Cytokinesis in fission yeast: Modeling the assembly of the contractile ring Nikola Ojkic, Dimitrios Vavylonis Department of Physics, Lehigh University Damien Laporte, Jian-Qiu Wu Department of Molecular Genetics and Department of Molecular and Cellular Biochemistry The Ohio State University UBC May 25, 2012
Projects in actin cytoskeleton dynamics Regulation of actin assembly at the leading edge (with Naoki Watanabe, Tohoku University) Cell polarizarion, model of Cdc42 oscillations (with Fulvia Verde, Univ of Miami) LifeAct-mCherry G. L. Ryan, H. Petroccia, N. Watanabe and D. Vavylonis Biophys. J. (2012). CRIB-GFP M. Das, T. Drake, D. Wiley, P. Buchwald, D. Vavylonis and F. Verde (Science, 2012). Image analysis methods to quantify patterns use of model biological systems coarse-grained modeling Jfilament, Cytoskeleton 2010 ImageJ plugins Speckle TrackerJ Biophys. J. 2011
Actin Cytoskeleton in Cell Division fission yeast cdc25-22 cell CHD-GFP binds to sides of actin filaments spindle poles Spb1 Vavylonis, Wu, et al. Science 2008
Contractile ring assembles from ~ 65 myosin II nodes in ~ 10 min Rlc1p-3GFP 10 min spinning disk confocal microscopy Vavylonis, Wu, Hao, O Shaughnessy, Pollard, Science 2008 ~ 40 myosin II (Myo2p) molecules/node ~ 2 formin Cdc12p dimers/node Mid1p (annilin homologue) Wu and Pollard, Science 2005
Formins nucleate actin filaments and remain associated with the growing end actin formin dimer FH2 FH1 Kovar, Pollard. PNAS (2004) profilin actin purified fluorescent actin + formin Bni1p Vavylonis, Kovar, O Shaughnessy, Pollard Mol. Cell 2006
Model with static connections condenses nodes into clumps numerical simulation 0 100 200 300 400 500 600 sec time pattern and dynamics of connections is important
Nodes move in stochastic manner, making many starts, stops and changes of direction 15x time lapse 10 minutes each frame: average of 6 typical node speed v 30 nm/sec typical duration 20 sec Vavylonis, Wu, Hao, O Shaughnessy, Pollard, Science 2008
(nm 2 ) Diffusive motion of stationary nodes mean square displacement 15x, 200s total diffusive stage thermal (?) motion D 2 30 nm /sec condensation stage: velocity in response to force v 30 nm/sec kt F v 4 pn D consistent with force exerted by few molecular motors
Actin filament assembly and disassembly around nodes GFP-CHD, Rlc1p-tdTomato (static) single confocal slice 4x time lapse (0.3 s/frame) GFP-CHD Rlc1p-tdTomato cdc25-22 cell 2X time lapse 0.2 s/frame
Search, capture, pull and release model actin filament polymerization actin filament capture ~ 0.2 m/sec r c ~ 100 nm lifetime of connections ~ 20 sec F 4pN traction on filaments between nodes lifetime of filaments ~ 20 sec Dynamic reestablishment of connections plasticity of network
Ring formation by SCPR and dependence on parameter values v pol 0.2 m/sec v pol 0.04 m/sec red: nodes green: actin filaments
Many mutant cells form clumps Wild type: robust ring formation ring Formin Mutant: clump formation clumps Hachet and Simanis, Genes and Development, 2008 Chen and Pollard, JCB 2011: cofilin mutants form clumps Theory of clump formation kinetics: Ojkic and Vavylonis, Phys Rev Lett 2010
Linear myosin structures appear near the end of ring assembly Rlc1p-3GFP WT Rlc1p-mRFP1 1µm Ojkic, Wu, Vavylonis J. Phys. Cond Matt (2011) 1µm
SCPR + LOCAL NODE ALIGNMENT Ojkic, Wu, Vavylonis J. Phys. Cond. Matt. (2011) WT SCPR Point A Point B cdc25-22 Point A (100 nodes, w=3.2 m) Point B (200 nodes, w=3.2 m)
Normal node condensation depends on cross-linkers In α-actinin deletion, myosin nodes condense into clumps. Rlc1-3GFP Radial projections: D. Laporte, N. Ojkic, D. Vavylonis and J.-Q. Wu (submitted)
Overexpressing α-actinin makes nodes condense into meshworks CHD-GFP actin marker
Actin filaments modeled as semi-flexible polymers using Langevin dynamics l 0 dr b dt i F thermal i F sp i F no long range hydrodynamics l 0 represents 70 actin monomers bend i model of actin filament Thermal force: Spring force: Bending force: Kim, T., W. Hwang, and R.D. Kamm, 2009. Experimental Mechanics, 49:91-104. Pasquali, M. and D.C. Morse, 2002, J. Chem. Phys. 116:1834-1838.
Probability Curvature distribution P 2D ( ) Validation of semiflexible polymer dynamics 2l p l 0 e l p l 0 2 / 2 0.08 0.06 0.04 l p sim = 10.6 (3) m l p theo = 10 m 0.02 Relaxation Dynamics 0.00 0 1 2 3 4 m 1 curvature, m -1 10 n b L 4 ( ) ( n 1/ 2) flexural rigidity n (s) 1 0.1 0.01 0.001 in water B D simulation theory Gittes, F., B. Mickey, J. Nettleton, and J. Howard. 1993. J. Cell Biol. 120:923 0.0001 1 10 Fourier mode, n
Revised SCPR with semi-flexible filaments search & capture pull cross-linking U(r) slowing down of movement inside a bundle 0 r cross r maintainance of angle of polymerization r 0 cross 50 nm stiffness k cross We assume a simple spring potential is sufficient to capture morphological changes
Simulations as function of cross-linking strength a = 0 a = 0.7 a = 1
Simulations as function of cross-linking strength
simulation results experimental results D. Laporte, N. Ojkic, D. Vavylonis and J.-Q. Wu (submitted).
Optimal values of k cross and α make bundles of parallel+antiparallalel filaments
Simulations suggest that cross-linkers bind dynamically to actin filaments, contributing to alignment and damping In vitro, dissociation rate of a-actinin ~ 1 s -1 or faster (Xu et al., JBC 1998; Strehle et al., Eur. Biophys. J. 2011 Friction force per cross-linked filament bead a k cross l 0 0.7pN Estimate of drag force per a-actinin from in vitro experiments ~ 0.012 pn Greenberg and Moore Cytoskeleton 2010 Suggests a few cross-linkers per micron in fission yeast
2x myo2 41nmt1-ain1 Myosin overexpression can rescue cross-linker overexpression phenotype simulation results experimental results
Myosin activity is essential for node condensation
Acknowledgments Nikola Ojkic (Lehigh) Damien Laporte (OSU) Jian-Qiu Wu (OSU) Support: NIH (Wu, Vavylonis)