Use of fluorescent sphingolipid analogs to study lipid transport along the endocytic pathway.
Sphingolipids (SLs) are concentrated at the plasma membrane where they play important roles in cell-cell communication, host-pathogen interactions, and cell signaling events. Our laboratory has used fluorescent SL analogs and SL-binding toxins to elucidate mechanisms by which SLs are internalized by endocytosis and subsequently sorted and transported to various intracellular compartments. These studies have relied on the use of temperature shift protocols, co-localization studies with compartment-specific markers, selective biochemical treatments that inhibit specific endocytic mechanisms, and the expression of dominant negative proteins (e.g., rabs) that block specific steps in transport. These methods are presented here so that they can be utilized by others for the study of endocytic trafficking of lipids and other molecules.
Marks
David L
DL
Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Singh
Raman Deep
RD
Choudhury
Amit
A
Wheatley
Christine L
CL
Pagano
Richard E
RE
D001692:Biological Transport; D001896:Boron Compounds / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D003470:Culture Media; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D008956:Models, Chemical; D011485:Protein Binding; D021381:Protein Transport; D012680:Sensitivity and Specificity; D013107:Sphingolipids / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors; D020761:rab1 GTP-Binding Proteins / Q000378:metabolism
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D003470:Culture Media; D008055:Lipids; D013107:Sphingolipids; D049452:Green Fluorescent Proteins; D020761:rab1 GTP-Binding Proteins