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  • br Disp Mediated Hh Membrane

    2022-06-15


    Disp-Mediated Hh Membrane Recycling Due to its cholesterol modification, Drosophila Hh enriches on sphingolipid-rich apical membranes of polarized epithelial ER 27319 maleate mg 33., 38., 39.. Genetic studies examining Hh release from wing imaginal disc epithelia suggest that Hh must be endocytosed from the apical cell surface in a Disp-dependent manner prior to its eventual release for long-range signaling (Figure 2B,C) 33., 38.. Based on observed subapical colocalization with the early endosomal marker Rab5, Disp was proposed to collect Hh from the cell surface and into recycling endosomes (Figure 2C). Expression of a dominant-negative Rab5 mutant blocked Hh endocytosis, and decreased long-range target gene expression, consistent with Disp-mediated endosomal recycling being required for long-range signaling activity [33]. The effects of Disp were specific for endocytosis of Hh because internalization of the endocytic reporters FM-64 and dextran were unaffected by Disp loss. Hh was also found to localize to Rab4-positive recycling endosomes, further supporting that Hh undergoes membrane recycling as part of its release mechanism (Figure 2D) [38]. How Disp selectively promotes Hh membrane recycling is not currently understood, but one logical hypothesis is that Disp might couple Hh to the endocytic machinery. Unfortunately, due to a lack of information regarding cellular Disp binding partners, prediction of candidates for Disp-mediated endocytic coupling is not currently feasible. The ability of Disp to prompt Hh internalization was lost on mutation of the conserved transporter motifs in TM4 and TM10 [33]. This result could suggest that either: (i) Disp uses the proton motive force to promote Hh recycling; or (ii) the TM4 SSD is required for recycling to occur. The second hypothesis may be suggested by the function of another SSD family member, the endoplasmic reticulum (ER) cholesterol monitor sterol regulatory element-binding protein cleavage-activating protein (SCAP). Cholesterol membrane depletion prompts SCAP to transport sterol regulatory element-binding protein (SREBP) transcription factors from the ER to the Golgi [40]. The SCAP response to changing sterol levels is dependent on its SSD, which releases SCAP protein from INSIG ER tethers on sensing sterol reduction 26., 40.. Insig release allows SCAP to associate with coatomer II (COPII) proteins to load SREBP into budding, Golgi-bound vesicles [41]. It is tempting to speculate that the Disp SSD might respond to a specific sterol environment surrounding apically localized Hh to load ligand into recycling endosomes for membrane cycling and subsequent release. Although genetic support for Disp-mediated Hh membrane recycling in Drosophila is relatively clear, why Hh reinternalization is required for Disp to release the long-range signal is not. Overexpression studies in cultured cells suggest that Hhs are deployed on lipoproteins, so it is possible that endosomal recycling could allow Disp-mediated loading of ligand onto these complexes [42]. More recent experimental evidence indicates that Hhs are released from producing cells in exovesicles in both flies and vertebrates, raising the possibility that Disp-controlled recycling facilitates Hhs exovesicle packaging (Figure 2E) 39., 43., 44., 45.. Immunoelectron microscopy revealed Disp- and Hh-positive puncta in multivesicular bodies (MVBs) and exovesicles along basolateral membranes in Drosophila epithelia [39]. Disp was therefore hypothesized to initiate the internalization of Hh from apical membranes to redirect it basolaterally for exovesicle-mediated release. An alternative model posits that, rather than directing Hh basally, Disp recycles Hh back to the apical membrane to be released for long-range signaling. In a study supporting this model, expression of dominant-negative Rab4 drove the accumulation of Hh basolaterally and reduced long-range target gene induction, suggesting that Hh cannot deploy from basolateral membranes for long-range transport [38]. Nevertheless, the two studies agree that Hh must be packaged into exovesicles and undergo Disp-dependent membrane recycling for long-range signaling to occur 33., 38., 39., 43.. Further investigation will be needed to clarify whether Disp preferentially directs Hh for apical or basolateral membrane release through the recycling process.