The unique enzymatic and mechanistic propertiesof plant myosinsArnon Henn1 and Einat Sadot2

Available online at www.sciencedirect.com

ScienceDirect

Myosins are molecular motors that move along actin-filament

tracks. Plants express two main classes of myosins, myosin VIII

and myosin XI. Along with their relatively conserved sequence

and functions, plant myosins have acquired some unique

features. Myosin VIII has the enzymatic characteristics of a

tension sensor and/or a tension generator, similar to functions

found in other eukaryotes. Interestingly, class XI plant myosins

have gained a novel function that consists of propelling the

exceptionally rapid cytoplasmic streaming. This specific class

includes the fastest known translocating molecular motors,

which can reach an extremely high velocity of about

60 mm s�1. However, the enzymatic properties and

mechanistic basis for these remarkable manifestations are not

yet fully understood. Here we review recent progress in

understanding the uniqueness of plant myosins, while

emphasizing the unanswered questions.

Addresses1 Faculty of Biology, Technion-Israel Institute of Technology, Haifa

3200003, Israel2 The Institute of Plant Sciences, Volcani Center, PO Box 6, Bet-Dagan

5025000, Israel

Corresponding authors: Henn, Arnon (arnon.henn@technion.ac.il) and

Sadot, Einat (vhesadot@agri.gov.il)

Current Opinion in Plant Biology 2014, 22:65–70

This review comes from a themed issue on Cell biology

Edited by Shaul Yalovsky and Viktor Zarsky

http://dx.doi.org/10.1016/j.pbi.2014.09.006

1369-5266/# 2014 Elsevier Ltd. All rights reserved.

IntroductionLive-cell imaging of plant cells reveals strikingly rapid

movement of organelles and vesicles, rearrangement of

the endoplasmic reticulum (ER) and constant remodeling

of the tonoplast. Under certain conditions slower move-

ment of the chloroplasts and nucleus can also be observed.

This intracellular dynamic is believed to enable the immo-

tile plant to respond quickly to abiotic and biotic stresses.

The machinery underlying these vigorous movements

involves actin-based molecular motors, the myosins and

actin microfilaments that together drive the formation of

streaming patterns in plant cells. Myosins utilize ATP to

perform mechanical work such as muscle contraction and

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the translocationofcargoalongactinfilaments ineukaryotic

cells [1]. Across all eukaryotes there are 35 known classes of

myosins [2]. In plants there are two main classes, VIII, XI

[3], which are widely distributed in higher and lower plants.

Plant myosins are involved in various organelles’ motility

[4,5��,6–14], ER remodeling [15–18] and cytoplasmic

streaming [19,20,21��,22��]. They have been found in

the plasmodesmata [23–25,26��] and can be involved in

targeted RNA transport [27], viral spread from cell to cell

[28–31] and in a plant’s size and shape determination

[6,22��,32]. All known myosins share a highly conserved

amino-terminal motor domain followed by a neck (also

referred to as the ‘lever arm’) and the tail regions. The

neck region, which contains conserved IQ repeats (as many

as one to six in tandem), serves as the light chains binding

domains and the tail region which displays diverse inter-

acting domains such as cargo and membrane binding motifs

[1]. The myosin actin-dependent ATPase cycle includes at

least six nucleotide-linked, biochemical intermediates

(Figure 1a). The cycling of myosin on actin is driven by

ATP binding, hydrolysis and product release, and can be

described as follows (Figure 1a from left to right): ATP

binding to actomyosin causes myosin head to dissociate

from actin. Detached myosin hydrolyzes ATP to ADP and

Pi, which drives the repriming of the lever arm to the ‘pre-

power stroke’ state. This post hydrolysis state can rebind

actin with a higher affinity than before the hydrolysis of

ATP. Upon the rebinding of myosin-ADP-Pi state to actin

conformational changes within the myosin motor domain

are coupled to the release of Pi and the lever arm rotation

relative to the actin-bound myosin head. This lever arm

rotation transition is the force-generating step, or the

‘power-stroke’, of the myosin ATPase cycle, which is next

followed by ADP release, and the formation of the myosin-

actin nucleotide free state. Thereafter, the catalytic cycle

begins again with ATP binding. Specific nucleotide states

dictate the affinity of the myosin motor domain towards

actin. The strength of the myosin nucleotide binding states

towards actin follows from strong to weak binding affinities

as such: actomyosin > actomyosin-ADP > actomyosin-

ADP-Pi > actomyosin-ATP. Although this ATPase cycle

kinetic pathway is highly conserved among all myosins

studied to date, the enzymatic properties of the catalytic

motor domain display very large deviations in their kinetic

and thermodynamicproperties, that is, each myosin isoform

displays different rate and equilibrium constants for the

biochemical transitions described in Figure 1a [33,34].

These different rate and equilibrium constants give raise

to different ATPase cycling-times, affinities toward actin

and motility rates among myosins. They are the hallmarks

Current Opinion in Plant Biology 2014, 22:65–70

66 Cell biology

Figure 1

“Strongly Bound”

“Strongly Bound”

AM AM AMADP ADP’ ADP

“Dissociated States” “Thermodynamic Coupling box”

AM

M

(a)

(b)

ATP M ADP M ADP

Low TC for Myosin V, VIIIHigh TC for Myosin XIChara.

Myosin XIChara:Very fast ADP release

Myosin V, VIII:Rate limiting Stepduring ATPasecycling

Second strongly boundactomyosin-ADP state

Slow isomerization

M ADP+Pi

AM ATP AM ADP AM AMADPPiK’Pi

KAD KA

KD

KA

M + ATP

KAPi

KHKT

K’D1 K’D2

K’H

KPi

ATP+

+

ADP+

“Strongly Bound ”“Weakly Bound ”

Current Opinion in Plant Biology

The actomyosin ATPase cycle. (a) Schematic representation of myosin’s conserved chemomechanical energy transduction pathway is shown. The

reaction pathway in the black frame is the predominant pathway for the myosin ATPase cycle as described in the text. Key features in plant myosins

VIII and XIChara in relation to the ATPase reaction scheme are shown. The dashed blue frame contains the key parameters for determining

thermodynamic coupling (TC) between actin and ADP affinities toward myosin. Black and blue callouts mark unique features among measured

parameters of plant myosins. Blue ovals highlight extremely rapid rate constants for Chara myosin XI. Red ovals mark rate and equilibrium constants

that have not been measured for any plant myosin. (b) Expansion of the minimal kinetic scheme showing the transition from actomyosin-ADP to

actomyosin with additional intermediate ADP state namely actomyosin-ADP0 indicative of tension-sensor myosins.

of the diverse enzymatic behavior among the myosin family

classesandisoforms. Inturns, theseuniquefeaturesenables

each myosin isoform to be enzymatically adapted to its

cellular functions. Here we describe the findings related to

the motor properties of the two main classes of plant

myosins and discuss their enzymatic adaptation to the

relevant unique cellular functions in plants.

Class VIII myosinsThe first plant myosin to be cloned and sequenced was

Arabidopsis thaliana myosin 1 (ATM1). Phylogenetic

analysis based on its motor domain placed ATM1 in a

new myosin class VIII [35]. Specific antibodies against its

tail domain revealed its subcellular localization in the

plasmodesmata and newly formed cell plates in the roots

of maize and cress (Lepidium sativum) [36]. Subsequent

green fluorescent protein (GFP) fusions confirmed these

observations [25,26��,37], and also suggested its localiz-

ation in ER junctions [25]. Analysis of the knockout plant

of all five myosin VIII transcripts of P. patens revealed

that this class has a role in development and hormone

Current Opinion in Plant Biology 2014, 22:65–70

homeostasis [32]. Recent detailed transient kinetic and

thermodynamic studies of myosin VIII have revealed its

enzymatic properties [26��]. It was found that ADP

release (at physiological levels of Mg2+) [26��] is the

rate-limiting step (the slowest transition within one com-

plete ATPaes cycle, Figure 1a) in myosin VIII’s ATPase

cycle, which contributes to the long-lived and strongly-

bound actomyosin-ADP state during the ATPase cycle

and to high duty ratio (DR — see below). The cycling

time is defined as a single complete cycle of ATP binding,

hydrolysis and product release (Figure 1a). Myosin VIII

displays a relatively slow to moderate ATPase cycle, with

a slow motility rate that is highly regulated by Mg2+

[26��]. The latter characterize local tension-bearing myo-

sins rather than fast moving transporters [33,38]. In

addition, the two states, with fast and slow dissociation

rates (Figure 1b), observed in myosin VIII [26��] suggest

that additional work can be achieved with the slower step

of ADP dissociation [38]. This is a unique feature that is

found in several tension-sensor myosins [33]. Further-

more, myosin VIII displays a very weak thermodynamic

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Plant myosins Henn and Sadot 67

coupling (TC) ratio of 2.3 [26��]. TC is a measure of how

the affinity of myosin to actin is affected by its binding to

ADP, in comparison to how its binding to ADP is affected

by actin. This simply reflects the degree of communi-

cation between the nucleotide and actin binding sites,

and is derived from the free energy detailed balance of

the ADP states. Detailed balance requires that in the

absence of external energy input or consumption the

product of the four equilibrium constants equal unity

and, therefore, K 0D=KD ¼ KA=KAD (Figure 1a). Tension-

sensor myosins have been shown to display very weak TC

or a low ratio close to unity between the equilibrium

constants [33,38]. A weak TC permits strong binding to

actin in the presence of ADP, and hence to bear tension in

the strongly bound ADP states. Lastly, the kinetic adap-

tation of plant myosin VIII for its function also stems from

its measured high DR of 0.9 [26��]. The DR is defined as

the fraction of time that myosin spends strongly bound to

actin during the complete ATPase cycle. Thus, the

detailed enzymological studies of myosin VIII strongly

suggest that it is enzymatically adapted to maintaining

tension, such as that required for ER tethering to the

plasma membrane for proper plasmodesmatal function

[39]. However, the latter assumption requires in-vivo

validations.

Class XI myosinsClass XI myosins from Chara corallina are the fastest

actin-based motors characterized to date, even faster than

myosin XI members in higher plants. Class XI myosins

show sequence and structural similarities to vertebrates’

transporter class V myosins [40]. Both contain a long lever

arm composed of six IQ motifs, indicative of a large step

size, and a coiled-coil motif, which favors a double-

headed (dimer) myosins’ oligomeric state [41]. Despite

these similarities to myosin class V, the velocity measure-

ments by in vitro motility assay for plant myosins have

demonstrated remarkably higher rates. For purified C.corallina myosin XI, the measured velocities range from

20 mm s�1 up to 60 mm s�1 [42–44], and for myosin XI

from Nicotiana tabacum and Arabidopsis this is about

7 mm s�1. These measurements are up to 150-folds and

20-folds respectively, faster than myosin V [45]. Purified

myosin XI from N. tabacum was found to have a step size

of �35 nm [46]. This step size was similar to that

measured for myosin V, which has an identical lever

arm length of six IQ motifs [45]. The step size measured

for the faster Chara myosin XI was �19 nm [44]. The

extraordinarily high velocity of myosin XI does not result

from an unusually large step size. Calculations combining

velocity with step size suggested that the ATPase cycling

time for the slower measured velocity (myosin XI from N.tabacum and Arabidopsis) should be �200 ATP s�1 h�1

(s�1 h�1 is define as moles of ATP hydrolyzed per second

per head, that is, a head is a single motor domain, 200 is

calculated from the measured values of 7 mm s�1 per

35 nm stepsize) and �1052 ATP s�1 h�1 (20 mm s�1 per

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19 nm) for the faster measured velocity (Chara myosin

XI). Interestingly, the fastest measured actin-activated

ATPase rate for N. tabacum myosin XI was

76 ATP s�1 h�1 [46], and for Chara myosin XI,

670 � 20 ATP s�1 h�1 [47]. Detailed enzymology for N.tabacum myosin XI is still lacking, but it will be interesting

to see how it supports not only rapid velocity but also a

sufficiently high DR (�0.8 [46]) for its processive move-

ment (the ability to take multiple steps along actin before

dissociating). Detailed transient kinetic measurements

were performed on the motor domain of Chara myosin

to reveal its ATPase reaction mechanism; some interest-

ing features were found that might support the rapid

ATPase cycling time [48]. Chara myosin, that was shown

to posses a �19 nm step size exhibits an extremely rapid

ADP-dissociation rate (>2800 s�1), fast ATP-binding rate

(36 mM�1 s�1) and ATP-induced dissociation from actin

(2200 s�1), as well as very rapid ATP hydrolysis

(>530 s�1). However, it dwells in the strongly bound

state for less than 0.82 ms, which is a very short lifespan

with respect to its full ATPase cycling time of 2.5 ms

calculated from the ATPase rate (kcat of 390 s�1). Further-

more, its low DR of <0.3 may not support high proces-

sivity or long run length, as seen for N. tabacum [33,34].

For Chara myosin XI, the super-fast transitions could

support rapid ATP turnover within the range of the

suggested actin-gliding velocity. A structural molecular

basis for the extremely short cycling time was tested

experimentally by mutating the properties of loops

2 and 3 in the motor domain of Chara myosin XI which

are known to be part of the actin-binding site. Yamamoto

and co-workers [47] identified a net charge of zero for loop

2, with no lysine cluster, unlike in myosin V. In addition,

it was found that loop 3 had a high positive charge and was

the largest in size of all known myosins [47]. Loop 2 is also

important for the myosin-ADP-Pi state binding to actin

[47]. In this study the authors provide new insights

regarding these two loops. Firstly, the interaction of Charamyosin XI with actin is mediated mainly by loop 3 and

secondly, it does not hinder rapid release of ADP. In

contrast, they found that lengthening loop 2 slows down

ADP release, which had not been shown before. There-

fore, the authors suggest that it is this unique combination

of zero net charge of a short sequence in loop 2 with a very

long loop 3 which facilitates the high ATPase rate necess-

ary to support the very high velocity [47]. However, more

work is needed to elucidate additional unique features of

the extremely fast ATPase cycle. Based on the above

data, the myosin XI ATPase cycling time is <1 ms while

that of myosin V is on the order of �65 ms [49]. This time

scale for Chara myosin requires that most kinetic tran-

sitions and binding events in the cycle (Figure 1a) be near

diffusion-limited reaction rate constants. In other words,

some Chara class XI myosins exhibit near kinetic perfec-

tion, which is often assessed by the specificity constant,

kcat/Km in the order of 108–109 M�1 s�1 [50]. For the

fastest Chara, the calculated specificity constant is on

Current Opinion in Plant Biology 2014, 22:65–70

68 Cell biology

the order of �50 � 106 M�1 s�1 [44,50], which is con-

siderably slower than for the lower limit for such diffu-

sion-limited reaction rate constants. Thus, this alone

cannot account for the high efficiency and fast ATPase

cycling of Chara myosin. Furthermore, structural confor-

mational changes, such as opening and closing of the

nucleotide-binding and actin-binding clefts, take on the

order of milliseconds and are therefore too slow to allow

such rapid ATPase cycling [51]. More structural studies

and dynamic investigation of the actin-binding and

nucleotide-binding clefts may provide further structural

basis for the observed rapid transition and binding of

nucleotides and actin to Chara myosin from class XI.

The mechanism by which myosins propelcytoplasmic streamingIn comparison to other myosin classes the biomechanical

properties of plant myosins are not their only unique

feature, since the way they propel the movement of large

organelles might also be different. It is believed that the

motion of organelles associated with myosin motor

proteins along actin cables propels the viscous cyto-

plasmic fluid [21��,52]. The speed of cytoplasmic stream-

ing in Arabidopsis, N. benthamiana, and in Chara,

resembles the speed of myosin XI from these plants in

in vitro motility assays [53]. However, it is still not clear

which organelles are directly associated with myosins to

propel the cytoplasmic streaming. While some studies

have found that GFP fusions to fragments from various

myosin XI tail domains colocalize to known organelles

[54–56], other studies have shown no such colocalization

[5��,11,13,14]. An active full-length myosin XIK from

Arabidopsis fused to GFP was found to interact solely

with specific unknown transport vesicles via specific

myosin-binding proteins, myoB1/2 [5��]. Moreover, the

movement of the transport vesicles was fast and constant

along the actin fibers, whereas that of known organelles

was characterized by stochastic changes in speed and

direction [11,12,14]. Thus, cytoplasmic streaming was

suggested to be created by the special transport vesicles

associated with myosin XIK, while the other organelles

merely drifted in the hydrodynamic flow or, alternatively,

were transiently associated with the transport vesicles

[5��]. Since load is known to affect myosin functioning

[33], it would be intriguing to determine whether the

specific transport vesicles are adjusted to facilitate the

super speed velocity of class XI plant myosins, thus

constituting another component of the fast and efficient

mechanism.

ConclusionsMyosins are divided into two groups: conventional, in-

cluding class II myosins and unconventional, which in-

clude plant myosins, among many others. However, plant

myosins seem to be highly unconventional within the

group of unconventional myosins. Some of the evolved

biological, biochemical and biophysical optimizations

Current Opinion in Plant Biology 2014, 22:65–70

that underlie the manifestations of plant myosins remain

to be determined.

AcknowledgementsThe authors apologize to all colleagues whose important contributions couldnot be highlighted or discussed herein due to space limitations. We thankour group members for their critical reading of the manuscript andinvaluable comments. ES acknowledges a grant from the Israeli ScienceFoundation (ISF) 401/09. AH acknowledges the Marie Curie CareerIntegration Grant 1403705/11.

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