Figure 1:   Schematic representation of proposed torsion gear mechanism and microtubule (MT) catch-bonding. The six member ring of CDC’s motor domain is shown in a relaxed state in orange and during high load conditions the ring is compressed as shown by the red model. During very high load conditions, CDC’s microtubule binding domain clamps down onto the MT as shown by the purple model. 

There are several methods utilized by motors to move along MTs; these are single head one step, double head step over step, stutter step, and linear diffusion^8-10. The most well-known movement is the step over step method utilized by double head motor proteins like kinesin heavy chain (KHC, kinesin-1 sub-family) and cytoplasmic myosin. These motors move without speed or gear control and more frequently fall off MTs compared to CDC under similar loads. In contrast, CDC is a different case; for example, in vitro, it moves in steps as large as 24-36 nm, which is equivalent to the length of 3 to 4 tubulin heterodimers¹¹. This is surprising because CDC exists as a dimer with a spacer distance (motor to MT) of 9 nm each, but the motor domains are not bound closely to each other as they are in myosin and kinesin, and there is additional spacer length from the cargo binding spacer which is 15 nm long (Figure 1). In vitro, there is not much resistance placed on the motor in the form of cargo dragging through a cellular matrix. In vivo, the CDC movement is very different, with step length reduced to 8 nm when under load, which corresponds to the length of a tubulin heterodimer¹². Thus, CDC can change its step size and maintain MT contact under different loads.

In recent years, some exquisite live cell microscopic work by several groups ^11-15 has revealed how dynein moves and maintains control of movement in vivo. The groups improved on earlier work by a) utilizing laser-captured vesicles to measure force, b) by limiting their data to only straight tracking (<5% angles) particles, c) by more accurately defining what was a stall and a run in one direction, d) measuring the width of a stalled vesicle as an indication of opposing motors working against each other, and e) utilizing late endosome vesicles, which show more uniform motor protein components, i.e., KHC and CDC only.   

When MT minus-end-directed vesicles are observed in vivo, they move in plus and minus-end directions and also stall; in effect the vesicles are changing from KHC to CDC driven motion and back again. This scenario is non-intuitive and we might expect only the presence of CDC. Mallik’s group studied this movement in more detail, initially utilizing a laser trap to calculate the force generated on a moving vesicle¹². In the majority of cases, the authors measured a force of 5-6 piconewtons (pN) in the plus direction (KHC) and a 5-8 pN force in the minus direction (CDC). From in vitro measurements, it is known that these forces reflect the presence of one KHC dimer and 5 to 10 CDC complexes. The length of motion after starting a run was also measured, yielding a ratio of 5:1 (6 nm CDC/1.2 nm KHC), which is highly in favor of minus-end movement. Furthermore, the speed and step size after stall were measured and both were in favor of CDC in the minus-end direction by a factor of 1.5 to 2.0 fold. All of these aspects combined to produce overall motion in the minus-end direction. The authors summarized their findings by concluding that dynein is a tenacious team worker where each molecule contributes 1 pN in force12, extending others’ findings that dynein catch-bonds MTs and prohibits movement in the plus direction¹⁶.    

In summary, dynein represents an evolutionarily unique mechanochemical-geared motor which applies strong forces by combining the effects of multiple motors. Dynein is tenacious and catch-bonds tightly to MTs to oppose KHC motor force. At Cytoskeleton, we are developing load and non-load MT stimulated CDC ATPase assays which can be used to identify inhibitors and enhancers such as that reported by Firestone¹⁷. Dynein protein is also available as a custom protein preparation (see New proteins... table), contact tservice@cytoskeleton.com for more information. 

Related Research Tools and Services