Supplementary Methods
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Kamei et al., MS# 2006-01-00604A
SUPPLEMENTARY METHODS
Preparation of fli1:cdc42wt-EGFP vector
The EGFP-cdc42wt fusion protein along with the SV40 early mRNA polyadenylation
signal was amplified from the pEGFP-C2 vector (BD Biosciences) containing wild-type
cdc42 generated previously1 using the primers 5'AGGCTAGCGCCACCATGGTGAGCAAGGGCGAGGAGC-3' and
5'-AGGGCGCGCCGACAAACCACAACTAGAATGC-3'. Inserts and pfli15L vector
were digested with NheI and AscI (New England Biolabs) and cloned using standard
methods. Positive clones were verified by sequence analysis.
Preparation of mRFP1-F and fli1:mRFP1-F vectors
Monomeric red fluorescent protein (mRFP1)2 and was modified to attach a membrane
localization farnesylation signal3,
4
to the 3’ end The membrane localization signal
farnesylation signal was attached to the 3’ end of the mRFP1 through successive rounds
of
PCR
with
following
primers:
on
the
5’
end,
primer
5660
(5’-
aagcttatggcctcctccgaggacgtcatcaag-3’), common in all reactions. On the 3’ end, primer
4471
(5'-cagcttagatctgagtccggaTGCGGCGCCGGTGGAGTGGCGGCCCTCG-3’),
primer 4472 (5'-cggggccactctcatcaggagggttcagcttagatctgagtccggatgcg-3'), primer 4473
(5'-agcacacacttgcagctcatgcagccggggccactctcatcaggagggt-3')
and
tctagatcaggagagcacacacttgcagctcatgc-3')
successive
were
used
in
primer
5661
order.
(5'This
manipulation altered the stop codon of the mRFP1 to an alanine residue, and attached a
farnesylation site. The PCR product was gel-purified and cloned into the pCRII-TOPO
vector (Invitrogen) to generate pTOPO-mRFP1-F. The sequencer-verified mRFP1-F
insert was excised from the vector by double digesting with HindIII and XbaI, gel-
Supplementary Methods
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Kamei et al., MS# 2006-01-00604A
purified, and cloned into the pXex plasmid5 to generate pXex-mRFP1-F (containing a
polyA sequence). PCR was performed using pXex-mRFP1-F as template and primers
5766 (5'-gctagcatggcctcctccgaggacgtcatcaag-3') and 5656 (5'GGCGCGCCcatacacatacgatttaggtgacactata-3'). The resulting PCR product was cloned
into pCRII-TOPO vector for sequence verification. The modified mRFP1-F sequence
was then excised by double digesting with NheI and AscI and cloned into the pFli1
vector6 to generate the fli1:mRFP1-F construct.
Generation of stable ECs expressing membrane-localized mRFP1-F and EGFP-F
To establish stable EC cell lines expressing membrane-localized mRFP1 (mRFP1-F) or
EGFP (EGFP-F), the mRFP1 and EGFP sequences were TOPO cloned into the
pLenti6/V5-D-TOPO lentiviral vector (Invitrogen, Carlsbad, CA). The mRFP1-F insert
was amplified from vector pTOPO-mRFP1-F by PCR using the following primers:
mRFP1-F UP: 5’-CACCATGGCCTCCTCCGAGGACGTC-3’ and mRFP1-F DN: 5’ACTCAGGAGAGCACACACTTGCAGC-3’. The EGFP-F insert was amplified from
vector pCS2+membraneEGFP7 using the following primers: EGFP-F UP: 5’CACCATGGTGAGCAAGGGCGAGGAG-3’
ACTCAGGAGAGCACACACTTGCAGC-3’.
and
EGFP-F
DN:
5’-
Inserts were confirmed by sequence
analysis and stable EC lines were generated by infection with recombinant lentiviruses
and selection with blasticidin according to the manufacturer’s instructions. Similar
strategies were utilized to obtain stable EC lines carrying EGFP or mRFP1 alone with
no modifications.
Supplementary Methods
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Kamei et al., MS# 2006-01-00604A
Preparation of EGFP-RalA recombinant adenovirus
Full length wild type human RalA was amplified (Guthrie cDNA Resource Center)
using 5'-AGAGATCTCGATGGTCGACTACCTAGCAAATAAGC-3' (BglII) and 5'AGAAGCTTTTATAAAATGCAGCATCTTTC-3' (HindIII) primers and cloned into
the pEGFP-C2 vector (BD Clontech). Recombinant adenovirus was constructed by
amplifying
the
EGFP-RalA
fusion
AGGGTACCGCCACCATGGTGAGCAAGGGCGAG-3'
construct
(KpnI)
with
and
5'HindIII
downstream primer and inserting into the pAdTrack-CMV vector8.
Endothelial Cell Culture and 3D Collagen Vasculogenesis Assays
Details of our human umbilical vein endothelial cell culture and serum-free, 3D type I
collagen assays have been reported previously 9 except that 3.75 mg/ml of collagen type
I was used. In some cases, ECs were infected with either GFP-Cdc42 or GFP-RalA
recombinant adenoviruses as described 1. In order to perform time-lapse imaging, 15 µl
cultures were established in black 384 well plates (VWR, West Chester, PA) or as 2 µl
dot cultures in black 96 well plates (VWR). Once 3D cultures were plated, they were
allowed to equilibrate for 30 minutes at 5% CO2 before the addition of serum-free
culture media which contained Medium 199, a 1:250 dilution of the Reduced Serum
supplement II, 50 μg/ml of ascorbic acid, 50 ng/ml of phorbol ester, 40 ng/ml of
recombinant VEGF-165 (Upstate Biochemical, Lake Placid, NY), and 40 ng/ml of
FGF-2 (Upstate Biochemical).
essentially as described
1, 10
Labeling of pinocytic vacuoles was performed
except using a Dextran tetramethylrhodamine conjugate
(MW 3000, Molecular Probes, Eugene, OR) was utilized to label vacuoles at a final
concentration of 0.1 mg/ml.
Supplementary Methods
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Kamei et al., MS# 2006-01-00604A
Zebrafish Husbandry and Generation of Transgenic Lines
Zebrafish husbandry and maintenance were conducted as described11. Microinjection
and generation of transgenic lines were carried out as described6. The pfli1-EGFPcdc42wt construct was linearized with NotI and injected into freshly laid eggs at 150 pg
per egg. Embryos were examined the next day for expression of the transgene in the
vasculature. Embryos exhibiting robust vascular fluorescence were raised to adulthood
and screened for germline transmission by mating siblings and scoring the resulting
embryos for animals with strong green fluorescence throughout the entire vasculature.
Embryos obtained from one particular injected founder fish were raised to establish the
Tg(fli1:EGFP-cdc42wt)y48 line. Using Western blotting with a cdc42 antibody (Cell
Signaling Technology, Beverly, MA)
we estimate that fli1-positive cells in
Tg(fli1:EGFP-cdc42wt)y48 animals have about 5 times as much GFP-cdc42 fusion
protein as endogenous cdc42.
Microscopy and Imaging
A Nikon Eclipse TE2000-U fluorescent, inverted microscope was used to visualize
vacuole formation, coalescence, and lumen formation in EC in vitro.
Time-lapse
imaging of living cells was performed using a temperature controlled chamber (Solent
Scientific, Segensworth, UK) set to 37°C with continuous flow of 5% CO2. Time-lapse
fluorescence imaging was performed at the lowest possible excitation levels. Images of
vacuole forming cells were captured every 10 minutes in single Z planes or in 10-15
planes per stack at a spacing of 5-10 µm with a Cool-Snap HQ monochromatic camera
with a 6.45 X 6.45-µm pixel pitch (Photometrics, Tucson, AZ) using Metamorph
software (Molecular Devices, Downingtown, PA).
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Kamei et al., MS# 2006-01-00604A
Intravascular injection of zebrafish was performed as described12, 13. Qtracker 605 nontargeted quantum dots used as tracer dye for the patent embryonic vasculature were
purchased from the Quantum Dot Corporation (Hayward, CA).
For fluorescence
imaging of zebrafish a Bio-Rad Radiance-based system with either 960 nm two-photon
(for EGFP-cdc42wt and Qtracker 605 quantum dots) or standard confocal (for mRFP1F) excitation was employed. Time-lapse imaging was performed using a continuousflow chamber devised for extremely long-term imaging of developing zebrafish as
described14. Time-lapse two-photon imaging was performed with an external direct
detection system for increased sensitivity, at the lowest possible excitation light levels.
Image stacks were collected every 3-5 minutes, with 12-20 planes per stack at a spacing
of 2 µm. 3D reconstructions were generated using Metamorph.
References
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Bayless, K. J. & Davis, G. E. The Cdc42 and Rac1 GTPases are required for
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2.
Campbell, R. E. et al. A monomeric red fluorescent protein. Proc Natl Acad Sci
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3.
Aronheim, A. et al. Membrane targeting of the nucleotide exchange factor Sos is
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Hancock, J. F., Cadwallader, K. & Marshall, C. J. Methylation and proteolysis
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Johnson, D. I. Cdc42: An essential Rho-type GTPase controlling eukaryotic cell
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Supplementary Methods
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Lawson, N. D. & Weinstein, B. M. In vivo imaging of embryonic vascular
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Moriyoshi, K., Richards, L. J., Akazawa, C., O'Leary, D. D. & Nakanishi, S.
Labeling neural cells using adenoviral gene transfer of membrane-targeted GFP.
Neuron 16, 255-60 (1996).
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He, T. C. et al. A simplified system for generating recombinant adenoviruses.
Proc Natl Acad Sci U S A 95, 2509-14 (1998).
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Davis, G. E. & Camarillo, C. W. An alpha 2 beta 1 integrin-dependent pinocytic
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capillary lumen and tube formation in three-dimensional collagen matrix. Exp
Cell Res 224, 39-51 (1996).
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Davis, G. E. & Bayless, K. J. An integrin and Rho GTPase-dependent pinocytic
vacuole mechanism controls capillary lumen formation in collagen and fibrin
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Westerfield, M. The zebrafish book (University of Oregon Press, Eugene, 1995).
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Kamei, M., Isogai, S. & Weinstein, B. M. Imaging blood vessels in the
zebrafish. Methods Cell Biol 76, 51-74 (2004).
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Weinstein, B. M., Stemple, D. L., Driever, W. & Fishman, M. C. Gridlock, a
localized heritable vascular patterning defect in the zebrafish. Nat Med 1, 11437 (1995).
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Kamei, M. & Weinstein, B. M. Long-term time-lapse fluorescence imaging of
developing zebrafish. Zebrafish 2, 113-123 (2005).
Supplementary Methods
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Kamei et al., MS# 2006-01-00604A
GRANT SUPPORT
This work was supported in part by a grant from the NIH (HL 59373) to G.E.D.
B.M.W. is supported by the intramural program of the NICHD.