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Fanni Gergely Cancer Research UK Cambridge Research Institute Tuesday Case Study I. CDK5RAP2: Study of the centrosome and microtubule cytoskeleton in DT40 cells •generation of knockouts •detecting truncated proteins •finding phenotype(s) : time-lapse light microscopy •proliferation and clonogenic assays •microtubule cytoskeleton assays (depolymerisation, regrowth, microtubule spin-down) •centrosome isolation and microtubule assembly assays in vitro How to use DT40 for cell biology? The centrosome microtubule organising centre microtubules γ-tubulin ring complex pericentriolar matrix (PCM) mother/daughter centrioles Joo-Hee Sir Centrosome duplicates once and only once per cell cycle procentrioles S G1 G2 Centriole disengagement M Bipolar spindle formation Centrosome separation Centrosome maturation Centrosomes have multiple roles centrosomes provide structural support for cytoskeletal elements (mitotic spindle, cilia, flagella) centrosomes are signalling platforms (Chk1, Mcph1) centrosomes have cell-type/tissue specific functions centrosomes may be instructive in stem cell division (age of centrioles) Our questions how does the centrosome organise microtubules during interphase and mitosis? how does the centrosome assemble? are there subdomains within the centrosome? >200 proteins centriole microtubule assemby? anhcoring? microtubule nucleation? Advantages of DT40 cells for our studies: centrosomal proteins are very stable; siRNA does not offer a good option centrosome proteins are large and modular; DT40 offers a system to study specific domains the majority of centrosomal proteins are not essential; knockouts survive little is known about the regulation of centrosome proteins; manipulate regulatory sites in situ in DT40 centrosomal protein complexes have not been explored; purify protein complexes from DT40 cells in which proteins are tagged in situ Centrosome dysfunction is linked with human diseases Bardet-Biedl-syndrome Polycystic kidney disease Lissencephaly Primordial Dwarfism Autosomal Primary Recessive Microcephaly brain is 20-30% of normal size sloping forehead all MCPH proteins are spindle pole- or centrosome-associated (AspM, CenpJ, CDK5RAP2, MCPH1, Stil) MCPH proteins are highly conserved; do they define a special centrosomal complex or a pathway? CDK5RAP2 in fission yeast Mto1 and Pcp1 are required for interphase cytoplasmic and mitotic microtubule organisation, respectively (Sawin et al 2004, Sawin and Tran, 2006 and Fong et al, 2010) in Drosophila, centrosomin (CNN) is required for proper centrosome function (Megraw at et al, 1999, Zhang and Megraw, 2007, Lucas and Raff, 2007, Dobbealere et al, 2008) CNN has 2 human orthologues (Myomegalin and CDK5RAP2) CDK5RAP2 is mutated in primary microcephaly (Bond et al, 2005) Targeting strategy to disrupt the cdk5rap2 gene cdk5rap2 locus 89kb 3.8kb 5.2kb 2 1 3 4 5 6 39 40 7 8 Targeting constructs cnn1 cnn2 Neo(D)/ Puro(E) cnn1-/- targeted locus 1 2 6 7 8 44 Neo(D)/ Puro(E) A B 41 42 cnn2-/- targeted locus 1 38 39 40 A 1 A 2 6 F 7 8 G 44 Neo(C)/ Puro(D) STOP STOP C D/E cnn1lox targeted locus 43 43 44 Neo(C)/ Puro(D) STOP STOP 41 B C/D cnn1loxcnn2-/- targeted locus 44 1 2 6 39 40 41 43 44 Neo(C)/ Puro(D) STOP STOP Finding your truncated product Antibody wt cnn1 cnn1lox cnn2 CDK5RAP2 merged cnn1loxcnn2 CDK5RAP2 merged wt wt cnn1-/- cnn1lox wt cnn2+/- cnn2-/210 kDa 110kDa 55kDa * * CDK5RAP2 ΔCNN2 wb: CDK5RAP2 wb: α-tubulin cnn2-/interphase mitosis Disrupted alleles of cdk5rap2 gene Antibody wt cnn1 cnn1lox cnn2 cnn1loxcnn2 ? Protein-tagging in DT40 cells Uses of in situ tagging replaces need for chicken-specific antibody reports on how complete gene disruption is allows detection of truncated proteins after gene disruption (western and immunofluorescence) Another truncated product tag-wt tag-cnn1lox protein G (tagCDK5RAP2) merged w t ta gcn wt n ta 1 lox gcn n1 lo x protein G (tagCDK5RAP2) merged tag-wt tag-CDK5RAP2/ tag-ΔCNN1 210kDa GsTAP (protein G+strep bp) protein G protein G (tag-ΔCNN1) merged (tag-ΔCNN1) merged wb: protein G tag-cnn1lox 72kDa Bürckstümmer et al, 2006 Nat Methods cell lines protein product wt CDK5RAP2 tag-wt lox c1 cnn1-/- tag-CDK5RAP2 tag-cnn1lox cnn1lox cnn2-/- tag- CNN1 cnn1lox cnn2-/- wb: α-tubulin 55kDa CNN1 interphase Immunoblot Subcellular Localisation protein detectable interphase mitotic centrosome centrosome ? ? (35%) CNN2 CNN1 CNN2 ? (16%) ? ? ? yes no ? not known mitosis So far... ΔCNN1 and ΔCNN2 truncated products are expressed in knockouts ΔCNN1 and ΔCNN2 truncated products are absent from interphase centrosomes but are present at low levels in mitotic centrosomes Are the knockout lines as ʻfitʼ as wild-type DT40? (assaying proliferative potential of cells) DIC microscopy late passages Late passages (3-4 weeks in culture) film cells for 22-26 hours (CO2 chamber is a must) cellcell numbers numbers 1.0E+08 wt 1.0E+07 het 1.0E+06 del 1.0E+05 0 24 48 72 time (hrs) time (hours) Arno Alpi determine # of cell divisions # of cell deaths length of cell division etc. Are the knockout lines as ʻfitʼ as wild-type DT40? (assaying proliferative potential of cells) DIC microscopy late passages Late passages (3-4 weeks in culture) film cells for 22-26 hours (CO2 chamber is a must) cellcell numbers numbers 1.0E+08 wt 1.0E+07 het 1.0E+06 del 1.0E+05 0 24 48 72 time (hrs) time (hours) determine # of cell divisions # of cell deaths length of cell division etc. Arno Alpi 26 hours wt cnn1 2 cell divisions 42% 17% death 1% 11% Assaying proliferative potential of cells % cells with colony forming ability 100 Single cell Colony 7 or 10 days 80 60 40 20 0 CNN2+/+ CNN2+/- CNN2-/- CNN1-/- CNN1-/CNN2+/- CNN1-/CNN2-/- BUT WHY? find out whether the centrosome and the microtubule cytoskeleton are intact Microtubule depolymerisation and regrowth microtubules in DT40 are cold-stable depol use 2 μg/ml nocodazole for depolymerisation for regrowth wash cells with DMSO+medium B cnn1-/- wt cnn1-/- wt α-tubulin merged 0 min 5 min volume of α-tubulin associated with centrosome (µm3) regrowth 9 8 7 6 5 4 3 2 1 0 p<0.27 (N.S) wt wt cnn1 cnn1-/--/- 5 min Microtubule assembly on centrosomes in vitro synchronise harvest lyse (0.25% Triton) pellet centrosomes onto coverslip Buffer only centrin α-tubulin wt add tubulin, GTP and taxol to centrosomes and incubate at 30C End of Part I. * centrosomes detach from spindle poles in cnn1-/- cells DIC imaging shows that cnn1-/- cells divide less and die more often Clonogenic potential of cnn1-/- cells is reduced centrosomes can assemble microtubules in cnn1-/- cells in vivo many unanswered questions....are centrosomes intact otherwise in cells? how does centrosome detachment arise? how dynamic is centrosome detachment? we need microscopy... Seeing is believing...microscopy in DT40 cells Thursday Case Study II. CDK5RAP2: the use of microscopy in DT40 cells •zooming in: electron microscopy •finding phenotype(s) in mitotic cells: confocal microscopy (fixed samples) and spinning disk confocal microscopy (live) •tagging proteins in mutant backgrounds •identifying and correlating mitotic spindle and centrosome phenotypes Centrosomes detach from the mitotic spindle poles in cnn1-/- cells centrin TACC3 merged wt Centrin (centrioles) * cnn1-/- TACC3 (PCM +spindle) * * cnn2-/- partially detached centrosome % prometaphase/ metaphase cells 70 detached centrosome 60 50 40 30 20 10 0 wt cnn1-/- cnn1lox cnn2-/- cnn1lox cnn2-/- Detective work starts here... Why do centrosomes fall off in cnn1-/- cells? NuMA dynein/dynactin abnormal centrosome structure? abnormal spindle pole? Transmission Electron Microsopy in DT40 harvest cells fix in 1% warm glutareldehyde post-fix in osmium tetroxide dehydrate pellet in increasing 30-100% EtOH washes cut sections (50-100nm) embed using Epoxy kit Transmission electron microsopy Joo-Hee Sir Joo-Hee Sir Serial sections in electron microsopy +/+ -/- Alexis Barr -/- 1nnc tw cnn1 1nnc cnn1 -/- wt Why do centrosomes fall off in cnn1-/- cells? NuMA dynein/dynactin abnormal centrosome structure? NO! abnormal spindle pole? Fixing cells for fluorescent microsopy seed cells onto poly-lysine coated coverslips (allow cells to settle for 15 minutes); avoid centrifugation if possible What do I want to see? Find the right fixation conditions for your antigen/antibody... cytoskeleton: 100% -20C methanol; 3% formaldehyde/paraformaldehyde (extraction 0.1-0.5% Tween or Triton); 0.5-1% glutaraldehyde/1-3% paraformaldehyde mix (quench afterwards!) chromatin-binding/nuclear protein: 1-4%paraformaldehyde (extraction 0.1-0.8% Triton) make sure that your antibody is specific!!!!! block cells in 5-10%BSA or in 5-10%FCS for 5-30 minutes prior to primary antibody mount in antifade medium Laser-scanning confocal microscopy A point light source for illumination (laser) A point light focus within the specimen A pinhole at the image detecting plane These three points must be aligned accurately to each other in the light path of image formation to obtain confocal image. Confocal effects result in supression of out-of-focal-plane and stray light in the final image. Confocal allows optical sectioning. The architecture of cnn1-/- spindles 5μm 5μm Why do centrosomes fall off in cnn1-/- cells? NuMA dynein/dynactin abnormal centrosome structure? NO! abnormal spindle pole? NO! NuMA dynein/ dynactin (p150) Abnormal attachment? Centrosome purification Purified centrosomes 3. % sucrose: Fractions: 70 2 50 3 70 40 4 5 6 7 WCE 2. WCE 1. 2 50 3 4 40 5 6 7 19kDa centrin 1 55kDa 60% sucrose 70%, 50%, 40% sucrose collect fractions 41kDa γ-tubulin 62kDa Plk1 210kDa protein G (tagAKAP450) 111kDa p150glued tagAKAP-cnn1lox DT40 tagAKAP-wt DT40 pellet centrosomes from each fraction Fraction 3 Fraction 4 γ-tub 421.8 288.7 Plk1 471.4 803.1 AKAP450 5.6 48.1 p150 0 0 % of protein in cnn1lox compared to wt centrosomes Immunofluorescence confirms lack of tagged AKAP450 in mitotic cnn1-/- centrosomes akap450 locus 1 48 49 50 51 52 GsTAP Neo targeting construct STOP tag-akap locus 1 48 49 50 51 52 GsTAP Neo STOP Protein G (AKAP450) γ-tubulin Protein G (AKAP450) merged wt tagAKAPcnn1lox tagAKAPwt tagAKAPcnn2-/- γ-tubulin in situ tag replaces need for chicken-specific antibody merged Our final model CDK5RAP2 is required for efficient cell proliferation CDK5RAP2 links centrosome with spindle poles CDK5RAP2 recruits dynactin and AKAP450 into the centrosome NuMA, Dynein spindle pole AKAP450 dynactin (p150) CDK5RAP2 centrosome Centrosome detachment from spindle poles does not prevent bipolar spindle formation Centrosome within the spindle pole contributes to maintaining spindle length and proper chromosome alignment Fixed cells are not ideal to study a dynamic process... Spinning disc confocal microscope * Rotating disc has thousands of pinholes field of view is scanned in single exposure, thus allowing the capture of confocal images at high speed (great for z-stacks) multiple points are collected simultaneously, sample receives less laser light spinning disk confocal microscopy is well suited to high speed 3D imaging of living systems disadvantages: a huge amount of light is lost through the Nipkow disc; cannot image low fluorescent intensity; background can be high due to pinhole crosstalk Time-lapse microscopy on DT40 cells Leibowitz media ibidi dish