Technical Reference #288

288.

Rapid; Endoplasmic Reticulum-independent Diffusion of the Mitotic Golgi Haze Magnus A. B. Axelsson and Graham Warren, Yale University, Molecular Biology of the Cell, 15(288), (2004)
Link To Paper

Abstract:
Early in mitosis the mammalian Golgi apparatus disassembles and fluorescence microscopy reveals Golgi clusters andan extensive nonresolvable haze that either represents scattered vesicles or a merged endoplasmic reticulum (ER)-Golgicompartment. To

Materials & Methods:
Chemicals The following working concentrations (and stock solutions) were used: BFA (Epicenter Technologies Madison WI) 5 g/ml (5 mg/ml in ethanol); filipin III (Sigma-Aldrich St. Louis MO) 5 g/ml (10 mg/ml in methanol); BODIPY FL ceramide (Molecular Probes Eugene OR) 5 M (5 mM in methanol) and ilimaquinone (IQ; Sigma-Aldrich) 25 M (5 mg/ml in dimethyl sulfoxide). BODIPY ceramide was used as described by Sciaky et al. (1997) and ilimaquinone as described by Takizawa et al. (1993). Plasmid Constructs The plasmid encoding GalNAc-T2-YFP a yellow fluorescent protein (YFP) version of GalNAc-T2-fluorescent protein (Storrie et al. 1998; White et al. 2001) was obtained from Dr. J. White (European Molecular Biology Laboratory Heidelberg Germany). It consists of the stalk region of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) sufficient for correct Golgi localization (Storrie et al. 1998 and references therein) inserted into the vector pEYFP-N1 (BD Biosciences Clontech Palo Alto CA). The cyan fluorescent protein (CFP) version of Sec61 (VLP 25; Rolls et al. 1999) was obtained from Dr. M. Rolls (Harvard Medical School Boston MA). CFPSec61 -Hygro was generated by the ligation of a SnaBI-NotI fragment from this construct into the SnaBI-NotI digested vector pcDNA3.1/Hygro() (Invitrogen Carlsbad CA). Cell Culture and Transfection BS-C-1 cells (ATCC CCL 26) were routinely cultured in minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum (Gemini Irvine CA) 100 U/ml penicillin and 100 g/ml streptomycin (Invitrogen) and 2 mM l-glutamine (Invitrogen). Transfection (plasmids prepared using EndoFree maxi kit; QIAGEN Valencia CA) was performed using the Super- Fect transfection reagent (QIAGEN) according to the manufacturer’s protocol. Cells were transferred to selective medium after 24 h and after 2–3 wk colonies were picked by repetitive pipetting of trypsin and expanded. Single stable GalNAc-T2-YFP transfectants were first selected in geneticin (G-418 sulfate 800 g/ml; Invitrogen) an appropriate clone was further transfected with CFP-Sec61 -Hygro and double-stable transfectants were then selected and maintained using Hygromycin B (130 g/ml; Calbiochem San Diego CA) in addition to geneticin. Confocal Microscopy and Quantitative FRAP Microscopy was performed in phenol red-free minimum essential medium (Invitrogen) with 10% fetal bovine serum 2 mM l-glutamine and 25 mM HEPES buffer pH 7.3 on 60% confluent cells in 0.17-mm glass bottom petri dishes (MatTek Ashland MA). A 37°C environment was maintained using a stage heater (Carl Zeiss Thornwood NY) except during filipin III treatment and the following imaging or FRAP which were performed at room temperature. Single confocal planes were collected with an inverted laser scanning confocal microscope (LSM510; Carl Zeiss) by using 458-nm laser excitation for CFP and 514 nm for YFP and BODIPY ceramide a C-Apochromat 40 objective lens 6 zoom and a pinhole equivalent to one Airy disk diameter. CFP YFP and BODIPY ceramide were imaged together by using 458- and 514-nm laser excitations and the META detector system in the range 467–606 nm with 10.7-nm steps. Signals were separated based on reference spectra from live cells expressing only CFP or YFP or wild-type cells stained with BODIPY ceramide. All imaging and FRAP of BODIPY ceramide was performed within 30–60 min after the dye was washed away and the cells were returned to 37°C as described by Sciaky et al. (1997). Photobleaching was performed using high laser intensity (100% transmission two scans for BODIPY ceramide 20 for YFP and 50 for CFP) on a 2.5- by 2.5-m region (selected to be free of resolvable MGCs when viewing Golgi). The FRAP was monitored by continuous scanning of the whole cell at low transmission and values were obtained from the bleached area and an unbleached control area by using the LSM510 software (Carl Zeiss). Values were corrected for background using a cell-free region of the field of view and values from the bleached area were compensated for bleaching during monitoring by using values from the control area. As for BODIPY ceramide the monitoring affected ER and Golgi differently and bleached and control areas were selected to represent the same organelle as judged by positional criteria. Values are given as percentage of the prebleach values which are averages of two measurements.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
BS-C-1