In an RNAi screen to interrogate the function of transmembra

In an RNAi screen to interrogate the function of transmembrane proteins in ISCs/EBs, we identified the SJ protein tetraspanin 2A (Tsp2A) as a tumor suppressor. Further characterization reveals that Tsp2A expression initiates in ISCs and Tsp2A protein assembles at the SJs in the progenitor ABT-199 sale that are differentiating toward ECs. Importantly, we found that Tsp2A undergoes active internalization from the SJs and mediates the degradation of the Hippo-antagonizing protein aPKC. Therefore, endocytic regulation by Tsp2A couples the process of EC maturation with the downregulation of Yki activity. Tsp2A belongs to the large family of four-pass transmembrane proteins that often function as scaffolding co-receptors. Similar to our observation with Tsp2A, previous studies have documented the endocytosis of a putative Tsp2A ortholog CD81 and claudins (TJ proteins) (Farquhar et al., 2012, Matsuda et al., 2004). While the internalization of Tsp2A ortholog and claudins has long been an intriguing observation in cultured cells, our finding about the Tsp2A-aPKC signaling uncovers a physiological function for the internalization of occluding junction (SJ or TJ) protein in vivo.
Results
Discussion Characterization of Tsp2A in the adult Drosophila midgut reveals a pivotal link between EC maturation and the restriction of ISC proliferation (Figure S7). Progenitor cells differentiating toward ECs undergo a series of changes (Xu et al., 2018), which include the increase of nucleus ploidy and cell size, the formation of SJs, as well as the loss of concentrated aPKC staining at the cell surface. aPKC activity in ISCs/EBs, which increases following tissue damage, can help promote and sustain proliferation via Yki-JAK-Stat signaling. Tsp2A assembly at SJs and its subsequent internalization facilitate aPKC or Rack1 degradation and downregulate aPKC activity. Therefore, in the regeneration process following tissue damage, Tsp2A-SJ assembly not only allows EC maturation but also signals ISCs to reduce proliferation activity when enough mature ECs have been produced for tissue repair. Defects in Tsp2A expression cause excessive aPKC-Yki-JAK-Stat activity and make the midgut epithelia highly proliferative, like a wound that cannot heal.
STAR★Methods
Acknowledgments We thank Allison Bardin, Steve Hou, and Ben Ohlstein for sharing fly stocks and Yasushi Izumi, Richard Fehon, Kim McCall, and Julie Kadrmas for antibodies. We thank Martin Hemler and Fernando Camargo for discussions, the Microscopy Resources on the North Quad (MicRoN) core at Harvard Medical School for confocal imaging support, and Maria Ericsson and the Harvard Medical School EM Facility for electron microscopy support. We also thank Perrimon lab members, especially Young Kwon, Phillip Karpowicz, Mary-Lee Dequéant, and Richelle Sopko, for discussion and technical instructions; Christians Villalta for transgenic fly injections; and Stephanie Mohr, Afroditi Petsakou, Richard Binari, David Doupé, and Li He for comments on the manuscript. Work in the Perrimon lab is supported by NIGMS (grant GM067761), the STARR consortium, and HHMI. N.P. is an Investigator of the Howard Hughes Medical Institute. H.-W.T. is supported by the Human Frontier Science Program. R.-J.H. is supported by the Jane Coffin Childs Foundation.
Hippo Pathway MST kinases are conserved homologues of yeast Sterile 20 (STE20) kinase and, despite millions of years of divergent evolution, exert similar functions across eukaryotes. The MST family consists of five different proteins that are conserved in all metazoans [1]. They can be divided into two subgroups: the first group containing MST1 [serine/threonine protein kinase 4 (STK4)] and MST2 (STK3); the second group containing MST3 (STK24), MST4 (STK26 or MASK) and MST5 [STK25 or YSK1] (Table 1) [2]. Cell division cycle 15 (Cdc15) in Saccharomyces cerevisiae was the first MST kinase characterised 2, 3; later, Hippo (Hpo) was discovered in Drosophila melanogaster[4]. Both Cdc15 and Hpo are crucial in controlling cell proliferation and are homologues of mammalian MST1 and MST2, collectively called MST1/2 (Table 1).