Dynein harnesses active fluctuations of microtubules for faster movement

Abstract

Motor proteins take part in the organization and division of eukaryotic cells by using their ability to move unidirectionally along the cytoskeletal tracks. While kinesin and myosin motor families have members that move towards either end of actin and microtubules, respectively, all dynein motors exclusively move towards the minus-end of microtubules. Previous studies reported that dynein asymmetrically responds to external forces, moving faster when pulled forward, while resisting backward movement under hindering forces. I hypothesized that this asymmetry enables dynein to harness energy from external force fluctuations for faster movement towards the minus-end. In my doctoral work, I have shown that dynein can harness energy from cytoskeletal fluctuations. Using optical trapping techniques, I have characterized how external forces affect the velocity of dynein motility in the presence and absence of ATP. The results demonstrated that dynein forms an asymmetric slip bond with the microtubule. Using an oscillatory optical trapping assay, I showed that dynein can rectify force fluctuations to move towards the microtubule minus-end in the absence of ATP. Dynein was capable of moving towards the minus-end, even when the net force is in the plus-end direction. In the presence of ATP, dynein was able to move faster, generate power from force fluctuations, and stand against higher resistive forces. I developed a mathematical model that connects the force-induced release rate of dynein monomers with the force-velocity relationship of dynein dimers to describe dynein's reaction to force. i This dissertation is dedicated to my mom, Meryem Ezber, who has sacrificed all she has for her four children. She is the most patient person I know, and her unconditional love is the best gift I have received in my life. She will always be the person that I will seek the support of.

The regulatory function of the AAA4 ATPase domain of cytoplasmic dynein

“…While a recent study modeled the apo-state bond behavior of a single S. cerevisiae dynein head as an asymmetric slip bond for forces up to 14 pN 65 , a closer look at the measured data shows an excellent agreement with our results for forces up to ~5 pN, including the slip bonding up to ~2 pN and an ideal bonding up to ~5 pN under backward load. Our data also suggest an increasing unbinding rate above ~5 pN so that the bond appears best described by an overall slip-ideal-slip behavior for the 0–10 pN force range (Figs.…”

The regulatory function of the AAA4 ATPase domain of cytoplasmic dynein

“…A recent study reported that a single S. cerevisiae dynein head exhibited slipbonding in both MT directions 77 . Although the authors confirmed that the unbinding rate in the backward direction increased more slowly than in the forward direction 38,58,66,78 , the unbinding rates increased exponentially.…”

“…3 & 4). As we have already discussed 38 , the reason for this discrepancy is primarily due to the different unbinding-force assays that were used: while Ezber et al 77 used the "oscillatory assay" where the trap rapidly switched between two positions 66 , we used the "constant-pulling assay" where a MT attached to a coverslip moved at a constant velocity past the stationary trapped bead 38,58,79 . Extremely high loading rates up to ~25,000 pN/s can develop in the oscillation assay 38 ; therefore, it is possible that the high loading rates affect the dynein-MT bond and contribute to increased unbinding rates with larger forces.…”

Processivity of molecular motors under vectorial loads

“…The present model can be extended to study the effects of the vectorial character of the applied load on the mechanics of dynein and myosin motors, since the load-velocity and -detachment rate behaviours of kinesins, dyneins, and myosins are similar [14][15][16][17]. This can help to understand the mechanics of collective function of different molecular motors, owing to the force-dependent interactions of the motors.…”

“…They measured the vertical load component F z , where the horizontal component F x was resisting (<0, applied against the stepping direction). More recently, single-molecule optical trapping experiments [14][15][16][17] have studied the function of molecular motors under assisting loads F x (>0, applied in the stepping direction) as well. They have shown that the motors exhibit similar responses to the applied load direction: i) their velocity decreases with resisting loads, but changes slightly when the load is assisting, and ii) motors favor faster detachment rate under assisting loads than resisting ones.…”