That alternative splicing does occur in plant myosin transcripts has recently been shown by the sequencing of two cDNAs corresponding to alternatively spliced transcripts of a single rice myosin XI gene. In plants, myosin transcript data is still quite limited even in plant systems with abundant genomic resources. In animals, alternative splicing allows the same gene to encode different myosins that have different cargo-binding capabilities. There may actually be more than 13 myosin XIs present in the Arabidopsis cell, as myosin genes are quite large, with many exons and introns that might undergo alternative splicing. Class VIII myosins were predicted to be involved in new cell wall formation and transport in the plasmodesmata, while class XI myosins, which are closely related to animal and fungal myosin class V, were considered likely to be involved in vesicle and organelle movement. In the complete rice genome sequence, 2 class VI and 12 class XI myosins were detected. They fall into 2 classes: myosin class VIII containing 4 genes and myosin class XI containing 13 members. When the Arabidopsis genome sequence became available, a total of 17 myosin-like genes were identified. Myosins have three common domains: a highly conserved motor domain located at the N-terminus which interacts with actin and hydrolyses ATP an IQ domain which binds calmodulin or calmodulin-related proteins a tail which varies by length and structure and which contains a coiled-coil domain consisting of alpha-helices for protein dimerization. Myosins are molecular motors carrying cargoes on actin filaments in eukaryotic cells. Though inhibitor studies indicate that the actin cytoskeleton is important for motility of all of these organelles, little information is available on the motor proteins responsible for movement of particular cargoes in plants. The dynamic nature of mitochondria, chloroplasts, non-green plastids, peroxisomes, and Golgi bodies has been documented through chlorophyll or fluorescent protein labeling of the organelles. Intracellular motility of organelles and transport vesicles is critical for optimization of photosynthesis and metabolism. YFP-myosin tail fusions provide specific labeling for vesicles of unknown composition, whose identity can be investigated in future studies. Although comparable constructs lacking the motor domain result in a dominant negative effect on organelle motility in animal systems, the plant organelles remained motile. ConclusionĦ 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. 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. 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. Tail cropping experiments demonstrated that the coiled-coil region was required for specific localization and shorter tail regions were inadequate for targeting. Despite the absence of a motor domain, the fluorescently-labeled organelles were motile in most cells. All YFP-myosin-tail fusion proteins were targeted to small organelles ranging in size from 0.5 to 3.0 μm. 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. 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. We investigated 6 of the 13 class XI Arabidopsis myosins. Little information is available concerning the remaining 12 class XI myosins. 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. Class XI myosins are related to animal and fungal myosin class V that are responsible for movement of particular vesicles and organelles. The myosin genes can be divided into two plant-specific subfamilies, class VIII with four members and class XI with 13 members. Seventeen myosin genes have been identified in the nuclear genome of Arabidopsis.
Myosins are molecular motors that carry cargo on actin filaments in eukaryotic cells.