9 C)

9 C). as defects in cell migration. Structural characterization of Sac2 revealed a unique pleckstrin-like homology Sac2 domain name conserved in all Sac2 orthologues. Collectively, our findings provide evidence for the tight regulation of PIs by Sac2 in the endocytic recycling pathway. Introduction Phosphoinositides (PIs) are unique and specialized lipids in that their headgroup, myo-inositol ring, can be reversibly phosphorylated at the 3, 4, and 5 positions to generate seven distinct biologically active PI isoforms. Although comprising 10% of total phospholipids, PIs are essential regulators of many cellular processes such as cell signaling, cytoskeleton dynamics, and membrane trafficking (Odorizzi et al., 2000; De Matteis and Godi, 2004; Di TGFβRI-IN-1 Paolo and De Camilli, 2006). PIs have a heterogeneous distribution in different membranes, thus allowing selective recruitment of proteins containing PI recognition modules to specific organelle membranes. The maintenance of the selective distribution of specific PI species, as well as the dynamic control of PI composition in response to acute signaling inputs is usually achieved by a large number of PI kinases and phosphatases (Balla, 2013). The Sac1 domainCcontaining proteins constitute one essential family of the PI phosphatases. In vertebrates, five genes have been identified to contain the Sac1 homology domain name, which include Sac1, Sac2, Sac3/Fig4, and synaptojanin 1 and 2. The founding member of this family, Sac1, is usually a transmembrane protein localized to the ER and the Golgi apparatus and plays a major role in the homeostasis of phosphatidylinositol 4-phosphate (PI(4)P; Whitters et al., 1993; Nemoto et al., 2000). Sac3/Fig4 has been shown to regulate PI(3,5)P2 levels at lysosomes or yeast vacuoles (Rudge et al., 2004; Duex et al., 2006) and genetic mutations in Sac3/Fig4 lead to several diseases, TGFβRI-IN-1 including an autosomal recessive Charcot-Tooth disorder (CMT4J) and a subset of amyotrophic lateral sclerosis in human (Chow et al., 2007, 2009). Synaptojanin 1 and 2 are unique members in that each contains a Sac1 domain name, which dephosphorylates PI(3)P and PI(4)P, and a 5-phosphatase domain name, which dephosphorylates PI(4,5)P2 and PI(3,4,5)P3 (McPherson et al., 1996; Guo et al., 1999). Among the Sac1 domainCcontaining proteins, Sac2 remains the least well comprehended. Sac2 is usually a 128-kD protein encoded by the gene species and = 5,000 in each time point; mean SEM). *, P 0.05; **, P 0.01. CRISPR-mediated knockout of Sac2 delays TfnR recycling To further investigate the role of Sac2 in Tfn recycling, we generated N2A mutant cell lines with a complete disruption of Sac2 expression by the CRISPR technique (Horvath and Barrangou, 2010; Cong et al., 2013; Fig. S3). Similar to cells transiently expressing Sac2CS, Sac2 null cells showed a reduced surface distribution of TfnR (Fig. 5, A and B). In agreement with the notion that Sac2 plays a role in Tfn recycling, pulse-chase flow cytometry analyses showed a delayed recycling of Tfn in Sac2 null cells (Fig. 5 C). More importantly, this delay of Tfn recycling was restored by the reexpression of WT GFP-Sac2 in Sac2 null cells (Fig. 5 C). The effect on Tfn recycling in Sac2 null cells was visualized by confocal microscopy. Cells were labeled with Alexa Fluor 647CTfn and chased at Rps6kb1 the indicated time points (Fig. 5 D). A prominent retention of intracellular Tfn signals was observed in Sac2 null cells at later time points, which again suggests a delay in Tfn recycling (Fig. 5 D). Notably, the reduced surface signal and delayed Tfn recycling in Sac2 null cells were rescued by expressing WT Sac2 (Fig. 5 D). Together, these data demonstrate that Sac2 is an important regulator in the Tfn/TfnR recycling pathway. Open in a TGFβRI-IN-1 separate window Physique 5. Defects of Tfn recycling in Sac2 null cells. (A) Western blot analysis of biotin surface-labeled TfnR. (B) Quantification was performed as in Fig. 4 B. Values are normalized to WT cells (surface/total). Data are from three replicate experiments (mean SEM). (C) Flow cytometry analysis of Tfn recycling in WT and Sac2 null N2A cells. Data are from three replicate experiments (= 5,000 in each time point; mean SEM). (D) Representative images of Tfn recycling at the indicated times points. (bottom) Cells transfected with Flag-Sac2 (green). Bars, 10 m. *, P 0.05; **, P 0.01. CRISPR-mediated knockout of Sac2 perturbs integrin distribution and recycling To assess whether Sac2 has a general impact on the recycling of other endocytic cargos, we analyzed integrin recycling in Sac2 null cells. We first compared the surface distribution of 1 1 integrin in WT and Sac2 cells by flow cytometry. Surface labeling revealed that.