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					SUPPLEMENTARY MATERIAL AND METHODS


Nematode Strains
The wild type strain used was the C. elegans Bristol strain N2. The genotypes of baf-1
mutant worm strains are: pna-1(t1499) (GE2249) or pna-1(t1639) (GE2576), unc-
32(e189)/qC1 dpy-19(e1259ts), glp-1(q339); him-3(e1147) (Gönczy et al, 1999).
Based on our findings we re-named pna-1 baf-1. Strains XA3556 (unc-119(ed3)
qaEx3556[unc-119(+), pie-1::GFP::baf-1], XA3557 (unc-119(ed3) qaEx3557[unc-
119(+), pie-1::GFP::baf-1(t1639)], BN5 (unc-119(ed3) bqEx5[unc-119(+), pie-
1::GFP::vrk-1], BN8 and BN9 (unc-119(ed3) bqEx8/9[unc-119(+), pie-1::LAP::vrk-
1] were generated by microparticle bombardment of DP38 unc-119(ed3) (Praitis et al,
2001) with plasmids pPAG20-baf-1, pPAG20-baf-1(t1639), pID vrk-1 and pPGLv.1
vrk-1, respectively. Strains XA3558 (baf-1(t1639); GFP::-tubulin), XA3559 (baf-
1(t1639); YFP::LMN-1) and XA3560 (baf-1(t1639); GFP::LEM-2) were obtained by
crossing GE2576 with WH204 (GFP::-tubulin, (Strome et al, 2001)), XA3502
(YFP::LMN-1), and XA3507 (GFP::LEM-2) (Galy et al, 2003), respectively. Other
worm strains used: AZ212 (GFP::hisH2B) (Praitis et al, 2001), XA3501 (GFP::-
tubulin; GFP::hisH2B), XA3504 (GFP::EMR-1) (Askjaer et al, 2002), and XA3506
(GFP::NPP-5, (Franz et al, 2005)). All strains were cultured using standard C.
elegans methods (Stiernagle, 2006).


Plasmid Construction
PCR-amplified wild type baf-1 and baf-1(t1639) genomic DNA sequences were
inserted downstream of the gfp sequence in the pie-1-based germline expression
vector pPAG20 (Askjaer et al, 2002) to generate vectors pPAG20-baf-1 and pPAG20-
baf-1(t1639). PCR-amplified vrk-1 genomic DNA sequence was inserted into pID-
3.01B (Pellettieri et al, 2003) and pPGLv.1 by Gateway cloning (Invitrogen) to
generate pID vrk-1 and pPGLv.1 vrk-1, respectively. Plasmid pPGLv.1 was
constructed by inserting a PCR fragment encoding TEV-S-peptide amplified from
pIC26 (Cheeseman et al, 2004) into the unique SgrAI site of pID-3.01B.
For RNAi constructs the following sequences were obtained by PCR and inserted into
pPD129.36 L4440 vector: Full-length baf-1 genomic DNA, baf-1 genomic DNA




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corresponding to 3’UTR nucleotides (nt) 1-336 downstream of the stop codon, and
vrk-1 ORF nt 835-1625. As a negative control, the empty pPD129.36 vector was used.
Plasmids pQE30-VRK-1 and pQE60zz-VRK-1 were made by insertion of RT-PCR-
amplified full-length vrk-1 cDNA into BamHI/HindIII sites of pQE30 and BamHI site
of pQE60zz, respectively. pGEX-VRK-1-His was constructed by insertion of a
BamHI/XbaI fragment from pQE30-VRK-1 and a XbaI/HindIII fragment from
pQE60zz-VRK-1 into BamHI/HindIII sites of pGEX-KG.
Plasmid pQE82l-BAF-1 was generated by insertion of RT-PCR amplified full-length
baf-1 cDNA into BamHI site of pQE82l. Vector zz80N-BAF-1 was made by insertion
of baf-1 cDNA into BamHI site of zz-80N.


Recombinant Proteins and Antibodies
His-VRK-1 was purified under denaturing conditions according to standard
procedures (Qiagen), dialyzed and injected into rabbits. For affinity purification of
anti-VRK-1 antibodies, GST-VRK-1-His purified under native conditions with
Glutathione Sepharose 4 (Amersham Pharmacia) was immobilized on a CNBr-
activated Sepharose 4B column (Amersham Pharmacia) and incubated with anti-
VRK-1 antiserum. Anti-VRK-1 antibodies were eluted with 0.1 M glycine pH 2.5.
For protein kinase assays native GST-VRK-1-His was tandem purified over
glutathione and cobalt (BD Bioscience Clontech) columns before dialysis against
phosphate buffered saline (PBS) containing 8.7 % (v/v) glycerol.
His-BAF-1 was purified under denaturing conditions in the presence of 6 M guanidine
hydrochloride, dialyzed against PBS containing 8.7 % (v/v) glycerol and was used for
immunizing rabbits. zz-BAF-1-His for kinase assays was purified under native
conditions according to standard procedures and dialyzed in PBS containing 8.7 %
(v/v) glycerol. All proteins were expressed in E. coli.


Immunofluorescence
Antibodies were diluted as follows: Anti-VRK-1 antiserum and affinity purified
antibodies, 1:200; anti-BAF-1 antiserum, 1:250; anti-Nup96 antiserum (Galy et al,
2003), 1:500; anti-LEM-2 antiserum 3597 (Lee et al, 2000), 1:100; anti-EMR-1
antiserum 3598 (Lee et al, 2000), 1:100; monoclonal antibody 414 against
nucleoporins (Jackson Immunoresearch Laboratories, West Grove, PA), 1:250-1:400;
Cy5-conjugated donkey anti-rat antibody (Jackson), 1:500; goat anti-mouse Alexa


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Fluor 488 and 546 and goat anti-rabbit Alexa Fluor 633 antibodies (Molecular Probes,
Eugene, OR), 1:1000. Hoechst 33258 (Hoechst, Germany) was used at 1-1.25 g/ml
final concentration. Confocal images were obtained with a Leica AOBS SP2
microscope.


Transmission Electron Microscopy
C. elegans hermaphrodites fed bacteria expressing either control or vrk-1 dsRNA
were cryo-immobilized immediately using a Leica EM PACT high-pressure freezer
(Leica, Vienna, Austria) and processed as described (Franz et al, 2005). Analysis of
baf-1(t1639) and baf-1(RNAi) embryos was performed with the following
modifications. Worms were transferred to planchettes filled with 20% BSA and were
rapidly frozen using an EM PACT2 (Leica, Vienna, Austria). Specimens were freeze
substituted for 48 h at –90oC in acetone containing 2% osmium tetroxide, 0.1% uranyl
acetate and 5% water (Walther and Ziegler, 2002). They were then brought to –30oC
for 3 h and then gradually to room temperature (5oC/h).


Co-immunoprecipitation
Affinity purified anti-VRK-1 antibody (25 µg) or rabbit control IgG (25 µg, Santa
Cruz Biotechnology) was coupled to 50 µl AffiPrep Protein A support (BioRad) using
20 mM dimethylpimelimidate and washed in the following order with 1) 0.2 M
ethanolamine, 0.2 M NaCl, pH 8.5, 2) lysis buffer (50 mM HEPES, 1 mM EGTA, 1
mM MgCl2, 100 mM KCl, 10% glycerol, 0.05% Nonidet P40, pH 7.4), 3) 0.1 M
glycine, pH 2.5, 4) lysis buffer. Approximately 600 µl of embryos obtained by
bleaching N2 adults (Stiernagle, 2006) were resuspended in lysis buffer (without
Nonidet P40) and frozen in N2. Embryos were ground in a pre-chilled mortar in a total
volume   of   1.2   ml   lysis   buffer   containing   protease   inhibitors   (1   mM
phenylmethylsufonyl fluoride, 1 mM benzamidine, Roche Complete Mini EDTA-
free) and 0.05% Nonidet P40 followed by mild sonication on ice until visual
inspection under a dissection stereomicroscope revealed complete breakage of
embryos. Extracts were cleared by centrifugation at 10,000 g for 15 min and 250 µl
supernatant were incubated with each antibody column by rotation for 12 h at 4C.
Unbound material was collected and columns were washed with 80 bed volumes of
lysis buffer before bound proteins were eluted with 100 µl 3.5 M MgCl2, precipitated



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with acetone and separated by 15% SDS-PAGE. Proteins were transferred to
Immobilon P membrane (Millipore), which was probed with anti-BAF-1 antiserum
diluted 1:500 in PBS containing 0.05% Tween-20 and 3% low-fat milk.


2-Dimensional Gel Analysis
Worms were grown as specified in Results and bleached to obtain the embryos.
Embryos were frozen in N2 and ground for 10 min, re-suspended in buffer containing
7 M urea, 2 M thio-urea, 4% CHAPS, 5 mM tris-butyl-phosphine (TBP), 0.25% SDS
and 2% biolyte. Proteins were precipitated with acetone and methanol overnight at -
80°C and centrifuged at 6000 g for 30 min. The pellets were washed twice in acetone
and re-suspended by vortexing at 23°C for 4 h in buffer containing 9 M urea, 4%
CHAPS, 100 mM DTT, 1% biolyte, protease inhibitors (Roche Protease Inhibitors
Cocktail, 1:100 v/v) and phosphatase inhibitors (Sigma Phosphatase Inhibitors I and
II, 1:100 v/v). Non-soluble proteins and debris were removed by centrifugation at
4000 g for 30 min. 10 µg total proteins of each sample were applied to 7 cm 3-10 IPG
strips (Linear gradient, GE Healthcare, Little Chalfont, UK), re-hydrated and focused
in a Protean IEF Cell (BioRad, Hercules, CA). For the second dimension, strips were
first equilibrated for 15 min in equilibration buffer (6 M urea, 30% glycerol, 5% SDS,
0.05 M Tris) containing 1% DTT, followed by 15 min in equilibration buffer
containing 4% (w/v) iodoacetamide, then resolved on 15% SDS-PAGE gels. Proteins
were transferred to nitrocellulose membranes that were probed with anti-BAF-1
antiserum diluted 1:1000 in PBS containing 0.1% Tween-20 and 3% low-fat milk.
For  phosphatase treatment wild type frozen embryos were ground and re-suspended
in phosphatase buffer (50 mM Tris (pH 7.5), 50 mM NaCl, 0.1 mM MnCl2, 2 mM
DTT, 0.075% NP-40 and protease inhibitors) and split in two halves. Phosphatase
inhibitors were added to the control sample, while 1000 units  protein phosphatase
(New England Biolabs) and 2 mM MnCl2 (final) was added to the other half. Samples
were incubated with gentle shaking at 30°C for 2 h followed by precipitation and 2-D
gel-electrophoresis.


In vitro kinase assays
Kinase assays containing 0 or 0.5 µM GST-VRK-1-His and 20 µM of zz-BAF-1-His
were performed at room temperature in 50 µl reactions containing 20 mM Tris (pH



                                          4
7.5), 5 mM MgCl2, 1 mM DTT, protease and phosphatase inhibitors. Reaction was
initiated with 0.5 µl of 10 µCi/µl -32PATP and stopped after 60 or 120 min by
addition of SDS-PAGE sample buffer. Proteins were resolved by 12% SDS-PAGE
and phosphorylation was detected by autoradiography.


SUPPLEMENTARY REFERENCES


Askjaer P, Galy V, Hannak E, Mattaj IW (2002) Ran GTPase cycle and importins
alpha and beta are essential for spindle formation and nuclear envelope assembly in
living Caenorhabditis elegans embryos. Mol Biol Cell 13: 4355-4370


Cheeseman IM, Niessen S, Anderson S, Hyndman F, Yates JR 3rd, Oegema K, Desai
A (2004) A conserved protein network controls assembly of the outer kinetochore and
its ability to sustain tension. Genes Dev 18: 2255-2268


Franz C, Askjaer P, Antonin W, Iglesias CL, Haselmann U, Schelder M, de Marco A,
Wilm M, Antony C, Mattaj IW (2005) Nup155 regulates nuclear envelope and
nuclear pore complex formation in nematodes and vertebrates. EMBO J 24: 3519-
3531


Galy V, Mattaj IW, Askjaer P (2003) Caenorhabditis elegans nucleoporins Nup93
and Nup205 determine the limit of nuclear pore complex size exclusion in vivo. Mol
Biol Cell 14: 5104-5115


Gönczy P, Schnabel H, Kaletta T, Amores AD, Hyman T, Schnabel R (1999)
Dissection of cell division processes in the one cell stage Caenorhabditis elegans
embryo by mutational analysis. J Cell Biol 144: 927-946


Lee KK, Gruenbaum Y, Spann P, Liu J, Wilson KL (2000) C. elegans nuclear
envelope proteins emerin, MAN1, lamin, and nucleoporins reveal unique timing of
nuclear envelope breakdown during mitosis. Mol Biol Cell 11: 3089-3099




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Pellettieri J, Reinke V, Kim SK, Seydoux G (2003) Coordinate activation of maternal
protein degradation during the egg-to-embryo transition in C. elegans. Dev Cell 5:
451-462


Praitis V, Casey E, Collar D, Austin J (2001) Creation of low-copy integrated
transgenic lines in Caenorhabditis elegans. Genetics 157: 1217-1226


Stiernagle T (2006) Maintenance of C. elegans. WormBook, ed. The C. elegans
Research       Community,         WormBook,         doi/10.1895/wormbook.1.101.1,
http://www.wormbook.org.


Strome S, Powers J, Dunn M, Reese K, Malone CJ, White J, Seydoux G, Saxton W
(2001) Spindle dynamics and the role of gamma-tubulin in early Caenorhabditis
elegans embryos. Mol Biol Cell 12: 1751-1764


Walther P, Ziegler A (2002) Freeze substitution of high-pressure frozen samples: the
visibility of biological membranes is improved when the substitution medium
contains water. J Microsc 208: 3-10




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