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There exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin …


Biology Articles » Cell biology » Actin polymerisation at the cytoplasmic face of eukaryotic nuclei » Methods

Methods
- Actin polymerisation at the cytoplasmic face of eukaryotic nuclei

Cell lines and cell culture

HeLa (ATCC) were cultured in complete DMEM (DMEM Glutamax (Gibco BRL), 10% FCS, 100 U ml-1 streptomycin, 100 μg ml-1 penicillin) at 37°C; 5% CO2. H-PomGFP cells: nuclear pore labelling in HeLa cells was achieved by transfecting cells (Fugene6, Boehringer-Mannheim) with a plasmid containing POM121-GFP (kind gift of E. Hallberg, University College, Huddinge). Citrin-actin (kind gift of A. Hoppe, University of Michigan) was expressed where stated in HeLa cells using the same transfection method. Citrin-actin is a fusion construct of actin with an improved YFP variant [36]; the plasmid used in the current study has been described elsewhere [24].

Constructing HeLa cells stably expressing LaminA-dsRed

Prelamin A cDNA cloned into pSVK3 plasmid was amplified by PCR, with an EcoRI restriction site engineered at the 5' end of the sense primer (5'-GCG AAT TCT ATG GAG ACC CCG TCC CAG CGG-3') and a KpnI restriction site engineered at the 5' end of the antisense primer (5'-GC GGT ACC TTA CAT GAT GCT GCA GTT CTG-3') (as described in [37]). The reaction product was digested with EcoRI and KpnI and ligated in frame into purified, linearised pDsRed-C1 (Clontech, Inc, Palo Alto, CA) after digestion with EcoRI and KpnI. HeLa cells cultured to 80% confluency were transfected in chamber slides with Lipofectamine PLUS (Life Technologies, Inc., Gaithersburg, USA.), following the manufacturer's instructions. Stable transfected cells were selected in the presence of antibiotic (1 mg ml-1 G418; Gibco Life Technologies Ltd). Selection of dsRed-positive cells was performed with FACS (fluorescence-activated cell sorter) analysis.

Actin-binding drug treatment of HeLa cells

HeLa cells were grown on cover-slides and treated for 30 minutes with cytochalasin D 1μM (Sigma), latrunculin A 1μM (Molecular Probes) or jasplakinolide 200 nM (Molecular Probes) at 37°C. Then cells were washed with PBS, fixed for 5 minutes with 3%PFA at RT, stained with Hoechst 33342 (Sigma-Aldrich) (10 min, 0.001 mg ml-1) and mounted on slides. The nuclei were imaged with a widefield microscope (see below) and nuclear shape was evaluated and counted in two independent experiments. For each experimental condition at least 150 nuclei from at least ten microscopic fields were counted.

In vitro actin polymerisation assay on isolated rat liver nuclei

Nuclei were purified from rat liver [38]: Rats were asphyxiated with CO2, and were then decapitated. The livers were isolated, placed into ice-cold low sucrose buffer (0.25 M sucrose, 50 mM Tris-HCl pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5 M PMSF and 1 μg ml-1 leupeptin/aprotinin/pepstatin) and minced. Then, 1 volume of minced liver and 2 volumes of the same buffer were homogenised in a Potter-Elvehjem homogeniser (0.025 cm clearance) for 15 full strokes at 1,700 rpm. The homogenate was filtered through 4 layers of cheese cloth and centrifuged at 800 g for 15 minutes at 4°C. 1 volume of loose pellet was mixed with 2 volumes of high sucrose buffer (2.3 M sucrose, 50 mM Tris-HCl pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5 M PMSF and 1 μg ml-1 leupeptin/aprotinin/pepstatin) before overlaying 1 part of high sucrose buffer with 5 parts of the initial mixture and ultra-centrifuged at 28000 rpm for 1 hour at 4°C in a SW-28 rotor (Beckman, USA). The nuclei containing pellet was re-suspended in the low sucrose buffer and was again homogenised with 2 full strokes as described above. This homogenate was centrifuged at 800 g for 15 minutes at 4°C and the pellet contained the highly purified rat liver nuclei. These nuclei were re-suspended in the low sucrose buffer, and aliquots were stored at-80 °C. The number of nuclei was determined by measuring the OD260 (OD260 = 1 equals 3 × 106 nuclei). The quality and entireness of isolated nuclei was assured by light microscopy.

Fluorescent actin-Alexa488 (Molecular Probes) in G-buffer (0.2 mM ATP, 0.2 mM CaCl2, 1 mM DTT, 10 mM Tris-Hcl, pH 8) was polymerised at 0.5 μM by addition of a 1:9 dilution of 10x KMEI (500 mM KCl, 10 mM MgCl2, 10 mM EGTA, 100 mM imidazole, pH 7) in the presence of 1 unit of isolated rat liver nuclei (~3 × 106 nuclei). Isolated nuclei were pre-incubated with antibodies overnight at 4°C with continuous, gentle agitation. Antibodies used in this assay were mAb414 (Eurogentec, 1/1000), mAb anti-Nuance (kind gift of A. Noegel, University of Cologne; 1/10), anti-Emerin (kind gift of H. Hermann, DKFZ Heidelberg; 1/200) and mAb anti c-Myc (9E10 Santa-Cruz Biotechnologies, 1/1000). Additionally, we incorporated the non-specific nuclear transport inhibitor wheatgerm agglutinin (Invitrogen) in this assay. An aliquot of the reaction mix was sealed under a glass cover slip (coated with 2% cold water fish skin gelatin) on a microscope slide and then incubated for 1 hour at 37°C to allow time for actin polymerisation to occur. Finally, the number of nuclei displaying actin microfilaments was counted using a widefield microscope.

Microinjection

Single cells were cytoplasmically microinjected with either 10 μM actin-Alexa568 (Molecular Probes) or 2.5 mg ml-1 dextran-rhodamine 70 kDa (Molecular Probes) using a Femtojet/Injectman micromanipulator (Eppendorf, Hamburg, Germany).

CD-BODIPY in vitro measurements

Purified, native and fluorescent (FITC) conjugated rabbit skeletal muscle actin (Cytoskeleton, Denver, U.S.A.) in 2 μl stock aliquots (10 mg ml-1; 222 μM) containing 2 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT, 2% sucrose, and 0.005% NaN3 (pH 8) were prepared at working dilutions (0.5–0.75 mg ml-1; 11–17 μM) by addition of low salt buffer (5 mM Tris-HCl, 0.2 mM Na-ATP, 0.5 mM DTT, 0.2 mM CaCl2, pH 8). For each experiment a single fresh actin stock was prepared. Drugs were added, yielding the final concentrations stated. Samples were vigorously mixed by pipetting, vortexed and then left on ice for 1 hour before centrifugation (100,000x g, 4°C, 30 minutes). Supernatants were added to 5x concentrated high salt polymerisation buffer giving final concentrations of 100 mM KCl and 10 mM Mg-ATP. After mixing, a 5 μl aliquot was sealed under a glass cover slip on a microscope slide and incubated at 37°C for at least 3 hours to allow actin to fully polymerise to a steady state [17] before semi-quantitative observation using fluorescence microscopy. Each experiment (triplicate samples) was performed at least twice yielding comparable results.

Image acquisition, processing, and analysis

For live cell imaging, cells cultured on 35 mm glass bottom dishes (MatTek Corporation, USA) were exchanged into OptiMEM (Gibco BRL), and transfered to the microscope stage that was maintained at 37°C (Carl Zeiss "Tempcontrol 37–2 digital" incubator support). For experiments using CD-BODIPY, experimental medium was freshly prepared by serial dilution of a 1 mM stock solution (DMSO) to the stated final concentrations (2–50 nM). Facile and sufficient staining of cells was achieved by incubation of cells in this solution for 20 minutes before recordings began. In the presence of such very low concentrations, we found that cells could be maintained for several hours, or even days, without any deleterious effects. Using bright-field observation, we routinely ensured that observed cells were flat, adherent and healthy. Fluorescent imaging was performed using a high-speed spinning disk confocal system (UltraView RS Perkin-Elmer, USA) equipped for dual wavelength excitation (Kr-Ar laser; 488 nm and 568 nm) and axial z-stack sampling (PI objective piezo-drive). The system was built around a Carl Zeiss (Germany) Axiovert 200 microscope. Cells were visualised using a Hamamatsu (Japan) ERII camera (exposure time 200 ms) using a 100x NeoFluor NA 1.3 oil-immersion objective (Carl Zeiss). Fast axial (through-stack) sampling used a piezo-objective drive stepping at 200 nm throughout the cell volume.

For analysis, all axial image datasets were treated identically. First using geometric light reconstruction (deconvolution) based on a calculated point-spread-function, followed by a "maximum likelihood estimate" (MLE) iterative algorithm (Huygens software; Scientific Volume Imaging, Netherlands), in "Nipkow-disk" mode with parameters for the Yokagawa CSU-22 scan head. The deconvolved 3D through-stack results were then analysed for signal coincidence using the "Coloc" plug-in of Imaris (Bitplane, Switzerland). For all results we set a threshold based on the averaged background signal detected outside the region of interest in the channel used for NE-membrane detection (POM121-GFP, or dsRed-Lamin), such that the threshold identified a binary mask region delineating the NE-membrane signal. The second channel (Alexa568-actin, citrin-actin, or CD-BODIPY-TMR) was then similarly thresholded and masked. Quantification of signal co-incidence between two channels was then measured as a function of the percentage of pixels detected in the NE-membrane signal channel that were also positive for binary masked pixels derived from the actin dependent signal. This quantification process was applied to each image in any given stack, yielding an estimate of three dimensional signal co-incidence in a given cell. As a systematic control for this 3D signal coincidence we used exactly the same acquisition and analysis protocol except H-PomGFP cells were microinjected cytoplasmically with red fluorescent Alexa568-dextran. This provided us a relative measure of the microscope's ability to distinguish the NE-membrane (green) from diffusible non-specific cytoplasmic signal (red). On average (n = 5), throughout the imaged 3D volume we found that just ~12% of pixels containing specifically localised NE-membrane signal were spatially coincident with pixels also positive for red dextran signal. Given that the dimensions of the NE-membrane were below the optical resolution limits of the microscope, and that all two-color, three-dimensional analysis used the same imaging hardware/analysis this value served as a systematic threshold limit below which the NE-membrane and cytoplasmic signals could be considered "non-coincident".

Images of isolated intact nuclei for the in vitro actin polymerisation assay used a standard Zeiss Axiovert 200 M microscope equipped with a 40x objective, NA 1.3. The microscope was controlled by Till Vision (Till photonics, Germany) designed for fast live cell imaging and equipped with a xenon lamp, polychromator (PolychromeIV) for wavelength selection and a Till Photonic Imago QE CCD camera.


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