Cleary FB , Dewitt MA , Bilyard T , Htet ZM , Belyy V , Chan DD , Chang AY , Yildiz A .

R01 GM094522 NIGMS NIH HHS , GM094522 NIGMS NIH HHS

	Figure 2. Dynein processivity requires a single motor domain(a) Possible ring-ring communication between the two heads is abolished by replacing one of the monomers with a SRS-MTBD chimera. (b) A kymograph of SRS85:82/WT showing that it moves processively along MTs (c) The average velocity (± SD) and run length (± 95% CI) of the SRS-MTBD/WT constructs compared to WT/WT. (d) An example high-resolution tracking trace (black) and stepping fit (red) of the SRS85:82 head. (e) Histograms of the step sizes for the SRS (upper) and WT (lower) heads (mean ± SD).
	Figure 3. The linker provides force to drive the motility of a dynein dimer(a) Dimerization of the C-terminal ring of one head to the N-terminal tail of the other results in a dimer of a free-linker head (FLH) and a bound-linker head (BLH). (b) Kymograph showing that the FLH/BLH heterodimer is capable of processive motility. (c) Run length histogram of FLH/BLH at 2 mM ATP, with maximum likelihood fit (± 95% CI). (d) The average velocities (± SEM) of FLH/BLH constructs carrying an ATPase mutation in either the AAA1 or AAA3 site in one head (ND: motility not detected). (e) Kymographs of ATPase mutants of the FLH or BLH at 2 mM ATP. The AAA1K/A mutation on BLH abolishes directional motility, whereas AAA1E/Q mutation leads to non-directional diffusion along the MT. The same mutations on FLH do not stop motility, indicating that BLH monomer is responsible for FLH/BLH motility.
	Figure 4. Tension on the linker inhibits nucleotide-dependent release of dynein from MTs(a) Dynein monomers are attached to a polystyrene bead either through the N-terminus of the linker via a GFP-antibody linkage, or through the C-terminus of the ring via a 74 bp long DNA tether (not to scale). (b) A representative trace showing bead position (blue) and trap position (green). (c) Force-dependent release rates of dynein monomers when the trapped bead is attached to the N-terminal linker domain. The distribution of the forward and backward release rates are unaffected by the presence (red, n = 3003) and absence (blue, n = 2335) of ATP. Shaded regions indicate 95% confidence intervals. Inset: A representative example of the distribution of measured MT-bound times at 0.94 pN average force towards the MT-minus end at 1 mM ATP. Black curve represents maximum likelihood fit to the dwell time histogram. (d) Force-dependent release rates when dynein is pulled through the C-terminus. The rate is similar to the N-terminal attachment case (dotted black line) in the absence of ATP (blue, n = 1105), but increases several fold in the presence of 2 mM ATP (red, n = 1652).
	Figure 5. Tension gating of a dynein head is observed at high interhead separations in a motile dimer(a) A representative trace showing simultaneous tracking of AAA1K/A/WT stepping properties with two colors of quantum dots at saturating (1 mM) ATP. (b) The scatter plots represent the step sizes of WT (top) and AAA1K/A (bottom) heads as a function of interhead separation. Interhead separation is positive when the stepping head is in the lead. Slope and y intercept of the linear fit (red line) reveal the change in step size per nm extension between the heads and the net bias to move towards the minus-end, respectively. Both heads take smaller steps when they release the MT in the leading position, similar to native dynein. The AAA1K/A head takes mostly backward steps when stepping from the leading position (probability of backward stepping, pBW = 0.63) (c) The AAA1K/A head remains in the trailing position 72% of the time. Distance to the WT head is positive when the AAA1K/A head is in the lead. (d) Histogram of the dwell time between steps of the WT (left) or AAA1K/A (right) head, independent of the action of the other head, with exponential fit (± 95% CI). (e) Stepping rates of the WT and AAA1K/A heads as a function of interhead separation (shaded region is the 95% CI). The WT head steps more frequently when it is positioned close to the AAA1K/A head, and is otherwise as a similar rate to the AAA1K/A head, indicating that the WT head is gated at high interhead separations.
	Figure 6. A model for dynein motility(a) Two proposed mechanisms of dynein stepping. (Left) When the heads are close to each other, either head can release the MT and step forward upon binding ATP. (Right) When the heads are far apart, tension on the linker prevents ATP-dependent MT release, and the asymmetry of the release rates under tension favors the trailing head to take a step. (b) A representative Monte-Carlo simulation of dynein motility shows stepping of the two head domains (blue and red). (c) The trailing head is more likely to take a step in simulated traces as the interhead separation increases. The data shown is the average of 200 simulations (± SD). (d) The average velocity of 200 100 s simulated traces (± SEM) agrees well with measured velocities for various dynein mutants (± SEM, n > 100). The results of the model are within ±15% of the experimental data.

Asbury, Kinesin moves by an asymmetric hand-over-hand mechanism. 2003, Pubmed

Asbury, Kinesin moves by an asymmetric hand-over-hand mechanism. 2003, Pubmed
Banaszynski, Characterization of the FKBP.rapamycin.FRB ternary complex. 2005, Pubmed
Block, Kinesin motor mechanics: binding, stepping, tracking, gating, and limping. 2007, Pubmed
Burgess, Dynein structure and power stroke. 2003, Pubmed
Capitanio, Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke. 2012, Pubmed
Carminati, Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex. 1997, Pubmed
Carter, Structure and functional role of dynein's microtubule-binding domain. 2008, Pubmed
Carter, Crystal structure of the dynein motor domain. 2011, Pubmed
Cashikar, Defining a pathway of communication from the C-terminal peptide binding domain to the N-terminal ATPase domain in a AAA protein. 2002, Pubmed
Cho, Regulatory ATPase sites of cytoplasmic dynein affect processivity and force generation. 2008, Pubmed
DeWitt, Cytoplasmic dynein moves through uncoordinated stepping of the AAA+ ring domains. 2012, Pubmed
Dunn, Contribution of the myosin VI tail domain to processive stepping and intramolecular tension sensing. 2010, Pubmed
Gee, An extended microtubule-binding structure within the dynein motor domain. 1997, Pubmed
Gennerich, Walking the walk: how kinesin and dynein coordinate their steps. 2009, Pubmed
Gennerich, Force-induced bidirectional stepping of cytoplasmic dynein. 2007, Pubmed
Gerdes, Microtubule transport defects in neurological and ciliary disease. 2005, Pubmed
Gibbons, The affinity of the dynein microtubule-binding domain is modulated by the conformation of its coiled-coil stalk. 2005, Pubmed
Griffis, Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore. 2007, Pubmed
Hafezparast, Mutations in dynein link motor neuron degeneration to defects in retrograde transport. 2003, Pubmed
Hancock, Kinesin's processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains. 1999, Pubmed
Huang, Lis1 acts as a "clutch" between the ATPase and microtubule-binding domains of the dynein motor. 2012, Pubmed
Imamula, The coordination of cyclic microtubule association/dissociation and tail swing of cytoplasmic dynein. 2007, Pubmed
Kaseda, Alternate fast and slow stepping of a heterodimeric kinesin molecule. 2003, Pubmed
Klumpp, Kinesin's second step. 2004, Pubmed
Kodera, Video imaging of walking myosin V by high-speed atomic force microscopy. 2010, Pubmed
Kon, X-ray structure of a functional full-length dynein motor domain. 2011, Pubmed
Kon, ATP hydrolysis cycle-dependent tail motions in cytoplasmic dynein. 2005, Pubmed
Kon, Helix sliding in the stalk coiled coil of dynein couples ATPase and microtubule binding. 2009, Pubmed
Kon, The 2.8 Å crystal structure of the dynein motor domain. 2012, Pubmed
Kon, Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein. 2004, Pubmed
McKenney, LIS1 and NudE induce a persistent dynein force-producing state. 2010, Pubmed
Morales-Mulia, Spindle pole organization in Drosophila S2 cells by dynein, abnormal spindle protein (Asp), and KLP10A. 2005, Pubmed
Nicastro, The molecular architecture of axonemes revealed by cryoelectron tomography. 2006, Pubmed , Echinobase
Qiu, Dynein achieves processive motion using both stochastic and coordinated stepping. 2012, Pubmed
Rasnik, Nonblinking and long-lasting single-molecule fluorescence imaging. 2006, Pubmed
Reck-Peterson, Single-molecule analysis of dynein processivity and stepping behavior. 2006, Pubmed
Roberts, AAA+ Ring and linker swing mechanism in the dynein motor. 2009, Pubmed
Schmidt, Insights into dynein motor domain function from a 3.3-Å crystal structure. 2012, Pubmed
Shastry, Neck linker length determines the degree of processivity in kinesin-1 and kinesin-2 motors. 2010, Pubmed
Shima, Head-head coordination is required for the processive motion of cytoplasmic dynein, an AAA+ molecular motor. 2006, Pubmed
Shima, Two modes of microtubule sliding driven by cytoplasmic dynein. 2006, Pubmed
Spudich, Molecular motors take tension in stride. 2006, Pubmed
Thoresen, Processive movement by a kinesin heterodimer with an inactivating mutation in one head. 2008, Pubmed
Uemura, Kinesin-microtubule binding depends on both nucleotide state and loading direction. 2002, Pubmed
Vallee, Dynein: An ancient motor protein involved in multiple modes of transport. 2004, Pubmed
Vallee, Multiple modes of cytoplasmic dynein regulation. 2012, Pubmed
Visscher, Versatile optical traps with feedback control. 1998, Pubmed
Yildiz, Intramolecular strain coordinates kinesin stepping behavior along microtubules. 2008, Pubmed , Echinobase
Yildiz, Kinesin walks hand-over-hand. 2004, Pubmed
Yildiz, Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. 2003, Pubmed