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Stochastic simulation method

Our simulation technique belongs to the kinetic Monte Carlo (MC) class of models as
individual molecules are sampled randomly in our model and they are attempted either a
diffusion or a reaction move. In our model, not only is the explicit spatial location of
signaling molecules considered but also the mutual exclusion of signaling molecules and
the slow diffusion of large molecular complexes are taken into account to simulate
crowding. One novel aspect of our stochastic model is that instead of using a free energybased Metropolis scheme (1), we map our probabilistic parameters to the experimentally
measurable on and off kinetic rates of signaling reactions (2) and thereby satisfy the
detailed balance condition at any point in space. The probability of diffusion is separately
mapped to the macroscopic diffusion constant.
At the beginning of the simulation all the
signaling
molecules
are
randomly
distributed inside a 3 dimensional cubic
box. Surface bound signaling molecules are
placed on one of the six surfaces of the box
and they are allowed to diffuse only on the
2 dimensional plane. We pick up a
molecule randomly from the system and
then decide between diffusion move and a
reaction move using an unbiased coin toss.
If we choose the diffusion move, the
molecule attempts to move to one of its
empty neighbor sites with a predefined
Schematics of the 3D simulation lattice
probability of diffusion Pdiff. Complex
molecules composed of three or more
molecules are chosen to be immobile (3,4). If the chosen move is a reaction, three
possible reactions can take place (i) complex formation from two free molecules with a
predefined probability Pon (ii) dissociation of an already formed complex molecule into
constituent free molecules with a given probability Poff, and (iii) catalysis of an already
formed enzyme-substrate complex molecule into the product and enzyme molecules with
a given probability Poff. One of the noteworthy features of our model is its treatment of
reaction events where multiple binding sites of a single molecule are considered
separately at the nanoscale (~10 nm) detail.
Finally, a simple yet rigorous mapping scheme between our simulation parameters and
those observed in experiments is also established (2). One rudimentary way of choosing
the size of the time step in stochastic simulations is to match the simulation time scale
with the already-known time scale of the relevant biological process. Instead of this
approach, the time-step t of our simulation is set by mapping the macrosopic diffusion
constant D, calculated using single molecule (low density) diffusion simulations for a
given pdiff, to experimentally observed diffusion constants. By using such a mapping
scheme we were able to successfully match the time scale of our simulation to realistic
values. Once we have obtained the time scale mapping, it is straightforward to map poff to
koff : koff= t.poff. Once we know poff from this equation, the value of pon can be determined

by calculating the association constant Ka from a separate set of simulations (only for
binding-unbinding type reactions) and then mapping that to experimentally measured Ka.
The association constant Ka is measured using the equilibrium molecule numbers of free
and complex molecules (either averaged over many runs, or over a long time after
equilibrium is reached). Calculation of pon using Ka from simulations crucially depends
on the spatial dimension (surface bound = 2d or cytosolic = 3d).
Apoptosis signaling reactions
Our stochastic computational model considers apoptotic signaling through two distinct
pathways (a) direct activation of caspase-3 by caspase-8 (type 1) and (b) activation of
caspase-3 by mitochondrial cytochrome-c release and apoptosome formation (type 2)
(Figure 1). Intracellular apoptosis signaling in our model is triggered by the activation of
caspase-8 molecules at the cell surface, which in turn diffuse in the cytosol and activate
both pathways of apoptosis signaling (Figure 1). In the type 1 pathway, caspase-8
molecules directly catalyze the cleavage reaction of procaspase-3 to generate caspase-3.
In the type 2 pathway, caspase-8 binds with Bid and catalyzes its truncation to form tBid,
which in turn binds to Bax to generate Bax2 complex molecules. Bcl2, an anti-apoptotic
molecule, can inhibit both tBid and Bax, thus creating a local loop structure in the type 2
signaling cascade. The Bax2 complex leads to cytochrome c release from mitochondria
and ultimately to the activation of caspase-3 (Figure 1). Hence, caspase-3 activation at
the end of both the type 1 and the type 2 pathways creates a global loop structure in
apoptosis signaling. We describe below the major signaling reactions in the apoptoticsignaling cascade that are simulated in our model.
Kon = 3.5 x 10-3 nM-1 s-1

Disc + ProCaspase8

DiscProCaspase8

Koff = 1.8 x 10-2 s-1

DiscProCaspase8 + ProCaspase8

Kon = 3.5 x 10-3 nM-1 s-1

Disc2 ProCaspase8

Koff = 1.8 x 10-2 s-1

Disc2 ProCaspase8
P4341

Kcat = 3.0 x 10-1 s-1

Kcat = 1.0 x 10-1 s-1

Disc + 2 P4341
Caspase8

Kon = 5 x 10-3 nM-1 s-1

Caspase8 + Bid

Caspase8 Bid
Koff = 5 x 10-3 s-1

Caspase8 Bid

Kcat = 1 x 10-1 s-1

Caspase8 + t Bid

Caspase8 + ProCaspase 3

Caspase8 ProCaspase3

Kon = 1.0 x 10-5 nM-1 s-1

Caspase8 ProCaspase3

Koff = 6.0 x 10-2 s-1

Caspase8 + Caspase3

Kcat = 1 x 10-1 s-1

Kon = 2.0 x 10-4 nM-1 s-1

t Bid + Bax

t Bid Bax
Koff = 2.0 x 10-2 s-1
Kon = 2.0 x 10-4 nM-1 s-1

t Bid Bax + Bax

-2

Koff = 2.0 x 10 s

t Bid Bax_2

Bcl2 + t Bid

t Bid Bax_2

-1

t Bid + Bax_2

Koff = 2.0 x 10-2 s-1

Kon = 2.0 x 10-4 nM-1 s-1

Bcl2 t Bid

Koff = 2.0 x 10-2 s-1


Kon = 2.0 x 10-3 nM-1 s-1

Bcl2 + Bax

Bcl2 Bax
Koff = 2.0 x 10-2 s-1

Kon = 2.8 x 10-7 nM-1 s-1

Cytochrome C + Apaf

Apoptosome + ProCaspase9

Cytochrome CApaf

Koff = 5.7 x 10-3 s-1

Kon = 2.8 x 10-4 nM-1 s-1


-2

Koff = 7.5 x 10 s

Apoptosome2ProCaspase9

ApoptosomeProCaspase 9

-1

K cat = 7 x10-1 s-1

Apoptosome + Caspase9

Kon = 2.0 x 10-5 nM-1 s-1

Caspase9 + ProCaspase3

Caspase9-ProCaspase3
-2

Koff = 5.7 x 10 s

-1

Caspase9-ProCaspase3

Kcat = 4.8 s-1

Caspase9 + Caspase3

Cytochrome C is released from mitochondria once Bax complex (Bax2) concentration


reaches a threshold (set as 10 nm in our simulations). Kinetic reaction constants shown
above are taken from previous studies (3,5).
Concentration of signaling molecules
Initial concentrations (in nanomolar unit) for the signaling molecules (3,4,5,6) are given
below:
DISC = 165; Bid = 25; Bax = 83.33; Bcl2 = 75; Cytochrome C = 100; Apaf = 100;
ProCaspase-9 = 20; Procaspase-3 = 200; ProCaspase-8 concentration is varied between
33 333 nm in our simulations. Concentrations of other molecules such as ProCaspase3
can vary as well depending on the specifics of cell line and cellular condition such as the
progression in the cell cycle. Two fold variations in concentrations did not change our
main findings.
Initial concentrations of signaling molecules, which are generated in the course of
signaling, are taken to be zero.
Lattice size of simulation box and spatial-temporal scales
We have used a 50 x 50 x 50 box to simulate apoptosis signaling reactions. Lattice
spacing is assumed to be ~ 20 nm so the total volume of the simulation box is ~ 1 m3.
We assume a typical cell volume of ~ 500 m3 and the number of molecules in our 1 m3
simulation box were scaled down accordingly. Using a bigger simulation box resulted in
qualitatively similar results. Each Monte Carlo (MC) time step is assumed to be ~ 10-4
seconds (using diffusion constants of signaling molecules ~ 0.1 m2/sec.).
References
1. Newman, M. E. J, and G. T. Barkema. 1999. Monte Carlo Methods in Statistical Physics. Oxford University Press, USA.
2. Tsourkas, P., N. Baumgarth, S. I. Simon, and S. Raychaudhuri. 2007. Mechanism of B-cell synapse formation predicted by
Monte Carlo simulation. Biophys J. 92:1-13.
3. Hua, F., M. G. Cornejo, M. H. Cardone, C. L. Stokes, D. A. Lauffenburger. 2005. Effects of Bcl-2 Levels on Fas signalinginduced caspase-3 activation: molecular genetic tests of computational model predictions. J. Immunol. 175:985-995.
4. Eising, T. H., E. D. Conzelman, F. Allgower, E. Bullinger, and P. Scheurich. 2004. Bistability analysis of a caspase activation
model for receptor-induced apoptosis. J. Biol. Chem. 279:36892-36897.
5. Bagci, E. Z., Y. Vodovotz, T. R. Billiar, G. B. Ermentrout, and I. Bahar.2006. Bistability in apoptosis: roles of Bax, Bcl-2, and
mitochondrial permeability transition pores. Biophys. J. 90:1546-1559.
6. Sun, X., S. B. Bratton, M. Butterworth, M. MacFarlane, and G. M. Cohen. 2002. Bcl-2 and Bcl-xL inhibit CD95-mediated
apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of x-linked Inhibitor-of-apoptosis
protein. J. Biol. Chem. 277, 11345-11351.