news and views

Pulling out the coordination mechanism of myosin-VI
Martin Lindén© 2009 Nature America, Inc. All rights reserved.

Efficient transport by single two-headed motor proteins requires coordination of the motor domains. A new
single-molecule study sheds light on an important coordination mechanism by demonstrating an asymmetricstrain dependence of the weak-to-strong binding transition in myosin-VI heads.

Many intracellular transport processes arepowered by two-headed motor proteins,which literally walk along the cytoskeletal
filaments in cells1. The walk is powered byATP-driven conformational changes in themotors’ heads (the feet of these motors arefor historical reasons called ‘heads’, while
the mode of walking, which is reminiscentof the way humans walk, is referred to as the‘hand-over-hand’ model). For a two-headed
motor to walk efficiently, the heads must act
in a coordinated fashion. The heads of twoheaded myosins are separated by a few tensof nanometers a large part of the time, whichcorresponds to the repeat distance of actin.
This indicates that the heads coordinate
using intramolecular strain mediated by thelever arms (the myosin equivalent of legs).
In this issue, a single-molecule study by
Iwaki et al.2 shows that the strong bindingof myosin-IV heads to actin is greatly accelerated by backward strain. This mechanism
would allow the diffusing free head to act asa binding ratchet: it increases the probability of forward stepping by favoring strong
attachment to forward-facing binding sites.
The myosins are a large superfamily ofactin-based motor proteins that perform awide array of cellular functions. Myosin-VI
seems to have both anchoring and trafficking roles in vivo3. It forms two-headed structures when bound to endocytotic vesicles4,and dimers in vitro are capable of processive
unidirectional motion over several microns3.
In the myosin crowd, it stands out by walking
in the opposite direction relative to all otherknown myosins and by taking what appear
to be the largest steps despite not having thelongest lever arms3,5. To understand how
the motor achieves unidirectional and processive walking, several mechanisms must
be explained. First, at least one of the heads
should be kept tightly bound to actin. Second,
the rear head should release first when bothMartin Lindén is in the Departmentof Environmental Science, Policy andManagement, University of California,Berkeley, California, USA.
e-mail: linden@nature.berkeley.edu

376

VesicleForward

Strain12

3
4

Figure 1 Forward stepping with asymmetric binding rates. The rear head releases (1) and undergoes
Brownian motion, occasionally binding and unbinding weakly (2) with the actin filament. Once boundweakly far enough in front of the other head (3), backward strain transmitted through the lever armaccelerates strong binding (4).

heads are bound. This is an example of a gating mechanism: one head is effectively stalled
in some state until the other head reaches acertain state (the gate). Third, a free headshould bind to a site in front of the other
head—a binding reaction that proceeds via anintermediate weakly bound state. This could
be achieved by mechanically positioning thehead near the forward binding site and/or bybiasing the actin binding reaction to favor
forward binding in the spirit of the Huxleymodel for muscle6. Myosin-VI has a lot ofstructural flexibility7, which might be usefulwhen navigating complex actin networks invivo8. This points toward some kind of biased
binding5, but until now, there was no direct
evidence to support that assumption.
In their experiment, Iwaki et al.2 attachedsingle-headed motors to small polystyrene
beads. They then used a laser trap to rapidly
scan a bead back and forth above an actin filament suspended between microfabricated pillars. During the scans, the motors sometimes
interacted with the filament, producing brief
attachment events that transiently pulled thebead away from the moving trap. Two kinds ofattachment events, with two orders of magnitude difference in average dwell time, could
be discerned. The authors interpreted this interms of weakly and strongly bound states.

Supposedly, a free head first becomes weakly
bound to actin, and then either detaches
directly (short event) or binds strongly before
eventually detaching (longer event). The frequencies of long and short events depend on
scanning speed and direction, in a way thatstrongly indicates that the weak-to-strong rateincreases with increasing backward strain. This
would lead to the biased binding mechanism
for forward stepping discussed above, as thefree head is strained backward when interacting with a forward binding site (Fig. 1). At
the highest scanning speed (most strain), theauthors could fit their data to a kinetic modeland compute the weak-to-strong rate to beabout 30 times higher than the (strain-free)value extracted from a bulk assay9.
This result fills an important gap in ourqualitative understanding of myosin-IV. It
also points toward a mechanism that could
be at work in other motor proteins as welland adds to the list of strain-dependent gating mechanisms. Previous data show thatbackward strain favors binding of ADP
over ATP in myosin-VI (ref. 10). Because
ATP binding induces head detachment, thishints at a gating mechanism where intramolecular strain hinders the leading head fromdetaching before the rear head3. The theoretical framework for understanding motor

volume 5 number 6 JUne 2009 nature chemical biology

news and views
proteins is often called mechanochemistry,referring to the fact that reactions that produce movement must depend on applied load.
However, putting together a coherent pictureof flexible two-headed motors requires a characterization not only of how different transitions depend on strain but also of how thestrain is distributed among the various parts

and joints. It seems this purely mechanicalaspect is becoming more important the more
we learn about these systems.
1. Vale, R.D. Cell 112, 467–480 (2003).
2. Iwaki, M., Iwane, A.H., Shimokawa, T., Cooke, R. &Yanagida, T. Nat. Chem. Biol. 5, 403–405 (2009).
3. Sweeney, H.L. & Houdusse, A. Curr. Opin. Cell Biol. 19,57–66 (2007).
4. Altman, D., Goswami, D., Hasson, T., Spudich, J.A. &Mayor, S. PLoS Biol. 5, e210 (2007).
5. Ali, M. et al. Biophys. J. 86, 3804–3810 (2004).
6. Huxley, A.F. Prog. Biophys. Biophys. Chem. 7, 255–318(1957).
7. Yildiz, A. et al. J. Biol. Chem. 279, 37223–37226 (2004).
8. Ali, M.Y. et al. Proc. Natl. Acad. Sci. USA 104, 4332–4336 (2007).
9. De La Cruz, E.M., Ostap, E.M. & Sweeney, H.L. J. Biol.
Chem. 276, 32373–32381 (2001).
10. Altman, D., Sweeney, H.L. & Spudich, J.A. Cell 116,737–749 (2004).

S1P1 signaling just keeps going and going and going…

© 2009 Nature America, Inc. All rights reserved.

Stuart Cahalan & Hugh Rosen
Synthetic agonists of sphingosine-1-phosphate receptor subtype 1 are able to generate long-term signalingthat persists for hours after ligand-induced receptor internalization. These signals are independent of agonist
potency, can be reversed after receptor internalization by specific antagonists, and show a distinct acyl-chainlength structure-activity relationship.
Sphingosine-1-phosphate (S1P) is a pleiotropic signaling lipid that activates a family of G
protein–coupled receptors (S1P1–S1P5). The
S1P receptor prodrug agonist FTY720 (fingolimod) is currently in phase 3 clinical trials for relapsing-remitting multiple sclerosis.
After phosphorylation by sphingosine kinase
2, the active agonist FTY720-phosphate(FTY720-P) elicits some of its therapeutic
effects by causing systemic retention of lymphocytes in lymph nodes, thus preventing
autoreactive T cells from causing pathology.
In this issue, Mullershausen et al.1 provide
new chemical insights into the propensity of
FTY720 and its analogs to induce and maintain sustained, and perhaps tonic, signalingof the S1P1 receptor despite its complete
internalization, and they also highlight theimportance of differing hydrophobic interactions in these processes (Fig. 1).
The in vivo mechanism leading to the phenotypes of S1P1 agonist treatment has beenlong debated. Initially, FTY720-P was demonstrated to be a potent agonist of S1P1, S1P3,S1P4 and S1P5 (ref. 2). It was later shown thatFTY720-P could lead to rapid, sustained internalization and degradation of the S1P1 receptor3. Genetic studies established that S1P1 on
thymocytes was required for their exit fromthe thymus into blood, and when thymocytes
lacking S1P1 were transferred intravenously
into recipient mice, they were sequestered fromthe blood and the lymph4. These findings led
Stuart Cahalan and Hugh Rosen are in theDepartments of Chemical Physiology andImmunology, The Scripps Research Institute,La Jolla, California, USA.
e-mail: hrosen@scripps.edu

to a proposed mechanism by which FTY720-P
causes a loss of surface S1P1 expression on thelymphocytes, rendering them functionally
null and unable to respond to an S1P gradient to leave the secondary lymphoid organs.
However, it has also been shown that synthetic

S1P1-specific agonists, some of which cause
similar receptor internalization and recycling as S1P itself, can also cause lymphocyte
sequestration5. Additionally, S1P1-specificantagonists do not induce sequestration, andcan in fact rapidly reverse agonist-induced

S1P1

HOAFD-R
HOO
PO
POH

OHOOPOHS1PNH2

H 2NOH

HOOO

HOOH

OP

FTY720-P OO

H 2N

H2NOH

OH

OC5OOH

Fsk

Fsk[cAMP][cAMP]

5-h washout

S1PFsk

S1PFsk[cAMP][cAMP]
Ca2+Figure 1 Effect of S1P1 signaling and trafficking in response to different agonists. Upon agonist
treatment, S1P1 induces Gi-mediated inhibition of cAMP production by forskolin (Fsk) andinternalization of the receptor. 5 h after removal of agonist, cells treated with FTY720-P and AFD-R
still inhibit forskolin-dependent cAMP production through persistent signaling of internalized receptors,
whereas cells treated with S1P and OC5 respond normally to forskolin. After removal of agonist, cells
treated with FTY720-P and AFD-R are unable to release Ca2+ in response to S1P treatment due toreceptor internalization, whereas cells treated with S1P and OC5, which have membrane-associated
S1P1, release Ca2+ in response to S1P treatment.

nature chemical biology volume 5 number 6 JUne 2009377