Exploring plant endomembrane dynamics using the photoconvertible protein Kaede.
Photoactivatable and photoconvertible fluorescent proteins capable of pronounced light-induced spectral changes are a powerful addition to the fluorescent protein toolbox of the cell biologist. They permit specific tracking of one subcellular structure (organelle or cell subdomain) within a differentially labelled population. They also enable pulse-chase analysis of protein traffic. The Kaede gene codes for a tetrameric protein found in the stony coral Trachyphyllia geoffroyi, which emits green fluorescence that irreversibly shifts to red following radiation with UV or violet light. We report here the use of Kaede to explore the plant secretory pathway. Kaede versions of the Golgi marker sialyl-transferase (ST-Kaede) and of the vacuolar pathway marker cardosin A (cardA-Kaede) were engineered. Several optical devices enabling photoconversion and observation of Kaede using these two constructs were assessed to optimize Kaede-based imaging protocols. Photoconverted ST-Kaede red-labelled organelles can be followed within neighbouring populations of non-converted green Golgi stacks, by their gradual development of orange/yellow coloration from de novo synthesis of Golgi proteins (green). Results highlight some aspects on the dynamics of the plant Golgi. For plant bio-imaging, the photoconvertible Kaede offers a powerful tool to track the dynamic behaviour of designated subpopulations of Golgi within living cells, while visualizing the de novo formation of proteins and structures, such as a Golgi stack.
Brown
Spencer C
SC
Laboratoire Dynamique de la Compartimentation Cellulaire, CNRS, Institut des Sciences du Végétal, Centre de recherche de Gif (FRC3115), 91198, Gif-sur-Yvette Cedex, France.
Bolte
Susanne
S
Gaudin
Marie
M
Pereira
Claudia
C
Marion
Jessica
J
Soler
Marie-Noëlle
MN
Satiat-Jeunemaitre
Béatrice
B
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism; D020126:Brefeldin A / Q000494:pharmacology; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D007834:Lasers; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D056004:Molecular Dynamics Simulation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000528:radiation effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology
C513582:Kaede protein, Trachyphyllia geoffroyi; D008164:Luminescent Proteins; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D012799:Sialyltransferases; D016282:Aspartic Acid Endopeptidases; C097455:aspartic proteinases, Cynara cardunculus