Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane

Endocytosis in yeast requires actin and clathrin. Live cell imaging has previously shown that massive actin polymerization occurs concomitant with a slow 200-nm inward movement of the endocytic coat (Kaksonen, M., Y. Sun, and D.G. Drubin. 2003. Cell. 115:475–487). However, the nature of the primary endocytic profile in yeast and how clathrin and actin cooperate to generate an endocytic vesicle is unknown. In this study, we analyze the distribution of nine different proteins involved in endocytic uptake along plasma membrane invaginations using immunoelectron microscopy. We find that the primary endocytic profiles are tubular invaginations of up to 50 nm in diameter and 180 nm in length, which accumulate the endocytic coat components at the tip. Interestingly, significant actin labeling is only observed on invaginations longer than 50 nm, suggesting that initial membrane bending occurs before initiation of the slow inward movement. We also find that in the longest profiles, actin and the myosin-I Myo5p form two distinct structures that might be implicated in vesicle fission

Analysis of CK2-dependent regulation of Myo5-induced actin polymerization during the endocytic uptake in S.cerevisiae

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Function and regulation of Saccharomyces cerevisiae myosins-I in endocytic budding

Myosins-I are widely expressed actin-dependent motors which bear a phospholipid-binding domain. In addition, some members of the family can trigger Arp2/3 complex (actin-related protein 2/3 complex)-dependent actin polymerization. In the early 1990s, the development of powerful genetic tools in protozoa and mammals and discovery of these motors in yeast allowed the demonstration of their roles in membrane traffic along the endocytic and secretory pathways, in vacuole contraction, in cell motility and in mechanosensing. The powerful yeast genetics has contributed towards dissecting in detail the function and regulation of Saccharomyces cerevisiae myosins-IMyo3 and Myo5 in endocytic budding from the plasma membrane. In the present review, we summarize the evidence, dissecting their exact role in membrane budding and the molecular mechanisms controlling their recruitment and biochemical activities at the endocytic sites. ©The Authors Journal compilation ©2011 Biochemical Society.This work was supported by the Ministerio de Ciencia e Innovación [grant numbers BFU2008-03500 and CSD2009-00016 (to M.I.G.)].Peer Reviewe

Coupled sterol synthesis and transport machineries at ER-endocytic contact sites

Sterols are unevenly distributed within cellular membranes. How their biosynthetic and transport machineries are organized to generate heterogeneity is largely unknown. We previously showed that the yeast sterol transporter Osh2 is recruited to endoplasmic reticulum (ER)–endocytic contacts to facilitate actin polymerization. We now find that a subset of sterol biosynthetic enzymes also localizes at these contacts and interacts with Osh2 and the endocytic machinery. Following the sterol dynamics, we show that Osh2 extracts sterols from these subdomains, which we name ERSESs (ER sterol exit sites). Further, we demonstrate that coupling of the sterol synthesis and transport machineries is required for endocytosis in mother cells, but not in daughters, where plasma membrane loading with accessible sterols and endocytosis are linked to secretion

Crosstalk between PI(4,5)P2 and CK2 Modulates Actin Polymerization during Endocytic Uptake

© 2014 Elsevier Inc. A transient burst of actin polymerization assists endocytic budding. How actin polymerization is controlled in this context is not understood. Here, we show that crosstalk between PI(4,5)P2 and the CK2 catalytic subunit Cka2 controls actin polymerization at endocytic sites. We find that phosphorylation of the myosin-I Myo5 by Cka2 downregulates Myo5-induced Arp2/3-dependent actin polymerization, whereas PI(4,5)P2 cooperatively relieves Myo5 autoinhibition and inhibits the catalytic activity of Cka2. Cka2 and the PI(4,5)P2-5-phosphatases Sjl1 and Sjl2, the yeast synaptojanins, exhibit genetic interactions indicating functional redundancy. The ultrastructural analysis of plasma membrane invaginations in CK2 and synaptojanin mutants demonstrates that both cooperate to initiate constriction of the invagination neck, a process coupled to the remodeling of the endocytic actin network. Our data demonstrate a holoenzyme-independent function of CK2 in endocytic budding and establish a robust genetic, functional, and molecular link between PI(4,5)P2 and CK2, two masters of intracellular signaling.This work was supported by grants BFU2011-30185 and CSD2009-00016 to M.I.G. J.P.G. is a recipient of a Ramon y Cajal contract (RYC-2011-08589)Peer Reviewe

A cross-talk between PI(4,5)P2 and CK2 modulates actin remodeling during endocytic uptake

Trabajo presentado en la Gordon Conference on Lysosomes & Endocytosis, celebrada en Andover, New Hampshire, Estados Unidos, del 15 all 20 de junio de 2014A transient burst of actin polymerization assists endocytic budding. How actin polymerization is controlled in this context is not understood. Here, we show that a crosstalk between PI(4,5)P2 and the CK2 catalytic subunit Cka2, controls actin polymerization at endocytic sites. We find that phosphorylation of the myosin-I Myo5 by Cka2 down-regulates Myo5-induced Arp2/3-dependent actin polymerization whereas PI(4,5)P2 cooperatively relieves Myo5 autoinhibition and inhibits the catalytic activity of Cka2. Cka2 and the PI(4,5)P2 5-phosphatases Sjl1 and Sjl2, the yeast synaptojanins, exhibit genetic interactions indicating functional redundancy. The ultrastructural analysis of plasma membrane invaginations in CK2 and Synaptojanin mutants demonstrate that both cooperate to initiate constriction of the invagination neck, a process coupled to the remodeling of the endocytic actin network. Our data demonstrate a novel holoenzyme-independent function of CK2 in endocytic budding and establish a robust genetic, functional and molecular link between PI(4,5)P2 and CK2, two masters of intracellular signaling.Peer Reviewe

Localized synthesis and transport of sterol at ER endocytic contact sites initiates actin polymerization

Trabajo presentado en las Lysosomes and Endocytosis Gordon Research Conference: Molecules, Pathways, and Physiology of Endosomes, Lysosomes and Lysosome-Related Organelles, celebradas en Andover, New Hampshire (USA), del 17 al 22 de junio de 2018Oxysterol Binding Proteins (OSBP) are a family of conserved lipid binding proteins, enriched at endoplasmic reticulum (ER) contacts sites. OSBPs promote non-vesicular lipid transport to other organelles and work as lipid sensors in the context of multiple cellular tasks, but the determinants of their distinct localization and function are still not understood. Using a combination of Time Resolved Electron Microscopy (TREM) and life-cell imaging in yeast, we demonstrate that the endocytic invaginations associate with the cortical endoplasmic reticulum as they mature, and that this association requires the OSBPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Using mutations that specifically disrupt the myosin-I/OSBP/VAP link, as well as a reticulon mutant with extended cER-free plasma membrane subdomains, we show that ER contact to the endocytic sites has a dual function initiating actin-dependent membrane invagination and promoting vesicle scission. Further, we show that ER-induced actin polymerization requires the localized transfer of sterols by the endocytic OSBPs.N

OSBP-mediated ER contact with endocytic sites initiates actin polymerization

Trabajo presentado en el CNRS - Conférences Jacques Monod: “Molecular basis for membrane remodelling and organization”, celebrado en Roscoff (Francia), del 3 al 7 de abril de 2017Oxysterol Binding Proteins (OSBP) are a family of conserved lipid binding proteins, enriched at endoplasmic reticulum (ER) contacts sites. OSBPs promote non-vesicular lipid transport to other organelles and work as lipid sensors in the context of multiple cellular tasks, but the determinants of their distinct localization and function are still not understood. Using a combination of Time Resolved Electron Microscopy (TREM) and life-cell imaging in yeast, we demonstrate that the endocytic invaginations associate with the cortical endoplasmic reticulum as they mature, and that this association requires the OSBPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Using mutations that specifically disrupt the myosin-I/OSBP/VAP link, as well as a reticulon mutant with extended cER-free plasma membrane subdomains, we show that ER contact to the endocytic sites has a dual function initiating actin-dependent membrane invagination and promoting vesicle scission. Further, we show that ER-induced actin polymerization requires the localized transfer of sterols by the endocytic OSBPs.N

OSBP-mediated ER contact with endocytic sites initiates actin polymerization

Trabajo presentado en el CNRS - Conférences Jacques Monod: “Molecular basis for membrane remodelling and organization”, celebrado en Roscoff (Francia), del 3 al 7 de abril de 2017Oxysterol Binding Proteins (OSBP) are a family of conserved lipid binding proteins, enriched at endoplasmic reticulum (ER) contacts sites. OSBPs promote non-vesicular lipid transport to other organelles and work as lipid sensors in the context of multiple cellular tasks, but the determinants of their distinct localization and function are still not understood. Using a combination of Time Resolved Electron Microscopy (TREM) and life-cell imaging in yeast, we demonstrate that the endocytic invaginations associate with the cortical endoplasmic reticulum as they mature, and that this association requires the OSBPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Using mutations that specifically disrupt the myosin-I/OSBP/VAP link, as well as a reticulon mutant with extended cER-free plasma membrane subdomains, we show that ER contact to the endocytic sites has a dual function initiating actin-dependent membrane invagination and promoting vesicle scission. Further, we show that ER-induced actin polymerization requires the localized transfer of sterols by the endocytic OSBPs.N

ORP-mediated ER contact with endocytic sites facilitates actin polymerization

Oxysterol binding protein-related proteins (ORPs) are conserved lipid binding polypeptides, enriched at ER contacts sites. ORPs promote non-vesicular lipid transport and work as lipid sensors in the context of many cellular tasks, but the determinants of their distinct localization and function are not understood. Here, we demonstrate that the yeast endocytic invaginations associate with the ER and that this association specifically requires the ORPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Disruption of the ER contact with endocytic sites using ORP, VAP, myosin-I, or reticulon mutants delays and weakens actin polymerization and interferes with vesicle scission. Finally, we provide evidence suggesting that ORP-dependent sterol transfer facilitates actin polymerization at endocytic sites.This work was supported by the MINECO (BFU2014-59765-P) and IJCI-2014-21318 to J.E.d.D. We thank C.R. Vázquez de Aldana and S. Orellana (IBFG, Salamanca) and J. Correa (Unex) for testing imaging conditions, and H. Valdivieso (IBFG, Salamanca) for critical reading. The LSM Zeiss780 system at the IBMB was funded by FEDER (CSIC-4E-2065). We thank T. Zimmerman for advice on STED imagingPeer reviewe