal4.
All rab-Gal4 lines were crossed to UAS-CD8-GFP. (A) Larval tissues showing GFP expression in green, 3×P3-RFP (positive marker of the Gal4 knock-in cassette) and nuclear Toto3 in blue. (B) Pupal (P+30% +/−5%) and 1-day adult brains (top panel: anterior; bottom panel: posterior. All scale bars represent 100 µm.
Figure 3.
Subcellular Localization Features of YFP-Rab30, YFP-Rab40, YFP-RabX5, and YFP-RabX6.
(A) Double immunolabelings of the posterior larval brain ventral ganglion at high resolution are shown for the four YFP-Rabs driven by their respective rab-Gal4 lines. Left column: YFP-Rab (green), anti-Rab11 (red, recycling endosomes), anti-Rab5 (blue, early endosomes); right column: YFP-Rab (green), anti-CSP (red, synaptic vesicles), anti-Rab7 (blue, late endosomes). Cell bodies are peripherally and synaptic neuropils centrally located. Scale bar for all panels represents 20 µm. (B) Corresponding Gal4-lines drive the expression of wild type YFP-tagged Rabs in the left column, constitutively active (GTP-bound) YFP-tagged Rabs in the middle column and dominant negative (GDP-bound) YFP-tagged Rabs in the right column. Toto-3 labels nuclei (blue). Scale bar for all panels represents 20 µm.
Figure 4.
Analysis of Cellular Expression in Developing and Adult Tissues.
Qualitative analysis of rab-Gal4>UAS-CD8-GFP expression (columns) for specific cells and tissues (rows). If expression was found to be particularly strong compared to other cells and tissues it was designated ‘YS’ for ‘Yes Strong’. If expression was found to be present but very weak it was designed ‘YW’ for ‘Yes Weak’.
Figure 5.
Analysis of Subcellular Localization Features.
Qualitative analysis of subcellular localization features based on rab-Gal4>UAS-YFP-rab expression in the L3 larval ventral ganglion. ‘Synaptic localization’ and ‘Cell body localization’ indicate the presence in the respective compartments with no distinction of pre- versus post-synaptic compartments. ‘Rab11, Rab5, Rab7 and CSP colocalization’ indicate clearly recognizable individual punctae positive for both the YFP-Rab and one of the four antibody labelings. ‘Punctate ad DN or CA’ indicate whether the dominant negative (GDP-bound) or constitutively active (GTP-bound) YFP-Rab variants were clearly recognizable as distinct compartments (punctae). Labeling as in Figure 4, except ‘L – Lethal’ marks cases were L3 larvae could not be obtained because the dominant negative proteins caused lethality.