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First published online April 15, 2009; 10.1104/pp.109.136853 Plant Physiology 150:700-709 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
A Comparative Study of the Involvement of 17 Arabidopsis Myosin Family Members on the Motility of Golgi and Other Organelles1,[W],[OA]Institute of Plant Sciences, Volcani Center, Bet-Dagan 50250, Israel (D.A., M.A.-A., E.B., E.S.); and School of Life Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom (C.H., I.A.S.)
Gene families with multiple members are predicted to have individuals with overlapping functions. We examined all of the Arabidopsis (Arabidopsis thaliana) myosin family members for their involvement in Golgi and other organelle motility. Truncated fragments of all 17 annotated Arabidopsis myosins containing either the IQ tail or tail domains only were fused to fluorescent markers and coexpressed with a Golgi marker in two different plants. We tracked and calculated Golgi body displacement rate in the presence of all myosin truncations and found that tail fragments of myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K were the best inhibitors of Golgi body movement in the two plants. Tail fragments of myosins XI-B, XI-F, XI-H, and ATM1 had an inhibitory effect on Golgi bodies only in Nicotiana tabacum, while tail fragments of myosins XI-G and ATM2 had a slight effect on Golgi body motility only in Nicotiana benthamiana. The best myosin inhibitors of Golgi body motility were able to arrest mitochondrial movement too. No exclusive colocalization was found between these myosins and Golgi bodies in our system, although the excess of cytosolic signal observed could mask myosin molecules bound to the surface of the organelle. From the preserved actin filaments found in the presence of enhanced green fluorescent protein fusions of truncated myosins and the motility of myosin punctae, we conclude that global arrest of actomyosin-derived cytoplasmic streaming had not occurred. Taken together, our data suggest that the above myosins are involved, directly or indirectly, in the movement of Golgi and mitochondria in plant cells.
The Arabidopsis (Arabidopsis thaliana) myosin gene family contains 17 members: myosin group XI, which includes 13 members (myosins XI-A, -B, -C, -D, -E, -F, -G, -H, -I, -J, and -K, MYA1, and MYA2), and myosin group VIII, which includes four members (ATM1, ATM2, myosin VIIIA, and myosin VIIIB). Both groups are related to unconventional myosin V (Berg et al., 2001  ; Foth et al., 2006). The Arabidopsis myosins contain a conserved motor domain with ATPase and actin-binding activities, a number of IQ domains that bind myosin light chains, a coiled-coil domain for dimerization, and a specific tail that binds different cargo (Kinkema and Schiefelbein, 1994; Tominaga et al., 2003). Using these functional domains, myosins convert chemical energy from ATP hydrolysis into physical movement along actin fibers, carrying with their tails membrane-bound organelles or RNA/protein complexes (Li and Nebenführ, 2008b).
Plant myosins have been implicated in various cellular activities, such as cytoplasmic streaming (Shimmen and Yokota, 2004
The information that exists regarding specific roles of each plant myosin is rather limited. Immunolocalization studies indicated that myosin XIs are associated with various particles in lily (Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes (Yokota et al., 1995 We have determined the role of all 17 genes through transient overexpression of dominant negative forms in leaf epidermal cells. Fluorescent dominant negative fusions not only provide data on the subcellular location but also provide a relatively easy way of determining expression. Additionally, overexpression of dominant negative forms can expose a role of an individual member, which might be masked by redundant activity, if it was silenced. In order to undertake such a large-scale study, we needed to choose an efficient, fast, and reproducible expression system. Therefore, Agrobacterium tumefaciens-mediated transient expression in Nicotiana leaves was suitable.
Isolation and Generation of eGFP and mRFP Fusions to the 17 Arabidopsis Myosins
In order to compare the involvement of all annotated Arabidopsis myosins with Golgi movement, we isolated, sequenced, and subcloned a fragment containing the IQ, coiled-coil, and tail domains of all of them downstream to enhanced green fluorescent protein (eGFP) or containing the coiled-coil and tail domains downstream of mono red fluorescent protein (mRFP; Fig. 1
). The idea was to create dominant negative clones of each myosin family member that lacks the head actin-binding domain but should still be able to bind cargo. This should saturate the binding sites and compete out the function of endogenous wild-type myosin molecules. Sequence analysis of several independent clones in both laboratories revealed the following differences in translation compared with the annotated database sequences. In myosin XI-D, instead of 1,256-KSLDLFVFMYLFQ-1,268, we found 1,256-VSFTRPP-1,262, which probably results from a different splicing site and an A-to-V change in position 1,082. In myosin XI-G, nucleotides 3,217 to 3,261 are missing; again, this seems to be a different splicing site prediction. In myosin VIIIB, we found that the predicted 932-VVFLPDVC-939 is 932-ELLSEQFE-939; again, this is probably a result of different splicing. The clone of ATM1 in our hands contains an 865G-to-865R change (Knight and Kendrick-Jones, 1993
Myosin Tail and IQ Tail Fusions Are Located to Various Subcellular Structures in Tobacco Leaf Epidermal Cells First, the subcellular localizations of tail and IQ tail myosin truncations were compared with each other and with Golgi bodies. Myosin tails were coexpressed with a fluorescent Golgi body marker (sialyl transferase [ST]-XFP) in N.t. epidermal cells (Fig. 2 ); in addition, the localization of the two different myosin fusions, mRFP-tail and eGFP-IQ tail, were compared by coexpression in N.t. (Supplemental Fig. S3) and the eGFP-IQ tail fusions were expressed in N.b. (Supplemental Fig. S2). A general observation was that while class VIII fusions mainly located to the plasma membrane (ATM1, ATM2, VIIIA, and VIIIB; Fig. 2; Supplemental Fig. S2) and/or the nucleolus (ATM2 and VIIIB; Fig. 2), class XI fusions tended to be in more motile punctate structures (MYA1, MYA2, XI-E, XI-H, and XI-I) or diffuse (XI-C, XI-D, XI-F, XI-J, and XI-K) throughout the cytoplasm (Fig. 2; Supplemental Fig. S2). XI-A, XI-B, and XI-G tail/IQ tail expression tended to vary from motile puncta to diffuse. Interestingly, both XI-I truncations collocated to punctate structures and also collocated to the nuclear envelope in both plant species (Fig. 2; Supplemental Figs. S2, S3, and S5).
None of the myosin fusions solely colocated with Golgi bodies, but the surrounding cytosolic location could represent a small proportion of bound myosin fusions (Fig. 2). Upon coexpression, most of the IQ tail and tail fusions of the same myosin gene colocated to the same structures in N.t. with few exceptions: the ATM2 IQ tail fusion did not colocate to the nucleolus unlike the shorter tail fusion, XI-A and XI-C tail and IQ tail fusions were diffuse throughout the cytosol, and the IQ tail fusion was also in the nucleoplasm (Supplemental Fig. S3). Thus, it is suggested that subcellular localization is similar whether or not the IQ domain is present.
In order to study the effect of each myosin on the motility of Golgi, we coexpressed each myosin fragment with a Golgi marker in two different Nicotiana species. Tail fusions were expressed in N.t. and IQ tail fusions were expressed in N.b. Myosin fusions that behaved differently in the two Nicotiana species were expressed and compared in N.b. This comparison allowed us to verify variations derived from either the plant or the length of the myosin fusion. Owing to the variability of organelle movement within a cell (i.e. fast, slow, and stationary), large samples of organelles were analyzed from at least 10 movies from three to five independent experiments. Volocity software was used to track organelles and calculate velocity and displacement rates of each tracked organelle over time. Displacement rate is the final distance an organelle moves between the first and last frame of the time-lapse sequence over time. We found that organelles that were undergoing short-range oscillations in one spot, but appeared by eye to have stopped moving (Supplemental Movies S1–S4), obtained somewhat unexpectedly high values of velocity calculated by the software. The two data sets from N.t. and N.b. were acquired using different rates of image acquisition, which had a different influence on velocity values determined for such oscillatory movements (i.e. a higher frame rate measured more oscillations, resulting in higher velocities than those captured using a lower frame rate). However, this did not affect displacement rate measurements, since this is a measure of movement away from the starting point and is not significantly affected by short-range oscillatory motion. The two data sets generated from N.b. and N.t., therefore, were compared using the displacement rate values, as they are a better reflection of the perturbation of directional organelle movement and allow comparisons to be drawn between data sets from the two species. To confirm that the different rates of image acquisition did not affect the velocity or displacement rate calculated for organelles in similar plant species, high and low rates of image capture were compared in N.b., but no significant differences were found (Supplemental Table S2.). Figure 3 shows the mean displacement rate (normalized against the respective controls) and cumulative distribution frequency graphs that describe the distribution of displacement rates within a Golgi population in the presence of each truncated myosin fusion. Comparisons between the two data sets, from N.t. (Fig. 3A) and from N.b. (Fig. 3C), reveal that both tail and IQ tail fusions of myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K significantly inhibited Golgi displacement rates in both plants (see Supplemental Table S1 for statistical analysis; Supplemental Movies S1 and S2). Figure 3 also shows that myosins XI-B, XI-F, XI-H, and ATM1 significantly interfered with Golgi movement only as tail fusions in N.t. (Fig. 3, A and B) but not as IQ tail fusions in N.b. (Fig. 3, C and D). In addition, myosins XI-G and ATM2 partially interfered with Golgi movement only as IQ tail fusions in N.b. plants (Fig. 3, C and D). In order to understand the origin of the differences obtained in the two systems, the tail fusions of myosins XI-G, ATM2, XI-B, XI-F, XI-H, and ATM1 were analyzed in N.b. Figure 4 shows that except for the ATM2 tail fusion, all other tail fusions had a similar effect on Golgi body motility to the IQ tail fusions in N.b. The tail fusion of ATM2 did not affect Golgi movement, although its IQ tail fragment did to some extent (Supplemental Table S1). Importantly, when IQ tail mutants of XIB, XIF, and ATM1 were expressed in N.t., they exhibited an inhibitory effect on Golgi body movement similar to their tail fusion counterparts in this plant (data not shown). Thus, it seems that the source of variability between the two expression systems is the plant species itself and not an intrinsic difference in the myosin fragment fused to a fluorescent marker protein.
Myosin Inhibitors of Golgi Motility Affect Mitochondrial Movement as Well Next, we addressed the question of whether myosin truncations specifically perturbed Golgi movement only or whether they affected other organelle classes. Assessing the displacement rates of mitochondria in the presence of myosin IQ tail mutants in N.b. showed that the same myosins that inhibited Golgi movement in the two systems were able to arrest mitochondrial motility as well (Fig. 5 ; Supplemental Movie S3; Supplemental Table S1). The rest of the putative dominant negative mutants of myosins XI-A, XI-B, XI-D, XI-F, XI-G, XI-H, XI-J, ATM1, ATM2, VIIIA, and VIIIB had no significant effect on the movement of mitochondria in N.b. leaves (Fig. 5; Supplemental Table S1). The inhibitory IQ tail myosin fusions did not specifically colocalize to Golgi or mitochondria in N.b. (Supplemental Fig. S5).
Myosin-Truncated Fragments Do Not Indiscriminately Arrest Cytoplasmic Streaming
To ensure that myosin-truncated fragments are not nonspecifically interfering with actomyosin-derived cytoplasmic streaming, actin was labeled by the DsRed FABD2 marker (Voigt et al., 2005
These data provide strong evidence that the myosin mutants are not arresting all cytoplasmic motion in the cells but are specifically inhibiting the motility of the tested Golgi and mitochondria.
Using dominant negative myosin mutants lacking the head motor domain, we found six myosins (MYA1, MYA2, and myosins XI-C, XI-E, XI-I, and XI-K) that were more efficient in inhibiting the motility of Golgi bodies in two different Nicotiana species either as IQ tail or tail truncations. Additionally, IQ tail fragments of these myosins also inhibited mitochondrial motility in N.b. This suggests that these myosins are involved in transporting these organelles in plant cells either in a collaborative, synergistic manner or simply a redundant manner. Although transient expression of Arabidopsis genes in other plant systems is an accepted, widely used method, the data presented here still have to be reconfirmed, in the future, in Arabidopsis plants. The calculated organelle velocity obtained in N.b. was smaller than that obtained in N.t. and smaller than reported previously (Nebenführ et al., 1999 ; Avisar et al., 2008b; Sparkes et al., 2008). The lower image frame rate used in N.b. movies in contrast to the higher frame rate image acquisition used in N.t. raised the possibility of biased analysis toward slow-moving organelles in N.b. However, when fast and slow frame rates of image acquisition were compared in N.b., no differences in measured organelle motility were found. This suggested that the differences found in organelle velocity between the two plants are the result of different growth conditions of N.b. and N.t. in the two laboratories. Interestingly, mutants of ATM1, ATM2, XI-B, XI-G, XI-H, and XI-F exhibited conflicting results in the two species of Nicotiana plants: while myosins XI-B, XI-F, XI-H, and ATM1 were inhibitory only as tail fusions in N.t., myosins XI-G and ATM2 slightly inhibited Golgi movement only as IQ tail fusions in N.b. However, when all of these myosin constructs were expressed side by side in N.b., they affected Golgi movement in a similar way (except for the slight effect observed with IQ tail of ATM2). Thus, it may be concluded that species-specific differences rather than intrinsic differences in the myosin gene fusions expressed resulted in the different rates observed in the different plants (i.e. N.t. versus N.b.). Again, this also emphasizes the conclusion that the major players in Golgi movement are myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K.
The slight effect of ATM1 (tail in N.t.) and ATM2 (IQ tail in N.b.) with Golgi movement is in agreement with our previous observation that ATM1 colocalized with the ER (Golomb et al., 2008
Our findings regarding IQ tail truncations of Arabidopsis myosins XI-K and XI-E in N.b. as inhibitors of Golgi and mitochondrial movement are in agreement with previous publications (Avisar et al., 2008b
The subcellular location of tail and IQ tail fusions vary from plasma membrane (ATM, ATM2, VIIIA, and VIIIB), diffuse in the cytosol (XI-A, MYA2, XI-B, XI-C, XI-D, XI-E, XI-F, XI-G, XI-I, XI-J, and XI-K), nuclear envelope (XI-I), nucleolus (VIIIB and ATM2 tail only), to motile puncta (MYA1, MYA2, XI-A, XI-B, XI-E, XI-G, XI-H, XI-I, and XI-K). Previous expression studies of fluorescent fusions to class VIII (Golomb et al., 2008 Interference of a dominant negative myosin mutant with the function of other myosins could be executed in several putative ways: (1) competition for myosin-binding sites, (2) competition among adaptor proteins that mediate myosin-cargo interactions, (3) competition for a limited pool of light chains, (4) dimerization of the mutant headless myosin with wild-type myosin, and/or (5) nonspecific inhibition of total cytoplasmic streaming by the toxic dominant negative mutant.
Taken together, the data presented here and in the literature suggest that option 2 is more likely; however, more data are required to draw discerning conclusions. It was shown that a fragment of N.b. myosin XI-K including the IQ domain, coiled coil, and tail was able to arrest the motility of Golgi, mitochondria, and peroxisomes in N.b. leaves (Avisar et al., 2008b
Gross inhibition of cytoplasmic streaming by dominant negative myosin mutants is unlikely, because continuous movement of myosin puncta is presented here. Significant motility of eGFP-myosin fusions was also shown previously (Reisen and Hanson, 2007
Apart from mutants of myosin XI-K and MYA2, all other family members of myosin XI had no observable phenotypes, excluding a moderate decrease in peroxisome movement observed in mya1 knockout plants and in plants expressing the MYA1 dominant negative globular tail domain (Peremyslov et al., 2008 ). Therefore, we conclude that myosins XI-K, MYA1, and MYA2 are major players in organelle motility in leaves and root hairs (Peremyslov et al., 2008; Prokhnevsky et al., 2008). Myosins XI-C and XI-E may be major players in pollen tubes, where they are highly expressed, but they are only minor players in leaves and root hairs. This might be the reason that no phenotypes were observed in the published knockout plants (Peremyslov et al., 2008). In addition, we discovered that the myosin XI-I mutant is very potent in inhibiting Golgi and mitochondrial movement. Despite the fact that its level of expression in all tissues is similar to or higher than that of MYA1 and MYA2 and myosin XI-K, no phenotype was observed in its knockout plant. One explanation may be that its dominant negative mutant interferes directly or indirectly with myosin XI-K, MYA2, and MYA1 activities.
Generation of IQ Tail and Tail Constructs
IQ tail fusions were amplified by PCR from first-strand cDNA synthesized using oligo(dT) and reverse transcriptase from Arabidopsis (Arabidopsis thaliana) whole plant (including flowers) RNA. The ATM1 clone was kindly provided by Dieter Volkmann from the University of Bonn (Reichelt et al., 1999
Myosin tail fusions were amplified using IQ tail fusions as templates or by reverse transcription-PCR using the SuperScript III one-step reverse transcription-PCR platinum Taq HiFi kit (Invitrogen) from mRNA extracted from various Arabidopsis tissues using the Nucleospin RNA II kit (Macherey-Nagel). The ATM tail clone was amplified from the clone received from J.K. Jones (Knight and Kendrick-Jones, 1993
Nicotiana benthamiana plants were grown in peat in a controlled growth room at 25°C with optimum light of 16 h daily. Nicotiana tabacum plants were grown according to Sparkes et al. (2005)
N.b. leaf epidermal cells were infiltrated as follows. A. tumefaciens strain GV3101 was transformed with the plasmid and grown at 28°C for 24 h. The bacteria were precipitated and resuspended to a final optical density at 600 nm of 0.5 in the following buffer: 10 mM MES, pH 5.6, 10 mM MgCl2, and 100 µM acetosyringone (Sigma Aldrich). Leaves of 3-week-old N.b. plants were infiltrated with the bacterial culture using a 1-mL syringe as described previously (Golomb et al., 2008
N.t. leaf epidermal cells were infiltrated as outlined by Sparkes et al. (2006)
For N.b., an Olympus IX81/FV500 laser-scanning microscope was used to observe fluorescently labeled cells with the following filter sets. To observe eGFP, we used the eGFP channel, 488-nm excitation, and BA505-525; to observe mRFP, we used 543-nm excitation and BA610. The objective used was PlanApo 60X1.00 WLSM For N.t., a Zeiss LSM META 510 confocal microscope was used with the following settings: dual imaging of eYFP and CFP were captured using multitracking in-line switching mode. CFP was excited with 458 nm and eYFP with 514 nm using a 458/514 dichroic mirror, and the subsequent emission was detected using 470- to 500-nm and 560- to 615-nm band-pass filters, respectively. Similarly, eGFP and mRFP dual imaging was captured using multitracking in-line switching mode. eGFP was excited with 488 nm and mRFP with 543 nm using the 488/543 dichroic mirror, and the subsequent emission was detected using 505- to 530-nm and 570- to 650-nm band-pass filters, respectively.
For N.b., ST-mRFP was used as a Golgi marker (Saint-Jore-Dupas et al., 2006
For N.t., Golgi bodies were tracked and analyzed according to Sparkes et al. (2008)
The following materials are available in the online version of this article.
We thank J.K. Jones and D. Volkmann for providing us with the ATM construct, Nicholas Teanby for help with the cumulative distribution frequency analysis, and Janet Evins for technical support at Oxford Brookes University. Received February 9, 2009; accepted April 8, 2009; published April 15, 2009.
1 The work was supported by the Israeli Science Foundation (grant no. 752/05), by BARD, the United States-Israel Binational Agricultural Research and Development Fund (grant no. IS–4038–07 to E.S.), and by the Leverhulme Trust (grant no. F/00 382/G to I.A.S.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Einat Sadot (vhesadot{at}agri.gov.il).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.136853 * Corresponding author; e-mail vhesadot{at}agri.gov.il.
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-2,6-ST-cyan fluorescent protein [CFP]/GFP). Images were taken 48 to 72 h after infiltration. 
/0.17. When eGFP and mRFP were detected in the same sample, we used dichroic mirror 488/543. In all cases, where more than one color was monitored, sequential acquisition was performed. 
