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Fig. S1. Comparison of autophagic organelles in young and old Drosophila central brain neurons. Representative images of autophagosomes in young wild type (A-E) and p35 mutant (F-J) neurons. Young neurons have less mature and smaller AOs. Note developing ATG in A and F, growing MLB in B, D, G and I. With age the size of ATGs and MLBs increased, reaching up to 2000 nm in old wild type control (K-M) and p35 mutants (N-P). Black arrow for ATGs, white arrow for MLBs and black arrowhead for macromolecular depositions; m for mitochondria. Scale bar, 500 nm.
Fig. S2. Autophagic organelles accumulate in glia and neurites upon aging. Electron micrographs of glia surrounding MB neurons (A-B) in p35 mutants. Old glial cells accumulate autophagosome-lysosomal organelles (black arrow for ATGs and white arrow for MLBs) including macromolecular depositions (black arrowhead) characteristic to old neurons. Prominent example of dystrophic neurite (C) containing multiple autophagic organelles. Scale bar, 500 nm.
Fig. S3. Electron micrographs showing variety in morphology of depositions in old Drosophila neurons. Rare example of cytoplasmic deposition (black arrowhead) in young wild type neuron. Representative electron micrographs of depositions in old wild type (B-E) and p35 mutant (F-J) neurons. Old neurons accumulate a variety of multimolecular depositions with geometric symmetry in cytoplasm, single (double black arrowhead) or double membrane limited organelles (black arrow), and those depositions persist into later ATG (white arrow). Often, symmetrically organized units were surrounded by disorganized units (B, D, E, and F) or amorphic cytoplasmic material (D and E). Fusion with mitochondria was observed only in old neurons (D and H). Scale bar, 100 nm.
Fig. S4. p35 is required for normal survival under starvation stress. Young wild type and p3520c/DfC2 mutant males were exposed to the wet starvation test. Axis Y- average survival in hours alive for each genotype. Results are shown for 3 independent experiments ± s.d.
Fig. S5. Characterization of developmental defects in p35 mutant MB neurons. MARCM clones were analyzed in late larval stage. Images are Z-stack projections of sequential confocal slices covering the whole MB area. Midline is on the left, brain dorsal side is up. (A-B) Big and small wild type clone exhibit normal morphology. (C-D) p35 mutant neurons extend their dendrites outside (white arrow) of the calyx (ca, orange dotted line) area. Note that dendrites overshoot for a short distance in the direction of Fas2 positive nerves and these extensions do not carry claw-like tips (insert in C). This phenotype was briefly described in previous publication (Trunova et al., 2011). Position of different parts of MB neuron is shown: somatic part (s), peduncle (p), dorsal (d) and medial (m) lobes. mCD8-GFP in green, Fas2 in red, and NCad in blue. Scale bar, 30 µm. (E) Frequency of the dendritic phenotype in single cell count and per calyx (brain hemisphere).
Fig. S6. Longevity of MARCM lines. Life span chart for MARCM lines, related to Figure S4. Note that controls and p35 MARCM line that carry one copy of p3520C null allele have very similar life span.
