Identification and functional characterization of cargo binding sites of plant myosins

Myosins are actin-based molecular motors of eucaryotic organisms that play fundamental roles in many forms of motility. In addition to the well known muscle myosin a large number of myosins exist, that are essential for movement at the cellular and subcellular level. Such processes are for instance cell migration, cytokinesis, endo-/phagocytosis, exocytosis, growth cone extension, remodelling of cell shape and morphology, and organelle/particle trafficing. More recent evidence implicates myosins as important actors even in processes such as signal transduction and polymerization of actin. Based on alignment and tree production by comparing the core motor domain sequences of the myosins (myosin heavy chains) from the public databases, all known myosins were grouped into 17 or 18 classes. Remarkably, the myosins from higher plants exclusively fall into two classes, VIII and XI. In an additional class, class XIII, myosins from the alga Acetabularia cliftonii are grouped. Knowledge about structure and in particular about function of plant myosins is very limited. Major sources of information about higher plant myosin genes are the completed genome sequences of the model plants Arabidopsis thaliana and Oryza sativa (rice). 17 myosin genes were identified in the Arabidopsis genome, 4 of them belong to class VIII and 13 to class XI; the rice genome encodes for 14 genes (2 class VIII and 12 class XI). Besides the fact that plant myosins appear to form a clade of their own, the available sequence data nevertheless suggest that they follow the domain pattern typical for all myosins: the highly conserved N-terminal head (motor) domain responsible for ATP hydrolysis, binding to actin and production of force, the neck domain containing characteristic repeat motifs (IQ repeats), and the C-terminal tail domain. The IQ motifs of the neck domain are binding sites for calmodulin or regulatory light chains, which are unknown for plant myosins. The tail domain usually contains one or more coiled-coil regions responsible for dimerization. In the case of class XI myosins the tail domain contains additionally the DIL domain at the C-terminal end. In mammals and yeast this domain is present in class V myosins. Data from yeast shed light on its function, since it was demonstrated that this domain contains a distinct binding region for movement of vesicular cargo. However, cargo binding sites must not necessarily be localized only within the DIL domain, since also in yeast another region, putatively binding vacuoles, was found considerably upstream. In this dissertation a survey of the subcellular localization, the dynamic behavior, the transported cargoes and their potential binding site(s) in myosin tails was obtained. For this investigation transient overexpression of selected A. thaliana class VIII and XI myosins and a variety of respective subregions fused to fluorescent proteins as markers was used as assay system. The approach combined high resolution in vivo microscopy, molecular biology and in vivo biochemistry. Such an integrated approach was so far not applied for plant myosins. The data showed that specific binding sites for Golgi stacks/vesicles and peroxisomes which are highly conserved among all A. thaliana class XI myosins reside within the C-terminal part of class XI myosins (DIL domain). In addition, studying the tail domain of barely class XI myosins revealed that the identified cargo binding sites are highly conserved in monocots and dicots. The peroxisomal localization of the DIL domain of the yeast myosin V (Myo2p) in plant cells was a strong indication for the involvement of the DIL domain as cargo binding site among class V and XI myosins. The phenotypic characterization of 10 out of 13 class XI homozygous knock-out lines revealed no drastic effects with respect to growth and development or pathogen defence. Only various degrees of stunted growth, changes in seed morphology and a slight increase in susceptibility to bacterial infection were observed. Alltogether, these results demonstrate considerable functional redundancy among class XI myosins. For two selected class VIII myosins (ATM1 and ATM2), each as a representative for one myosin VIII subclass, it appeared that only ATM2 localized on vesicles which later showed partial colocalization with endosomes. The detailed analysis of the ATM2 tail domain showed that in contrast to class XI myosins, ATM2 probably binds only one cargo. For the involvement of ATM2 in such an important process like endocytosis would also speak the fact that the homozygous knock out of this gene results in lethality. The other class VIII myosin investigated, ATM1, showed plasma membrane localization in agreement with previous studies where by immunocytochemistry this myosin was found in the cell plate associated with the plasma membrane in particular at plasmodesmata. These data suggest a role in the formation of the new cell wall and the generation and maintenance of plasmodesmata. Thus, the functions of class VIII myosins might be quite diverse

Arabidopsis myosin XI sub-domains homologous to the yeast myo2p organelle inheritance sub-domain target subcellular structures in plant cells

Myosin XI motor proteins transport plant organelles on the actin cytoskeleton. The Arabidopsis gene family that encodes myosin XI has 13 members, 12 of which have sub-domains within the tail region that are homologous to well-characterized cargo-binding domains in the yeast myosin V myo2p. Little is presently known about the cargo-binding domains of plant myosin XIs. Prior experiments in which most or all of the tail regions of myosin XIs have been fused to yellow fluorescent protein (YFP) and transiently expressed have often not resulted in fluorescent labeling of plant organelles. We identified 42 amino-acid regions within 12 Arabidopsis myosin XIs that are homologous to the yeast myo2p tail region known to be essential for vacuole and mitochondrial inheritance. A YFP fusion of the yeast region expressed in plants did not label tonoplasts or mitochondria. We investigated whether the homologous Arabidopsis regions, termed by us the “PAL” sub-domain, could associate with subcellular structures following transient expression of fusions with YFP in Nicotiana benthamiana. Seven YFP::PAL sub-domain fusions decorated Golgi and six were localized to mitochondria. In general, the myosin XI PAL sub-domains labeled organelles whose motility had previously been observed to be affected by mutagenesis or dominant negative assays with the respective myosins. Simultaneous transient expression of the PAL sub-domains of myosin XI-H, XI-I, and XI-K resulted in inhibition of movement of mitochondria and Golgi