ARTS (Sept4_i2) is a pro-apoptotic tumor suppressor protein that functions as an antagonist of X-linked IAP (XIAP) to promote apoptosis. It is generally thought that mitochondrial outer membrane permeabilization (MOMP) occurs before activation of caspases and is required for it. Here, we show that ARTS initiates caspase activation upstream of MOMP. In living cells, ARTS is localized to the mitochondrial outer membrane. In response to apoptotic signals, ARTS translocates rapidly to the cytosol in a caspase-independent manner, where it binds XIAP and promotes caspase activation. This translocation precedes the release of cytochrome C and SMAC/Diablo, and ARTS function is required for the normal timing of MOMP. We also show that ARTS-induced caspase activation leads to cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation that can promote MOMP. This leads to the subsequent release of additional mitochondrial factors, including cytochrome C and SMAC/Diablo, which then amplifies the caspase cascade and causes apoptosis. Apoptosis is important for regulating cell numbers and maintaining tissue homeostasis. The main executioners of apoptosis are caspases, a family of cysteine proteases that cleave substrates after aspartate. 1 In the mitochondrial pathway, release of pro-apoptotic factors, including cytochrome C (cytoC) and Smac/Diablo (SMAC), from the mitochondrial intermembrane space (IMS) to the cytosol promotes caspase activation. This release requires mitochondrial outer membrane permeabilization (MOMP). 2 A holoenzyme complex known as the 'apoptosome' is formed when cytoC is released from mitochondria and binds to apoptotic protease-related factor-1 (Apaf-1) to activate procaspase-9. 3 The best-studied family of caspase inhibitors is the inhibitor of apoptosis (IAP) proteins. 4 IAP proteins contain at least one baculoviral IAP repeat (BIR) domain which can directly interact with caspases and inhibit their apoptotic activity, a RING domain that bestows E3-ubiquitin ligase activity and an Ubiquitin-associated (UBA) domain, which enables the binding of polyubiquitin conjugates via lysine 63. 5-7 X-linked IAP (XIAP) directly inhibits caspases-3, -7 and -9. 8 XIAP is considered to be the most potent inhibitor of caspases in vitro, and elevated levels of this protein are found in human cancers. 9 Although XIAP-null mice are viable, it was recently shown that loss of XIAP function causes elevated caspase-3 activity and sensitizes certain primary cells toward apoptosis. 7 In dying cells, apoptosis can be overcome through the release of caspases from their binding to IAP proteins. [10][11][12][13] Several mammalian XIAP antagonists have been identified, including SMAC, 14,15 Omi/HtrA2 16 and ARTS. 17,18 SMAC and Omi/HtrA2 are located in the mitochondrial IMS, contain a conserved IAP-binding motif (IBM) and are released to the cytosol upon apoptotic induction. 14,15 Genetic inactivation of SMAC and Omi/HtrA2 has fail...

Developmental axon pruning is essential for normal brain wiring in vertebrates and invertebrates. How axon pruning occurs in vivo is not well understood. In a mosaic loss-of-function screen, we found that Bsk, the Drosophila JNK, is required for axon pruning of mushroom body γ neurons, but not their dendrites. By combining in vivo genetics, biochemistry, and high-resolution microscopy, we demonstrate that the mechanism by which Bsk is required for pruning is through reducing the membrane levels of the adhesion molecule Fasciclin II (FasII), the NCAM ortholog. Conversely, overexpression of FasII is sufficient to inhibit axon pruning. Finally, we show that overexpressing other cell adhesion molecules, together with weak attenuation of JNK signaling, strongly inhibits pruning. Taken together, we have uncovered a novel and unexpected interaction between the JNK pathway and cell adhesion and found that destabilization of cell adhesion is necessary for efficient pruning.

ARTS (Sept4_i2), is a pro-apoptotic protein localized at the mitochondria of living cells. In response to apoptotic signals, ARTS rapidly translocates to the cytosol where it binds and antagonizes XIAP to promote caspase activation. However, the mechanism of interaction between these two proteins and how it is regulated remained to be explored. In this study, we show that ARTS and XIAP bind directly to each other, as recombinant ARTS and XIAP proteins co-immunoprecipitate together. We also show that over expression of ARTS alone is sufficient to induce a strong down-regulation of XIAP protein levels and that this reduction occurs through the ubiquitin proteasome system (UPS). Using various deletion and mutation constructs of XIAP we show that ARTS specifically binds to the BIR3 domain in XIAP. Moreover, we found that ARTS binds to different sequences in BIR3 than other IAP antagonists such as SMAC/Diablo. Computational analysis comparing the location of the putative ARTS interface in BIR3 with the known interfaces of SMAC/Diablo and caspase 9 support our results indicating that ARTS interacts with residues in BIR3 that are different from those involved in binding SMAC/Diablo and caspase 9. We therefore suggest that ARTS binds and antagonizes XIAP in a way which is distinct from other IAP-antagonists to promote apoptosis.

The mechanisms controlling wiring of neuronal networks are not completely understood. The stereotypic architecture of the Drosophila mushroom body (MB) offers a unique system to study circuit assembly. The adult medial MB c-lobe is comprised of a long bundle of axons that wire with specific modulatory and output neurons in a tiled manner, defining five distinct zones. We found that the immunoglobulin superfamily protein Dpr12 is cell-autonomously required in c-neurons for their developmental regrowth into the distal c4/5 zones, where both Dpr12 and its interacting protein, DIP-d, are enriched. DIP-d functions in a subset of dopaminergic neurons that wire with c-neurons within the c4/5 zone. During metamorphosis, these dopaminergic projections arrive to the c4/5 zone prior to c-axons, suggesting that c-axons extend through a prepatterned region. Thus, Dpr12/DIP-d transneuronal interaction is required for c4/5 zone formation. Our study sheds light onto molecular and cellular mechanisms underlying circuit formation within subcellular resolution.

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