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Hill Kirschner 1982 Subunit Treadmilling of Microtubules or Actin in The Presence of Cellular Barriers Possible
Hill Kirschner 1982 Subunit Treadmilling of Microtubules or Actin in The Presence of Cellular Barriers Possible
USA
Vol. 79, pp. 490-494, January 1982
Cell Biology
ABSTRACT Free microtubule or actin filaments, along with free, although the details are different (to be published
the monomeric forms of the protein, hydrolyze GTP or ATP to elsewhere).
produce a flux of subunits through the polymer. This flux, called Our object in this paper is to outline the basic bioenergetic
treadmilling, produces no useful work. In the cell, however, these theory applicable to a polymer that is treadmilling against bar-
filaments are likely to be constrained between nucleating sites and riers at both ends, with or without an attached resisting force.
other barriers that will limit polymer growth. We study here the With the resisting force, work can be done. As a preliminary,
effects of a small compression of the filaments resulting from poly- however, we consider an equilibrium polymer (i.e., one with
merization against such barriers. If subunits can still exchange at no NTPase activity) that has grown and encountered barriers,
the two ends, treadmillingwill take place here as well. Under these and consequently is under a compressive force. This equilib-
conditions, the filament system can do useful work. The free en- rium treatment would apply to other cases directly, for example,
ergy of NTP hydrolysis can be used to transport materials, at- to sickle-cell hemoglobin (Hb S) aggregation.
tached to the filament, against a resisting force. This process can
in principle take place at high efficiency and bears a resemblance EQUILIBRIUM POLYMER BETWEEN BARRIERS
in a bioenergetic sense to the utilization of ATP free energy in
muscle contraction. The same general principles apply to a poly- We consider the aggregation of a linear polymer with no NTPase
mer in which one end is anchored and one end is free. activity. Let Ce be the "critical" concentration of monomers (sub-
units) in equilibrium with an infinite polymer in solution. If the
Steady-state treadmilling (1-3) of subunits in microtubules or monomer concentration c is slightly <Ce, finite polymers are
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actin microfilaments in solution uses GTP or ATP, respectively, stable (4, 6, 7). If c > ce, the polymers will grow steadily. We
but none of the NTP free energy loss is converted into me- are interested here in the case in which c > ce, but the polymer,
chanical work or other form offree energy (4). Ifthe free subunit in its growth, encounters barriers a distance L apart that limit
concentration is increased above the steady-state concentration, its length. Monomer exchange is assumed to be possible at one
these polymers will grow (5). In a cell, a growing polymer, with or both ends despite the barriers. The polymer is somewhat
one or both ends free, will eventually encounter a barrier of one compressible. The assumed compressibility, including bend-
kind or another (e.g., the cell membrane) at the free end or ing, if any, is expressed by F = a(l - lo) (Hooke's law), where
ends. At this point, further addition of a relatively small number 10 is L/No, 1 is LIN, F is the force on the polymer (negative for
of subunits will produce sufficient compression of the polymer compression), a is a force constant, N is the number of mono-
to stop the net growth. This important effect is a consequence mers in the polymer, and No is the value of N at zero force (F
of the increase in the chemical potential of the subunits in the = 0). Hooke's law uses the first term in an expansion in powers
polymer; there is some resemblance here to the pressure-in- of I - 10; it is adequate for small compressions, as in the present
duced chemical potential increase of the solvent (in the solution) problem.
in an osmotic system. Although growth has ceased, a new re- The system (polymer) is characterized by the independent
gime of treadmilling will now be possible if subunits can still variables c, L, a(T), 10(T), and the temperature T (N is a depen-
be exchanged between the solution and both ends of the poly- dent variable that fluctuates). The independent thermodynamic
mer. In this paper we show that, if a chromosome or some other variables are ,u, L, and T, where ,u is the chemical potential of
structure (or structures) is attached to the polymer, treadmilling monomers in the polymer (g) or in the solution (.t8):
between barriers, and exerts a force that resists the directional /40 + kT ln c.
= /L8 = [1]
motion of the subunits of the polymer, then some of the NTP
free energy of hydrolysis, expended in the treadmilling, will be For microtubules, lo = 80/13 = 6.15 A; a is not known, but we
converted into mechanical work. In fact, this latter system, in make reasonable guesses below. Strictly, we should use the
a formal bioenergetic sense, resembles the actin-myosin-ATP grand partition function ,(pi,L,T) and small system thermo-
system in muscle contraction. For example, there will be a dynamics (6, 8) for this open finite system (with end effects in-
force-velocity curve: As the resisting force is increased, the cluded). However, No is fairly large, of order 104 or 105, so with
treadmilling velocity decreases. very small error we can use the canonical (or any) partition func-
Treadmilling and free energy transfer of the above type is also tion and macroscopic thermodynamics.
possible if one end of the polymer is anchored and one end is The canonical partition function for the above defined sys-
tem, omitting end effects, is
The publication costs ofthis article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertise- Q(NLT) = {q exp[ a(L -
i0) 2kT]} [2]
ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
490
Cell Biology: Hill and Kirschner Proc. Natl. Acad. Sci. USA 79 (1982) 491
where Q = qN when the polymer is under no compression (c 1/a. For example, if C/Ce = e = 2.72 and My = 0.01, than in =
= Ce). The chemical potential in this case is 100 and or,, = 10. If y = 0.05, n = 20 and orn = 4.5. Recall that
-kT In q p0 = uo + kT In ce, [3] No is of order 104 or 105 and that in = 13 would correspond to
adding one subunit to each strand of a microtubule. The ad-
so that dition of 10-30 subunits for C/Ce of order 3-5 seems intuitively
reasonable.
A - AO = kT ln(c/ce). [4] The estimation of the actual compressibility ofa microtubule
(or of a Hb S aggregate) is not easy. As an average for both tor-
From the thermodynamic relation sional and center-of-mass vibrational motion in Hb S, using an
dA = - SdT + FdL + udN [5] Einstein model, Ferrone et al. (10) chose a frequency 3 X 109
s l. For the center-of-mass motion itself, we then estimate 8
and Eq. 2, we deduce X 109 s-'. Then, by a straightforward argument to be published
F =a(l -10) [6] elsewhere, taking No = 2 x 104, we find y -0.05.
If we write Eq. 2 as Q = qNe-W/kT, then W is the work of
inserting in subunits. W/kT is easily seen to be yni2/2. Then the
A- =-2 (12-12) = -[10F + (F2/2a)]. [7] work per subunit added is
2
For a given value of C/Ce > 1, Eqs. 4 and 7 determine the W/ = y-nkT/2 = (kT/2)ln(C/ce). [13]
equilibrium (compressive) force F. The corresponding value of If C/Ce = 2.72, W/n = kT/2 300 cal mol'. Note that W/in
N is also given by Eq. 7 because 1 = L/N. If the monomer con- is independent of y.
centration is increased from Ce to C > Ce, -F and N increase from For reference below, we now consider this system from a
-F = 0 and N = No until p in the polymer becomes equal to more detailed stochastic point of view (in the same approxi-
ps(c) in the solution (Eq. 1). mation). We are interested in the rate constants for the arbitrary
The detailed balance relations for on-off transitions, at either polymerization step (at one end only) n . n + 1. With no
end of the polymer, are ac = a' for Eq. 1 and aOCe = a0 for compression, these rate constants are a0c and a' (Eq. 8). The
Eq. 3, where the as are rate constants. These rate constants are work of insertion for n -* n + 1 is
related by (Eq. 4)
AW/kT = (y/2)[(n + 1)2 - n2] = (2n + l)y/2. [14]
a/a' = (alao)e A [8]
On including this effect, the two rate constants become (Eq.
The separate rate constants can be written as 9)
a = aoef(w-;L)kT
[9] ciocx
(2n + 1)f and a.X(2n+l)(f-l), [15]
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4 and 7). In this case, L refers to the polymer length when in the present case (Eq. 2),
growth stops (equilibrium). A cap is a barrier that offers no
mechanical resistance at all (F = 0), although rate constants will Pn (q9A) x, P.+ /Pn = qX2n+l+l [17]
be affected. (This case will be discussed elsewhere.) On comparing this with Eqs. 4 and 16, we verify that
As already mentioned, N actually fluctuates, so that N in Eq.
7 is really the mean value N. The variance in N, when L is held qA = c/ce= aoc/ao, [18]
fixed, is (using Eq. 7)
which is a self-consistency check.
oN2 =~-3
N= -P2 2
= kT(aN/Za)LT = kTN3/aL2. [10] The equilibrium distribution P. will be substantially confined
to positive n if ii > 3oa-that is, if
The Principal Approximation. There are approximate forms
ofthe above relations, probably good to 1% or better (depending ln(c/ce) > 3i/2. [19]
on a). We define n (number of subunit insertions) by N = No This will usually be the case.
+ n. Then, if n << No (i.e., very little compression), which is The parameterf in Eqs. 9 and 15 could in principle be a func-
presumably the case, the above equations simplify to tion of n (or of F), but it is a reasonable approximation to treat
(p - ,wo0)/kT = ln(c/ce) =-10F/kT = 'yn it as a constant for n > 0 (9). This is done below; here, at equi-
[11] librium, the choice is immaterial.
2 =
2 = 1/i, V a/N~kT,
[12] TREADMILLING BETWEEN BARRIERS
where 'y is a dimensionless force constant (Eq. 11) for subunit We now consider a polymer with on-off NTPase activity at each
insertions (n) that is more convenient as a parameter than a. end (1, 4) but without a resisting force (Fr = 0 in Fig. 1). The
Because ln(c/ce) is of order unity, the approximation is valid if monomer or subunit concentration c is large enough (c > c=;
No >> 'yf. Eqs. 8, 9, and 11 are equivalent to equations 5-8 see below) so that the polymer grows until it is restrained by
of ref. 9. barriers, as discussed above, and as shown schematically in Fig.
For a given value of c/ce, both W and o-2 vary as I/fy or as 1. This is presumably a common situation in vivo for both tu-
492 Cell Biology: Hill and Kirschner Proc. Natl. Acad. Sci. USA 79 (1982)
A n fl%
I A
a442/a° la%2 = /30132/03-113
XT AT D -P,
2 = eT/kT
[23]
ei =
OE
(3-1 (30
steady-state result. Also, we can make a correction to JT and Jm
(Eqs. 25 and 26) to take care of the fact that this is actually an
02 802E (3-2 =
o
2-E1' open system with fluctuations in n. We find, after a short
where four differentfs are introduced for generality and E = calculation,
elOF/kT in the approximation above (n << No) that we shall con- a=
c(a +
fA
I Pn (2n+1)fi
tinue to use (Eq. 7 gives the correction term in the definition n
[30]
of E if the approximation is not used). In principle, the fs can
all be functions (9) of F, but we shall treat them as constants =J .(c/c, )f2/(f1 +f2)e-fif2/2(f1 +f2)
an appendage C that moves with the subunits against a resisting On substituting Eq. 20 and using Eqs. 32, we find
force Fr, There has been much .interest in possible ways that
treadmilling NTPase activity might be put to some use (5). The JT JA +IB +IC + JD, E JJC JD + JE + JF, [34]
scheme in Fig. 1, with Fr included, allows the transfer of some
(or all, in a limiting case) NTPase free energy of hydrolysis into where the subscripts identify the cycle (Fig. 2) and
the mechanical work of moving C against Fr. For example, C
might be a vesicle or chromosome that is transported by a mi- 1A= ala2(1 e -XTIkT)/D, lB = BI32(1 - e-XT/kT)/D
crotubule and Fr its viscous drag [although Fr in this case would
be very small, -10-8 dyne (1 dyne = 0.01 mN) for a particle IC = al,2(l exTkTr)/D, ID = a2,81(1 - e-XT/kTr-')/D
-
the size of a chromosome (12, 13)]. The position of C, specified
by the fraction 6 in Fig. 1, does not appear in the final results JE = alP(1 -r)ID, IF = a2j32(1- r)/D [35]
(see below). C could also represent a composite of a number of
attachments. D =
a, + ,(1 + a-2 + 1P-2-
Our primary interest here is not in particular applications but
in general principles: we shall consider that Fr can be made The terms in e-Xr/kT are generally negligible as are the rate con-
arbitrarily large, even large enough to stop or to reverse the stants that have negative subscripts.
treadmilling direction. The efficiency offree energy transduction and the rate offree
If one subunit is inserted at the + end ofthe polymer in Fig. energy dissipation are
1, C moves to the left essentially a distance 01o against the force
Fr. From this and similar considerations for other possible cases, 7 = JmIJr/ITXT [36]
we must now generalize Eqs. 22 to read
T(dAS/dt) = JTXT - JmloFr [37]
aloeor
Of~ej a~ a1 fi1-1 r 0(1-f'l)
0
al = ar- =
Using this F (Eq. 39), we then find for the steady-state fluxes Eq. 47 is very close to linear [unlike the force-velocity curve
in muscle (14)]. The value of Fr ("isometric force," Eq. 43) at
JT = (ao + 3or-f2) [c(a° + Pl3rf')/(aO + por-f2)]f2l(fW+f2) [41] whichJm = 0 is 1.3 x 10-6 dyne (25TC), compared with lo-'
(a?2°r f2 af131rfi) [c(a' + 80rfi)1(aO dyne for the drag of a chromosome (12, 13). The maximum
m = -
treadmilling velocity (i.e., at F. = 0) in this example is 9.1 x
+ p0r-f2)]f2/(f1+f2)/(aO + 3°r0fi). [42] 6.15 = 56 A-s- compared with 20 Aims-1 for Vmax in muscle
contraction and 170 Ais-1 (or 1 tum-min-') for a chromosome
J1 and Jm do not depend on (see the discussion of Eqs. 31). in anaphase. The efficiency Iq (Eq. 36) is zero at Fr = 0 and at
Jm is zero when Im = 0. In between, 'q has a maximum of only 0.81% (using XT/
kT = 23.0) at r = 2.67: most of the GTP hydrolysis is wasted.
r= r0--(alf32Ja°/ )l0(f +f2) [43]
At this r, J1JT = 0.19 and VoFATX = 0.043. The low efficiency
A plot of ln r againstJm is essentially the "force-velocity curve" is implicit in this example at the outset because of the use of one-
(14) for this system. way (irreversible) cycles and the contributions -of the inefficient
In the tight-coupling special case (13,l = a? = 0), cycles A, B, and D. The corresponding efficiency in muscle
contraction is of order 50% (15).
JT = Jm = PB2(c/c,,)fi2(fi1+f2)r-f1fr2/(fi1+f2), [44] At r = 2.67, c' = 2.14 ,uM for 6 = 1/2 (Eq. 40). Then -IF/
kT = 0.90 (Eq. 39). Also, ii = 18.0 if we take -y = 0.05 (Eq. 39).
where c. = 132/a?.
We conclude with a numerical example that illustrates Eqs. 1. Wegner, A. (1976) J. Mol Biol 108, 139-150.
39-43. We take, for a microtubule, f1 = f2 = 1/2, c = 5.25 ,uM, 2. Margolis, R. L. & Wilson, L. (1977) Proc. NatL Acad. Sci. USA 74,
and 3466-3470.
3. Bergen, L. G. & Borisy, G. G. (1980)J. Cell BioL 84, 141-150.
a° = 7 ,uM-1 s-, ao = 7 s1, [45] 4. Hill, T. L. (1980) Proc. NatL Acad. Sci. USA 77, 4803-4807.
5. Kirschner, M. W. (1980)J. Cell Biol; 86, 330-334.
010 = 1 ,UM-1-S-1, IP2= 7 s-1. 6. Hill, T. L. (1964) Thermodynamics of Small Systems, Part II
(Benjamin, New York).
Then, 7. Oosawa, F. & Asakura, S. (1975) Thermodynamics of the Poly-
merization of Protein (Academic, New York).
Jo = 14 s-1 [46] 8. Hill, T. L. (1963) Thermodynamics of Small Systems, Part I (Ben-
cx - 1.75 ,uM, jamin, New York).
9. Hill, T. L. (1981) Proc. Natl Acad. Sci. USA 78, 5613-5617.
Jo = 5.25 s-1, c/cX 3, ro-= 7. 10. Ferrone, F. A., Hofrichter, J., Sunshine, H. R. & Eaton, W. A.
(1980) Biophys. J. 32, 361-377.
For the fluxes at c, 11. Hill, T. L. (1981) Biophys. J. 33, 353-371.
12. Taylor, E. W. (1965) in Proceedings of the Fourth International
r =1, Fr = 0:JT= 24.2 s', Jm = 9.1 s1, [47] Congress on Rheology, ed. Copley, A. L. (Interscience, New
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