Chapter II Materials and methods 2.1 Strains, reagents and genetic methods 2.1.1 Schizosaccharomyces pombe strains S. pombe strains used in this study and their genotype are listed in the table below. Table 1: Strains used in this study. Name SO5 SO11 SO34 SO76 SO77 SO87 SO115 SO116 SO132 SO197 SO201 SO249 SO502 SO504 SO646 SO1222 SO1248 SO1287 SO1387 SO1490 SO1491 SO1618 SO1620 SO1760 SO1762 SO1778 SO1795 Genotype mia1-GFP::ura4+ ura4-D18 leu1-32 ade6-M210 hpREP1-mia1 ade6-210 ura4-D18 leu1-32 pREP1-mia1 clp1-EGFP::KanR ura4-D18 leu1-32 pREP1- α -tubulin-GFP ura4-D18 leu1-32 ade6-M210 pREP1-α-tubulin-GFP mia1Δ::ura4+ ura4-D18 leu1-32 ade6-M210 hpREP1-mia1 rlc1p-EGFP::ura4+ ura4-D18 leu1-32 pREP1-mia1 uch2p-EGFP::ura4+ ura4-D18 leu1-32 pREP1-mia1 cut11p-EGFP::ura4+ ura4-D18 leu1-32 pREP41-mia1-GFP ura4-D18 leu1-32 ade6-M210 hpREP1-mia1 uch2p-GFP::ura pim1-1 ura4-D18 leu1-32 pREP1-mia1 LacO(lys1+)Dis1promoter LacI-EGFPNLS(his7+) leu1-32 ura4-D18 Source SO collection This study This study SO collection SO collection This study This study This study This study This study SO collection From K.Gould and J. Tasto pREP1-α-tubulin-GFP uch2-GFP::ura4+ ura4-D18 leu1-32 This study This study pREP1-α-tubulin-GFP uch2-GFP::ura4+ mia1Δ::ura4+ This study mto1/mbo1-GFP::kanR mia1Δ::ura4+ alp4-GFP::ura4+ ura4-D18 leu1-32 ade6-M210 hThis study This study alp4-GFP::ura4+ mia1Δ::ura4+ ura4-D18 rsp1-GFP::kanR leu1-32 ura4-D18 ade6-M2x From F.Chang This study rsp1-GFP::kanR mia1Δ::ura4+ leu1-32 ura4-D18 This study mto1-GFP::kanR mad2Δ::ura4+ This study mto1/mbo1-GFP::kanR nda3-KM311 mad2Δ::ura4+ This study pREP1-α-tubulin GFP sid2-GFP::ura4+ This study pRep1-α-tubulin GFP sid2-GFP::ura mia1Δ::ura4+ pREP1-α-tubulin-GFP uch2-EGFP::ura4+ pim1-1 ura4This study D18 leu1-32 pREP1-α-tubulin-GFP cut11-EGFP::ura4+ ade6-704 leu1This study 32 ura4-D18 This study mto1/mbo1-GFP::kanR alp14Δ::ura4+ This study pREP-α-tubulin-GFP sid2-GFP::ura4+ alp14Δ::ura4+ mto1/mbo1-GFP::kanR ura4-D18 leu1-32 ade6-M2x h(KGY2434) 33 SO1797 mto1-GFP::KanR mal3Δ::his3+ SO1952 pREP1-mia1 Sad1p-GFP::ura4 ura4-D18 leu1-32 pREP1-mia1 uch2-GFP::ura4+ sad1-DsRed::KanR ura4SO2002 D18 leu1-32 SO2037 pREP1-mia1 ase1Δ::ura ura4-D18 leu1-32 ade6-216 pREP1-mia1 pcp1-GFP::kanR uch2-GFP:ura4+ pim1-1 SO2069 ura4-D18 leu1-32 SO2090 pREP1-mia1 cut7-GFP::ura4+ ura4-D18 leu1-32 SO2098 pREP1-mia1 ase1-GFP::ura4+ ura4-D18 leu1-32 SO2111 pREP1-mia1 mad2-GFP::ura4+ ura4-D18 leu1-32 SO2131 pREP1-mia1 pcp1-GFP::KanR leu1-32 SO2244 pREP1 ura4-D18 leu1-32 ade6-210 pREP1-α-tubulin-GFP uch2-EGFP::ura4+ dis1Δ::KanR SO2318 leu1-32 ura4-D18 SO2323 pREP1-mia1 mis6-GFP::ura4+ leu1-32 ura4-D18 pREP1-mia1 nmt81-atb2-GFP::KanR uch2-GFP::ura4+ SO2341 leu1-32 pREP1-mia1 alp4p-GFP::ura sad1-DsRed::KanR leu1-32 SO2363 ura4-D18 pREP1-mia1 dis1promoter-cen1-GFP sad1-DsRed::kanR SO2379 pim1-1 leu1-3 pREP1-mia1 ark1-GFP::ura4+ cut7-mCherry::ura4+ leu1SO2440 32 ura4-D18 pREP1-mia1 alp4-GFP::ura cut7-mCherry::ura4+ leu1-32 SO2434 ura4-D18 pREP1-mia1 cut7-mCherry pcp1-GFP::kanR uch2SO2444 GFP::ura4+ leu1-32 This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study 2.1.2 Growth, media and conditions Media for vegetative growth (YES or EMM2 with suitable supplements) and genetic methods were as described in Moreno et al., 1991. Genetic crosses and sporulation were performed on YPD agar plates. Tetrad dissections were performed using a Singer MSM micromanipulator. Double mutants were isolated from non-parental ditype (NPD) tetrad. To induce Mia1p-overexpression, cells were grown in EMM2 lacking thiamine at 30oC for 20h. 2.1.3 Plasmid 34 pREP1 α-tubulin-GFP plasmid was a gift from Dr. Y. Hiraoka (Kansai Advanced Research Center, Kobe, Japan). Plasmid pSO56 pREP1 Mia1p was constructed by introducing Mia1-ORF at BamHI-SmaI restrictive sites of the pREP1 vector (Maundrell, 1993). Plasmid pSO pJK210-GFP was constructed by introducing GFP at restrictive sites between BamHI-NotI of the pJK210 vector (Keeney and Boeke, 1994). 2.1.4 Enzymes antibodies and drugs used Restriction enzymes used were purchased from New England Biolabs (Beverly, MA, USA). Both anti-mouse and anti-rabbit secondary antibodies used for immunoflorescence were product of Sigma Chemical Company (St. Louis, USA). Zymolyse was obtained from US Biological (Swampscott, MA, USA). The anti-tubulin antibody, TAT-1 was a gift from Dr. K. Gull (University of Oxford, Oxford, United Kingdom). Poly-lysine (Sigma-Aldrich) at 2mg/ml concentration was used to mount cells on glass for chamber experiments. The microtubule-destabilizing drug methyl-1(butylcarbamoyl)-2-benzimidazolecarbamate (Carbendazim; MBC) from Aldrich Chemical was used to depolymerize microtubules at 50 μg/ml final concentration. Thiamin was used to repress the transcription from nmt1 promoter at the final concentration of 5μg/ml. 2.2 Molecular methods and yeast methods 2.2.1 Recombinant DNA techniques 35 Recombinant DNA techniques in this study were carried out as described in Molecular Cloning, Maniatis T., Fritsch E. F., Sambrook J. 2.2.2 Transformation of E.coli by electroporation was performed as described in REF: Prentice HL. Nucleic Acid Research. 1992 20(3) 621. the procedure consists of the following: 1. 50 μl E.coli competent cells were thawed on ice. 2. 1-3μl of DNA (about 1μg) was mixed with the cells and incubated on ice for 2-4 min. 3. Cells were transfer to pre-cooled E.coli Pulser Cuvette from BIO-RAD. 4. Place the cuvette to the chamber of E.coli Pulser apparatus with 2.50KV and plus once. 5. Immediately add 1ml of SOC medium to the cuvette and transfer the cell suspension to the eppendorf tube and incubate at 37oC for 1hour. 6. Plate on selective medium. 2.2.3 LiAc transformation of S. pombe was performed as described in Fission Yeast Handbook. The procedure consists of the following: 1. Grow cells in 20ml YES (for one transformation) overnight to 107 (OD595=0.5). 2. Wash cells with equal volume of water and spin down. 3. Wash cells with 5ml LiAc/TE buffer and resuspend in 100μl of LiAc/TE [make 10 times dilution from 10X LiAc (1M LiAc, ph 7.5) and 10X TE (0.1M Tris-HCl, 0.01M EDTA, ph 7.5) solutions]. 36 4. Add 2μl of carrier DNA and 2-10μl of plasmid DNA (10-20μg) into 100μl of cells and incubate at RT for 10 min. 5. Add 240μl of PEG/LiAc/TE solution (for 20ml solution, dissolved 8g of PEG 4000 in 2ml 10X LiAc and 2ml 10X TE and 9.75 ml water and filer sterilize. This solution can be stored up to one month at 4oC) to the cells, gently mix and incubate at 30oC for 10-30 min. 6. Add 43μl of DMSO into the cells and mix by swirling. Cells are heated at 42oC for min. 7. Wash cell pellet with 1ml water and resuspend in 0.1ml water to plate on selective plate. 2.2.4 Mia1p/Microtubule association experiments E.coli BL21 was used to express glutathione S-transferase (GST)-Mia1p. Pelleted bacterial cells were lysed in the SDS-PAGE sample buffer. The lysate was separated on SDS-PAGE gel, and then transferred to nitrocellulose filter and incubated in the PEMG buffer [100mM piperazine-N, N’-bis(2-ethanesulfonic acid), PH6.9, 2mM EGTA, 1mM MgSO4 and 1mM GTP] containing 5% milk and then with taxol-stabilized bovine brain 3X microtubules (50μg/ml) in the same buffer, followed by washing and immunoblotting withTAT1 anti-tubulin antibodies. 2.2.5 Extraction of S.pombe genomic DNA 1. 5ml S. pombe cells were grown overnight at 24oC. 2. Cells were spun down at 3000rpm and washed once with 0.5ml TE buffer. 37 3. Cells were resuspended in 0.5ml digestion buffer (1.2M sorbitol, 0.1 M EDTA, 2mg/ml Zymolyse, 1% BME) and incubated at 37oC for 30min. 4. Spheoplasts were spun down for at 3000rpm. The pellet was resuspended in 0.5ml lysis buffer (100mM NaCl, 50 mM EDTA, 50mM Tris pH9.0, 50μl 10%SDS and 10μl 20mg/ml proteinase K was added shortly buffer before use) and incubated at 65oC for 30min. 5. Samples were incubated on ice for 10min. 200μl 5M Kac was added to the sample and mixed by inversion. Samples were kept on ice for 30min. 6. Samples were spun at 14,000rpm for 30min at 4oC to remove proteins, Supernatant was recovered and equal volume isopropanol was added to precipitate DNA. DNA was spun down at 14,000rpm for 10min at 4oC. The pellet was air dried for 10min and resuspended in 40μl TE buffer containing RNAse and incubated at 37oC for 20min. 7. DNA was stored at -20oC. 2.3 Construction of knock out mutants and epitope tagging of genes 2.3.1 Construction of knock out mutants To create null mutant of S. pombe strains, gene coding regions were replaced with ura4+ gene. The primer pairs containing flanking 5’ and 3’ sequences of target genes were used to amplify the ura4+ gene by PCR. The purified PCR products were introduced into uracil autotroph cells by LiAC procedure (for details, see 2.2.3). Ura+ transformants were checked for correct integration by PCR assay and/or nucleotide sequence determination of the genomic DNA. 38 2.3.2 Construction of epitope tagging of genes All chromosomally tagged genes were created through homologous recombination. Green fluorescent protein (GFP) was the predominant tags used in this study. pJK210based plasmids, containing the required gene fused to tags at its 5’ or 3’ end, were used for integration at the genomic locus as described previously (Keeney and Boeke, 1994). Putative integrants were selected based on uracil prototrophy conferred by pJK210 vector and confirmed by PCR. 2.3.3 Generation of cells overexpressing Mia1p The open reading frame of Mia1+ was cloned into a pREP1 plasmid which contains an inducible nmt1 promoter. In the presence of thiamine, the nmt1 promoter is repressed, yet there is a low level based expression. In the absence of thiamine, the nmt1 promoter is fully induced. Thus expression of Mia1p is controlled by addition of exogenous thiamine into the medium. The resulting plasmid was then transformed into S. pombe cells and leu prototrophy conferred by pREP1 plasmid was used to select the transformants. 2.4 Cell biology and microscopy 2.4.1 Immunofluorescence staining were carried out as described by Fission Yeast handbook, the procedure consists of the following: 1. 50ml S. pombe cells were cultured overnight until OD595=02.-0.5. 39 2. For formaldehyde fixation, 37% formaldehyde was added into liquid cell culture to the final 7.4% concentration. Samples were immediately mixed by inversion and incubated at the growth temperature for 12min. For methanol fixation, a volume of cell culture were spun down and cell pellet was resuspended in 8ml cold methanol (prechilled to -20oC) at -20oC for 8min. 3. Cells were spun down and pellet was washed with 20ml PBS buffer twice and kept on ice. 4. Cells were spun down, washed once with 1ml of 1.2 M sorbitol dissolved in PBS and resuspended in 140μl of 1.2M sorbitol disolved in PBS. 5. 60μl of protoplasting buffer (50mg/ml Lysing enzyme, 30mg/ml Zymolase, 1.2 M sorbitol in PBS) was added to the samples and incubated at room termperature for 5-10 min. Progress of cell wall digestion was checked by viewing cells under the microscope. 6. The reaction was stopped by 15-50ml PBS + 1% Triton. Spheroplasts were spun down at 3000rpm and transferred to an eppendorf. 7. Spheroplasts were washed with PBS and then with PBAL buffer (1% BSA, 100 mM Lysine-Hydrochloride, 50 μg/ml Carbenicilin, 1mM Sodium Azide, in PBS). Block in 1ml PBAL at RT for 1h. 8. Spheroplasts were resuspended in 200μl PBAL with 0.5μl primary antibodies. The mixture was incubated at room temperature with shaking for 3hrs or overnight at 4oC. 9. Spheroplasts were washed times with 500μl PBAL and resuspended in 200μl PBAL with 1μl secondary antibodies. They were incubated at RT with shaking 40 for 1h. Spheroplasts were then washed times with 500μl PBAL and resuspended in 30μl of PBAL. 10. 5μl of cells were spread on a coverslip evenly with the pipette tip and were dried for several min. 2.4.2 Flow chamber experiments Poly-lysine (2mg/ml) was used to mount cells (which were washed with EMM medium) on the cover glass with two parallel pieces of double-stick tape mounted on it. The flow chamber was created by overlaying the cover glass with a coverslip. Flowthrough could be achieved by adding medium on one side of the chamber and absorbing the liquid from the opposite by tissue paper. 2.4.3 Time-lapse fluorescent microscopy Time-lapse images were generated on a Zeiss Axiovert 200M microscope equipped with Ultra View RS confocal system: CSU21 confocal optical scanner, 12 bit digital cooled Hammamatsu Orca-ER camera (OPELCO, Sterling, VA), and kryptonargon triple line laser illumination source (488, 568 and 647 nm). Still images were collected on a Zeiss Axiovert 200M microscope using Cascade:650 camera (Photometrics/Roper Scientific, Trenton, NJ) and Uniblitz shutter driver (Photonics, Rochester, NY) under the control of MetaMorph software package (Universal Imaging, Sunnyvale, CA). For imaging of microtubule dynamics, S. pombe cells expressing αtubulin-GFP were grown in appropriate selective medium and placed in sealed growth chambers containing 2% agarose media. For three-dimensional time-lapse imaging, each 41 image stack consisted of 13 sections of 0.5μm spacing and 15-s intervals between stacks. For single-section time-laspe analyses, images were collected at 5s intervals. 2.4.4 Electron microscopy techniques Cells were rapidly frozen by high-pressure freezing (BAL_TEC HPM-010; Technotrade International, Manchester, NH) and freeze-substituted at -90oC in 0.2% glutaraldehyde plus 0.01% uranyl acetate in acetone for 96h in an EM-AFS device (Leica, Vienna, Austria). The cells were warmed over 25h to -40oC and then infiltrated with HM20 (Electron Microscopy Sciences, Hat-field, PA) resin over a period of 5d. The cells were embedded under UV light at -40oC in HM20 for 3d and then warmed to room temperature over a 6h period. Embedded cells were sectioned and immunostained as follows: sections on gold grids (Electron Microscopy Sciences) were floated on blocking buffer (0.02% Tween 20, 0.8% bovine serum albumin, and 0.1% fish gelatin in PBS) for 1h, immunostained with anti-GFP antibodies overnight at 4oC as described previously, rinsed with PBS plus 0.1% Tween 20 three times, incubated with 10nm gold goat-antirabbit secondary antibodies (BB international, Cardiff, United Kingdom) for 2h at room temperature, rinsed and fixed with 1.0% glutaraldehyde for min, stained with aqueous uranyl acetate and lead citrate, and imaged in a Philips Tecnai TF20 FEG electron microscope operating at 80KeV. 2.4.5 Laser microsurgery Laser microsurgery was conducted on a custom-assembled microscopy workstation centered around a Nikon TE2000-E2 microscope (Nikon Instruments, 42 http://www.inkon.com). Laser pulses (532nm for 8ns) were generated by Q-switched Nd:YAG laser (Diva II; Thales Lasers, http://thales.nuxit.net) run at a 20-Hz repetition rate. Collimated laser beam was expanded to approximately mm to fill the aperture of a 100X1.4 NA PlanApo lens and delivered through a dedicated epi-port. Fluorescence images were recorded with a Cascade512B back-illuminated EM-CCD camera (Photometrics) attached to the left microscope port (100% transmission) in confocal mode (spindding disk confocal; Perkin-Elmer, http://www.perkinelmer.com) as threedimensional datasets at 0.25μm Z steps. Differential interference contrast (DIC) images were recorded with a CoolSnap CF CCD camera attached to the right port of the microscope (modified to 100% transmission). All light sources were shuttered by either fast mechanical shutters (Vincent Associates, http://www.uniblitz.com) or AOTF (Solamere Technology Group, http://www.solameretech.com) so that cells were exposed to light only during laser operations and/or image acquisition. 43 [...]... (100% transmission) in confocal mode (spindding disk confocal; Perkin-Elmer, http://www.perkinelmer.com) as threedimensional datasets at 0.25μm Z steps Differential interference contrast (DIC) images were recorded with a CoolSnap CF CCD camera attached to the right port of the microscope (modified to 100% transmission) All light sources were shuttered by either fast mechanical shutters (Vincent Associates,...http://www.inkon.com) Laser pulses ( 532 nm for 8ns) were generated by Q-switched Nd:YAG laser (Diva II; Thales Lasers, http://thales.nuxit.net) run at a 20-Hz repetition rate Collimated laser beam was expanded to approximately 8 mm to fill the aperture of a 100X1.4 NA PlanApo lens and delivered through a dedicated epi-port Fluorescence images were recorded with a Cascade512B back-illuminated EM-CCD... fast mechanical shutters (Vincent Associates, http://www.uniblitz.com) or AOTF (Solamere Technology Group, http://www.solameretech.com) so that cells were exposed to light only during laser operations and/ or image acquisition 43 . 33 Chapter II Materials and methods 2.1 Strains, reagents and genetic methods 2.1.1 Schizosaccharomyces pombe strains S. pombe strains used in this study and their genotype are listed in. and then with taxol-stabilized bovine brain 3X microtubules (50μg/ml) in the same buffer, followed by washing and immunoblotting withTAT1 anti-tubulin antibodies. 2.2.5 Extraction of S .pombe. 10min at 4 o C. The pellet was air dried for 10min and resuspended in 40μl TE buffer containing RNAse and incubated at 37 o C for 20min. 7. DNA was stored at -20 o C. 2 .3 Construction of