Abstract
Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin-Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor-like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.
Highlights
Plasma membrane protrusions, such as Second, we observed that in vivo knockdown primary cilia, as well as extracellular vesicles of prom3 in zebrafish alters the number and derived thereof
This posttranslational modification promotes the interaction of prom1 with the p85 subunit of phosphoinositide 3-kinase (PI3K) thereby activating the transformation of phosphatidylinositol[4,5]-bisphosphate (PIP2) into phosphatidylinositol[3,4,5]trisphosphate (PIP3) at the inner leaflet of the plasma membrane [36], which plays a pivotal role in the organization of plasma membrane protrusions [28]
Mutation of tyrosine 819/828 in human PROM1 reduces ciliary length We recently demonstrated that the mutation Y819F or Y828F in human PROM1.s1 and s2 splice variants, respectively, impairs tyrosine phosphorylation and the elongation of microvilli in epithelial Madin-Darby canine kidney (MDCK) cells [28,39]
Summary
Plasma membrane protrusions, such as Second, we observed that in vivo knockdown primary cilia, as well as extracellular vesicles of prom3 in zebrafish alters the number and derived thereof. These data differ from those previously obtained with the expression of wild-type PROM1 where an increase in ciliary length was observed for s1 [3.1 ± 1.4 μm (n = 569)] and s2 [3.1 ± 1.4 μm (n = 953)] variants (Fig. 1C; 7 dpc, see Ref. 33).
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