Single molecule biophotonicshttp://hdl.handle.net/2117/238082024-03-19T08:42:25Z2024-03-19T08:42:25ZThe ER cholesterol sensor SCAP promotes CARTS biogenesis at ER–Golgi membrane contact sitesWakana, YuichiHayashi, KaitoNemoto, TakumiWatanabe, ChiakiTaoka, MasatoAngulo-Capel, JessicaGarcia-Parajo, Maria F.Kumata, HidetoshiUmemura, TomonariInoue, HirokiArasaki, KoheiCampelo, FelixTagaya, Mitsuohttp://hdl.handle.net/2117/3484392022-05-19T11:36:47Z2021-07-05T13:49:13ZThe ER cholesterol sensor SCAP promotes CARTS biogenesis at ER–Golgi membrane contact sites
Wakana, Yuichi; Hayashi, Kaito; Nemoto, Takumi; Watanabe, Chiaki; Taoka, Masato; Angulo-Capel, Jessica; Garcia-Parajo, Maria F.; Kumata, Hidetoshi; Umemura, Tomonari; Inoue, Hiroki; Arasaki, Kohei; Campelo, Felix; Tagaya, Mitsuo
In response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here, we show that in cholesterol-fed cells, ER-localized SCAP interacts through Sac1 phosphatidylinositol 4-phosphate (PI4P) phosphatase with a VAP–OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER–Golgi membrane contact sites (MCSs). SCAP knockdown inhibited the turnover of PI4P, perhaps due to a cholesterol transport defect, and altered the subcellular distribution of the VAP–OSBP complex. As in the case of perturbation of lipid transfer complexes at ER–Golgi MCSs, SCAP knockdown inhibited the biogenesis of the trans-Golgi network–derived transport carriers CARTS, which was reversed by expression of wild-type SCAP or a Golgi transport–defective mutant, but not of cholesterol sensing–defective mutants. Altogether, our findings reveal a new role for SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis via ER–Golgi MCSs, depending on the ER cholesterol.
2021-07-05T13:49:13ZWakana, YuichiHayashi, KaitoNemoto, TakumiWatanabe, ChiakiTaoka, MasatoAngulo-Capel, JessicaGarcia-Parajo, Maria F.Kumata, HidetoshiUmemura, TomonariInoue, HirokiArasaki, KoheiCampelo, FelixTagaya, MitsuoIn response to cholesterol deprivation, SCAP escorts SREBP transcription factors from the endoplasmic reticulum to the Golgi complex for their proteolytic activation, leading to gene expression for cholesterol synthesis and uptake. Here, we show that in cholesterol-fed cells, ER-localized SCAP interacts through Sac1 phosphatidylinositol 4-phosphate (PI4P) phosphatase with a VAP–OSBP complex, which mediates counter-transport of ER cholesterol and Golgi PI4P at ER–Golgi membrane contact sites (MCSs). SCAP knockdown inhibited the turnover of PI4P, perhaps due to a cholesterol transport defect, and altered the subcellular distribution of the VAP–OSBP complex. As in the case of perturbation of lipid transfer complexes at ER–Golgi MCSs, SCAP knockdown inhibited the biogenesis of the trans-Golgi network–derived transport carriers CARTS, which was reversed by expression of wild-type SCAP or a Golgi transport–defective mutant, but not of cholesterol sensing–defective mutants. Altogether, our findings reveal a new role for SCAP under cholesterol-fed conditions in the facilitation of CARTS biogenesis via ER–Golgi MCSs, depending on the ER cholesterol.Shear forces induce ICAM-1 nanoclustering on endothelial cells that impact on T cell migrationPiechocka, Izabela K.Keary, SarahSosa-Costa, AlbertoLau, LukasMohan, NitinStanisavljevic, JelenaBorgman, Kyra J.E.Lakadamyali, MelikeManzo, CarloGarcia-Parajo, Maria F.http://hdl.handle.net/2117/3468362022-05-17T17:12:32Z2021-06-08T08:59:03ZShear forces induce ICAM-1 nanoclustering on endothelial cells that impact on T cell migration
Piechocka, Izabela K.; Keary, Sarah; Sosa-Costa, Alberto; Lau, Lukas; Mohan, Nitin; Stanisavljevic, Jelena; Borgman, Kyra J.E.; Lakadamyali, Melike; Manzo, Carlo; Garcia-Parajo, Maria F.
The leukocyte specific β2-integrin LFA-1, and its ligand ICAM-1 expressed on endothelial
cells (ECs), are involved in the arrest, adhesion and transendothelial migration of
leukocytes. Although the role of mechanical forces on LFA-1 activation is wellestablished, the impact of forces on its major ligand ICAM-1, has received less attention.
Using a parallel-plate flow-chamber combined with confocal and super-resolution
microscopy, we show that prolonged shear-flow induces global translocation of ICAM-1
on ECs upstream of flow direction. Interestingly, shear-forces caused actin rearrangements and promoted actin-dependent ICAM-1 nanoclustering prior to LFA-1
engagement. T-cells adhered to mechanically pre-stimulated ECs or nanoclustered
ICAM-1 substrates, developed a pro-migratory phenotype, migrated faster and exhibited
shorter-lived interactions with ECs than when adhered to non-mechanically stimulated
ECs, or to monomeric ICAM-1 substrates. Together, our results indicate that shearforces increase ICAM-1/LFA-1 bonds due to ICAM-1 nanoclustering, strengthening
adhesion and allowing cells to exert higher traction forces required for faster migration.
Our data also underscores the importance of mechanical forces regulating the nanoscale
organization of membrane receptors and their contribution to cell adhesion regulation.
2021-06-08T08:59:03ZPiechocka, Izabela K.Keary, SarahSosa-Costa, AlbertoLau, LukasMohan, NitinStanisavljevic, JelenaBorgman, Kyra J.E.Lakadamyali, MelikeManzo, CarloGarcia-Parajo, Maria F.The leukocyte specific β2-integrin LFA-1, and its ligand ICAM-1 expressed on endothelial
cells (ECs), are involved in the arrest, adhesion and transendothelial migration of
leukocytes. Although the role of mechanical forces on LFA-1 activation is wellestablished, the impact of forces on its major ligand ICAM-1, has received less attention.
Using a parallel-plate flow-chamber combined with confocal and super-resolution
microscopy, we show that prolonged shear-flow induces global translocation of ICAM-1
on ECs upstream of flow direction. Interestingly, shear-forces caused actin rearrangements and promoted actin-dependent ICAM-1 nanoclustering prior to LFA-1
engagement. T-cells adhered to mechanically pre-stimulated ECs or nanoclustered
ICAM-1 substrates, developed a pro-migratory phenotype, migrated faster and exhibited
shorter-lived interactions with ECs than when adhered to non-mechanically stimulated
ECs, or to monomeric ICAM-1 substrates. Together, our results indicate that shearforces increase ICAM-1/LFA-1 bonds due to ICAM-1 nanoclustering, strengthening
adhesion and allowing cells to exert higher traction forces required for faster migration.
Our data also underscores the importance of mechanical forces regulating the nanoscale
organization of membrane receptors and their contribution to cell adhesion regulation.PLANT: A Method for Detecting Changes of Slope in Noisy TrajectoriesSosa-Costa, AlbertoPiechocka, Izabela K.Gardini, LuciaPavone, Francesco S.Capitanio, MarcoGarcia-Parajo, Maria F.Manzo, Carlohttp://hdl.handle.net/2117/1175842022-05-17T12:35:54Z2018-05-28T09:07:25ZPLANT: A Method for Detecting Changes of Slope in Noisy Trajectories
Sosa-Costa, Alberto; Piechocka, Izabela K.; Gardini, Lucia; Pavone, Francesco S.; Capitanio, Marco; Garcia-Parajo, Maria F.; Manzo, Carlo
Time traces obtained from a variety of biophysical experiments contain valuable information on underlying processes occurring at the molecular level. Accurate quantification of these data can help explain the details of the complex dynamics of biological systems. Here, we describe PLANT (Piecewise Linear Approximation of Noisy Trajectories), a segmentation algorithm that allows the reconstruction of time-trace data with constant noise as consecutive straight lines, from which changes of slopes and their respective durations can be extracted. We present a general description of the algorithm and perform extensive simulations to characterize its strengths and limitations, providing a rationale for the performance of the algorithm in the different conditions tested. We further apply the algorithm to experimental data obtained from tracking the centroid position of lymphocytes migrating under the effect of a laminar flow and from single myosin molecules interacting with actin in a dual-trap force-clamp configuration.
2018-05-28T09:07:25ZSosa-Costa, AlbertoPiechocka, Izabela K.Gardini, LuciaPavone, Francesco S.Capitanio, MarcoGarcia-Parajo, Maria F.Manzo, CarloTime traces obtained from a variety of biophysical experiments contain valuable information on underlying processes occurring at the molecular level. Accurate quantification of these data can help explain the details of the complex dynamics of biological systems. Here, we describe PLANT (Piecewise Linear Approximation of Noisy Trajectories), a segmentation algorithm that allows the reconstruction of time-trace data with constant noise as consecutive straight lines, from which changes of slopes and their respective durations can be extracted. We present a general description of the algorithm and perform extensive simulations to characterize its strengths and limitations, providing a rationale for the performance of the algorithm in the different conditions tested. We further apply the algorithm to experimental data obtained from tracking the centroid position of lymphocytes migrating under the effect of a laminar flow and from single myosin molecules interacting with actin in a dual-trap force-clamp configuration.Optical Antenna-Based Fluorescence Correlation Spectroscopy to Probe the Nanoscale Dynamics of Biological MembranesWinkler, Pamina M.Regmi, RajuFlauraud, ValentinBrugger, JürgenRigneault, HervéWenger, JérômeGarcia-Parajo, Maria F.http://hdl.handle.net/2117/1724442022-10-13T10:04:02Z2018-05-14T15:03:21ZOptical Antenna-Based Fluorescence Correlation Spectroscopy to Probe the Nanoscale Dynamics of Biological Membranes
Winkler, Pamina M.; Regmi, Raju; Flauraud, Valentin; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; Garcia-Parajo, Maria F.
The plasma membrane of living cells is compartmentalized at multiple spatial scales ranging from the nano- to the mesoscale. This nonrandom organization is crucial for a large number of cellular functions. At the nanoscale, cell membranes organize into dynamic nanoassemblies enriched by cholesterol, sphingolipids, and certain types of proteins. Investigating these nanoassemblies known as lipid rafts is of paramount interest in fundamental cell biology. However, this goal requires simultaneous nanometer spatial precision and microsecond temporal resolution, which is beyond the reach of common microscopes. Optical antennas based on metallic nanostructures efficiently enhance and confine light into nanometer dimensions, breaching the diffraction limit of light. In this Perspective, we discuss recent progress combining optical antennas with fluorescence correlation spectroscopy (FCS) to monitor microsecond dynamics at nanoscale spatial dimensions. These new developments offer numerous opportunities to investigate lipid and protein dynamics in both mimetic and native biological membranes.
2018-05-14T15:03:21ZWinkler, Pamina M.Regmi, RajuFlauraud, ValentinBrugger, JürgenRigneault, HervéWenger, JérômeGarcia-Parajo, Maria F.The plasma membrane of living cells is compartmentalized at multiple spatial scales ranging from the nano- to the mesoscale. This nonrandom organization is crucial for a large number of cellular functions. At the nanoscale, cell membranes organize into dynamic nanoassemblies enriched by cholesterol, sphingolipids, and certain types of proteins. Investigating these nanoassemblies known as lipid rafts is of paramount interest in fundamental cell biology. However, this goal requires simultaneous nanometer spatial precision and microsecond temporal resolution, which is beyond the reach of common microscopes. Optical antennas based on metallic nanostructures efficiently enhance and confine light into nanometer dimensions, breaching the diffraction limit of light. In this Perspective, we discuss recent progress combining optical antennas with fluorescence correlation spectroscopy (FCS) to monitor microsecond dynamics at nanoscale spatial dimensions. These new developments offer numerous opportunities to investigate lipid and protein dynamics in both mimetic and native biological membranes.TANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranesRaote, IshierOrtega-Bellido, MaríaSantos, António J MForesti, OmbrettaZhang, ChongGarcia-Parajo, Maria F.Campelo, FelixMalhotra, Vivekhttp://hdl.handle.net/2117/1149252022-10-13T10:06:36Z2018-03-08T12:52:44ZTANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranes
Raote, Ishier; Ortega-Bellido, María; Santos, António J M; Foresti, Ombretta; Zhang, Chong; Garcia-Parajo, Maria F.; Campelo, Felix; Malhotra, Vivek
Collagen export from the endoplasmic reticulum (ER) requires TANGO1, COPII coats, and retrograde fusion of ERGIC membranes. How do these components come together to produce a transport carrier commensurate with the bulky cargo collagen? TANGO1 is known to form a ring that corrals COPII coats and we show here how this ring or fence is assembled. Our data reveal that a TANGO1 ring is organized by its radial interaction with COPII, and lateral interactions with cTAGE5, TANGO1-short or itself. Of particular interest is the finding that TANGO1 recruits ERGIC membranes for collagen export via the NRZ (NBAS/RINT1/ZW10) tether complex. Therefore, TANGO1 couples retrograde membrane flow to anterograde cargo transport. Without the NRZ complex, the TANGO1 ring does not assemble, suggesting its role in nucleating or stabilising of this process. Thus, coordinated capture of COPII coats, cTAGE5, TANGO1-short, and tethers by TANGO1 assembles a collagen export machine at the ER.
2018-03-08T12:52:44ZRaote, IshierOrtega-Bellido, MaríaSantos, António J MForesti, OmbrettaZhang, ChongGarcia-Parajo, Maria F.Campelo, FelixMalhotra, VivekCollagen export from the endoplasmic reticulum (ER) requires TANGO1, COPII coats, and retrograde fusion of ERGIC membranes. How do these components come together to produce a transport carrier commensurate with the bulky cargo collagen? TANGO1 is known to form a ring that corrals COPII coats and we show here how this ring or fence is assembled. Our data reveal that a TANGO1 ring is organized by its radial interaction with COPII, and lateral interactions with cTAGE5, TANGO1-short or itself. Of particular interest is the finding that TANGO1 recruits ERGIC membranes for collagen export via the NRZ (NBAS/RINT1/ZW10) tether complex. Therefore, TANGO1 couples retrograde membrane flow to anterograde cargo transport. Without the NRZ complex, the TANGO1 ring does not assemble, suggesting its role in nucleating or stabilising of this process. Thus, coordinated capture of COPII coats, cTAGE5, TANGO1-short, and tethers by TANGO1 assembles a collagen export machine at the ER.Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living CellsRegmi, RajuWinkler, Pamina M.Flauraud, ValentinBorgman, Kyra J. E.Manzo, CarloBrugger, JürgenRigneault, HervéWenger, JérômeGarcia-Parajo, Maria F.http://hdl.handle.net/2117/1724222022-10-13T10:05:07Z2018-02-19T12:50:50ZPlanar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells
Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; Garcia-Parajo, Maria F.
Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 μs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.
2018-02-19T12:50:50ZRegmi, RajuWinkler, Pamina M.Flauraud, ValentinBorgman, Kyra J. E.Manzo, CarloBrugger, JürgenRigneault, HervéWenger, JérômeGarcia-Parajo, Maria F.Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 μs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.Transient Nanoscopic Phase Separation in Biological Lipid Membranes Resolved by Planar Plasmonic AntennasWinkler, Pamina M.Regmi, RajuFlauraud, ValentinBrugger, JürgenRigneault, HervéWenger, JérômeGarcia-Parajo, Maria F.http://hdl.handle.net/2117/1724432022-10-13T10:04:19Z2018-01-11T14:53:55ZTransient Nanoscopic Phase Separation in Biological Lipid Membranes Resolved by Planar Plasmonic Antennas
Winkler, Pamina M.; Regmi, Raju; Flauraud, Valentin; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; Garcia-Parajo, Maria F.
Nanoscale membrane assemblies of sphingolipids, cholesterol, and certain proteins, also known as lipid rafts, play a crucial role in facilitating a broad range of important cell functions. Whereas on living cell membranes lipid rafts have been postulated to have nanoscopic dimensions and to be highly transient, the existence of a similar type of dynamic nanodomains in multicomponent lipid bilayers has been questioned. Here, we perform fluorescence correlation spectroscopy on planar plasmonic antenna arrays with different nanogap sizes to assess the dynamic nanoscale organization of mimetic biological membranes. Our approach takes advantage of the highly enhanced and confined excitation light provided by the nanoantennas together with their outstanding planarity to investigate membrane regions as small as 10 nm in size with microsecond time resolution. Our diffusion data are consistent with the coexistence of transient nanoscopic domains in both the liquid-ordered and the liquid-disordered microscopic phases of multicomponent lipid bilayers. These nanodomains have characteristic residence times between 30 and 150 μs and sizes around 10 nm, as inferred from the diffusion data. Thus, although microscale phase separation occurs on mimetic membranes, nanoscopic domains also coexist, suggesting that these transient assemblies might be similar to those occurring in living cells, which in the absence of raft-stabilizing proteins are poised to be short-lived. Importantly, our work underscores the high potential of photonic nanoantennas to interrogate the nanoscale heterogeneity of native biological membranes with ultrahigh spatiotemporal resolution.
2018-01-11T14:53:55ZWinkler, Pamina M.Regmi, RajuFlauraud, ValentinBrugger, JürgenRigneault, HervéWenger, JérômeGarcia-Parajo, Maria F.Nanoscale membrane assemblies of sphingolipids, cholesterol, and certain proteins, also known as lipid rafts, play a crucial role in facilitating a broad range of important cell functions. Whereas on living cell membranes lipid rafts have been postulated to have nanoscopic dimensions and to be highly transient, the existence of a similar type of dynamic nanodomains in multicomponent lipid bilayers has been questioned. Here, we perform fluorescence correlation spectroscopy on planar plasmonic antenna arrays with different nanogap sizes to assess the dynamic nanoscale organization of mimetic biological membranes. Our approach takes advantage of the highly enhanced and confined excitation light provided by the nanoantennas together with their outstanding planarity to investigate membrane regions as small as 10 nm in size with microsecond time resolution. Our diffusion data are consistent with the coexistence of transient nanoscopic domains in both the liquid-ordered and the liquid-disordered microscopic phases of multicomponent lipid bilayers. These nanodomains have characteristic residence times between 30 and 150 μs and sizes around 10 nm, as inferred from the diffusion data. Thus, although microscale phase separation occurs on mimetic membranes, nanoscopic domains also coexist, suggesting that these transient assemblies might be similar to those occurring in living cells, which in the absence of raft-stabilizing proteins are poised to be short-lived. Importantly, our work underscores the high potential of photonic nanoantennas to interrogate the nanoscale heterogeneity of native biological membranes with ultrahigh spatiotemporal resolution.Sphingolipid metabolic flow controls phosphoinositide turnover at the trans Golgi networkCapasso, SerenaSticco, LuciaRizzo, RiccardoPirozzi, MarinellaRusso, DomenicoDathan, Nina A.Campelo, FelixGalen, Josse vanHölttä-Vuori, MaaritTuracchio, GabrieleHausser, AngelikaMalhotra, VivekRiezman, IsabelleRiezman, HowardIkonen, ElinaLuberto, ChiaraParashuraman, SeetharamanLuini, AlbertoAngelo, Giovanni Dhttp://hdl.handle.net/2117/1044992020-07-23T23:32:20Z2017-05-16T11:01:33ZSphingolipid metabolic flow controls phosphoinositide turnover at the trans Golgi network
Capasso, Serena; Sticco, Lucia; Rizzo, Riccardo; Pirozzi, Marinella; Russo, Domenico; Dathan, Nina A.; Campelo, Felix; Galen, Josse van; Hölttä-Vuori, Maarit; Turacchio, Gabriele; Hausser, Angelika; Malhotra, Vivek; Riezman, Isabelle; Riezman, Howard; Ikonen, Elina; Luberto, Chiara; Parashuraman, Seetharaman; Luini, Alberto; Angelo, Giovanni D
Sphingolipids are membrane lipids, which are globally required for eukaryotic life.
Sphingolipid composition varies among endomembranes with pre- and post-Golgi
compartments being poor and rich in sphingolipids, respectively. Thanks to this different
sphingolipid content, pre- and post-Golgi membranes serve different cellular functions.
Nevertheless, how subcellular sphingolipid levels are maintained in spite of trafficking and
metabolic fluxes is only partially understood. Here we describe a homeostatic control
circuit that controls sphingolipid levels at the trans Golgi network. Specifically, we show
that sphingomyelin production at the trans Golgi network triggers a signalling reaction
leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol, and
sphingolipid transport to the trans Golgi network, PtdIns(4)P consumption leads to the
interruption of this transport in response to excessive sphingomyelin production. Based on
this evidence we envisage a model where this homeostatic circuit maintains the lipid
composition of trans Golgi network and thus of post-Golgi compartments constant, against
instant fluctuations in the sphingolipid biosynthetic flow.
2017-05-16T11:01:33ZCapasso, SerenaSticco, LuciaRizzo, RiccardoPirozzi, MarinellaRusso, DomenicoDathan, Nina A.Campelo, FelixGalen, Josse vanHölttä-Vuori, MaaritTuracchio, GabrieleHausser, AngelikaMalhotra, VivekRiezman, IsabelleRiezman, HowardIkonen, ElinaLuberto, ChiaraParashuraman, SeetharamanLuini, AlbertoAngelo, Giovanni DSphingolipids are membrane lipids, which are globally required for eukaryotic life.
Sphingolipid composition varies among endomembranes with pre- and post-Golgi
compartments being poor and rich in sphingolipids, respectively. Thanks to this different
sphingolipid content, pre- and post-Golgi membranes serve different cellular functions.
Nevertheless, how subcellular sphingolipid levels are maintained in spite of trafficking and
metabolic fluxes is only partially understood. Here we describe a homeostatic control
circuit that controls sphingolipid levels at the trans Golgi network. Specifically, we show
that sphingomyelin production at the trans Golgi network triggers a signalling reaction
leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol, and
sphingolipid transport to the trans Golgi network, PtdIns(4)P consumption leads to the
interruption of this transport in response to excessive sphingomyelin production. Based on
this evidence we envisage a model where this homeostatic circuit maintains the lipid
composition of trans Golgi network and thus of post-Golgi compartments constant, against
instant fluctuations in the sphingolipid biosynthetic flow.Sphingomyelin metabolism controls the shape and function of the Golgi cisternaeCampelo, FelixGalen, Josse vanTuracchio, GabrieleParashuraman, SeetharamanKozlov, Michael M.Garcia-Parajo, Maria F.Malhotra, Vivekhttp://hdl.handle.net/2117/1044542022-05-17T10:14:52Z2017-05-15T14:50:57ZSphingomyelin metabolism controls the shape and function of the Golgi cisternae
Campelo, Felix; Galen, Josse van; Turacchio, Gabriele; Parashuraman, Seetharaman; Kozlov, Michael M.; Garcia-Parajo, Maria F.; Malhotra, Vivek
The flat Golgi cisterna is a highly conserved feature of eukaryotic cells, but how is this morphology achieved and is it related to its function in cargo sorting and export? A physical model of cisterna morphology led us to propose that sphingomyelin (SM) metabolism at the trans-Golgi membranes in mammalian cells essentially controls the structural features of a Golgi cisterna by regulating its association to curvature-generating proteins. An experimental test of this hypothesis revealed that affecting SM homeostasis converted flat cisternae into highly curled membranes with a concomitant dissociation of membrane curvature-generating proteins. These data lend support to our hypothesis that SM metabolism controls the structural organization of a Golgi cisterna. Together with our previously presented role of SM in controlling the location of proteins involved in glycosylation and vesicle formation, our data reveal the significance of SM metabolism in the structural organization and function of Golgi cisternae.
2017-05-15T14:50:57ZCampelo, FelixGalen, Josse vanTuracchio, GabrieleParashuraman, SeetharamanKozlov, Michael M.Garcia-Parajo, Maria F.Malhotra, VivekThe flat Golgi cisterna is a highly conserved feature of eukaryotic cells, but how is this morphology achieved and is it related to its function in cargo sorting and export? A physical model of cisterna morphology led us to propose that sphingomyelin (SM) metabolism at the trans-Golgi membranes in mammalian cells essentially controls the structural features of a Golgi cisterna by regulating its association to curvature-generating proteins. An experimental test of this hypothesis revealed that affecting SM homeostasis converted flat cisternae into highly curled membranes with a concomitant dissociation of membrane curvature-generating proteins. These data lend support to our hypothesis that SM metabolism controls the structural organization of a Golgi cisterna. Together with our previously presented role of SM in controlling the location of proteins involved in glycosylation and vesicle formation, our data reveal the significance of SM metabolism in the structural organization and function of Golgi cisternae.In-plane plasmonic antenna arrays with surface nanogaps for giant fluorescence enhancementFlauraud, ValentinRegmi, RajuWinkler, Pamina MartinaAlexander, Duncan T. L.Rigneault, HerveHulst, Niek F. vanGarcia-Parajo, Maria F.Wenger, JeromeBrugger, Juergenhttp://hdl.handle.net/2117/1008392022-05-17T11:29:01Z2017-02-10T12:14:28ZIn-plane plasmonic antenna arrays with surface nanogaps for giant fluorescence enhancement
Flauraud, Valentin; Regmi, Raju; Winkler, Pamina Martina; Alexander, Duncan T. L.; Rigneault, Herve; Hulst, Niek F. van; Garcia-Parajo, Maria F.; Wenger, Jerome; Brugger, Juergen
Optical nanoantennas have a great potential for enhancing light-matter interactions at the
nanometer scale, yet fabrication accuracy and lack of scalability currently limit ultimate
antenna performance and applications. In most designs, the region of maximum field
localization and enhancement (i.e., hotspot) is not readily accessible to the sample since it is
buried into the nanostructure. Moreover, current large-scale fabrication techniques lack
reproducible geometrical control below 20 nm. Here, we describe a new nanofabrication
technique that applies planarization, etch back and template stripping to expose the excitation
hotspot at the surface, providing a major improvement over conventional electron beam
lithography methods. We present large flat surface arrays of in-plane nanoantennas, featuring
gaps as small as 10 nm with sharp edges, excellent reproducibility and full surface
accessibility of the hotspot confined region. The novel fabrication approach drastically
improves the optical performance of plasmonic nanoantennas to yield giant fluorescence
enhancement factors up to 104-105 times, together with nanoscale detection volumes in the 20
zeptoliter range. The method is fully scalable and adaptable to a wide range of antenna
designs. We foresee broad applications by the use of these in-plane antenna geometries
ranging from large-scale ultra-sensitive sensor chips, to microfluidics and live cell membrane
investigations.
2017-02-10T12:14:28ZFlauraud, ValentinRegmi, RajuWinkler, Pamina MartinaAlexander, Duncan T. L.Rigneault, HerveHulst, Niek F. vanGarcia-Parajo, Maria F.Wenger, JeromeBrugger, JuergenOptical nanoantennas have a great potential for enhancing light-matter interactions at the
nanometer scale, yet fabrication accuracy and lack of scalability currently limit ultimate
antenna performance and applications. In most designs, the region of maximum field
localization and enhancement (i.e., hotspot) is not readily accessible to the sample since it is
buried into the nanostructure. Moreover, current large-scale fabrication techniques lack
reproducible geometrical control below 20 nm. Here, we describe a new nanofabrication
technique that applies planarization, etch back and template stripping to expose the excitation
hotspot at the surface, providing a major improvement over conventional electron beam
lithography methods. We present large flat surface arrays of in-plane nanoantennas, featuring
gaps as small as 10 nm with sharp edges, excellent reproducibility and full surface
accessibility of the hotspot confined region. The novel fabrication approach drastically
improves the optical performance of plasmonic nanoantennas to yield giant fluorescence
enhancement factors up to 104-105 times, together with nanoscale detection volumes in the 20
zeptoliter range. The method is fully scalable and adaptable to a wide range of antenna
designs. We foresee broad applications by the use of these in-plane antenna geometries
ranging from large-scale ultra-sensitive sensor chips, to microfluidics and live cell membrane
investigations.