Association of six YFP-myosin XI-tail fusions with mobile plant cell organelles

Abstract

Background: Myosins are molecular motors that carry cargo on actin filaments in eukaryotic cells. Seventeen myosin genes have been identified in the nuclear genome of Arabidopsis. The myosin genes can be divided into two plant-specific subfamilies, class VIII with four members and class XI with 13 members. Class XI myosins are related to animal and fungal myosin class V that are responsible for movement of particular vesicles and organelles. Organelle localization of only one of the 13 Arabidopsis myosin XI (myosin XI-6; At MYA2), which is found on peroxisomes, has so far been reported. Little information is available concerning the remaining 12 class XI myosins.

Results: We investigated 6 of the 13 class XI Arabidopsis myosins. cDNAs corresponding to the tail region of 6 myosin genes were generated and incorporated into a vector to encode YFP-myosin tail fusion proteins lacking the motor domain. Chimeric genes incorporating tail regions of myosin XI-5 (At MYA1), myosin XI-6 (At MYA2), myosin XI-8 (At XI-B), myosin XI-15 (At XI-I), myosin XI-16 (At XI-J) and myosin XI-17 (At XI-K) were expressed transiently. All YFP-myosin-tail fusion proteins were targeted to small organelles ranging in size from 0.5 to 3.0 mum. Despite the absence of a motor domain, the fluorescently-labeled organelles were motile in most cells. Tail cropping experiments demonstrated that the coiled-coil region was required for specific localization and shorter tail regions were inadequate for targeting. Myosin XI-6 (At MYA2), previously reported to localize to peroxisomes by immunofluorescence, labeled both peroxisomes and vesicles when expressed as a YFP-tail fusion. None of the 6 YFP-myosin tail fusions interacted with chloroplasts, and only one YFP-tail fusion appeared to sometimes co-localize with fluorescent proteins targeted to Golgi and mitochondria.

Conclusion: 6 myosin XI tails, extending from the coiled-coil region to the C-terminus, label specific vesicles and/or organelles when transiently expressed as YFP fusions in plant cells. Although comparable constructs lacking the motor domain result in a dominant negative effect on organelle motility in animal systems, the plant organelles remained motile. YFP-myosin tail fusions provide specific labeling for vesicles of unknown composition, whose identity can be investigated in future studies.

Figure 1

YFP::catalase and DsRed2::catalase label peroxisomes. A) Transient expression of YFP::catalase (yellow) in onion cells 24 h after bombardment. Maximum projection (cell depth = 92 μm) of 50 z-stack series. B) Transient expression of YFP::catalase (yellow) in tobacco leaves 48 h after Agrobacterium infiltration. Autofluorescence of chloroplasts is pseudo-colored in red. C) Transient expression of DsRed2::catalase (red) in tobacco leaves 48 h after Agrobacterium infiltration. Autofluorescence of chloroplasts is pseudo-colored in blue.

Figure 2

Protein sequence similarity of myosin tails. A) Protein sequence alignment of the six myosin tail constructs used in this study. Myosin XI-16 has the shortest tail. Identical and similar amino acids are highlighted in grayscale. Coiled-coil regions are marked in pink, and the dilute domains in yellow. Myosin gene numbers were derived from table 1 in Reddy and Day [15]. B) Cladogram based on similarity. Note the pairs myosinXI-17/XI-5 and myosinXI-6/XI-8. Myosin XI-15 is more similar to the myosinXI-17/XI-5 pair than to the other myosins. MyosinXI-16 is the most diverged. "100" refers to the confidence level of the analysis.

Figure 3

Transient expression of YFP-class XI-tail myosins in tobacco leaves. YFP-class XI-tail myosins were agroinfiltrated into tobacco leaves and the expression was observed 24 h later. A) Maximum projection (cell depth = 25 μm) of a z-stack series. B-F) Single confocal pictures. The pinhole was more opened in figures D and E. Yellow represents the YFP signal, chloroplasts are pseudo-colored in red. Bar = 10 μm.

Figure 4

Organelle Motility. A) Track length, track displacement and (B) average speed of labeled organelles were analyzed on 2D time series. The data represents the mean values from at least 3 different experiments and a range of 300–3000 organelles were analyzed by construct. The plant material used for the labeled organelle motility determination is marked in brackets.

Figure 5

Schematic representation of myosin constructs varying by tail length. Schematic representation of the fusion constructs with different tail lengths for myosin XI-5, myosin XI-6, myosin XI-15 and myosin XI-17. The position of the amino acid based on the full length protein sequence is shown for each construct.

Figure 6

Short myosin XI tail length fusion constructs are insufficient to be targeted to organelles. Tobacco leaf cells after Agrobacterium infiltration with different myosin tail length fusion constructs. A-D) Complete tail constructs. E-G) 1/2 coil constructs. H-J) Nocoil constructs. K-N) 1/2 tail constructs. O-R) Dilute constructs. The yellow signal is from the YFP fusion constructs, red is chlorophyll autofluorescence. The shorter the tail, the more unspecific cytoplasmic labeling is observed. Rather few punctuate structures were observed in cells with 1/2 tail or dilute constructs. n/d = not determined. Bar = 10 μm.

Figure 7

Co-localization experiment with YFP::Myosin XI-5-tail and YFP::Myosin XI-6-nocoil against peroxisome, mitochondrial and Golgi markers. Transient expression of (A) YFP::Myosin XI-5-tail or (B) YFP::Myosin XI-6-nocoil in tobacco leaves 48 h after Agrobacterium co-infiltration with either peroxisome marker DsRed2::catalase, or mitochondrial marker coxIV::GFP, or Golgi marker ERD2::GFP. All signals are pseudo-colored. Bar = 10 μm.

Figure 8

Co-localization experiment with YFP::Myosin XI-8-tail and YFP::Myosin XI-15-tail against peroxisome, mitochondrial and Golgi markers. Transient expression of (A) YFP::Myosin XI-8-tail or (B) YFP::Myosin XI-15-tail in tobacco leaves 48 h after Agrobacterium co-infiltration with either peroxisome marker DsRed2::catalase, or mitochondrial marker coxIV::GFP, or Golgi marker ERD2::GFP. All signals are pseudo-colored. Bar = 10 μm.

Figure 9

Co-localization experiment with YFP::Myosin XI-16-tail and YFP::Myosin XI-17-tail against peroxisome, mitochondrial and Golgi markers. Transient expression of (A) YFP::Myosin XI-16-tail or (B) YFP::Myosin XI-17-tail in tobacco leaves 48 h after Agrobacterium co-infiltration with either peroxisome marker DsRed2::catalase, or mitochondrial marker coxIV::GFP, or Golgi marker ERD2::GFP. All signals are pseudo-colored. Bar = 10 μm.

Figure 10

Appearance of filamentous structures labeled by myosin constructs. A) Maximum projection (z = 121 μm) of serial confocal sections of an onion epidermal cell transiently expressing YFP::myosin XI-5- 1/2 coil. B) Maximum projection (z = 18 μm) of serial confocal sections of an onion epidermal cell transiently expressing YFP::myosin XI-17-tail. C) Maximum projection (z = 24 μm) of serial confocal sections of an onion epidermal cell transiently expressing YFP::myosin XI-15-tail. Bar in A-C = 50 μm. D) Close-up of the microtubule-like structures in an onion epidermal cell transiently expressing YFP::myosin XI-15-tail.