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S. Portet, J.A. Tuszynski, J.M. Dixon and M.V. Sataric: Models of spatial and orientational self-organization of microtubules under the influence of gravitational fields

S. Portet, J.A. Tuszynski, J.M. Dixon and M.V. Sataric: Models of spatial and orientational self-organization of microtubules under the influence of gravitational fields

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PROOF COPY [EA8986] 021308PRE
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Models of spatial and orientational self-organization of microtubules under the influenceof gravitational fields
S. Portet
*
and J. A. Tuszynski
 Department of Physics, University of Alberta, Edmonton, Alberta, T6G 2J1 Canada
J. M. Dixon
 Department of Physics, University of Warwick, Coventry CV4 7AL, United Kingdom
M. V. Sataric
Faculty of Technical Sciences, University of Novi Sad, 21000 Novi Sad, Serbia, Federal Republic of Yugoslavia
Received 15 January 2003
Tabony and co-workers
C. Papaseit, N. Pochon, and J. Tabony, Proc. Natl. Acad. Sci. U.S.A.
97
, 8364
2000

showed that the self-organization of microtubules from purified tubulin solutions is sensitive to gravi-tational conditions. In this paper, we propose two models of spatial and orientational self-organization of microtubules in a gravitational field. First, the spatial model is based on the dominant chemical kinetics. Thepattern formation of microtubule concentration is obtained
1
in terms of a moving kink in the limit when thedisassembly rate is negligible, and
2
for the case of no free tubulin and only assembled microtubules present.Second, the orientational pattern of striped microtubule domains is consistent with predictions from a phenom-enological Landau-Ginzburg free energy expansion in terms of an orientational order parameter.DOI: 10.1103/PhysRevE.68.0219XX PACS number
s
: 82.20.
w, 82.70.
y, 82.39.
k, 89.75.Kd
I. INTRODUCTION
Microtubules
MT’s
are major cytoskeletal proteins andare present in virtually all eukariotic cells. They are involvedin many essential cell functions such as mitosis, maintenanceof cell shape, cell motility, signal transduction, and intracel-lular transport. MT’s exhibit a highly dynamical behaviorand specific spatial reorganizations which allow them to per-form their cellular roles. MT’s are long cylindrical polymersthat exhibit aggregation through the addition at their ends of 
 
,
 
-tubulin heterodimers
1
.Tabony and co-workers have conducted a series of experi-ments on the effects of the gravitational conditions on the
invitro
self-organization of MT’s under conditions of high tu-bulin concentration
2,3
. Preparations containing purifiedtubulin and GTP were heated from
7 °C to 37°C andMT’s were assembled in rectangular samples of particularsize (40
10
1 mm
3
). An enzymatic system was alsopresent to regenerate the GDP and to provide a continuoussource of GTP. The different samples were subjected to spe-cific gravitational conditions. Thus, progressive self-organization of MT’s was observed depending on the gravi-tation field strength and the orientation of samples withrespect to the gravity axis. These authors
2,3
observed thatafter about 5 h, the preparations had stabilized and the fol-lowing types of pattern of MT assembly had been identified:
1
under gravity
on Earth and in flight under 1
g
centrifu-gation
with the major axis of the sample parallel to thegravity axis, striped patterns of MT’s appeared, with twoadjacent stripes made up of highly oriented MT bundles at anangle of 45° and 135°, respectively, to the horizontal
Fig.1
;
2
under gravity and with the major axis of samplesperpendicular to the gravity axis, circular patterns were ob-served;
3
in weightlessness (10
4
g
), an isotropic patternappeared, and no preferential orientation was adopted by theMT’s.The effect of the gravitational field on the MT self-organization has been observed both for
in vitro
and
in vivo
experiments with mammalian and vegetal specimens. For ex-ample, in two different space flight experiments led by Lewis
4
and Vassy
5
, the structural organization of 
in vivo
MT’sshowed dramatic differences between the gravity and micro-gravity conditions. Instead of well-formed MT’s radiatingfrom organizing centers in the gravitational environment,cells in microgravity uniformly diffuse and exhibit shortenedMT’s without normal organization.In the present paper, we construct models in accordancewith the experimental conditions outlined in Ref.
6
, to de-scribe quantitatively the effects of the gravitational field onthe self-organization of MT’s. Two distinct approaches willbe presented dealing separately with the spatial distributionof the MT concentration and with orientational order withinthe MT assembly. The reason for this distinction is the pres-ence of the vastly different time scales of these two differentdynamical processes, the former being much faster than thelatter due to the significant differences between the diffusionconstants for tubulin and MT’s, respectively.
II. THE MODEL OF SPATIAL ORGANIZATION
To model the spatial self-organization of 
in vitro
MT’s,we consider the competition between different processes.These include nonlinear chemical kinetics of MT assembly
*
Author to whom correspondence should be addressed. Presentaddress: Department of Physics, University of Alberta, Edmonton,Alberta, T6G 2J1 Canada. Electronic address:sportet@phys.ualberta.caPHYSICAL REVIEW E
68
, 0219XX
2003
1063-651X/2003/68
2
 /0219XX
9
 /$20.00 ©2003 The American Physical Society
68
0219XX-1
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7,8
, diffusion processes of tubulin and MT
s accounting forsigni
cant differences in the diffusion coef 
cient as a resultof size and geometrical characteristics, and a hydrodynamicdrift process of tubulin dimers and MT
s resulting from theaction of the gravitational
eld with the buoyancy correction
9
. To develop a physical model, we use a reaction-diffusionapproach that accounts for the gravitational environment.The model equation is generally expressed by
1
where
S
is the two-vector of the concentrations
and
,
isthe number concentration of tubulin dimers, and
is thenumber concentration of MT
s.
D
is the diagonal matrix of positive diffusion constants.
u
is the diagonal matrix of driftvelocities of proteins induced by gravitational conditions.
R
is the two-vector of the reaction terms
(
,
 M 
) and
h
(
,
 M 
)that are described below in detail. The present model is con-sidered with the initial conditions
 x
, where
is thespatial domain,
(
 x
,0)
c
0
,
(
 x
,0)
0, and zero-
uxboundary conditions are used.In the development of the reaction term, we are guided bythe tools of chemical kinetics as applied to protein polymer-ization. The reader is referred to the seminal works of Hill
7
and Oosawa and Asakura
8
. Furthermore, in the presentstudy we wish to strongly emphasize the emergence of spa-tial patterns that were observed by Tabony and Job
3
.Hence, our focus is on the observed dynamics of MT aggre-gation. With the inclusion of free tubulin dynamics thatdrives MT aggregation, we therefore need to distinguish twoprincipal state variables: the assembled tubulin and the freetubulin. In our model, chemical kinetics equations are pro-posed in terms of number concentration for the free dimertubulin,
(
), and for MT
s,
(
).MT
s switch between assembly and disassembly phases.This behavior is called dynamic instability
1
, the transitionfrom disassembly to assembly phases is known as
rescue
andthe reverse process, i.e., the transition from assembly to dis-assembly, as
catastrophe
. Moreover, we assume the presenceof spontaneous
nucleation
from nuclei
can also be calledseeds
which initiate MT
s. The kinetics of MT nucleationwas studied in detail by Flyvbjerg
et al.
10
. We also in-clude the possibility of a nucleus elimination reaction, i.e.,the reverse chemical reaction that removes MT
s from thesolution because the nuclei may become structurally un-stable. Thus the dynamics of the number of MT
s will onlydepend on this reversible reaction. On the other hand, thedynamics of free tubulin will also be determined by the al-ternation of catastrophes and rescues, while the gradualshortening and elongation processes will be ignored due totheir shorter time scale and limited effect on the process of MT aggregation. While individual MT
s exhibit catastrophesand rescues, this polymerization behavior becomes smoothedout and is not so dramatic for ensembles at high concentra-tions. Nonetheless, there exist collective phases of assemblyand disassembly for MT aggregates and our objective in this
FIG. 1.
a
After about 5 h inthe experiments reported in Ref.
2
, striped patterns of MT
s ap-peared, with two adjacent stripesmade up of highly oriented MTbundles at an angle of 45
°
and135
°
, respectively, to the horizon-tal.
b
The characteristic spatialperiodicities developed withinstripes for the samples exposed togravity during the
rst 6 min, withthe major axis of the sample par-allel to the gravity axis.PORTET
et al.
PHYSICAL REVIEW E
68
, 0219XX
2003
0219XX-2
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paper is a coarse-grained approach that focuses on these col-lective phenomena. Somewhat similarly but in the study of adifferent effect, Jobs
et al.
11
developed an accurate modelfor MT oscillation dynamics. During disassembly events,MT
s release GDP tubulin which must be recycled as GTPtubulin to be used again to form a MT. Thus the inclusion of GTP and GDP concentrations as well as GDP-tubulin andGTP-tubulin species is necessary in the complete theoreticaldescription of MT dynamics. However, the experimentalconditions considered here, i.e., the presence of a GTP-regenerating system that provides a continuous source of GTP
3
, results in a constant GTP concentration in thesample. This leads us to consider, for the purpose of thepresent model, a simpli
cation of the recycling of GDP tu-bulin as an instantaneous process as well as to ignore thedifference between the GTP-tubulin and GDP-tubulin pools.Thus, we have opted to account for the recycling process,which also includes the effect of turnover, by introducing aneffective term that is proportional to the tubulin concentra-tion. As motivated above by the underlying chemical kinet-ics, we postulate the following reaction terms in Eq.
1
:
2a
and
2b
where
is the assembly rate,
is the disassembly rate,
n
is the nucleation rate,
n
is the rate of nucleus elimination,and
1
is the recycling rate for tubulin. Note that
and
should not be misconstrued to represent the related kineticscoef 
cients for a single MT. The present values jointly de-scribe the effective rate of the individual process and thefrequency of occurrence. Here,
n
is the critical number of tubulin dimers necessary for the MT nucleation. The processof MT nucleation is a slower process than assembly. Struc-turally speaking, it is a more nonlinear process than eitherassembly or disassembly. While nucleus elimination and dis-assembly terms in Eq.
2a
can be linked together math-ematically, they represent a different effect and are governedby a different dynamics. Nucleation processes, on the otherhand, cannot be combined with other effects. It should alsobe mentioned that the presence of recycling terms is analo-gous to an additional compartment in the so-called compart-mental models resulting in a delay of the assembly process.All the rate constants are positive.The drift velocity
u
i
for a particle of type
i
is calculatedaccording to
u
i
(
 D
i
 B
)
 f 
i
, where
D
i
is the diffusion co-ef 
cient of the molecule of type
i
,
 B
is the Boltzmann con-stant, and
is the temperature
in kelvin
. The term
i
rep-resents the net force
force of gravity and force of buoyancy
that acts on a molecule of type
i
:
i
m
i
(1
 
 
 
i
)
g
, where
m
i
is the molecular mass,
 
 
is the mean density of thesolution,
 
i
is the density of molecules, and
g
is the strengthof the gravitational
eld.The competition between hydrodynamic forces due to thesedimentation process and Brownian motion acting on eachtype of particle is characterized by the Peclet number Pede
ned as follows
12
:Pe
i
u
i
 D
i
i
 f 
i
 B
,
3
where
i
is the diameter of the molecule. When Pe
1, thediffusion process dominates over the directional transport,and the latter can be considered insigni
cant within thesample. It is assumed that the solution is an aqueous buffer,and that the
 
,
 
-tubulin heterodimer is approximated by aspherical particle of diameter
8 nm with a mass
m
100 kDa
1
. We estimate the MT mass by assuming that itis a hollow cylinder
the interior diameter of about 14 nmand the exterior diameter of about 25 nm
of 5
m length
2
, made up of 1625 dimers per 1
m length of a MT. ThePeclet number for tubulin dimers at 37
°
C is calculated asPe
10
10
, while for MT
s we obtain Pe
10
2
. Thus, thedrift induced by gravitational conditions can be neglected inthe case of tubulin dimers, but it is very relevant for theMT
s and dominates the diffusion process.Consequently, the model in Eq.
1
is now expressed inthe one-dimensional case
along the gravity axis
by twocoupled nonlinear partial differential equations
 
 
 D
 
2
 
 x
2
 f 
,
 M 
,
4a
 
 M 
 

 
 M 
 
 x
h
,
 M 
,
4b
where
D
is the diffusion coef 
cient of tubulin dimers. Ex-trapolating from the tubulin diffusion coef 
cient measuredfor
in vivo
sea urchin eggs at 25
°
C as 5.9
10
12
m
2
s
1
13
, we estimate
D
70
10
12
m
2
s
1
under the experi-mental conditions
2
considered here. In the experiments,the temperature was 37
°
C rather than 25
°
C, so to obtainthis estimate we have scaled the sea urchin diffusion con-stant, found in Ref.
13
, by the ratio of the two temperaturesand the ratio of the corresponding viscosities at these twotemperatures.The drift coe
cient
 
u
 M 
, where
u
 M 
(
 D
 M 
 B
)
m
 M 
(1
 
 
 
 M 
)
g
is the positive drift velocityfor a MT of 5
m in length. From the well-known Stokes-Einstein formula, using the combination of the parallel andperpendicular components of the drag coef 
cient, we esti-mate the MT diffusion coef 
cient to be
D
 M 
1.54
10
12
m
2
s
1
. Thus
u
 M 
is found to be equal to 5
10
10
ms
1
or 30 nm/min.The scaling parameter
 
models the coupling betweenMT
s and the so-called avalanche correlated clusters
14
.Tubulin dimers are negatively charged globular proteins that
MODELS OF SPATIAL AND ORIENTATIONAL SELF- . . . PHYSICAL REVIEW E
68
, 0219XX
2003
0219XX-3
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