Construction of expression constructs
Wild-type Arx cDNA was obtained from K. Kitamura (Institute of Life Sciences, Tokyo, Japan). Expansion of the first polyA tract was achieved by adding a short oligonucleotide duplex made by annealing two primers: 5′-ggccgctgctgctgctgctgccgc-3′ and 5′-ggccgcggcagcagcagcagcagc-3′ (IDT) into a unique NotI site within the region coding for the polyA tract. Wild-type and expanded Arx were individually subcloned in frame with 5′FLAG-, 5′Myc-, 5′GFP- and 3′V5-tagged expression constructs (pCMV-Tag2A (Stratagene), pCMV-Myc and pEGFP-C3 (CLONTECH Laboratories, Inc.), and pCDNA3.1-V5-His (Invitrogen), respectively; a). To generate the 3′deleted constructs, Arx cDNA was digested with SalI to remove the 203 3′ bp of the 241-bp exon 5, including the aristaless domain ( a). Hsp70 cDNA was provided by N. Bonini (University of Pennsylvania, Philadelphia, PA) and cloned into pCDNA3.1.
Cell culture and video microscopy
COS and 293T cells were maintained in DME with 10% FBS and transfected using Fugene6 (Roche) and 500 ng of plasmid DNA. The cells were acid-alcohol fixed 2 d after transfection.
For time-lapse video microscopy, COS cells transfected with GFP-tagged constructs were placed on a Nikon TE-300 microscope equipped with a deltaT4 environmental chamber (Bioptechs) and a motorized stage (Prior) 6–18 h after transfection. A Nikon Plan Fluor ELWD 40×/0.6 lens was used for image acquisition. Using a CCD camera (Hamamatsu) controlled by Phase 3 Imaging Software (Media Cybernetics), fields were selected at random and fluorescent images were acquired every 15 min for 12–24 h. Phase 3 Imaging software compiled images into AVI files which were converted to MOV files in Quicktime Player 6.5.1.
Immunocytochemistry, TUNEL, and immunohistochemistry
Fixed cells were blocked with 10% normal goat serum (Sigma-Aldrich) in PBS and incubated with primary antibody. Primary antibodies included: mouse anti-FLAG M2 1:250 (Sigma-Aldrich), mouse anti-Hsp70 (1:300; StressGen Biotechnologies), mouse anti-myc 1:300 (9E10), and mouse anti-ubiquitin 1:500 (CHEMICON International Inc.). Secondary antibodies were species-appropriate FITC- or Texas red–conjugated raised in goat (1:150; Jackson ImmunoResearch Laboratories). Nuclei were counterstained with DAPI (Molecular Probes). The percent of transfected cells with inclusions was determined as the number of cells containing inclusions divided by the total number of FLAG-positive cells.
TUNEL staining was performed as described previously (Minarcik and Golden, 2003
). In brief, fixed COS cells were blocked and incubated with TdT enzyme and UTP-biotin. Incorporation of UTP-biotin was labeled with streptavidin-Cy3 (Jackson ImmunoResearch Laboratories), and samples were immunostained as above. The percent of TUNEL-positivity in transfected cells was normalized to the percent of TUNEL-positivity in untransfected cells in order to control for the background rate of cell death for each transfection.
For immunohistochemistry of fixed embryonic brain slices, samples were blocked in PBS with 5% normal goat serum, 0.05% sodium azide, and 0.3% Triton X-100. They were incubated overnight at 4°C in primary antibody diluted in block, then incubated with secondary antibody in PBS. Nuclei were stained with DAPI.
Images were acquired on a DMR microscope (Leica) equipped with epifluorescence and an ORCA-ER CCD camera (Hamamatsu) connected to a Macintosh G4 running Openlab 3.1.5. The images were acquired using a Plan APO 40×/0.75 objective (Leica). Images were imported into Adobe Photoshop 7.0 and saved as TIF files. Some images were imported into Adobe Illustrator 10.0, labeled, and saved as EPS files.
Protein was extracted from cells using lysis buffer containing 25 mM Tris, pH 7.6, 1 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 1% PMSF, 50 μg/ml antipain, 2 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml peptastatin. The extract was homogenized using a 22-g needle and soluble protein was quantified with a BCA kit (Pierce Chemical Co.). These samples were then run on a 10% acrylamide SDS-PAGE gel with a 6% stacking gel. The separated proteins, including the stacking gel, were transferred to Immobilon membranes (Millipore). The membranes were incubated in block (5% NFDM, 0.1% Tween-20) followed by primary antibodies: anti-FLAG (1:1,000; Sigma-Aldrich) and anti-GAPDH (1:7500; CHEMICON International, Inc.) and HRP-conjugated secondary antibody (1:1,000; Amersham Biosciences). The blots were incubated in ECL buffer (Amersham Biosciences) and exposed to Kodak film. Quantification was performed with ImageJ.
Mouse brain electroporation
Whole brains from embryonic day 14.5–15 outbred CD1 mice were embedded in 4% low melting point agarose (Fisher Scientific). DNA was injected into one telencephalic ventricle and electroporated with three pulses of 40–60 mV using an ECM 830 electroporator and gold “Gene Paddle” electrodes (BTX). The brains were then cut at 250 μm on a vibrating microtome (model VT1000E; Leica). Brain slices were transferred to Millicell-CM filters (Millipore) and fed serum-containing medium for 2 h (1:1 DME/F12, Pen/Strep, 6.5 μM glucose, 10% FCS). Cultures were then grown for 2 d in serum-free medium (DME, Pen/Strep, 6.5 μM glucose, N2 supplement).
Online supplemental material
The dynamics of intranuclear inclusion formation were investigated by time-lapse video microscopy (Materials and methods). Mutant protein was first expressed throughout the nucleus at low levels followed by aggregation into dense inclusions. The development of inclusions was found to require many hours of mutant protein expression. Interestingly, the inclusions were dynamic, moving within the nucleus and able to form and disassemble. Video 1 is available at http://www.jcb.org/cgi/content/full/jcb.200408091/DC1