Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion

Abstract

The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migration and axon guidance in a wide range of organisms. During development, the Hh morphogen directs tissue patterning according to a concentration gradient. Lipid modifications on Hh are needed to achieve graded distribution, leading to debate about how Hh is transported to target cells despite being membrane-tethered. Cytonemes in the region of Hh signalling have been shown to be essential for gradient formation, but the carrier of the morphogen is yet to be defined. Here we show that Hh and its co-receptor Ihog are in exovesicles transported via cytonemes. These exovesicles present protein markers and other features of exosomes. Moreover, the cell machinery for exosome formation is necessary for normal Hh secretion and graded signalling. We propose Hh transport via exosomes along cytonemes as a significant mechanism for the restricted distribution of a lipid-modified morphogen.

Hedgehog signalling

“…Unlipidated active forms of Hh have been reported to be secreted by Drosophila and mammalian cells; however, there is good evidence that lipidated Hh forms are also released by cells, as monomers or multimers, or in association with lipoproteins or exosomes (Briscoe and Thérond, 2013;Gradilla et al, 2014;Matusek et al, 2014). The importance of exosomes for Hh signalling is further supported by recent findings in Drosophila demonstrating that ESCRT (endosomal sorting complex required for transport) proteins are necessary for Hh release and long-range signalling activity.…”

Essential basal cytonemes take up Hedgehog in the Drosophila wing imaginal disc

“…4A′-A‴); their lack of association with GRASP-marked extensions might indicate that cytonemes in the basolateral region do not track in the same manner as the basal cytonemes, that they have lower fluorescence or greater curvature, or that they are more sensitive to breakage and to the manipulations that were used to prepare the discs for imaging. Although we cannot dismiss the possibility that these points of fluorescence are free exosomes (Gradilla et al, 2014;Matusek et al, 2014), we favor the idea that they are puncta associated with unmarked cytonemes. Fig.…”

“…The path that Hh takes prior to release at cytoneme tips is complex, initially involving placement in the apical membrane of Hh-producing cells, and dynamin-dependent endocytosis prior to transit to the basolateral compartment in a vesicular form (Callejo et al, 2011 (Matusek et al, 2014), work from the Guerrero group suggests that Hhcontaining vesicles that are associated with cytonemes may appear to be 'exosomes' if the cytonemes are not marked and simultaneously imaged (Gradilla et al, 2014).…”

Adaptor Protein CD2AP and L-type Lectin LMAN2 Regulate Exosome Cargo Protein Trafficking through the Golgi Complex

“…These extracellular vesicles play numerous roles in intercellular communication over a distance by delivering such macromolecules as pathogen-derived peptides on MHC proteins (3,4); signaling molecules, including Wnt (5, 6), Hedgehog (7,8), and growth factors and their receptors (9 -11); and even genetic information in the forms of messenger RNAs and non-coding RNAs (12). When extracellular vesicles reach and are fused with the target cell membrane, the cargo molecules can elicit a variety of cellular responses ranging from activation of innate and adaptive immunity, body patterning, and stem cell regulation to tissue repair, tumorigenesis, and cancer metastasis (1,2).…”