Cytokinesis in fission yeast:

Modeling the assembly of the contractile ring

UBC

May 25, 2012

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

Cell polarizarion, model of Cdc42 oscillations

(with Fulvia Verde, Univ of Miami)

CRIB-GFP LifeAct-mCherry

Regulation of actin assembly at the leading edge

(with Naoki Watanabe, Tohoku University)

Projects in actin cytoskeleton dynamics

● use of model biological systems

● coarse-grained modeling

Image analysis methods to quantify patterns

Jfilament, Cytoskeleton 2010

Speckle TrackerJ Biophys. J. 2011 ImageJ plugins

M. Das, T. Drake, D. Wiley, P. Buchwald,

D. Vavylonis and F. Verde (Science, 2012). G. L. Ryan, H. Petroccia, N. Watanabe and

D. Vavylonis Biophys. J. (2012).

CHD-GFP

binds to sides of

actin filaments

spindle poles

Spb1

Vavylonis, Wu, et al. Science 2008

Actin Cytoskeleton in Cell Division

fission yeast cdc25-22 cell

Contractile ring assembles from ~ 65 myosin II nodes in ~ 10 min

~ 40 myosin II (Myo2p) molecules/node

~ 2 formin Cdc12p dimers/node

Mid1p (annilin homologue) Wu and Pollard, Science 2005

Rlc1p-3GFP

Vavylonis, Wu, Hao, O’Shaughnessy, Pollard, Science 2008 spinning disk confocal microscopy

10 min

Formins nucleate actin filaments and

remain associated with the growing end

purified fluorescent

actin + formin Bni1p

profilin actin

FH1 FH2

actin formin dimer

Kovar, Pollard. PNAS (2004)

Vavylonis, Kovar, O’Shaughnessy, Pollard Mol. Cell 2006

time

numerical simulation

pattern and dynamics of connections is important

0 100 200 300 400 500 600 sec

Model with static connections condenses nodes into clumps

15x time lapse

10 minutes

each frame:

average of 6

Nodes move in stochastic manner, making many

starts, stops and changes of direction

nm/sec30v

sec20

typical node speed

typical duration

Vavylonis, Wu, Hao, O’Shaughnessy, Pollard, Science 2008

Diffusive motion of stationary nodes

15x, 200s total

mean square displacement

(nm

2)

pNvD

kTF 4

consistent with

force exerted

by few molecular

motors

/secnm30 2Ddiffusive stage

thermal (?) motion

condensation stage:

velocity in response

to force

nm/sec30v

Actin filament assembly and disassembly around nodes

4x time lapse (0.3 s/frame) GFP-CHD, Rlc1p-tdTomato (static) single confocal slice

cdc25-22 cell

2X time lapse

0.2 s/frame

GFP-CHD

Rlc1p-tdTomato

actin filament

polymerization

m/sec2.0~

actin filament capture

nm 100~cr

traction on filaments

between nodes

pNF 4

lifetime of connections

lifetime of filaments

sec 20~

sec 20~

Dynamic reestablishment of connections plasticity of network

Search, capture, pull and release model

Ring formation by SCPR and dependence on parameter values

red: nodes

green: actin filaments

m/sec2.0 polv

m/sec04.0 polv

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

Formin Mutant: clump formation

Wild type: robust ring formation

Many mutant cells form clumps

ring

clumps

Linear myosin structures appear near the end of ring assembly

1µm

Rlc1p-mRFP1

Rlc1p-3GFP WT

1µm

Ojkic, Wu, Vavylonis J. Phys. Cond Matt (2011)

SCPR Point A Point B Point A

(100 nodes, w=3.2 m)

Point B (200 nodes, w=3.2 m)

WT cdc25-22

SCPR + LOCAL NODE ALIGNMENT

Ojkic, Wu,

Vavylonis

J. Phys. Cond.

Matt. (2011)

Radial projections:

In α-actinin deletion, myosin nodes

condense into clumps.

D. Laporte, N. Ojkic, D. Vavylonis and J.-Q. Wu (submitted)

Normal node condensation depends on cross-linkers

Rlc1-3GFP

Overexpressing α-actinin makes nodes condense into meshworks

CHD-GFP

actin marker

Actin filaments modeled as semi-flexible polymers using Langevin dynamics

bendspthermal

iiii

bdt

dFFF

r

0l

Thermal force:

Bending force:

model of actin filament

• no long range hydrodynamics

• l0 represents ≈ 70 actin monomers

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.

Spring force:

0.0001

0.001

0.01

0.1

1

10

1 10

B

D

n (

s)

Fourier mode, n

simulation theory

Gittes, F., B. Mickey, J. Nettleton, and J. Howard. 1993. J. Cell Biol. 120:923

4))2/1(

(

n

Lbn

flexural rigidity in water

2/0

2

20

2)(

llp

Dpe

llP

Curvature distribution

Relaxation Dynamics

0 1 2 3 40.00

0.02

0.04

0.06

0.08

m 1P

robab

ility

lpsim = 10.6 (3) m

lptheo = 10 m

curvature, m-1

Validation of semiflexible polymer dynamics

Revised SCPR with semi-flexible filaments

search & capture pull

cross-linking

nm500

cross r

crossr

)(rU

r0

stiffness kcross

We assume a simple spring potential is sufficient

to capture morphological changes

slowing down of movement inside a bundle

maintainance of angle of polymerization

a = 0

a = 0.7

a = 1

Simulations as function of cross-linking strength

Simulations as function of cross-linking strength

D. Laporte, N. Ojkic, D. Vavylonis and J.-Q. Wu (submitted).

simulation results

experimental results

Optimal values of kcross and α make bundles of parallel+antiparallalel filaments

Simulations suggest that cross-linkers bind dynamically

to actin filaments, contributing to alignment and damping

pN7.00cross lka

Friction force per cross-linked

filament bead 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

In vitro, dissociation rate of

a-actinin ~ 1 s-1 or faster

(Xu et al., JBC 1998;

Strehle et al., Eur. Biophys. J. 2011

Myosin overexpression can rescue cross-linker overexpression phenotype

2x m

yo

2 4

1n

mt1

-ain

1

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)