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Different subcellular localizations and functions of Arabidopsis myosin VIII.

Golomb L, Abu-Abied M, Belausov E, Sadot E - BMC Plant Biol. (2008)

Bottom Line: Interestingly, the punctate pattern was insensitive to brefeldin A (BFA) while in some cells closer to the root cap, ATM1 was found in BFA bodies.With the use of different markers and transient expression in Nicotiana benthamiana leaves, it was found that myosin VIII co-localized to the plasmodesmata and ER, colocalized with internalized FM4-64, and partially overlapped with the endosomal markers ARA6, and rarely with ARA7 and FYVE.Motility of ARA6 labeled organelles was inhibited whenever associated with truncated ATM1 but motility of FYVE labeled organelles was inhibited only when associated with large excess of ATM1.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Institute of Plant Sciences, The Volcani Center, Bet-Dagan 50250, Israel. lior_g@agri.gov.il

ABSTRACT

Background: Myosins are actin-activated ATPases that use energy to generate force and move along actin filaments, dragging with their tails different cargos. Plant myosins belong to the group of unconventional myosins and Arabidopsis myosin VIII gene family contains four members: ATM1, ATM2, myosin VIIIA and myosin VIIIB.

Results: In transgenic plants expressing GFP fusions with ATM1 (IQ-tail truncation, lacking the head domain), fluorescence was differentially distributed: while in epidermis cells at the root cap GFP-ATM1 equally distributed all over the cell, in epidermal cells right above this region it accumulated in dots. Further up, in cells of the elongation zone, GFP-ATM1 was preferentially positioned at the sides of transversal cell walls. Interestingly, the punctate pattern was insensitive to brefeldin A (BFA) while in some cells closer to the root cap, ATM1 was found in BFA bodies. With the use of different markers and transient expression in Nicotiana benthamiana leaves, it was found that myosin VIII co-localized to the plasmodesmata and ER, colocalized with internalized FM4-64, and partially overlapped with the endosomal markers ARA6, and rarely with ARA7 and FYVE. Motility of ARA6 labeled organelles was inhibited whenever associated with truncated ATM1 but motility of FYVE labeled organelles was inhibited only when associated with large excess of ATM1. Furthermore, GFP-ATM1 and RFP-ATM2 (IQ-tail domain) co-localized to the same spots on the plasma membrane, indicating a specific composition at these sites for myosin binding.

Conclusion: Taken together, our data suggest that myosin VIII functions differently in different root cells and can be involved in different steps of endocytosis, BFA-sensitive and insensitive pathways, ER tethering and plasmodesmatal activity.

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Related in: MedlinePlus

Subcellular localization of myosin VIII in abaxial leaf epidermis cells of N. benthamiana. Fluorescent chimeras were co-expressed by Agrobacterium infiltration. A. GFP-ATM1(IQ-tail). Scale bar 10 μm, 15 optic sections, 0.5 μm apart. B. GFP-ATM2 (IQ-tail) Scale bar 5 μm, 1 optic section. C. GFP-myosin VIIIA (IQ-tail). Scale bar 5 μm, 8 optic sections, 0.5 μm apart. D. GFP-ATM1(IQ-tail). E. RFP-ATM2(IQ-tail). F. Overlay of D and E. Scale bar 1 μm, 1 optic section. G. RFP-ATM1(IQ-tail). H. ERD2-GFP. I. Overlay of G and H. Scale bar 5 μm, 1 optic section. J. GFP-ATM1(IQ-tail). K. Aniline blue labeling of callose accumulated in pit fields. L. Overlay of J and K. Scale bar 5 μm, 1 optic section.
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Figure 4: Subcellular localization of myosin VIII in abaxial leaf epidermis cells of N. benthamiana. Fluorescent chimeras were co-expressed by Agrobacterium infiltration. A. GFP-ATM1(IQ-tail). Scale bar 10 μm, 15 optic sections, 0.5 μm apart. B. GFP-ATM2 (IQ-tail) Scale bar 5 μm, 1 optic section. C. GFP-myosin VIIIA (IQ-tail). Scale bar 5 μm, 8 optic sections, 0.5 μm apart. D. GFP-ATM1(IQ-tail). E. RFP-ATM2(IQ-tail). F. Overlay of D and E. Scale bar 1 μm, 1 optic section. G. RFP-ATM1(IQ-tail). H. ERD2-GFP. I. Overlay of G and H. Scale bar 5 μm, 1 optic section. J. GFP-ATM1(IQ-tail). K. Aniline blue labeling of callose accumulated in pit fields. L. Overlay of J and K. Scale bar 5 μm, 1 optic section.

Mentions: When fluorescent chimeras (GFP or RFP) of ATM1(IQ-tail) were transiently expressed in N. benthamiana leaves using Agrobacterium infiltration, fluorescence accumulated as dots (or aggregates of dots) on the plasma membrane of abaxial leaf epidermal cells (Figure 4A). To verify whether this is a general pattern of localization for myosin VIII members, we checked ATM2, myosin VIIIA and myosin VIIIB. ATM2 and ATM1 gave a similar pattern of dots (590 ± 180 nm in diameter) while myosin VIIIA formed smaller dots (330 ± 50 nm in diameter) (Figure 4B and 4C). Myosin VIIIB was very similar to myosin VIIIA (not shown). The vast majority of ATM1 fluorescent dots were stationary but rarely, less than 1% of the dots were motile (Additional file 3). The family members of myosin VIII exhibit different expression patterns as shown by genevestigator analysis (Additional file 4) [25]. While ATM1 and myosin VIIIA are similarly expressed in most organs, ATM2 and myosin VIIIB are more highly expressed in pollen and to a lesser extent in the stamen and root hairs (Additional file 4). It was thus interesting to determine whether the dots formed by ATM1 and ATM2 overlap in N. benthamiana leaves. GFP-ATM1 and RFP-ATM2 were therefore co-expressed in these leaves, and both localized to the same specific spots on the plasma membrane (Figure 4D–F). This suggests the existence of specific foci at the plasma membrane that are able to bind both myosins. Interestingly, when the ATM1 fluorescent chimera was co-expressed with an ER marker, ERD2-GFP [26], the punctate labeling pattern of ATM1 correlated with the ER (Figure 4G–I). The same picture was observed with ATM2 (not shown). Using aniline blue to stain callose [27], ATM1 was shown to accumulate in plasmodesmata-enriched pit fields (Figure 4J–L). To extend our view on the possible involvement of ATM1 in endocytosis the membrane dye FM4-64 was used to follow membrane internalization. ATM1 was found not only at the plasma membrane, but also in the cytoplasm where it co-localized with internalized FM4-64 (Figure 5A–C). In addition, fluorescent chimeras of ATM1 were co-expressed with the endosomal markers ARA6-GFP and GFP-ARA7, Rab5 orthologs from Arabidopsis [28], and DsRed-FYVE. The FYVE domain is a conserved protein motif characterized by its ability to bind with high affinity and specificity to phosphatidylinositol 3-phosphate (Pi(3)P), a phosphoinositide that is highly enriched in early endosomes [29] and has been shown to co-localize with ARA7 and with internalized FM4-64 in plants [30]. It was found that while FYVE and ARA7 labeled organelles were in the cytoplasm, in less than 1% of the labeled organelles, colocalization with ATM1 was observed (Figure 5D–F, G–I). Generally, the motility of FYVE and ARA7 labeled organelles was not affected by the presence of truncated ATM1 in the same cell, probably because most of them were not colocalized (Additional file 5). However, occasionally both motile organelles co-labeled with FYVE and ATM1 (Additional file 6), and motionless FYVE labeled bodies surrounded by excess of GFP-ATM1 could be detected (Additional file 7). About 80–90% of ARA6 labeled organelles colocalized with ATM1 at or close to the plasma membrane (Figure 5J–L). Importantly, while ARA6 labeled organelles were highly motile in the absence of GFP-ATM1 (additional file 8), in the presence of ATM1, all co-labeled organelles, became motionless and only those free from ATM1 remained motile (Additional file 9). The above suggests a major role for ATM1 in the function of ARA6 labeled endosomes and a minor role in the function of ARA7/FYVE labeled endosomes [31].


Different subcellular localizations and functions of Arabidopsis myosin VIII.

Golomb L, Abu-Abied M, Belausov E, Sadot E - BMC Plant Biol. (2008)

Subcellular localization of myosin VIII in abaxial leaf epidermis cells of N. benthamiana. Fluorescent chimeras were co-expressed by Agrobacterium infiltration. A. GFP-ATM1(IQ-tail). Scale bar 10 μm, 15 optic sections, 0.5 μm apart. B. GFP-ATM2 (IQ-tail) Scale bar 5 μm, 1 optic section. C. GFP-myosin VIIIA (IQ-tail). Scale bar 5 μm, 8 optic sections, 0.5 μm apart. D. GFP-ATM1(IQ-tail). E. RFP-ATM2(IQ-tail). F. Overlay of D and E. Scale bar 1 μm, 1 optic section. G. RFP-ATM1(IQ-tail). H. ERD2-GFP. I. Overlay of G and H. Scale bar 5 μm, 1 optic section. J. GFP-ATM1(IQ-tail). K. Aniline blue labeling of callose accumulated in pit fields. L. Overlay of J and K. Scale bar 5 μm, 1 optic section.
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Figure 4: Subcellular localization of myosin VIII in abaxial leaf epidermis cells of N. benthamiana. Fluorescent chimeras were co-expressed by Agrobacterium infiltration. A. GFP-ATM1(IQ-tail). Scale bar 10 μm, 15 optic sections, 0.5 μm apart. B. GFP-ATM2 (IQ-tail) Scale bar 5 μm, 1 optic section. C. GFP-myosin VIIIA (IQ-tail). Scale bar 5 μm, 8 optic sections, 0.5 μm apart. D. GFP-ATM1(IQ-tail). E. RFP-ATM2(IQ-tail). F. Overlay of D and E. Scale bar 1 μm, 1 optic section. G. RFP-ATM1(IQ-tail). H. ERD2-GFP. I. Overlay of G and H. Scale bar 5 μm, 1 optic section. J. GFP-ATM1(IQ-tail). K. Aniline blue labeling of callose accumulated in pit fields. L. Overlay of J and K. Scale bar 5 μm, 1 optic section.
Mentions: When fluorescent chimeras (GFP or RFP) of ATM1(IQ-tail) were transiently expressed in N. benthamiana leaves using Agrobacterium infiltration, fluorescence accumulated as dots (or aggregates of dots) on the plasma membrane of abaxial leaf epidermal cells (Figure 4A). To verify whether this is a general pattern of localization for myosin VIII members, we checked ATM2, myosin VIIIA and myosin VIIIB. ATM2 and ATM1 gave a similar pattern of dots (590 ± 180 nm in diameter) while myosin VIIIA formed smaller dots (330 ± 50 nm in diameter) (Figure 4B and 4C). Myosin VIIIB was very similar to myosin VIIIA (not shown). The vast majority of ATM1 fluorescent dots were stationary but rarely, less than 1% of the dots were motile (Additional file 3). The family members of myosin VIII exhibit different expression patterns as shown by genevestigator analysis (Additional file 4) [25]. While ATM1 and myosin VIIIA are similarly expressed in most organs, ATM2 and myosin VIIIB are more highly expressed in pollen and to a lesser extent in the stamen and root hairs (Additional file 4). It was thus interesting to determine whether the dots formed by ATM1 and ATM2 overlap in N. benthamiana leaves. GFP-ATM1 and RFP-ATM2 were therefore co-expressed in these leaves, and both localized to the same specific spots on the plasma membrane (Figure 4D–F). This suggests the existence of specific foci at the plasma membrane that are able to bind both myosins. Interestingly, when the ATM1 fluorescent chimera was co-expressed with an ER marker, ERD2-GFP [26], the punctate labeling pattern of ATM1 correlated with the ER (Figure 4G–I). The same picture was observed with ATM2 (not shown). Using aniline blue to stain callose [27], ATM1 was shown to accumulate in plasmodesmata-enriched pit fields (Figure 4J–L). To extend our view on the possible involvement of ATM1 in endocytosis the membrane dye FM4-64 was used to follow membrane internalization. ATM1 was found not only at the plasma membrane, but also in the cytoplasm where it co-localized with internalized FM4-64 (Figure 5A–C). In addition, fluorescent chimeras of ATM1 were co-expressed with the endosomal markers ARA6-GFP and GFP-ARA7, Rab5 orthologs from Arabidopsis [28], and DsRed-FYVE. The FYVE domain is a conserved protein motif characterized by its ability to bind with high affinity and specificity to phosphatidylinositol 3-phosphate (Pi(3)P), a phosphoinositide that is highly enriched in early endosomes [29] and has been shown to co-localize with ARA7 and with internalized FM4-64 in plants [30]. It was found that while FYVE and ARA7 labeled organelles were in the cytoplasm, in less than 1% of the labeled organelles, colocalization with ATM1 was observed (Figure 5D–F, G–I). Generally, the motility of FYVE and ARA7 labeled organelles was not affected by the presence of truncated ATM1 in the same cell, probably because most of them were not colocalized (Additional file 5). However, occasionally both motile organelles co-labeled with FYVE and ATM1 (Additional file 6), and motionless FYVE labeled bodies surrounded by excess of GFP-ATM1 could be detected (Additional file 7). About 80–90% of ARA6 labeled organelles colocalized with ATM1 at or close to the plasma membrane (Figure 5J–L). Importantly, while ARA6 labeled organelles were highly motile in the absence of GFP-ATM1 (additional file 8), in the presence of ATM1, all co-labeled organelles, became motionless and only those free from ATM1 remained motile (Additional file 9). The above suggests a major role for ATM1 in the function of ARA6 labeled endosomes and a minor role in the function of ARA7/FYVE labeled endosomes [31].

Bottom Line: Interestingly, the punctate pattern was insensitive to brefeldin A (BFA) while in some cells closer to the root cap, ATM1 was found in BFA bodies.With the use of different markers and transient expression in Nicotiana benthamiana leaves, it was found that myosin VIII co-localized to the plasmodesmata and ER, colocalized with internalized FM4-64, and partially overlapped with the endosomal markers ARA6, and rarely with ARA7 and FYVE.Motility of ARA6 labeled organelles was inhibited whenever associated with truncated ATM1 but motility of FYVE labeled organelles was inhibited only when associated with large excess of ATM1.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Institute of Plant Sciences, The Volcani Center, Bet-Dagan 50250, Israel. lior_g@agri.gov.il

ABSTRACT

Background: Myosins are actin-activated ATPases that use energy to generate force and move along actin filaments, dragging with their tails different cargos. Plant myosins belong to the group of unconventional myosins and Arabidopsis myosin VIII gene family contains four members: ATM1, ATM2, myosin VIIIA and myosin VIIIB.

Results: In transgenic plants expressing GFP fusions with ATM1 (IQ-tail truncation, lacking the head domain), fluorescence was differentially distributed: while in epidermis cells at the root cap GFP-ATM1 equally distributed all over the cell, in epidermal cells right above this region it accumulated in dots. Further up, in cells of the elongation zone, GFP-ATM1 was preferentially positioned at the sides of transversal cell walls. Interestingly, the punctate pattern was insensitive to brefeldin A (BFA) while in some cells closer to the root cap, ATM1 was found in BFA bodies. With the use of different markers and transient expression in Nicotiana benthamiana leaves, it was found that myosin VIII co-localized to the plasmodesmata and ER, colocalized with internalized FM4-64, and partially overlapped with the endosomal markers ARA6, and rarely with ARA7 and FYVE. Motility of ARA6 labeled organelles was inhibited whenever associated with truncated ATM1 but motility of FYVE labeled organelles was inhibited only when associated with large excess of ATM1. Furthermore, GFP-ATM1 and RFP-ATM2 (IQ-tail domain) co-localized to the same spots on the plasma membrane, indicating a specific composition at these sites for myosin binding.

Conclusion: Taken together, our data suggest that myosin VIII functions differently in different root cells and can be involved in different steps of endocytosis, BFA-sensitive and insensitive pathways, ER tethering and plasmodesmatal activity.

Show MeSH
Related in: MedlinePlus