Ionary history of this gene family and will greatly facilitate gene expression and comparative Title Loaded From File studies that focus on SERCA genes.(DOCX)AcknowledgmentsWe thank Bora Demiri, John Colbourne, Norman Yan, and two anonymous reviewers for their careful and insightful comments on the manuscript.Author ContributionsConceived and designed the experiments: IA MEC. Performed the experiments: IA JJV. Analyzed the data: IA JJV SX MEC. Contributed reagents/materials/analysis tools: MEC. Wrote the paper: IA JJV SX.Supporting InformationTable S1 List of protein sequences used for phylogeneticanalyses.
Title Loaded From File caveolae are bulb-shaped pits present in several mammalian cell types including adipocytes and muscle cells [1,2]. These structures play key roles in compartmentalization and organization of signaling pathways for cell growth and differentiation. In addition, caveolae were recently implicated in membrane-mediated mechanical responses [3,4]. Caveolin-1 (Cav1) is the main component of caveolae. Cav1 adopts a hairpin-like shape within the membrane bilayer with both the N and C-terminus facing the cytoplasm [1]. Recent studies showed that the Cav1 partner protein, Polymerase I and Transcript Release Factor (PTRF)/ cavin-1 selectively associates with mature caveolae at the plasma membrane and is involved in caveolae formation and function [1,5]. Total internal reflection fluorescence microscopy (TIRF-M) allowed characterizing the dynamics of individual caveolae and revealed that caveolae can be stored in stationary multi-caveolar structures at the plasma membrane, or undergo kiss and run processes without disassembling the caveolar coat [6]. Moreover, caveolae can undergo long-range cytoplasmic transport during diverse regulated processes such as mitosis and during loss of integrin-based adhesion to the extracellular matrix (ECM) [1,7,8]. All together, these data suggest some interplay between caveolar trafficking and cell adhesion [9]. Initially identified in Saccharomyces cerevisiae, the exocyst complex comprises eight subunits named Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84 [10,11]. This complex controls the docking and insertion of secretory and endocytic recycling vesicles to growing regions of the plasma membrane and impinges on diverse cellular 24195657 processes requiring polarization of membranetrafficking such as cell motility and neuronal development [12,13]. In yeast, Exo70 interacts with phospholipids at the plasma membrane and is crucial for tethering of secretory vesicles for exocytosis [14]. Phospholipid binding and plasma membrane localization are conserved in mammalian cells suggesting a similar role of Exo70 in targeting of the exocyst complex to the plasma membrane and regulation of vesicle docking [14?6]. Previous studies showed that detachment of cells from integrin-mediated adhesion triggers the caveolin-dependent internalization of lipid rafts [9]. Conversely, re-adhesion of cells to the ECM induces the re-insertion 11967625 of lipid rafts to the plasma membrane in a process that involves the Ras-related GTP binding proteins RalA and ARF6 and their downstream effectors, exocyst complex [17,18,19]. However, the specific contribution of Exo70 to Cav1 trafficking remains largely unknown. Here, using TIRF-M we could show that Exo70 accumulates in punctuate plasma membrane domains where it co-localized with Cav1. Upon cell detachment, Exo70 localized to Cav1-positive intracellular compartments. When cells were allowed to re-adhere, silencing of Exo7.Ionary history of this gene family and will greatly facilitate gene expression and comparative studies that focus on SERCA genes.(DOCX)AcknowledgmentsWe thank Bora Demiri, John Colbourne, Norman Yan, and two anonymous reviewers for their careful and insightful comments on the manuscript.Author ContributionsConceived and designed the experiments: IA MEC. Performed the experiments: IA JJV. Analyzed the data: IA JJV SX MEC. Contributed reagents/materials/analysis tools: MEC. Wrote the paper: IA JJV SX.Supporting InformationTable S1 List of protein sequences used for phylogeneticanalyses.
Caveolae are bulb-shaped pits present in several mammalian cell types including adipocytes and muscle cells [1,2]. These structures play key roles in compartmentalization and organization of signaling pathways for cell growth and differentiation. In addition, caveolae were recently implicated in membrane-mediated mechanical responses [3,4]. Caveolin-1 (Cav1) is the main component of caveolae. Cav1 adopts a hairpin-like shape within the membrane bilayer with both the N and C-terminus facing the cytoplasm [1]. Recent studies showed that the Cav1 partner protein, Polymerase I and Transcript Release Factor (PTRF)/ cavin-1 selectively associates with mature caveolae at the plasma membrane and is involved in caveolae formation and function [1,5]. Total internal reflection fluorescence microscopy (TIRF-M) allowed characterizing the dynamics of individual caveolae and revealed that caveolae can be stored in stationary multi-caveolar structures at the plasma membrane, or undergo kiss and run processes without disassembling the caveolar coat [6]. Moreover, caveolae can undergo long-range cytoplasmic transport during diverse regulated processes such as mitosis and during loss of integrin-based adhesion to the extracellular matrix (ECM) [1,7,8]. All together, these data suggest some interplay between caveolar trafficking and cell adhesion [9]. Initially identified in Saccharomyces cerevisiae, the exocyst complex comprises eight subunits named Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84 [10,11]. This complex controls the docking and insertion of secretory and endocytic recycling vesicles to growing regions of the plasma membrane and impinges on diverse cellular 24195657 processes requiring polarization of membranetrafficking such as cell motility and neuronal development [12,13]. In yeast, Exo70 interacts with phospholipids at the plasma membrane and is crucial for tethering of secretory vesicles for exocytosis [14]. Phospholipid binding and plasma membrane localization are conserved in mammalian cells suggesting a similar role of Exo70 in targeting of the exocyst complex to the plasma membrane and regulation of vesicle docking [14?6]. Previous studies showed that detachment of cells from integrin-mediated adhesion triggers the caveolin-dependent internalization of lipid rafts [9]. Conversely, re-adhesion of cells to the ECM induces the re-insertion 11967625 of lipid rafts to the plasma membrane in a process that involves the Ras-related GTP binding proteins RalA and ARF6 and their downstream effectors, exocyst complex [17,18,19]. However, the specific contribution of Exo70 to Cav1 trafficking remains largely unknown. Here, using TIRF-M we could show that Exo70 accumulates in punctuate plasma membrane domains where it co-localized with Cav1. Upon cell detachment, Exo70 localized to Cav1-positive intracellular compartments. When cells were allowed to re-adhere, silencing of Exo7.