Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy.
Combinations of molecular tags visible in light and electron microscopes become particularly advantageous in the analysis of dynamic cellular components like the Golgi apparatus. This organelle disassembles at the onset of mitosis and, after a sequence of poorly understood events, reassembles after cytokinesis. The precise location of Golgi membranes and resident proteins during mitosis remains unclear, partly due to limitations of molecular markers and the resolution of light microscopy. We generated a fusion consisting of the first 117 residues of alpha-mannosidase II tagged with a fluorescent protein and a tetracysteine motif. The mannosidase component guarantees docking into the Golgi membrane, with the tags exposed in the lumen. The fluorescent protein is optically visible without further treatment, whereas the tetracysteine tag can be reduced acutely with a membrane-permeant phosphine, labeled with ReAsH, monitored in the light microscope, and used to trigger the photoconversion of diaminobenzidine, allowing 4D optical recording on live cells and correlated ultrastructural analysis by electron microscopy. These methods reveal that Golgi reassembly is preceded by the formation of four colinear clusters at telophase, two per daughter cell. Within each daughter, the smaller cluster near the midbody gradually migrates to rejoin the major cluster on the far side of the nucleus and asymmetrically reconstitutes a single Golgi apparatus, first in one daughter cell and then in the other. Our studies provide previously undescribed insights into Golgi disassociation and reassembly during mitosis and offer a powerful approach to follow recombinant protein distribution in 4D imaging and correlated high-resolution analysis.
Gaietta
Guido M
GM
National Center for Microscopy and Imaging Research, Center for Research on Biological Structure, University of California at San Diego, La Jolla, CA 92093, USA.
Giepmans
Ben N G
BN
Deerinck
Thomas J
TJ
Smith
W Bryan
WB
Ngan
Lucy
L
Llopis
Juan
J
Adams
Stephen R
SR
Tsien
Roger Y
RY
Ellisman
Mark H
MH
D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron / Q000295:instrumentation / Q000379:methods*; D008938:Mitosis / Q000502:physiology*; D010084:Oxidation-Reduction; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D043323:alpha-Mannosidase / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D007527:Isoenzymes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D043323:alpha-Mannosidase; D003545:Cysteine