Drosophila clueless is highly expressed in larval neuroblasts, affects mitochondrial localization and suppresses mitochondrial oxidative damage

Abstract

Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu) has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, in this study we have identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. We show while mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, overall brain morphology appears to be normal. This is explained by our observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu's specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus Clu's role in mitochondrial function is not critical during larval development, but is important for pupae and adults.

Figure 1. Clu protein is highly expressed in NBs.

(A–C) Schematics of the larval brain and NB division. A) The larval brain, showing the outerproliferative center (OPC) and central brain neuroblasts. B) The OPC consists of the medulla and the lamina separated by the laminar furrow. C) Neuroblasts (NB) repeatedly divide asymmetrically to produce daughter ganglion mother cells (GMCs) that go on to differentiate and populate the adult central nervous system. (D–F) Wild type Clu protein localization in the third instar brain. D) Clu and Mir both label the same cells. E) Clu is highly expressed in the cytoplasm of large cells in the central brain, as well as medullar neuroblasts (bracket). F) Mir antibody labeling central brain NBs and medullar neuroblasts. G) WT NBs labeled with Clu antibody. NBs (round dotted outline) contain cytoplasmic Clu, as well as distinct Clu particles (arrow). Daughter GMCs (arrowhead, outline) have markedly lower amounts of Clu. H) clu^CA06604 Clu GFP-trap NBs labeled with anti-GFP antibody also show cytoplasmic Clu and Clu particles (arrow). (I–K) clu^d08713 mutant brains lack any detectable Clu expression. I) clu^d08713 mutant brain labeled with anti-Clu antibody, Mir and phalloidin. J) clu^d08713 lacks Clu expression but has normal Mir expression (K). (L–N) Clu is expressed in ectopic NBs formed in aur mutant larval brains. aur¹⁴⁶⁴¹ mutant brains have a greatly increased number of Mir positive NBs (N). These NBs also label with Clu antibody (L and M). (O) Clu protein levels are decreased in GMCs compared to NBs, even when the GMC is still connected to the NB. (O’–O’’’) Clu and Mir antibody labeling denotes the NB cell cycle stage examined for Clu expression. anti-Clu - green in D, G, H, L, O’–O’”, white in E, J, M. anti-Mir- magenta in D, I, L, O’–O’”, white in F, K, N. Phalloidin (green), I. Error bars: N for D–F, I–K, L–N. H for G and H.

Figure 2. Mitochondria are abundant, small spheres in neuroblasts.

A) Cartoon depicting microtubule organization (magenta), centrosomes (yellow) and DNA (blue) during the NB cell cycle. (B–E) Mitochondria during the NB cell cycle. B) During interphase, mitochondria are evenly dispersed in the cytoplasm, and small or slightly oblong. C) The majority of mitochondria aggregate around the first apical aster that forms before mitosis begins. D) Once the spindle forms mitochondria are evenly dispersed around the cell periphery and exclusively small spheres. E) During anaphase, only a small number of mitochondria segregate into the developing GMC. F) In contrast to the NB, the GMCs (dotted outline) have longer mitochondria (arrowhead). The surrounding glia have even longer mitochondria compared to either NBs or GMCs (arrow). (G–I) Mitochondria in aur¹⁴⁶⁴¹ mutant NBs. G) Mitochondrial shape is the same during mitosis in aur¹⁴⁶⁴¹ mutant NBs as wild type. H) In symmetric aur¹⁴⁶⁴¹ NB divisions, mitochondria appear to be evenly divided. I) This is in contrast to asymmetric aur¹⁴⁶⁴¹ NB cell divisions, which look similar to wild type. J) Mitochondria are very long and branched in the specialized glia that surround the brain and comprise the blood brain barrier. anti-CVα – green for B–J, microtubules – magenta for B–I, DAPI – blue for B–J, anti-phosphohistone H3 – white for B–J. Error bar  = 10 µm for B–J.

Figure 3. clu mutants cause mitochondrial mislocalization in NBs.

(A–C) Mitochondrial localization in clu^d08713 mutant NBs. A) During interphase, mitochondria clump. B) Abnormal mitochondrial clustering continues during mitosis. C) At anaphase, while mitochondria are still not normally dispersed, a few make it into the GMC (arrowhead). (D–F) Mitochondrial localization in sesB¹ mutant NBs. A) Mitochondria are localized normally during interphase (D), metaphase (E) and anaphase (F). During interphase (A), however, the mitochondria are consistently small and fragmented in contrast to wild type. (G–I) Mitochondrial localization in tko^25t. During interphase (G), mitochondria are evenly distributed around the cell periphery, but can be longer than wild type. Metaphase (H) and anaphase (I) have normal mitochondrial localization as well. However, during all phases of the cell cycle, a proportion of mitochondria in the NBs look round and swollen (I, inset, arrowhead). CVα – green, microtubules – magenta, DAPI – blue, phosphohistone H3 – white. Error bars: 10 µm in I for A–I, 2.5 µm in I inset.

Figure 4. Clu function is critical in adults, but not in larvae.

A) Western blot showing Clu is maternally deposited into wild type eggs (y w), but is absent in clu^d08713 mutants in 1st instar larvae, 3rd instar larvae and adults. emb  = 0–2 hr embryos, L1 =  first larval instar, L3 =  third larval instar. B) Hatching rates. clu mutant embryos hatch at normal rates regardless of maternal Clu contribution. Each bar represents hatching rates from progeny of the following parental crosses: 1) +/CyO GFP x +/CyO GFP, 2) clu^d08713/CyO GFP x clu^d08713/CyO GFP, 3) clu^d08713/CyO GFP X Df(2)Jp4/CyO GFP, 4) clu^d08713/clu^d08713 (germline clone) x clu^d08713/CyO GFP. C) Pupation rates. clu^d08713 mutant larvae experience delayed pupation, but ultimately pupate at numbers comparable to controls. AH = after hatching. D) Eclosion rates. Only 40% of clu^d08713 pupae eclose after a 2–3 day delay. E) Survivor curve. clu^d08713 homozygous and hemizygous mutant adults die after 3–4 days.

Figure 5. clu^d08713 mutant adults, but not larvae, have greatly reduced ATP and increased mitochondrial oxidative damage.

A) Mitochondrial aconitase activity in adults. clu^d08713 and SOD2^Δ2 mutants have greatly increased mitochondrial oxidative damage. B) Mitochondrial aconitase activity in larvae. clu^d08713 mutant larvae do not suffer from mitochondrial oxidative damage, in contrast to tko^25t and SOD2^Δ2 mutants. C) ATP levels in adults. After eclosion, clu^d08713 mutants experience decreased levels of ATP, that continue to go down before they die. sesB¹ and tko^25t mutants also have decreases in ATP, but not as severely as clu^d08713 mutants. D) ATP levels in larvae. clu^d08713 mutant larvae have normal ATP levels.