Two recent articles addressed the power-stroke of myosin VI molecules during stepping. Although both groups measured the angles of fluorescent probes attached on the myosin VI molecule lever arm using polarized fluorescence techniques, they differ about whether the myosin VI lever arm rotation is fixed1 or variable2. Here we discuss the causes of the discrepancy between the two studies and the implications for myosin VI processive motility

Harry Schroeder III

John Beausang

Kazuaki Homma

Mitsuo Ikebe

Yale Goldman

Yujie Sun

English

PubMed

Y. Sun

H. W. Schroeder

J. F. Beausang

K. Homma

M. Ikebe

Y. E. Goldman

Crossref

Myosin VI lever arm rotation: Fixed or variable?

Myosin VI Lever Arm Rotation: Fixed or Variable?

Harry  W. Schroeder III

John  F. Beausang

Yale  E. Goldman

Nature Precedings

 1
Myosin VI Lever Arm Rotation: Fixed or Variable? 
Yujie Sun,1 Harry W. Schroeder III,1 John F. Beausang,1 Kazuaki Homma,2 Mitsuo Ikebe,2 and Yale E. Goldman1 
1Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, PA 19104, USA 
2University of Massachusetts Medical School, North Worcester, MA 01655, USA 
 
Abstract: Two recent articles1,2 addressed the power-stroke of myosin VI molecules during stepping. 
Although both groups measured the angles of fluorescent probes attached on the myosin VI molecule lever 
arm using polarized fluorescence techniques, they differ about whether the myosin VI lever arm rotation is 
fixed1 or variable2. Here we discuss the causes of the discrepancy between the two studies and the 
implications for myosin VI processive motility. 
Introduction: Myosin VI, an unconventional myosin motor that walks toward the minus ends of actin 
filaments, has received great attention recently. Reifenberger et al.1 have presented evidence from 
Defocused Orientation and Positional Imaging (DOPI) assays that myosin VI lever arms tilt by an angle 
very close to 180° on each step. This contrasts with results from Sun et al.2 wherein we applied single 
molecule Polarized Total Internal Reflection Fluorescence (polTIRF) microscopy and found variable 
degrees of tilting both in the axial and azimuthal directions relative to actin. This variability is consistent 
with the known variable step size of myosin VI. There are several technical differences in the two papers 
that interested researchers should understand, and more importantly, substantive differences in the 
conclusions.  
Results and Discussion: Reifenberger et al.1 explained the difference in results between the two studies by 
a choice of analysis hemisphere, but their explanation is wrong. Fig. 1, here, shows lateral and axial views 
of a myosin molecule (M) that landed on actin (A) in a typical position away from the coverslip (blue). 
Although the fluorescence emission from the chromophore is symmetric about the dipole axis, the two 
ends of the bifunctional probe are different because they are linked to different cysteine residues (here 66 
and 73 on calmodulin, in the same construct used by both studies). To indicate this asymmetry we draw the 
probe as a directional arrow. Neither DOPI nor polTIRF can intrinsically distinguish between the two ends 
of the dipole, so the choice of which one to report requires additional considerations.  It is especially 
crucial to be sure that it's the same one before and after an angle change (e.g., after a motor step). For any 
measurement of polarized fluorescence, a hemisphere needs to be chosen that defines the range of valid 
angles reported. Fig. 1A shows the position of the probe before a step. Follow the pointed end of the arrow 
in Fig. 1 which is at the Cys66 end of the probe. If the lever arm rotates 180° axially (in a plane containing 
the actin), then the probe will be positioned as in Fig. 1B after the step. With an analysis hemisphere 
whose equator is parallel to the glass, as used by Reifenberger et al.1, the pointed end of the probe is 
positioned inside the hemisphere before (Fig. 1A middle panel) and outside the hemisphere after the step 
(Fig. 1B middle panel). In the latter case, the method will report the wrong angle. If the hemisphere is 
tilted around the axis of actin, as we did2, then the relevant end of the dipole is reported correctly before 
(Fig. 1A lower panel) and after (Fig. 1B lower panel) the step. This idea was explained in our original 
paper on DOPI (Suppl. Fig. 5)3 and formally shows the error in Reifenberger et al.'s categorical claim that 
a horizontal hemisphere should always be used.  
 
 
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Trailing
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AA
 
 
Figure 1. Views of myosin VI in the trailing (A) and leading (B) positions. In the upper panels of A, side views 
show the rigor state and bifunctional rhodamine bound to calmodulin residues 66 and 73. The rhodamine dipole 
is marked as a directional arrow to identify the end attached to Cys66. Myosin VI steps toward the “–” (pointed) 
end of actin, drawn to the right here. Note that the results of Sun et al.2 show that the rotation is variable, but for 
illustrative purposes, only the 180° rotated angle is shown in B. Middle and lower panels show an axial view 
from the “–” end of actin, looking toward the “+” end. The middle panels indicate what angles are reported if a 
horizontal hemisphere is used to select the range of allowable probe angles, as used by Reifenberger et al.1 In 
the trailing head, this hemisphere contains the Cys66 end of the dipole, but in the leading head, the opposite end 
of the dipole is within the hemisphere (B, middle panel), causing an erroneous angle to be reported [X!!]. The 
bottom panels show that a tilted hemisphere reports the correct angle (i.e., the same end of the dipole) in both 
leading and trailing positions. Thus, the analysis hemisphere cannot be horizontal for all molecules to obtain the 
correct angle from the DOPI or polTIRF methods. 
 
 The same point is shown in Fig. 3 of Reifenberger et al.1 (red and orange arrowheads). Molecules 
are also described in Reifenberger’s PhD thesis4 (pp. 138 – 143, Figs. 5.21 – 5.24) that artifactually 
jumped between the upper and lower hemispheres. A slight tilt of the equator of the analysis hemisphere 
would suppress this artifact, again showing that the correct hemisphere is not always horizontal. 
 Myosin VI molecules are shown to “wobble” on p. 136 of Reifenberger’s thesis4. That this point 
was omitted from the publication1 is surprising, inasmuch as it was a major conclusion of Sun et al2. 
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Reifenberger et al. point out that the 180° position of the leading head lever arm is compatible with 
the pre-powerstroke x-ray crystal structure containing ADP and a Pi analog5. However, Pi and probably 
ADP are both released rapidly from the leading head upon binding to actin6, so the crystal structure with 
both products bound is irrelevant in the present context. 
It is not possible to gauge from Reifenberger et al.'s paper1 whether their probe molecules were 
cross-linked between the two engineered Cys residues and thereby were constrained fully to lie along the 
axis between the two target Cys residues.  The DOPI technique has the advantage over polTIRF of 
simultaneously monitoring position and stepping, but this is offset by the disadvantage that it doesn't 
provide an independent estimate of probe wobble. polTIRF does report the wobble, both on the 
nanosecond and microsecond timescales, so we can be sure that all of the molecules analyzed in Sun et al. 
were immobilized on the calmodulin. Whether this accounts for some of the differences in the results is not 
clear at present. 
Myosin VI is known to have a broad distribution of step sizes giving a 25 to 40 nm span between 
the two heads. This point is confirmed by ours and earlier data7-9. Thus, the geometrical relationship 
between the two lever arms should depend on the distance between the two heads after any given step, a 
point made previously by both groups2,9. In Reifenberfer et al.1, the variable step size is dismissed by 
postulating that each molecule walks straight, rarely taking sideways steps, and the overall distance 
distribution is broad due to contributions from different molecules. This argument is fallacious for at least 
four reasons: a) if each molecule has a fixed step size, then certain molecules would need short steps and 
others long ones, both of these groups of molecules exhibiting strong helical (not straight) motion10,11; b) 
All individual recordings presented in papers from the authors’ lab1,4,9 and others7,12 show variable steps; c) 
Binding sites for myosin VI to a filament on the glass allow a broad distribution of azimuths (α) (Fig. 3 of 
Sun et al.2), but the α distributions (Fig. 2 of Reifenberger et al.1) show narrow peaks. This result implies 
severe restriction of myosin VI’s landing positions or else strongly biased selection of recordings; and d) 
Myosin V has been shown sometimes to take sideways steps13,14. If Reifenberger et al’s argument were 
correct, then step size distributions of myosin V for individual molecules would be wide, but they are 
narrow. Thus, negating the previous data showing the variable step size of individual myosin VI molecules 
on the basis of Reifenberger et al.’s DOPI results is untenable.  
If we retain the well established variable step size, and consider Reifenberger et al.’s proposal that 
the leading head lever arm is completely decoupled from the converter domain, it is difficult to understand 
how its angle, relative to actin, can be fixed at 180° independent of the (variable) distance between the two 
heads.  A mechanical model with flexible connections shows that the relative lever arm angles depend on 
the distance between the heads (Sun et al.2, Fig. 6). The azimuthal orientation between the two bound 
heads is determined by the actin helix, so it also has to vary with step size, as we found. 
Nevertheless, both studies conclude that the lever arm of myosin VI is not tightly coupled to the 
converter domain.  Reifenberger et al. suggest that the lever and converter are completely uncoupled in the 
leading head of a walking myosin VI molecule.  But, contradictorily, strain in the molecule is supposed by 
Reifenberger et al. to prevent the leading head from binding ATP in order to maintain processivity. This 
strain-gating conjecture is incompatible with complete lever arm-converter uncoupling, which would allow 
the leading head converter domain to rotate fully and rapidly to its rigor position, thereby posing no 
restraint on ATP binding. Although Reifenberger et al. do not explain how converter rotation is limited or 
how the 180° angle is maintained, they hint that the lever arm is tethered between two flexible hingepoints, 
the N-terminal hinge between the lever and the converter (the point of “complete uncoupling”) and the C-
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terminal hinge extending to an elastic segment, perhaps, for example, an unfolded 3-helix bundle. Linear 
strain (no torque) would then prevent ATP binding until the trailing head detaches. In that case, the lever 
would undergo large thermal rotational fluctuations about its own axis and the dipole at Cys66-Cys73 would 
exhibit much larger angular fluctuations in the leading than in the trailing position. Neither Reifenberger et 
al.’s nor our data shows signs of such extensive azimuthal wobble in the leading head.  
Conclusions: We conclude, as originally discussed in Sun et al.2, that a pliant region between the lever 
arm and converter (that is, not complete uncoupling), similar to that found by x-ray crystallography of 
scallop myosin15 enables partial rotation of the leading head converter domain. Torque on the converter 
would prevent full rotation and ATP binding until the trailing head detaches. The variable angle of the 
lever arm that we observed is set by the geometrical relationship of the two bound heads and partitioning 
of the total compliance between this rotational spring and the other flexible regions in the molecule. 
 
References: 
1. Reifenberger, J.G., Toprak, E., Kim, H., Safer, D., Sweeney, H.L. & Selvin, P.R. Myosin VI 
undergoes a 180° power stroke implying an uncoupling of the front lever arm. Proc. Nat. Acad. Sci. 
U.S.A. 106, 18255-18260 (2009). 
2. Sun, Y., Schroeder III, H.W., Beausang, J.F., Homma, K., Ikebe, M. & Goldman, Y.E. Myosin VI 
walks “wiggly” on actin with large and variable tilting. Mol. Cell 28, 954-964 (2007). 
3. Toprak, E., Enderlein, J., Syed, S., McKinney, S.A., Petschek, R.G., Ha, T., Goldman, Y.E. & Selvin, 
P.R. Defocused orientation and position imaging (DOPI) of myosin V. Proc. Nat. Acad. Sci. U.S.A. 
103, 6495-6499 (2006). 
4. Reifenberger, J.G. Doctor of Philosophy, Studies of Myosin Mechanics Using Fluorescence 
(University of Illinois at Urbana-Champaign, Urbana, Illinois). (2006) Available at  
 http://research.physics.illinois.edu/Publications/theses/copies/Reifenberger_Jeffrey.pdf 
5. Ménétrey, J., Llinas, P., Mukherjea, M., Sweeney, H. & Houdusse, A. The structural basis for the 
large powerstroke of myosin VI. Cell 131, 300-308 (2007). 
6. Sweeney, H.L., Park, H., Zong, A.B., Yang, Z., Selvin, P.R. & Rosenfeld, S.S. How myosin VI 
coordinates its heads during processive movement. EMBO J. 26, 2682-2692 (2007). 
7. Ökten, Z., Churchman, L.S., Rock, R.S. & Spudich, J.A. Myosin VI walks hand-over-hand along 
actin. Nat. Struct. Mol. Biol. 11, 884-887 (2004). 
8. Park, H., Ramamurthy, B., Travaglia, M., Safer, D., Chen, L.-Q., Franzini-Armstrong, C., Selvin, P.R. 
& Sweeney, H.L. Full-Length Myosin VI Dimerizes and Moves Processively along Actin Filaments 
upon Monomer Clustering. Mol. Cell 21, 331-336 (2006). 
9. Yildiz, A., Park, H., Safer, D., Yang, Z., Chen, L.-Q., Selvin, P.R. & Sweeney, H.L. Myosin VI Steps 
via a Hand-over-Hand Mechanism with Its Lever Arm Undergoing Fluctuations when Attached to 
Actin. J. Biol. Chem. 379, 37222-37226 (2004). 
10. Beausang, J.F., Schroeder III, H.W., Nelson, P.C. & Goldman, Y.E. Twirling of Actin by Myosins II 
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11. Ali, M.Y., Homma, K., Iwane, A.H., Adachi, K., Itoh, H., Kinosita Jr., K., Yanagida, T. & Ikebe, M. 
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12. Rock, R.S., Ramamurthy, B., Dunn, A.R., Beccafico, S., Rami, B.R., Morris, C., Spink, B.J., 
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13. Syed, S., Snyder, G.E., Franzini-Armstrong, C., Selvin, P.R. & Goldman, Y.E. Adaptability of 
myosin V studied by simultaneous detection of position and orientation. EMBO J. 25, 1795-1803 
(2006). 
14. Ali, M., Krementsova, E., Kennedy, G., Mahaffy, R., Pollard, T., Trybus, K. & Warshaw, D. Myosin 
Va maneuvers through actin intersections and diffuses along microtubules. Proc Natl Acad Sci USA 
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A Flexible Domain Is Essential for the Large Step Size and Processivity of Myosin VI.

Adaptability of myosin V studied by simultaneous detection of position and orientation.

Defocused orientation and position imaging (DOPI) of myosin V.

Doctor of Philosophy, Studies of Myosin Mechanics Using Fluorescence (University of Illinois at Urbana-Champaign,

How myosin VI coordinates its heads during processive movement.

Myosin Va maneuvers through actin intersections and diffuses along microtubules.

Myosin VI Steps via a Hand-over-Hand Mechanism with Its Lever Arm Undergoing Fluctuations when Attached to Actin.

Myosin VI undergoes a 180° power stroke implying an uncoupling of the front lever arm.

Myosin VI walks “wiggly” on actin with large and variable tilting.

Myosin VI walks hand-over-hand along actin.

N a t u r e P r e c e d i n g s : d o i : .

Twirling of Actin by Myosins II and V Observed via Polarized TIRF in a Modified Gliding Assay.

Unconstrained Steps of Myosin VI Appear Longest among Known Molecular Motors.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2867696

Myosin VI Lever Arm Rotation: Fixed or Variable?

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