Available online at www.sciencedirect.com

ScienceDirect

The molecular mechanism and physiological role of

cytoplasmic streaming

1,2 3

Motoki Tominaga and Kohji Ito

Cytoplasmic streaming occurs widely in plants ranging from Kamiya and Kuroda achieved a breakthrough pertaining

algae to angiosperms. However, the molecular mechanism and to this problem. They measured the flow velocities in the

physiological role of cytoplasmic streaming have long cells of the algae Nitella and found that they were high in

remained unelucidated. Recent molecular genetic approaches proximity to cell membranes and low in areas distant from

have identified specific myosin members (XI-2 and XI-K as cell membranes. Based on this observation and hydrody-

major and XI-1, XI-B, and XI-I as minor motive forces) for the namic considerations, they proposed the ‘sliding theory’,

generation of cytoplasmic streaming among 13 myosin XIs in by which the motive force in streaming is generated in

Arabidopsis thaliana. Simultaneous knockout of these myosin proximity to cell membranes [5]. Over the past several

XI members led to a reduced velocity of cytoplasmic streaming years, understanding concerning plant myosin has

and marked defects of plant development. Furthermore, the advanced progressively. Here we review recent advances

artificial modifications of myosin XI-2 velocity changed plant in our understanding of cytoplasmic streaming and plant

and cell sizes along with the velocity of cytoplasmic streaming. myosin.

Therefore, we assume that cytoplasmic streaming is one of the

key regulators in determining plant size.

Mechanism underlying cytoplasmic streaming

Addresses Actin filaments were found to be located in the proximity

1

Faculty of Education and Integrated Arts and Sciences, Waseda of cell membranes in cells of the alga Chara [6]. Chara is

University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan

2 an ancestor of land plants inhabiting ponds and have been

Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho

used for physiological research of cytoplasmic streaming

Kawaguchi, Saitama 332-0012, Japan

3

Department of Biology, Graduate School of Science, Chiba University, because of its large cell size (>10 cm). Pharmacological

1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan study using cytochalasin, a specific inhibitor of actin

polymerization, demonstrated reversible inhibition of

Corresponding authors: Tominaga, Motoki ([email protected])

cytoplasmic streaming in characean cells [7]. These

and Ito, Kohji ([email protected])

results strongly suggested that the ‘actin–myosin’ system

in proximity to cell membranes may provide the motive

Current Opinion in Plant Biology 2015, 27:104–110 force in cytoplasmic streaming (Figure 1). Although much

effort was made to purify plant myosins following the

This review comes from a themed issue on Cell signalling and gene

regulation discovery of plant actin, its purification took 20 years

owing to the difficulty in isolating active myosin from

Edited by Xiaofeng Cao and Blake C Meyers

plant tissue. The in vitro motility assay system, in which

For a complete overview see the Issue and the Editorial

fluorescently labeled actin filaments glide on a glass

Available online 17th July 2015

surface coated with myosin molecules, has enabled the

http://dx.doi.org/10.1016/j.pbi.2015.06.017 identification of plant myosins from pollen tubes of lily

(Lilium longiflorum) [8]. Using the same system, myosin

1369-5266/# 2015 The Authors. Published by Elsevier Ltd. This is an

open access article under the CC BY-NC-ND license (http://creative- was also purified from intermodal cells of Chara. The

commons.org/licenses/by-nc-nd/4.0/).

purified lily and Chara myosins moved actin filaments in

À1

the in vitro motility assay at 7.7 and 40–60 mm s , re-

spectively [8–10]. These were astonishing results, given

Introduction that these velocities are greater than those of animal fast

skeletal muscle myosins. Particularly, the velocity of

Animals can move, whereas plants cannot and remain at

Chara myosin was more than 10-fold that of the myosins

their place of germination. However, an active intracel-

of all other organisms. Actin sliding velocities with puri-

lular flow called ‘cytoplasmic streaming’ occurs in the

fied plant myosins were consistent with the cytoplasmic

plant cell. This was first reported in 1774 by the Italian

streaming velocities observed in plant cells from which

physicist Bonaventura Corti, who found the flow of cyto-

myosins had been isolated. Therefore, these results

plasm when he observed intermodal cells of algae Nitella

strongly suggested that these myosins indeed evoke

and Chara [1]. Because cytoplasmic streaming occurs

cytoplasmic streaming.

widely in plants ranging from algae to angiosperms, it

is supposed to be a primitive and essential system in

plants [2–4]. However, more than 200 years after its Molecular properties of plant myosins

discovery, the molecular mechanism for the force gener- Myosin is a motor protein that converts the chemical

ation in cytoplasmic streaming remains unclear. In 1956, energy liberated by ATP hydrolysis into directed

Current Opinion in Plant Biology 2015, 27:104–110 www.sciencedirect.com

The role of cytoplasmic streaming Tominaga and Ito 105

Figure 1

Plant Cell Cell wall

Myosin XI Golgi Vacuole Actin filaments ER

Vacuole Nucleus

Chloroplast

Late endosome

Early endosome Mitochondria

Current Opinion in Plant Biology

Cytoplasmic streaming is an active intracellular movement generated by organelle-associated plant-specific class XI myosins moving along the

actin filaments in plant cells. In Arabidopsis, there are 13 myosins in the class XI family. As reviewed in this paper, many studies indicated specific

and/or overlapping localization and function among class XI myosins. The localization and function of each class XI myosin are not fully

understood.

movement on actin filaments and is generally present in deduced from its cDNA is composed of an N-terminal

eukaryotes. Phylogenetic analyses of amino acid sequences motor domain that contains actin-binding and nucleotide-

of myosin have revealed at least 35 classes [11]. Motor binding sites, a neck domain comprising six tandem

functions such as motility and ATP hydrolysis vary greatly repeats of IQ motifs serving as binding sites for six

among classes, allowing each myosin to be finely tuned for a calmodulin-like myosin light chains, a a-helical coiled-

specific task [12]. Only class VIII and XI myosins are coil domain supporting dimer formation, and a globular

present in higher plants. Arabidopsis thaliana harbors four tail domain (GTD) (Figure 2). This molecular structure

and thirteen genes [13] and Oryza sativa (rice) two and resembles that of animal and fungus class V myosins [18].

twelve genes [14] encoding class VIII and XI myosins, By immunoscreening using antibodies raised against a

respectively. The first identified and sequenced plant purified Chara myosin whose velocity was similar to that

myosin was Arabidopsis class VIII myosin (Arabidopsis of cytoplasmic streaming of Chara cells, cDNA encoding

thaliana myosin 1, ATM1) [15]. Recently, the molecular Chara myosin was cloned. The sequence of the cloned

properties and intracellular localizations of class VIII myo- cDNA indicated that Chara myosin should be classified as



sin, ATM1, were elucidated [16 ]. The actin-activated a class XI myosin [19,20], showing that class XI myosin is

ATPase activities and actin sliding velocity of ATM1 were a generator of cytoplasmic streaming. Because yields of

very low (Vmax of actin-activated ATPase activities was native Chara myosin that could be purified to homogene-

À1 À1 À1

4 Pi head s and actin sliding velocity was 0.2 mm s ). ity were very low, biochemical analyses have employed

Green fluorescent protein (GFP)-fused full-length ATM1 recombinant Chara myosin XI. The Vmax of the actin-

expressed in Arabidopsis was localized to plasmodesmata, activated ATPase activities [21,22] and the ADP dissoci-

plastids, newly formed cell walls, and actin filaments in the ation rate from acto–myosin [23] of the recombinant

À1 À1 À1

cell cortex. The low velocity and localization of ATM1 Chara myosin XI were 500 Pi head s and 2800 s ,

show that class VIII myosin functions as a tension sensor/ respectively.

generator, but not as a generator of cytoplasmic streaming.

Intensive molecular characterization of higher plant class

The first identified and sequenced class XI myosins were XI myosin has been performed using Nicotiana tabacum

also from Arabidopsis (MYosin from Arabidopsis 1, MYA-1) 175-kDa class XI myosin (tobacco myosin XI) purified

[17]. The molecular structure of myosin XI of Arabidopsis from cultured tobacco BY-2 cells [24]. Single-molecule

www.sciencedirect.com Current Opinion in Plant Biology 2015, 27:104–110

106 Cell signalling and gene regulation

Figure 2

Organelle

Adaptor protein Tail domain Movement (cytoplasmic streaming) Neck domain

Motor domain Actin filament – end 35 nm + end

Current Opinion in Plant Biology

Myosin XI moves on actin filaments toward the plus-end with 35-nm steps. Class XI myosin consists of a motor domain, a neck domain

comprising six tandem repeats of IQ motifs and serving as binding sites for light chains, a tail domain comprising a a-helical coiled-coil domain

supporting dimer formation, and a globular tail domain that associates with specific adaptor proteins.

movement of tobacco myosin XI was observed by optical Inhibition is due to two conserved basic residues in the

trap nanometry. A single tobacco myosin XI molecule GTD, electrostatically binding to conserved acidic residues

moved processively toward the plus-end of an actin fila- in the motor domain. These same residues (Arg-1368 and

À1

ment in 35-nm steps at 7 mm s . This is the fastest Arg-1443) in the GTD of At (Arabidopsis) myosin XI-K were

progressive motor ever discovered [25]. Detailed kinetic found to be necessary for the dominant-negative effect of

analysis and molecular modeling using tobacco myosin XI tail overexpression, suggesting intramolecular regulation

revealed a new type of progressive mechanism for gener- via head–tail interaction for plant myosin XI [32].



ating high velocity [26 ]. These characteristics suggest

that tobacco myosin XI is suitable for transporting cargo Cargos carried by myosin XI

over a long distance with a small number of myosins at a Hydrodynamic considerations suggest that movement of

high velocity similar to that of cytoplasmic streaming. myosin alone is not enough to generate streaming. Myo-

sin XI should move with its tail attached to a large

Regulations of myosin XI organelle such as endoplasmic reticulum (ER) to produce

Cytoplasmic streaming in plant cells stops when cytosolic bulk flow of water [33,34]. Therefore, cargo binding (to

2+

calcium ion (Ca ) concentration is elevated [27,28]. In organelles, vesicles, and protein complex) is essential not

vitro motility assay using tobacco myosin XI revealed that only for intracellular transportation, but also for the gen-

the motility of this myosin is negatively regulated by eration of cytoplasmic streaming. Many cargos and adap-

2+

Ca -induced calmodulin dissociation from the neck do- tors for animal and fungi class V myosins that are

main [24,29]. Electron microscope observations showed homologs of plant class XI myosins have been identified

that the neck of tobacco myosin XI was shortened by 30% [35]. By contrast to class V myosins, little is known about

at pCa 4. Single-molecule analysis revealed that the step cargos of class XI myosins. In Arabidopsis, there are

size of myosin XI at pCa 4 was shortened to 27-nm under 13 class XI myosins: At myosin XI-1, XI-2, XI-A, XI-B,

low load and to 22-nm under high load, compared with XI-C, XI-D, XI-E, XI-F, XI-G, XI-H, XI-I, XI-J, and XI-



35-nm independent of the load for intact myosin XI. K [13,36 ]. This situation contrasts markedly with that of

These results indicate that the modulation of the me- class V myosins, which have two isoforms in fungi and

chanical properties of the neck domain is a key factor for three in animals. Fluorescent proteins-fused tail con-

2+

achieving the Ca -induced regulation of cytoplasmic structs expressed in Arabidopsis leaf epidermal cells



streaming [30 ]. showed that At XI-1 was localized at Golgi bodies and

peroxisomes and At XI-2 at peroxisomes [37]. Fluorescent

Several studies revealed that full-length animal myosin V, a proteins-fused tail constructs expressed in tobacco leaf

homolog of plant myosin XI, adopts a folded, inhibited epidermal cells showed that At XI-F was localized at

conformation [31]. Myosin V is both enzymatically and chloroplasts [38] and At XI-I at the nuclear envelope



mechanically ‘off’ when folded and ‘on’ when unfolded. [39,40 ]. However, it must be noted that these studies

Current Opinion in Plant Biology 2015, 27:104–110 www.sciencedirect.com

The role of cytoplasmic streaming Tominaga and Ito 107

were performed using tail domains that lacked motor and in vitro binding assay, At RabD1 and At RabC2a were

domains. It is possible that the localizations of native identified as adaptors that mediate between peroxisome

full-length myosins are different from those of only tail and At XI-2 [43], two uncharacterized transmembrane

domains. Recent mutant and biochemical studies suggest proteins (MyoB1/2) as adaptors that interact with At XI-



that the ER is a potential candidate for At XI-K cargo [41]. K [44 ], and P-body core component DECAPPING PRO-

Unexpectedly, the expression of GFP-fused full length TEIN1 (DCP1) as direct interactors between P-body with



At XI-K showed that the localization is not exclusive for At XI-1, XI-2, XI-I, and XI-K [45 ]. The mass spectrometry

the ER but for endomembrane vesicles [42]. These analysis of immunoprecipitated protein showed the bind-

results suggest that there is not a simple one-to-one ing of the tail domain of At XI-I with outer-nuclear mem-

but rather a complex relationship between plant myosin brane proteins (WIT1/20) that function in RanGAP,



and organelles. anchoring to the nucleus [40 ]. Because only a small

number of adaptors of class XI myosins have been identi-

There have also been a small number of reports describing fied to date, further studies are needed to identify the

adaptors of class XI myosins. By yeast two-hybrid screening individual roles of the 13 class XI myosins in Arabidopsis.

Figure 3

(A)

a. Chara myosin XI (50 µm/s) b. human myosin V (0.2 µm/s) c. Arabidopsis myosin XI-2 (5 µm/s)

d. High-speed myosin XI-2 (16 µm/s) e. Low-speed myosin XI-2 (0.2 µm/s)

(B)

Wild-type High-speed Low-speed

Current Opinion in Plant Biology

(A) Development of high-speed and low-speed myosin XI. (a) Chara myosin XI. (b) Human myosin Vb. (c) Wild-type (Arabidopsis myosin XI-2). (d)

High-speed myosin XI (Chara–Arabidopsis chimeric myosin XI-2). (e) Low-speed myosin XI (human–Arabidopsis chimeric myosin XI-2). High-speed

and low-speed myosin XI-2 were constructed by fusing the N-terminal region including the motor domain of Chara myosin XI and human myosin

Vb with the neck and tail region of Arabidopsis myosin XI-2, respectively. (B) Expression of chimeric myosin XI-2 in Arabidopsis: 25-day-old

plants, wild-type (left), expressing high-speed myosin XI (middle) and low-speed myosin XI (right).

www.sciencedirect.com Current Opinion in Plant Biology 2015, 27:104–110

108 Cell signalling and gene regulation

Biological function of myosins XI that cytoplasmic streaming is one of the key regulators



Development of molecular genetic approaches permits determining plant size [55 ].

identification of the biological function of each myosin XI

member. Gene knockout, dominant-negative, and RNAi Conclusion

studies indicate that the several class XI myosins (At XI-1,

Plant cells grow larger than animal cells. Consequently,

XI-2, XI-B, XI-I, and XI-K) are responsible for movement

positive cytoplasmic streaming by myosin is essential for

of organelles such as the ER, Golgi stacks, peroxisomes,

intracellular and intercellular material transport. The

and mitochondria in Arabidopsis [39,46–50]. In Arabidop-

acceleration of cytoplasmic streaming by high-speed my-

sis, single myosin knockout resulted in no macroscopic

osin XI-2 may contribute positively to the transport of

defects in phenotype, except for that of xi-2 and xi-k,

materials such as nutrients, plant hormones, carbon diox-

which led to shorter root hairs [47,51]. Triple and qua-

ide, and cell wall precursors. This result leads to a

druple myosin knockouts (among xi-1, xi-2, xi-b, xi-i, and

hypothesis that although land plants have the potential

xi-k) resulted in additive defects in plant development

to grow larger than their present sizes, cytoplasmic

[41,47–49,52]. These results suggest an intimate relation-

streaming velocity defines a plant size suitable for the

ship between cytoplasmic streaming and plant develop-

environmental conditions where they grow. In view of the

ment. However, it is difficult to elucidate this relationship

observation that cytoplasmic streaming is sensitive to

using such conventional methods as gene knockout,

external stimuli and that there are regulatory mechanisms

dominant-negative, and RNAi. Interestingly, a quadruple 2+

of class XI myosins (for example, Ca sensitivity and

myosin knockout (xi-1, xi-2, xi-i, and xi-k) showed

folding inhibition), cytoplasmic streaming may act as a

increased disease susceptibility to fungal pathogens, sug-

regulator of plant development and morphogenesis, sens-

gesting cytoplasmic streaming as a key regulators of plant

 ing temporary environmental conditions such as wind,

antifungal immunity [53 ]. A fact worthy of note that

rain, and physical contact.

myosin XI knockout (xi-f, and xi-k) or actin knockout

(act-8) exhibit hyperbending of stems in response to Acknowledgements

gravity, suggesting actin–myosin XI interactions act as

 The authors apologize to all colleagues whose work could not be included

a sensor for organ straightening and plant posture [54 ].

due to space limitations. MT acknowledges grants from the Japan Society

for the Promotion of Science KAKENHI Grants 20001009, 23770060, and

25221103; an Incentive Research Grant from RIKEN; and the Japan

Science and Technology Agency, PRESTO. KI acknowledges a grant from

Physiological function of cytoplasmic

the Japan Society for the Promotion of Science KAKENHI Grant 24658002;

streaming and the Futaba Electronics Memorial Foundation.

The physiological function of cytoplasmic streaming has

been a mystery since its discovery more than 200 years

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53. Yang L, Qin L, Liu G, Peremyslov VV, Dolja VV, Wei Y: Myosins

45. Steffens A, Jaegle B, Tresch A, Hulskamp M, Jakoby M:  XI modulate host cellular responses and penetration

 Processing-body movement in Arabidopsis depends on an resistance to fungal pathogens. Proc Natl Acad Sci U S A 2014,

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Plant Physiol 2014, 164:1879-1892. The rapid reorganization and polarization of actin filaments toward the

The authors showed that the movements of P-body depended on an pathogen penetration site is one of the earliest cellular responses. The

interaction of myosin XI-K and P-body core compornent DECAPPING authors showed that multiple disruption of myosin XIs responsible for

PROTEIN1 in Arabidopsis. This result (together with [43]) suggests multi- cytoplasmic streaming in Arabidopsis prevents dynamic reorganizations

ple functions of class XI myosin. of actin filaments, focal organelle accumulation and delivery of cell wall

defense compounds toward the pathogen penetration site. Their finding

46. Avisar D, Prokhnevsky AI, Makarova KS, Koonin EV, Dolja VV: shows a potential relationship between cytoplasmic streaming and plant

Myosin XI-K is required for rapid trafficking of Golgi stacks, antifungal immunity.

peroxisomes, and mitochondria in leaf cells of Nicotiana

benthamiana. Plant Physiol 2008, 146:1098-1108. 54. Okamoto K, Ueda H, Shimada K, Tamura K, Kato T, Tasaka M,

 Terao-Moritam M, Hara-Nishimura I: Regulation of organ

47. Peremyslov VV, Prokhnevsky AI, Avisar D, Dolja VV: Two class XI straightening and plant posture by an actin–myosin XI

myosins function in organelle trafficking and root hair cytoskeleton. Nat Plant 2015:15031.

development in Arabidopsis. Plant Physiol 2008, 146:1109-1116. Using Arabidopsis mutant lines defective in myosin XIs (specifically XI-F

and XI-K) or ACTIN8, the authors showed that actin–myosin XI cytoske-

48. Peremyslov VV, Prokhnevsky AI, Dolja VV: Class XI myosins are

leton regulates the organ straightening and plant posture against envir-

required for development, cell expansion, and F-Actin

onmental stimuli. They proposed a model that the long actin filaments in

organization in Arabidopsis. Plant Cell 2010, 22:1883-1897.

act as a bending tensile sensor for plant straightening system.

49. Prokhnevsky AI, Peremyslov VV, Dolja VV: Overlapping functions

55. Tominaga M, Kimura A, Yokota E, Haraguchi T, Shimmen T,

of the four class XI myosins in Arabidopsis growth, root hair

 Yamamoto K, Nakano A, Ito K: Cytoplasmic streaming

elongation, and organelle motility. Proc Natl Acad Sci U S A

velocity as a plant size determinant. Dev Cell 2013, 27:

2008, 105:19744-19749.

345-352.

The authors showed that cytoplasmic streaming is a key factor determin-

50. Sparkes IA, Teanby NA, Hawes C: Truncated myosin XI tail

ing plant size. They generated high-speed and low-speed chimeric

fusions inhibit peroxisome, Golgi, and mitochondrial

myosin by replacing the motor domains of Arabidopsis myoin XI-2 with

movement in tobacco leaf epidermal cells: a genetic tool for

those of Chara myosin XI and human myosin Vb, respectively. The plant

the next generation. J Exp Bot 2008, 59:2499-2512.

size of the transgenic Arabidopsis expressing high-speed and low-speed

51. Ojangu EL, Jarve K, Paves H, Truve E: Arabidopsis thaliana myosin XI-2 was larger or smaller, respectively than that of wild-type

myosin XIK is involved in root hair as well as trichome plant.

Current Opinion in Plant Biology 2015, 27:104–110 www.sciencedirect.com