each other, forming a linear chain called protofilament. Thus like actin, microtubule
has a structural polarity; beta tubulin lines at one end and alpha tubulin at the other
end. At the end of a protofilament exposing alpha tubulin, a beta subunit of a
heterodimer binds, whereas at the opposite end exposing beta tubulin, to which alpha
subunit in the heterodimer binds. The number of protofilament in a microtubule
varies between 10 and 15, but in the cell that is 14. When microtubule is grown
in vitro from a nucleus (a natural seed for polymerization, which is isolated from
protozoa such as Chlamidomonas), the number is 13 (see [59] for details).
In the microtubule consisted of 13 protofilament, 3-start, left-handed helices can
be recognized, as shown in Fig. 7.16b (shaded parallelogram). A tubulin in one
protofilament makes a contact with the tubulin of the same type (ie., alpha with alpha
or beta with beta) in the adjacent protofilament. However, since the rise of the 3-start
helix per turn is 1.5 times the length of the dimer, l, the alpha and beta tubulin in the
13
th protofilament each makes contact with different type of tubulin (ie., beta and
alpha) in the first protofilament. The contact thus formed along the microtubule is
called seam. In the case of 13 protofilaments, each protofilament is straight so that
the organelles transported by motor proteins walk on the same side of the
microtubule [60].
7.6.2 Microtubule Polymerization and Depolymerization
7.6.2.1 Basic Properties
A study on the polymerization of tubulin heterodimer has demonstrated that the
polymerization needs rather high temperature (37
C; [61, 62]); at 0
C it depolymerizes [63]. Actin polymerization does not exhibit such a strong dependence on
temperature. The degree of polymerization has been estimated from the amount of
tubulin that is pelleted by centrifugation (the plot of the amount of the pelleted
protein vs. total protein will provide empirical Cc value as the amount of protein
below which no pelleted polymer exist [64]). For the kinetic measurement of
polymerization, direct observation of individual microtubules by light microscopy
(differential-interference or dark-field) has been employed (see Sect. 7.6.3).
The critical concentration of tubulin heterodimer varies with the solution condition, especially with the temperature in the case of microtubule as mentioned above.
Typical values have been measured at 37
C to be ~5 μM for both ends [65]. The
existence of critical concentration suggests existence of energetically unfavorable
step in the polymerization. Electron microscopic study of polymerizing microtubules
revealed that the protofilaments at the end of microtubule are aligned as in a
microtubule, but making an open, sheet-like structure with outward curvature.
This is thought to reflect the structural feature of the heterodimer [59, 66]. Polymerization would have to proceed with the closure of the sheet, but this requires
transition of the sheet with outward curvature to more straight form that fits the
microtubule cylinder. It is likely that between these two conformations exists an
7.6 Microtubule
117
has a structural polarity; beta tubulin lines at one end and alpha tubulin at the other
end. At the end of a protofilament exposing alpha tubulin, a beta subunit of a
heterodimer binds, whereas at the opposite end exposing beta tubulin, to which alpha
subunit in the heterodimer binds. The number of protofilament in a microtubule
varies between 10 and 15, but in the cell that is 14. When microtubule is grown
in vitro from a nucleus (a natural seed for polymerization, which is isolated from
protozoa such as Chlamidomonas), the number is 13 (see [59] for details).
In the microtubule consisted of 13 protofilament, 3-start, left-handed helices can
be recognized, as shown in Fig. 7.16b (shaded parallelogram). A tubulin in one
protofilament makes a contact with the tubulin of the same type (ie., alpha with alpha
or beta with beta) in the adjacent protofilament. However, since the rise of the 3-start
helix per turn is 1.5 times the length of the dimer, l, the alpha and beta tubulin in the
13
th protofilament each makes contact with different type of tubulin (ie., beta and
alpha) in the first protofilament. The contact thus formed along the microtubule is
called seam. In the case of 13 protofilaments, each protofilament is straight so that
the organelles transported by motor proteins walk on the same side of the
microtubule [60].
7.6.2 Microtubule Polymerization and Depolymerization
7.6.2.1 Basic Properties
A study on the polymerization of tubulin heterodimer has demonstrated that the
polymerization needs rather high temperature (37
C; [61, 62]); at 0
C it depolymerizes [63]. Actin polymerization does not exhibit such a strong dependence on
temperature. The degree of polymerization has been estimated from the amount of
tubulin that is pelleted by centrifugation (the plot of the amount of the pelleted
protein vs. total protein will provide empirical Cc value as the amount of protein
below which no pelleted polymer exist [64]). For the kinetic measurement of
polymerization, direct observation of individual microtubules by light microscopy
(differential-interference or dark-field) has been employed (see Sect. 7.6.3).
The critical concentration of tubulin heterodimer varies with the solution condition, especially with the temperature in the case of microtubule as mentioned above.
Typical values have been measured at 37
C to be ~5 μM for both ends [65]. The
existence of critical concentration suggests existence of energetically unfavorable
step in the polymerization. Electron microscopic study of polymerizing microtubules
revealed that the protofilaments at the end of microtubule are aligned as in a
microtubule, but making an open, sheet-like structure with outward curvature.
This is thought to reflect the structural feature of the heterodimer [59, 66]. Polymerization would have to proceed with the closure of the sheet, but this requires
transition of the sheet with outward curvature to more straight form that fits the
microtubule cylinder. It is likely that between these two conformations exists an
7.6 Microtubule
117