Expression Constructs and Reagents:
A full length TEM4/ARHGEF17 (Genbank accession NM_014786) clone was obtained by subcloning the first exon (PCR amplified from human genomic DNA) encoding the first 456 aa into the KIAA1337 clone (Kazusa DNA Research Institute, Japan) using the internal SalI restriction site. cDNAs encoding full length TEM4/ARHGEF17 were subsequently cloned into pEGFP (Clontech) and pcDNA3-HA vectors. Point mutations (R1215A, N1252A) in the DH domain of TEM4 were introduced by site-directed
mutagenesis. Constructs containing wild-type MILO, in addition to the YA and YE mutant MILO constructs, were fluorescently tagged at the N-terminus with mCherry to allow for monitoring of MILO expression in single cells in parallel to the biosensor imaging. To generate these constructs, genomic clone KIAA1688 was amplified by PCR and ligated into the original source vector. The full-length cDNA encoding MILO was subsequently amplified by PCR and then ligated into a pTriEx-4 vector via the HindIII restriction site within the multiple cloning site. Proper orientation was verified first by PCR screening with subsequent sequencing. The mCherry fluorescent protein was amplified by PCR from template and ligated into these vectors via the BamHI site upstream of HindIII within the multiple cloning site. Again, proper orientation was verified first by PCR screening with subsequent sequencing. The Vav2 DH/PH domain plus C-terminal regulatory domain (amino acids 184-878) was a gift from Keith
Burridge, as was full-length RhoGDI-1.
Human recombinant TNFα was obtained from R&D Systems. All growth factors were used at a final concentration of 20 ng/ml unless otherwise indicated.
Cell Culture, Transient Transfections, and siRNA Transfection:
Human umbilical vein endothelial cells (HUVECs; Clonetics) were maintained in EGM-2 medium supplemented with 10% fetal bovine serum (FBS; HyClone) on gelatin- coated dishes. HUVECs were electroporated with expression constructs or siRNAs using Amaxa Nucleofection technology according to manufacturer’s instructions. All
experiments were carried out in cells between passages 4 and 5.
HeLa cells and 293T cells were obtained from the Lineberger Comprehensive Cancer Center tissue culture facility (UNC Chapel Hill), and tet-off mouse embryonic fibroblasts (MEFs) were acquired from Clontech, Inc. (Takara Biosciences), and were cultured in DMEM (high glucose, with glutamine, Mediatech) supplemented with 10% fetal bovine serum (HyClone) and antibiotics (Mediatech).
DNA plasmids were transfected into HeLa cells using Fugene6 according to the manufacturer’s instructions. For transfection of siRNAs into HeLa cells Oligofectamine reagent (Invitrogen) was used according to the manufacturer’s instructions. Briefly, HeLa cells were seeded on coverslips at 175,000-250,000 cells per 35mm dish. The next day, the cells were transfected with siRNA as follows: 11 μl Opti-MEM was combined with 4 μl Oligofectamine. In another tube, 175 μl Optimem was combined with 10 μL of siRNA stock to yield a final concentration of 20 nM. After 5 minutes, the two samples were combined and allowed to complex for 30 minutes, whereupon the solution was added dropwise to the cells, growing in 1 mL Opti-MEM. The cells were incubated with the siRNA for 5 hours at 37 °C, and then supplemented with 1 mL of Opti-MEM with 20% FBS. Cells were used for experiments between 48 and 72 hours post-transfection.
Lentivirus Production and Cellular Transduction:
293T cells (ATCC)were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS andantibiotics. For virus production, 293T cells (plated at 5 x 106 cells perT75 flask) were transfected using a standard calcium phosphate DNA precipitation method with a target vector and the ViraPower lentiviral packaging system (Invitrogen). Supernatant containing the virus was collected 48 h post transfection and the titer was determined by infecting fresh 293T cells.
HUVECs were transduced for 4-5 h in EGM-2 medium in the absence of serum. The transduction efficiency was close to 100% as determined by observing GFP or mCherry fluorescence marker expression. HUVECs were split 24 h after transduction and used for experiments 24-48 h later.
TEM4 RNA Interference:
Short hairpin (shRNA) oligos were cloned into pLL 5.0 GFP (Cai et al. 2007) or pLL 5.0 mCherry vector. Target sequences were as following: TEM4 #3 5’-
GCACCACTCTGAAGCGAA-3’, TEM4 #5 5’-GGAAATGACATGAGGAAA-3’, The control shRNA (NS; GATCGACTTACGACGTTAT) has no exact match in the human, mouse or rat genomes (Cai et al. 2007). TEM4 shRNA #5 consistently showed a higher degree of knockdown of TEM4 protein expression (about 50%) but caused cell death (same was observed for shRNA #4 and 6). TEM4 shRNA #3 decreased TEM4 expression by about 30% and did not cause cell death and therefore was used predominantly in this study.
Biosensor Designs:
Our analyses of the relationships between Rho GTPase activation and protrusion/retraction relied on three previously published and extensively evaluated biosensors, for Rac1, Cdc42, and RhoA. The designs of these sensors are summarized in Figure 3.2A-D. To facilitate the experimental procedures, the original FLAIR biosensor reporting Rac1 activity, which requires microinjection of cells, was modified as follows: The EGFP was replaced by CyPet and the Alexa-546 on the p21-binding domain of p21- Activated Kinase 1 (PBD) was replaced by YPet. Hence, biosensor constructs could be retrovirally co-transduced into MEF/3T3. A stable cell line was produced following selection with puromycin (10 μg/ml). We were concerned that restricting experiments to a fixed, optimized donor/acceptor ratio could result in a particular localization pattern of Rac1 activation and/or a particular timing behavior with respect to the protrusion cycle. Therefore, unlike the single-chain RhoA biosensor discussed below, we did not FAC sort the cell population for specific expression profiles of acceptor and donor components, but maximized the concentration variability of the two components. We then performed control experiments, described above, to examine how the reported Rac1 activation depended on the concentration and ratio of donor and acceptor constructs. For detection of Cdc42 activity, cells were microinjected with the meroCBD biosensor. Before
imaging, cells were allowed to recover for 30 minutes. As with the Rac1 sensor, we were concerned that variable amounts of microinjected meroCBD could affect the observed spatiotemporal relationships between Cdc42 activation and protrusion/retraction. To test the potential influence of biosensor concentration we examined the Cdc42 activities at meroCBD concentrations deliberately varied 15-fold. For detection of RhoA activity,
MEF/3T3 cells were stably transduced using a retroviral system with the RhoA FRET biosensor under the control of a tet-inducible promoter and FAC sorted for
lowexpressors, as described in(Pertz et al. 2006). Cells were kept under 1 μg/ml Doxycycline in the culture medium to repress biosensor expression. Forty-eight hours before experiments doxycycline was removed from the medium. These cells did not exhibit migration behaviors different from non-transfected MEF/3T3 cells.
Production of the Intermolecular FRET RhoA and Cdc42 Biosensors:
For controls a new intermolecular FRET RhoA biosensor was produced. mCyPet (Nguyen and Daugherty 2005) was subcloned upstream of full length RhoA, followed by a SGLRSELGS linker which contained a restriction site for BamHI. Full length RhoA was subcloned at the BamHI site within this linker sequence and the EcoRI site within the multiple cloning site (MCS) of pEGFP-C1 (Clontech, Inc.) using primer pairs 5’- GGATCCTCTATGGCTGCCATCCGGAAGAAAC-3’ and 5’-
GCGAATTCAGTTTCACAAGACAAGGCACCCAG-3’. The upstream restriction site for mCyPet was NcoI within the pEGFP MCS. For the binding domain portion and hence the FRET acceptor, the Rho binding domain (RBD; amino acids 7 to 89) from Rhotekin was used (Nalbant et al. 2004; Pertz et al. 2006). The binding domains were subcloned into the pTriEx backbone at the NcoI / BamHI sites within the MCS using the primer pairs 5’-GGATCCTGTCTTCTCCAGCACCTG-3’. mYPet (Nguyen and Daugherty 2005) was subcloned following the binding domain at BamHI and XhoI sites within the pTriEX MCS using the primer pair 5’-
ATATGGATCCGGAATGGTGAGCAAGGGCGAAGAGC-3’ and 5’- TTCTCGAGTCATTACTTGTACAGCTCGTCCATGC-3’.
For production of mouse embryonic fibroblasts (MEF) stably incorporating the biosensor DNA, we used the tet-OFF MEF/3T3 cells (Clontech). The biosensor components were cut out as cassettes from the pEGFP or pTriEx cloning constructs at NcoI/EcoRI sites for mCyPet-RhoA, and NcoI/XhoI sites for RBD-mYPet. Digested fragments were treated with Klenow fragment of the DNA polymerase I in the presence of 33μM dNTP for 15 minutes at room temperature to perform the end filling reaction of the 5’-overhangs to produce blunt ends. The pBabe-sin-Puro-tet-CMV was cut at an HpaI site to produce blunt ends and ligated with the blunt-ended inserts. Bacterial colonies were screened using polymerase chain reaction (PCR) for the proper directional incorporation of the biosensor DNA. Retroviral transduction was used to stably incorporate the biosensor DNA in MEF/3T3 under a tetracycline-repressible promoter and expression was inhibited via doxycycline at 1 μg/ml until induction for imaging experiments.
The new dual-chain Cdc42 biosensor uses an intermolecular design as reported by several groups (Itoh et al. 2002; Seth et al. 2003; Tzima et al. 2003; Hoppe and Swanson 2004), but here is further optimized by the use of different fluorescent proteins and of a Cdc42-binding domain from Neuronal Wiskott Aldrich Syndrome Protein (N-WASP), a fragment shown to provide good selectivity for activated Cdc42 in previously developed biosensors with a different design (Nalbant et al. 2004; Frantz et al. 2007; Koivusalo et al. 2010). The biosensor was generated by first constructing plasmids encoding either Cdc42 fused to the C terminus of CyPet, a CFP variant optimized for FRET (Nguyen and
Daugherty 2005), or the Cdc42-binding CRIB domain from WASP (CBD), amino acids 230–314, fused to the C terminus of YPet, a YFP variant optimized for FRET (Nguyen and Daugherty 2005). The EGFP coding region from the EGFP-C1 vector (Clontech, Inc.) was replaced with a PCR product containing the CyPet or YPet coding regions flanked by an NcoI restriction site and a SGLRSELGS linker containing a BamHI
restriction site. The PCR products of the Cdc42 and CBD coding sequences were inserted between the BamHI restriction site in the SGLRSELGS linker and an EcoRI restriction site in the downstream multiple cloning site of the vector.
Fluorometry Assays of GAP Activity on Cdc42 Activation:
To determine the ability of wild-type MILO and either phosphomimetic (YE) or non-phosphorylatable (YA) MILO mutants to affect Cdc42 activity, we performed FRET measurements in a spectrophotometer in a bulk cellular suspension assay in which 293 cells were expressing the Cdc42 activity FRET biosensor. In brief, 293 cells were plated on poly-L-lysine coated wells of a 6-well dish 36 hours before the assay was run. The following morning, 24 hours before the assay was run, the 293 cells were transfected with Lipofectamine according to the manufacturer’s instructions. A total of 650 ng of DNA was transfected per well, consisting of 100 ng of CyPet-Cdc42, 100 ng of YPet- CBD, and the balance consisted of mCherry-tagged MILO, MILO mutants, known regulators of Cdc42, or blank vector DNA. Each experimental condition was set up in triplicate. The following morning, the cells were checked for appropriate brightness, washed once with DPBS, gently trypsinized using 400 L trypsin which was removed after 15 seconds, and then resuspended in 500 L cold DPBS. 400 L of cell suspension
was then loaded into a spectrophotometer cuvette. Using a SPEX Fluorolog sensitive spectrophotometer, the cell suspension was excited at 433 nm, the excitation of CyPet, and the emission was monitored from 450 nm to 600 nm at 3 nm intervals to monitor the emission spectrum of YPet. For each test, a sample transfected with YPet-CBD only and monitored for FRET as above was monitored to account for differences in brightness and thus bleedthrough into the FRET channel by direct excitation of the acceptor fluorophore. This reading was subtracted from each measurement. For the calculation of FRET ratio, after bleedthrough correction, each sample was normalized such that the peak of CFP emission (475 nm) was set as an intensity value of 1.0. The FRET emission peak (525 nm) was then divided by the CFP peak for each sample, and the samples were averaged for each condition. FRET ratios could then be compared between each condition.
Positive and negative controls consisted of the Cdc42 sensor transfected with the catalytic DH/PH domain from Vav2, or GDI-1, respectively, to determine the maximum possible range of Cdc42 activation in the assay. Statistical significance between conditions was assessed by two-tailed students’ t-test assuming unequal variance.
Induction of Filopodia During Imaging:
To study filopodia growth, retraction, and dynamics, cells were transfected with control or MILO siRNA 48 hours prior to imaging, and then subsequently transfected with the Cdc42 biosensor with or without mutant GAP constructs 36 hours prior to imaging (Gadea et al. 2004). Cells were then plated on fibronectin-coated coverslips 24 hours prior to imaging. Six hours before imaging, cells were starved in Ham’s F12K medium containing 0.5% delipidated BSA plus glutamine. Cells were transferred to a
heated open chamber apparatus to allow for addition of TNFα and allowed to return to 37 C for 30 minutes before imaging. Cells were imaged for 5 minutes at 20 second intervals prior to the addition of 20 ng/mL TNFα and imaged for a subsequent 15 minutes post-stimulation to observe filopodia formation and retraction.
Imaging a Single Rho GTPase Activity in Cells:
Mouse embryonic fibroblasts (MEF/3T3, BD Biosciences, Clontech, # C3018-1) were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA) with 10% FBS. Rac1 activity was imaged using a modified version of the FLAIR biosensor (Kraynov et al. 2000), described above. Forty-eight hours post-induction of expression, cells were plated on fibronectin-coated glass coverslips for 3-4 hours prior to imaging. Imaging was performed in imaging medium (see below) with 2% fetal bovine serum. For emission ratio imaging, the following filter sets were used (Chroma): ECFP: D436/20, D470/40; FRET: D436/20, HQ535/30; EYFP: HQ500/20, HQ535/30. A dichroic mirror (“Quad-Custom” Lot# 511112038) was custom manufactured by Chroma Technology Corp. for compatibility with all of these filter sets. Cells were illuminated with a 200 W Hg arc lamp through a 10% transmittance neutral density filter. At each time point, three images were recorded with the following exposure times: CyPet (1.2 s), FRET (excitation of donor, observation of acceptor emission) (1.2 s), YPet (0.4 s) at binning 2x2. The image sets were taken at 10 s intervals. Ratio calculations to generate activity images were performed following bleed-through correction methods described previously (Kraynov et al. 2000). Briefly, Metamorph software (Universal Imaging) was used for image alignment and ratiometric calculation of activation signals. All images were
shading-corrected and background-subtracted. Binary masks with values equal to 1 inside the cell and 0 elsewhere were extracted by applying a threshold to the CyPet image, because it had the largest signal-to-noise ratio. Control cells expressing either CyPet alone or YPet alone were used to obtain bleed-through coefficients α and β in the following equation: CyPet YPet CyPet FRET R t (Eq. 1)
where R is the Ratio, FRETt is the total FRET intensity as measured, α is the bleed- through of CyPet into FRET channel, β is the bleed-through of YPet into the FRET channel and CyPet is the total CyPet intensity as measured. By calculating the linear slope of the relationship between FRET and CyPet intensities upon CyPet excitation of cells expressing only the CyPet, the bleed-through parameter α can be determined. Similarly, by determining bleed-through into the FRET channel upon CyPet excitation of cells expressing only the YPet, the bleed-through contribution of YPet excitation by CyPet into the FRET channel β can be determined. The α parameter was found to be within 0.4-0.5 and the β parameter was typically ~0.2 Both were dependent on the particular optical configuration of the microscope used. With these parameters, the ratio of corrected FRET over CyPet was calculated and used as a measure of Rac1 activation. In time-lapse experiments, CyPet and YPet typically bleach at different rates. Therefore, the ratio was corrected for photobleaching as described elsewhere (Hodgson et al. 2005). Briefly, by algebraic manipulation, (Eq.1) can be rearranged to:
CyPet YPet CyPet FRET R t (Eq.2)
where Γ is the fraction of FRET intensity over CyPet intensity, and Ψ is the fraction of YPet intensity over CyPet intensity, and α and β are the bleed-through constants described above. By taking double exponential fits of the decays of both Γ and Ψ, the correction function, R1, can be calculated following the methods outlined in (Hodgson
et al. 2005).
For detection of Cdc42 activity, cells were seeded on fibronectin-coated glass coverslips (10 g/ml fibronectin) for two to four hours. Endogenous Cdc42 activity was visualized using either the fluorescent biosensor MeroCBD (Nalbant et al. 2004;
Hodgson et al. 2006) or the genetically encoded probe described above. After
microinjection with MeroCBD biosensor, cells were allowed to recover for 30 minutes before imaging, otherwise for the genetically encoded probe, cells were treated just as for the Rac1 probe above. Cells were imaged in imaging medium (Ham’s F-12K without phenol red (Biosource), 10 mM HEPEs and 10 g/ml Oxyfluor reagent (Oxyrase Inc.) with 2 % fetal bovine serum in a heated closed chamber. Images were obtained using a Zeiss Axiovert 100TV microscope, a Zeiss 40 1.3 N/A EC-Plan NeoFluar DIC lens, a CoolSnapES CCD camera (Roper Scientific), and Metamorph software (Universal Imaging). The exposure times were ~900 ms for the ISO-dye and ~300 ms for EGFP at binning 2x2, with a 10% transmittance neutral density filter for MeroCBD, and for the genetically encoded probe, three images were recorded with the following exposure times: CyPet (1.2 s), FRET (excitation of donor, observation of acceptor emission) (1.2 s), YPet (0.4 s) at binning 2x2. The image sets were taken at 10 – 20 s intervals. For ratio imaging of MeroCBD, the following filter sets were used (Chroma): EGFP: HQ470/40, HQ525/50; ISO: HQ580/30, HQ630/40 for MeroCBD, or. The dichroic mirror (“Scripps
Custom” Lot#511111886) was custom manufactured by Chroma Technology Corp. for compatibility with EGFP and ISO fluorescence wavelengths. For emission ratio imaging of the genetically encoded Cdc42 probe, the following filter sets were used (Chroma): ECFP: D436/20, D470/40; FRET: D436/20, HQ535/30; EYFP: HQ500/20, HQ535/30. A dichroic mirror (“Quad-Custom” Lot# 511112038) was custom manufactured by Chroma Technology Corp. for compatibility with all of these filter sets. Image alignment, ratio calculations and correction for photobleaching were performed as described in (Nalbant et al. 2004).
RhoA activation imaging in MEF/3T3 cells was performed by expressing the previously described RhoA FRET biosensor (Pertz et al. 2006) under the control of a tet- inducible promoter. Cells were plated on fibronectin-coated glass coverslips for 3-4 hours prior to imaging. Imaging was performed in imaging medium with 2% fetal bovine serum. The filter sets and the dichroic mirror used for ratiometric imaging were identical to the sets used to image Rac1 above. Cells were illuminated with a 200 W Hg arc lamp through a 10% neutral density filter. At each time point, three images were recorded with the following exposure times: ECFP (1.2 s), FRET (0.6 s) at binning 2x2. The image sets were taken at 10 s intervals. Metamorph software (Universal Imaging) was used for image alignment, ratiometric calculation of activation signals, and photobleaching
corrections, as described above for Rac1 imaging. In contrast to Rac1 above, RhoA ratios did not require bleed-through corrections (Pertz et al. 2006) because the ECFP and EYFP are equimolar in any given pixel for this single-chain biosensor (Pertz et al. 2006). For RhoA imaging in HUVECs, cells were infected with a lentivirus-based TEM4 shRNA construct co-expressing an mCherry fluorescent protein marker. Next day, cells were
split onto gelatin-coated 25-mm glass slides at 25,000 cells/slide in EGM-2 medium with 10% FBS (HyClone). Five hours after plating, cells were infected overnight with an adenoviral vector-based single chain RhoA FRET construct (Pertz et al. 2006; Machacek et al. 2009). The next morning, the virus was removed and cells were transitioned to