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Master course Molecular and Cellular Life Sciences 16/10/2012 Cytokinesis Cytokinesis 1) How does it happen? (formation and constriction actin contractile ring) 1 How does it happen? 2 Where does it happen? 2) Where does it happen? (spindle determines cleavage plane position) 3 How is it controlled during development? 3) How is it controlled during development? (spindle positioning, asymmetric cell division) SvdH 1 SvdH 2 SvdH 4 Cytokinesis Cytokinesis Actin (cortex) Microtubules Sea urchin Sea urchin SvdH 3 The cytoskeleton is made up of three classes of protein filaments: Todayʼs topic: developmental control of the cleavage plane position# ! ! Players: actin and tubulin# motor proteins (myosin)# regulators (Rho GTPase)# ! ! # # # Model: C. elegans! Microtubules (α/β-tubulin) Microfilaments (actin filaments) Intermediate filaments (various, e.g. Keratin) α/β-tubulin G actin SvdH [email protected] 5 SvdH 6 SvdH 1 Master course Molecular and Cellular Life Sciences 16/10/2012 Dimers of α/β-tubulin assemble into large microtubules Figure 16-11 Molecular Biology of the Cell (© Garland Science 2008) SvdH Actin monomers assemble into filaments (microfilaments) 7 Formin promotes formation of actin filaments Figure 16-36 Molecular Biology of the Cell (© Garland Science 2008) Figure 16-12 Molecular Biology of the Cell (© Garland Science 2008) SvdH 8 The ARP complex nucleates the formation of side chains, which results in an actin web SvdH Myosin motor protein: binds and moves in association with actin 9 Figure 16-34c Molecular Biology of the Cell (© Garland Science 2008) SvdH 10 Myosin forms bundles (filaments) and is regulated by light chain phosphorylation Regulation by phosphorylation of light chains Figure 16-54a Molecular Biology of the Cell (© Garland Science 2008) [email protected] SvdH Figure 16-72a Molecular Biology of the Cell (© Garland Science 2008) SvdH 12 SvdH 1 Master course Molecular and Cellular Life Sciences 16/10/2012 The cytoskeleton is rapidly and dramatically reorganized during cell division Dynein a minus-end directed microtubule motor SvdH Figure 16-64 Molecular Biology of the Cell (© Garland Science 2008) The small GTPases Rho, Rac and Cdc42 control actin dynamics SvdH 14 Figure 16-2 Molecular Biology of the Cell (© Garland Science 2008) Small GTPases (Rho, Rac, CDC-42) switch between active and inactive states GEF Stress fibers (exchange factor) GTP GDP RhoA RhoA GAP (GTPase Activating Protein) Lamellipodia Filopodia Figure 16-97 Molecular Biology of the Cell (© Garland Science 2008) SvdH 15 Activated Rac promotes formation branched actin (meshwork) SvdH 16 Activated Rho promotes formation actin fibers (stress fibers and contractile ring) Actin fiber formation (formin) Myosin activation (ROCK) Cytokinesis! contractile ring formation and contraction Figure 16-98a Molecular Biology of the Cell (© Garland Science 2008) [email protected] SvdH 17 Figure 16-98b Molecular Biology of the Cell (© Garland Science 2008) SvdH 18 SvdH 1 Master course Molecular and Cellular Life Sciences 16/10/2012 Rapid and complete reorganization of the cytoskeleton during cell division Microtubules of the mitotic spindle control# the plane of cell division! Actin formation contractile ring: local Rho accumulation/activation Microtubules Figure 16-2 Molecular Biology of the Cell (© Garland Science 2008) SvdH 20 Conly Rieder SvdH 19 What is the role of microtubules in cleavage plane determination, and which microtubules are involved? “Equatorial stimulation” or “polar relaxation” ? SvdH 21 Both astral- and midzone microtubules (between segregating chromosomes) position cleavage plane Rappaport’s famous experiments with sand dollar eggs Cell cleavage takes place between spindle poles -> argument for “equatorial stimulation” by overlapping astral microtubules SvdH 22 In development, mitotic spindle positioning is used to change the orientation and plane of cell division change ! of axis! asymmetric! division! Mechanism only partly understood SvdH 23 [email protected] SvdH 24 SvdH 1 Master course Molecular and Cellular Life Sciences 16/10/2012 How is cell cleavage controlled in early C. elegans embryos? How can asymmetric division generate different daughter cells ? Note: asymmetric division ! 1) Polarity 2) Localization of Determinants Polarity determinants Stem cell lineage Chromosomes Germ line components 3) Correct plane of cell cleavage SvdH 25 SvdH 26 The cell cleavage plane is determined by the spindle: what determines the position of the spindle ? ! Asymmetric cell division Establishment of cell polarity: reorganization cytoskeleton# # Localization of cell determinants along the polarity axis# # GFP::Tubulin A. Desai # Asymmetric division in the early C. elegans embryo provides an attractive model Positioning of the mitotic spindle, which instructs # the plane of cell cleavage# -> Cell division generates unequal daughters # SvdH Mutations in par (partitioning abnormal) genes disrupt early embryonic asymmetries! Asymmetric cell division SvdH 28 Establishment of cell polarity: reorganization cytoskeleton: # -> MECHANISMS RELATED TO CYTOKINESIS# # Localization of cell determinants along the polarity axis# WT par-2 par-3 par-5 # Positioning of the mitotic spindle# # Division generates unequal daughters # SvdH 29 [email protected] SvdH 30 SvdH 1 Master course Molecular and Cellular Life Sciences 16/10/2012 Several PAR proteins localize to the cell cortex anterior-posterior! polarity In the fertilized egg, PAR-2 localizes to the posterior cortex, together with PAR-1# ! PAR-2::GFP Levitan et al. PNAS 2004 Movies: Cuenta et al. Dev 2003 Localization critical and dynamically controlled ! SvdH 31 SvdH 32 PAR-3/6 and PAR-2 localize to opposite domains and counteract each other Anterior PAR complex: PAR-3, PAR-6, PKC-3 and CDC-42 CDC-42 PAR-6 Conserved roles in polarized cells!! PKC-3 PAR-6 PAR-2 PAR-3 Counteract PAR-2, PAR-1 localization in C. elegans one-cell embryo SvdH 33 PAR-3/6 and PAR-2 localize to opposite domains and counteract each other PAR-2::GFP Initially: # PAR-3/PAR-6 are at the cortex, # PAR-2 cytoplasmic# When PAR-3/PAR-6 retract,# PAR-2 appears at the cortex # # In the absence of PAR-6, # PAR-2 is all over the cortex # WT par-6 mutant [email protected] # Cuenta et al. Dev 2003 SvdH 35 SvdH 34 The one-cell C. elegans embryos is polarized by a conserved set of PAR proteins PAR-1 (ser/thr protein kinase)# PAR-2 (a ring finger protein)# PAR-3 (a PDZ domain protein)# PAR-4 (ser/thr protein kinase related to LKB1)# PAR-6 (a PDZ domain protein)# PKC-3 (atypical protein kinase C)# Localizations regulated, dynamic and asymmetric!! SvdH 36 SvdH 1 Master course Molecular and Cellular Life Sciences 16/10/2012 ! Summary: model polarity establishment ! What triggers polarization of the fertilized egg? Drugs that prevent actin polymerization cause defects similar to those in par mutants Anterior PAR complex (PAR-3, PAR-6, PKC-3)# Posterior PARʼs PAR-1/PAR-2# SvdH 37 actin-myosin contracts towards anterior following fertilization with PAR-3/PAR-6 SvdH 38 Fertilization disrupts actin-myosin network, which triggers movement to opposite pole Munro et al. SvdH2004 39 Dev Cell, PAR-3/PAR-6 move with actin-myosin towards anterior pole Munro et al. SvdH2004 40 Dev Cell, ! Summary: model polarity establishment ! Sperm entry somehow destabilizes acto-myosin network collapses towards anterior Acto-myosin transports PAR-3/PAR-6/PKC-3 to anterior This allows PAR-2 to occupy posterior PAR-2 prevents the return of PAR-3/PAR-6 Acto-myosin Munro et al. SvdH2004 41 Dev Cell, [email protected] -> A/P axis created and fixed SvdH 42 SvdH 1 Master course Molecular and Cellular Life Sciences 16/10/2012 ! What is contribution sperm entry in polarity ! establishment Small GTPases (Rho, Rac, CDC-42) switch between active and inactive states GEF (exchange factor) GTP GDP Rho 1) 2) 3) Rho GAP Microtubule organizing centers# Need to mature (Cyclin E/CDK-2 dependent)# Regulation Rho GTPase -> Rho kinase -> # Myosin light chain phosphorylation (GTPase Activating Protein) SvdH 43 SvdH 44 The RHO-1 GTPase, ECT-2 GEF and CYK-4 GAP drive actin-myosin reorganization Small GTPases (Rho, Rac, CDC-42) switch between active and inactive states ECT-2 (exchange factor) GTP GDP RHO-1 RHO-1 CYK-4 (GTPase Activating Protein) SvdH 45 CYK-4 GAP is provided paternally and may trigger actin-myosin reorganization Jenkins et al. SvdH 2006 46 Science, Cortical exclusion of ECT-2 GEF by the centrosome may trigger actin-myosin movements CYK-4 RNAi Jenkins et al. SvdH 2006 47 Science, [email protected] Motegi and Sugimoto Nat. Cell Biol., SvdH 2006 48 SvdH 1 Master course Molecular and Cellular Life Sciences 16/10/2012 RHO-1 drives actin-myosin reorganization, sperm CYK-4 GAP provides cue ! Summary: model polarity establishment ! ECT-2 (exchange factor) GTP GDP RHO-1 RHO-1 1. define posterior Rho kinase 4. Position pronuclei Light Chain Phosphatase CONTRACTION Many aspects of the first cell division are asymmetric Cortex: # contractions become restricted to anterior domain # Spindle: # - moves to posterior, “rocks” and flattens# - rotates in P1# Cell division: unequal sizes # - planes opposite during second division# - timing different# Fate determinants become asymmetrically localized # 5. Position spindle and cleavage plane Anterior PAR complex: PAR-3, PAR-6, PKC-3# Posterior PARs: PAR-1/PAR-2# SvdH 49 3. Posterior PARs localize, Stabilize asymmetry! Myosin Light Chain Phosphorylation CYK-4 (GTPase Activating Protein) 2. actomyosin contraction, anterior PARs move (asymmetry!) Spindle positioning requires the function of the LIN-5 and GPR-1/2 proteins Meiosis Spindle movement Asymmetric division AB Spindle rotation P1 lin-5 RNAi gpr-1/2 RNAi SvdH 51 The LIN-5 and GPR proteins localize and function together, downstream of PAR proteins SvdH 50 SvdH 52 GPR-1,2 proteins contain a specific domain for interaction with Gαi/o subunits# TPR GPR/GoLoco GPR-1 Function spindle movement GPR-2 CTL LIN-5 GPR -1/2 AGS3 (Hs) PINS (Dm) Merge lin-5 (RNAi) LIN-5 GPR -1/2 The GoLoco peptide binds Gα⋅GDP! and competes with Gβγ! Merge SvdH 53 [email protected] GDI for Gαi spindle orientation, binds Gαi Siderovski! Nature 2002! SvdH 54 SvdH 1 Master course Molecular and Cellular Life Sciences 16/10/2012 Heterotrimeric G proteins are key components of trans-membrane signaling # An unusual Gα pathway controls the cell cleavage plane Similar loss of function phenotypes:# # LIN-5 (Mud, NuMA)# # GPR-1,2 (Pins, AGS3, LGN)# # GOA-1/GPA-16 Gαi/o # RIC-8 GEF# ! G-protein coupled receptor Gα • ! GDP! Gβγ! ligand (GEF)! Gα !• GTP! Gβγ! effectors! effectors! Wild type ric-8(RNAi) Gα(RNAi) SvdH 55 …..but here: independent of ligand and transmembrane receptor SvdH 56 A conserved Gα/GPR/LIN-5/dynein pathway controls spindle and cleavage plane positioning Gα subunit Dy nei n mic rot ubu le LGN, AGS3 Dm Pins NuMA/Mud Dynein Generates cortical pulling forces on astral microtubules during chromosome segregation and spindle positioning Van den Heuvel, Gonczy, SvdH Gotta [email protected] SvdH 1