ABSTRACT
The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.
Subject(s)
Secretory Vesicles/metabolism , Sphingolipids/metabolism , Sterols/metabolism , trans-Golgi Network/metabolism , Biological Transport , Cell Membrane/metabolism , Ergosterol/metabolism , Membrane Lipids/metabolism , Membrane Microdomains/physiology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructureABSTRACT
Microtubule behavior changes during the cell cycle and during spindle assembly. However, it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes Regulator of Spindle Assembly 1 (RSA-1), a targeting subunit for PP2A, and RSA-2, a protein that binds and recruits RSA-1 to centrosomes. In contrast to the multiple functions of the PP2A catalytic subunit, RSA-1 and RSA-2 are specifically required for microtubule outgrowth from centrosomes and for spindle assembly. The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors: the microtubule destabilizer KLP-7 and the C. elegans regulator of spindle assembly TPXL-1. By regulating a subset of PP2A functions at the centrosome, the RSA complex could therefore provide a means of coordinating microtubule outgrowth from centrosomes and kinetochore microtubule stability during mitotic spindle assembly.
Subject(s)
Caenorhabditis elegans/metabolism , Centrosome/metabolism , Multiprotein Complexes/metabolism , Phosphoprotein Phosphatases/metabolism , Spindle Apparatus/metabolism , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/embryology , Caenorhabditis elegans Proteins/metabolism , Carrier Proteins/metabolism , Catalysis , Dimerization , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/ultrastructure , Kinesins/metabolism , Microtubules/metabolism , Protein Binding , Protein Phosphatase 2 , Protein Subunits/metabolism , Protein TransportABSTRACT
Splitting of HOLZ lines on CBED patterns is systematically observed at the proximity of interfaces and prevents local strain measurements by monitoring of line shifts. It was previously suggested that such splitting occurs due to interface-strain relaxation in thin TEM lamella. Here we confirm this model by dynamical simulation of CBED patterns using the multislice algorithm.
