Limits...
Processivity of kinesin motility is enhanced on increasing temperature

View Article: PubMed Central - PubMed

ABSTRACT

Kinesin is a motor protein that processively moves step by step along a microtubule. To investigate the effects of temperature on run length, i.e., processivity of kinesin motility, we performed a single-molecular bead assay at temperature range of 20–40°C. An increase in the walking velocity of kinesin corresponded to the Arrhenius activation enthalpy of 48 kJ/mol, being consistent with the previous reports. Here, we found that the run length increased, that is, the kinesin processivity enhanced with increasing temperature. Then, we estimated the probability of detachment of kinesin from a microtubule per one 8-nm stepping event, and found that it diminishes from 0.014 to 0.006/step with increasing temperature from 20 to 40°C. And we noticed that prolonged incubation at 30, 35 and 40°C significantly slowed down the walking velocity, but further increased the run length and duration. Those results are interpreted according to the effect of temperature on the rate constants of some key kinetic steps in the ATPase cycle.

No MeSH data available.


Related in: MedlinePlus

Typical traces showing the time course of bead movement along a microtubule at various temperatures (Supplementary Movies 1–5. Note that those movies do not necessarily correspond to the data shown here). These traces were taken between 1 and 6 min of the incubation at each temperature. Attachment of the bead to a micro-tubule occurred at the zero point. Arrows indicate the time at which the kinesin-bound bead detached from the microtubule, detected by the disappearance of the fluorescent image of the bead. Run length was defined as the ordinate of the detachment point. Duration is the period of time between attachment and detachment of the bead. Walking velocity was estimated from the average slope of the time course of the bead movement obtained by the least squares method.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5036643&req=5

f1-2_13: Typical traces showing the time course of bead movement along a microtubule at various temperatures (Supplementary Movies 1–5. Note that those movies do not necessarily correspond to the data shown here). These traces were taken between 1 and 6 min of the incubation at each temperature. Attachment of the bead to a micro-tubule occurred at the zero point. Arrows indicate the time at which the kinesin-bound bead detached from the microtubule, detected by the disappearance of the fluorescent image of the bead. Run length was defined as the ordinate of the detachment point. Duration is the period of time between attachment and detachment of the bead. Walking velocity was estimated from the average slope of the time course of the bead movement obtained by the least squares method.

Mentions: Figure 1 shows typical traces of movement of a kinesin-bound bead at various temperatures we examined, i.e., 20, 25, 30, 35 and 40°C. We collected the data between 1 and 11 min after the beginning of incubation at each temperature. From these data, we obtained the values of run length, duration and velocity (parameters characterizing the processivity of molecular motors) of single kinesin molecules moving along a microtubule. Here, the run length is defined as the distance, which a bead travelled continuously before detachment. The duration is the period of time during which the bead continuously moved (the abscissa of the final position of raw data as shown in Fig. 1). The velocity was obtained from the average slope of the time course of bead displacement. As these data show, with increasing temperature both run length and velocity increased, whereas duration decreased. Note that only two parameters of these three are theoretically independent : the value of one parameter can be calculated from the others according to the relationship (run length) = (velocity) × (duration). However, this relationship is not exactly kept in practice, because the velocity is obtained as the average slope of the time course of bead movement, whereas both run length and duration are determined from the coordinates of the end point of the time course of the bead movement (see Fig. 1). In spite of the difference between the values obtained theoretically and experimentally, all the experimental data exhibit good agreement with the above relationship throughout the analysis. These results show therefore that the temperature dependence of the run length is weaker than that of the velocity, because the temperature dependence of duration is opposite.


Processivity of kinesin motility is enhanced on increasing temperature
Typical traces showing the time course of bead movement along a microtubule at various temperatures (Supplementary Movies 1–5. Note that those movies do not necessarily correspond to the data shown here). These traces were taken between 1 and 6 min of the incubation at each temperature. Attachment of the bead to a micro-tubule occurred at the zero point. Arrows indicate the time at which the kinesin-bound bead detached from the microtubule, detected by the disappearance of the fluorescent image of the bead. Run length was defined as the ordinate of the detachment point. Duration is the period of time between attachment and detachment of the bead. Walking velocity was estimated from the average slope of the time course of the bead movement obtained by the least squares method.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036643&req=5

f1-2_13: Typical traces showing the time course of bead movement along a microtubule at various temperatures (Supplementary Movies 1–5. Note that those movies do not necessarily correspond to the data shown here). These traces were taken between 1 and 6 min of the incubation at each temperature. Attachment of the bead to a micro-tubule occurred at the zero point. Arrows indicate the time at which the kinesin-bound bead detached from the microtubule, detected by the disappearance of the fluorescent image of the bead. Run length was defined as the ordinate of the detachment point. Duration is the period of time between attachment and detachment of the bead. Walking velocity was estimated from the average slope of the time course of the bead movement obtained by the least squares method.
Mentions: Figure 1 shows typical traces of movement of a kinesin-bound bead at various temperatures we examined, i.e., 20, 25, 30, 35 and 40°C. We collected the data between 1 and 11 min after the beginning of incubation at each temperature. From these data, we obtained the values of run length, duration and velocity (parameters characterizing the processivity of molecular motors) of single kinesin molecules moving along a microtubule. Here, the run length is defined as the distance, which a bead travelled continuously before detachment. The duration is the period of time during which the bead continuously moved (the abscissa of the final position of raw data as shown in Fig. 1). The velocity was obtained from the average slope of the time course of bead displacement. As these data show, with increasing temperature both run length and velocity increased, whereas duration decreased. Note that only two parameters of these three are theoretically independent : the value of one parameter can be calculated from the others according to the relationship (run length) = (velocity) × (duration). However, this relationship is not exactly kept in practice, because the velocity is obtained as the average slope of the time course of bead movement, whereas both run length and duration are determined from the coordinates of the end point of the time course of the bead movement (see Fig. 1). In spite of the difference between the values obtained theoretically and experimentally, all the experimental data exhibit good agreement with the above relationship throughout the analysis. These results show therefore that the temperature dependence of the run length is weaker than that of the velocity, because the temperature dependence of duration is opposite.

View Article: PubMed Central - PubMed

ABSTRACT

Kinesin is a motor protein that processively moves step by step along a microtubule. To investigate the effects of temperature on run length, i.e., processivity of kinesin motility, we performed a single-molecular bead assay at temperature range of 20–40°C. An increase in the walking velocity of kinesin corresponded to the Arrhenius activation enthalpy of 48 kJ/mol, being consistent with the previous reports. Here, we found that the run length increased, that is, the kinesin processivity enhanced with increasing temperature. Then, we estimated the probability of detachment of kinesin from a microtubule per one 8-nm stepping event, and found that it diminishes from 0.014 to 0.006/step with increasing temperature from 20 to 40°C. And we noticed that prolonged incubation at 30, 35 and 40°C significantly slowed down the walking velocity, but further increased the run length and duration. Those results are interpreted according to the effect of temperature on the rate constants of some key kinetic steps in the ATPase cycle.

No MeSH data available.


Related in: MedlinePlus