The role of unconventional myosins in Dictyostelium endocytosis

Dictyostelium discoideum is a simple eukaryote amenable to detailed molecular studies of the endocytic processes phagocytosis and macropinocytosis. Both the actin cytoskeleton and associated myosin motors are well-described and a range of mutants are now available that enable characterization of the role of the cytoskeleton in a range of cellular functions. Molecular genetic studies have uncovered roles for two different classes of Dictyostelium unconventional myosins in endocytosis. The class I myosins contribute to both macropinocytosis and phagocytosis by playing a general role in controlling actin-dependent manipulations of the actin-rich cortex. A class VII myosin has been shown to be important for phagocytosis. This brief review summarizes what is known about the role of these different myosins in both fluid and particle uptake in this system.     

Unconventional Myosins,Actin Dynamics and Endocytosis: A Ménage à Trois?

Ever since the discovery of class I myosins, the first nonmuscle myosins, about 30 years ago, the history of unconventional myosins has been linked to the organization and working of actin filaments. It slowly emerged from studies of class I myosins in lower eukaryotes that they are involved in mechanisms of endocytosis. Most interestingly, a flurry of recent findings assign a more active role to class I myosins in regulating the spatial and temporal organization of actin filament nucleation and elongation. The results highlight the multiple links between class I myosins and the major actin nucleator, the Arp2/3 complex, and its newly described activators. Two additional types of unconventional myosins, myosinIX, and Dictyostelium discoideum MyoM, have recently been tied to the signaling pathways controlling actin cytoskeleton remodeling. The present review surveys the links between these three classes of molecular motors and the complex cellular processes of endocytosis and actin dynamics, and concentrates on a working model accounting for the function of class I myosins via recruitment of the machinery responsible for actin nucleation and elongation .     

Unconventional myosins.

The unconventional myosins form a large and diverse group of molecular motors. The number of known unconventional myosins is increasing rapidly and in the past year alone two new classes have been identified. Substantial progress has been made towards characterizing the properties and functions of these motor proteins, which have been hypothesized to play fundamental roles in processes such as cell locomotion, phagocytosis and vesicle transport.     

Myosins in melanocytes: to move or not to move?

The actin network has been implicated in the intracellular transport and positioning of the melanosomes, organelles that are specialized in the biosynthesis and the storage of melanin. It contributes also to molecular mechanisms that underlie the intracellular membrane dynamics and thereby can control the biogenesis of melanosomes. Two mechanisms for actin-based movements have been identified: one is dependent on the motors associated to actin namely the myosins; the other is dependent on actin polymerization. This review will focus on to the role of the actin cytoskeleton and myosins in the transport and in the biogenesis of melanosomes. Myosins involved in membrane traffic are largely seen as transporters of organelles or membrane vesicles containing cargos along the actin networks. Yet increasing evidence suggests that some of the myosins contribute to the dynamics of internal membrane by using other mechanisms. The role of the myosins and the different molecular mechanisms by which they contribute or may contribute to the distribution, the movement and the biogenesis of the melanosomes in epidermal melanocytes and retinal pigmented epithelial (RPE) cells will be discussed.     

Myosins in melanocytes: to move or not to move?

The actin network has been implicated in the intracellular transport and positioning of the melanosomes, organelles that are specialized in the biosynthesis and the storage of melanin. It contributes also to molecular mechanisms that underlie the intracellular membrane dynamics and thereby can control the biogenesis of melanosomes. Two mechanisms for actin‐based movements have been identified: one is dependent on the motors associated to actin namely the myosins; the other is dependent on actin polymerization. This review will focus on to the role of the actin cytoskeleton and myosins in the transport and in the biogenesis of melanosomes. Myosins involved in membrane traffic are largely seen as transporters of organelles or membrane vesicles containing cargos along the actin networks. Yet increasing evidence suggests that some of the myosins contribute to the dynamics of internal membrane by using other mechanisms. The role of the myosins and the different molecular mechanisms by which they contribute or may contribute to the distribution, the movement and the biogenesis of the melanosomes in epidermal melanocytes and retinal pigmented epithelial (RPE) cells will be discussed.     

Functions of unconventional myosins

To date, fourteen classes of unconventional myosins have been identified. Recent reports have implicated a number of these myosins in organelle transport, and in the formation, maintenance and/or dynamics of actin-rich structures involved in a variety of cellular processes including endocytosis, cell migration, and sensory transduction. Characterizations of organelle dynamics in pigment cells and neurons have further defined the contributions made by unconventional myosins and microtubule motors to the transport and distribution of organelles. Several studies have provided evidence of complexes through which cooperative organelle transport may be coordinated. Finally, the myosin superfamily has been shown to contain at least one processive motor and one backwards motor.     

Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

Background

Three types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants.

Results

Using the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication.

Conclusions

Plant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined.     

Characterization of the unconventional myosin VIII in plant cells and its localization at the post-cytokinetic cell wall

Myosins are a large superfamily of motor proteins which, in association with actin, are involved in intra- cellular motile processes. In addition to the conventional myosins involved in muscle contractility, there is, in animal cells, a wide range of unconventional myosins implicated in membrane-associated processes, such as vesicle transport and membrane dynamics. In plant cells, however, very little is known about myosins. We have raised an antibody to the recombinant tail region of Arabidopsis thaliana myosin 1 (a class VIII myosin) and used it in immunofluorescence and EM studies on root cells from cress and maize. The plant myosin VIII is found to be concentrated at newly formed cross walls at the stage in which the phragmoplast cytoskeleton has depolymerized and the new cell plate is beginning to mature. These walls are rich in plasmodesmata and we show that they are the regions where the longitudinal actin cables appear to attach. Myosin VIII appears to be localized in these plasmodesmata and we suggest that this protein is involved in maturation of the cell plate and the re-establishment of cytoplasmic actin cables at sites of intercellular communication.     

Unconventional myosins: new frontiers in actin-based motors

The unconventional myosins are a superfamily of actin-based motors responsible for a rich array of intracellular motility events. Recent evidence suggests that these motors play important roles in cell migration, endocytosis and intracellular transport. Several genetic mutants have been identified whose abnormalities are the result of the loss of a specific myosin. This article describes how analysis of these mutants, coupled with basic studies of the intracellular localization and biochemical properties of individual myosins, is leading to a clearer understanding of the in vivo function of a number of these interesting motor proteins.     

New insights into the role of the cytoskeleton in phagocytosis of Entamoeba histolytica

Entamoeba histolytica , a human parasite, crosses the natural barriers of the intestine and, in turn, spreads into the deeper organs, resulting in amoebiasis. The motility of the parasite and its ability to lyse or phagocytose human cells facilitates passage of the amoeba through the intestinal epithelium. Little is known about the uptake of material by this parasite; nevertheless, the cytoskeleton is believed to play a role in phagocytosis. Myosin IB, an actin-binding protein, localizes to the phagocytic cup and, with time, surrounds the internalized phagosome itself. The role of unconventional myosins in phagocytosis has also been demonstrated in other cell types, suggesting that this molecular mechanism is a common denominator in phagocytic events. Here, we summarize the emerging view of the role of unconventional myosins as well as other cytoskeleton-associated proteins in pseudopod formation at early stages of phagocytosis and during the late step of this process in E. histolytica .     

Do unconventional myosins exert functions in dynamics of membrane compartments?

Unconventional myosins have now been identified in amoeba as well as in higher eucaryotic cells. Their cellular localization, their ability to bind membrane vesicles and their ability to produce in vitro movement suggest that they can generate forces on the plasma membrane relative to actin filaments as well as on membrane compartments relative to actin. Genetic approaches and biochemical analysis of cells over-producing nonfunctional domains of unconventional myosins have provided direct evidence for a role of unconventional myosins in movement of intracellular vesicles and have allowed us to formulate hypotheses about the possible mechanisms by which unconventional myosins could participate in the intracellular transport of membrane proteins and secretory proteins.     

Coupled myosin VI motors facilitate unidirectional movement on an F-actin network

Unconventional myosins interact with the dense cortical actin network during processes such as membrane trafficking, cell migration, and mechanotransduction. Our understanding of unconventional myosin function is derived largely from assays that examine the interaction of a single myosin with a single actin filament. In this study, we have developed a model system to study the interaction between multiple tethered unconventional myosins and a model F-actin cortex, namely the lamellipodium of a migrating fish epidermal keratocyte. Using myosin VI, which moves toward the pointed end of actin filaments, we directly determine the polarity of the extracted keratocyte lamellipodium from the cell periphery to the cell nucleus. We use a combination of experimentation and simulation to demonstrate that multiple myosin VI molecules can coordinate to efficiently transport vesicle-size cargo over 10 µm of the dense interlaced actin network. Furthermore, several molecules of monomeric myosin VI, which are nonprocessive in single molecule assays, can coordinate to transport cargo with similar speeds as dimers.     

Microfilaments and microtubules regulate recycling from phagosomes

It is clear that the uptake of large particles is driven by a finely controlled rearrangement of the actin cytoskeleton. Here, we present evidence that myosin motors and microtubules also participate in the Fcgamma-mediated internalization process in macrophages. During phagocytosis, a substantial amount of plasma membrane is internalized without a net reduction in cell surface area, implying an active mechanism for membrane recycling. Despite the importance of this recycling pathway in phagosome maturation and in the retrieval of immunogenic peptides from phagosomes, the cytoskeletal requirements are largely unknown. To study this vesicle-mediated recycling transport, we used a biochemical assay and we developed a method to follow this process by confocal fluorescence microscopy. Interestingly, recycling from the phagosomal compartment was increased when the actin cortex was thinned by inhibitors of F-actin. In contrast, depolymerization of microtubules diminished both phagocytosis and recycling from phagosomes. Our results suggest that actin and microtubules are needed not only for phagosome biogenesis but also at other steps along the phagocytic pathway.     

Unique sequences and predicted functions of myosins in Tetrahymena thermophila

Myosins are eukaryotic actin-dependent molecular motors that play important roles in many cellular events. The function of each myosin is determined by a variety of functional domains in its tail region. In some major model organisms, the functions and properties of myosins have been investigated based on their amino acid sequences. However, in protists, myosins have been little studied beyond the level of genome sequences. We therefore investigated the mRNA expression levels and amino acid sequences of 13 myosin genes in the ciliate Tetrahymena thermophila. This study is an overview of myosins in T. thermophila, which has no typical myosins, such as class I, II, or V myosins. We showed that all 13 myosins were expressed in vegetative cells. Furthermore, these myosins could be divided into 3 subclasses based on four functional domains in their tail regions. Subclass 1 comprised of 8 myosins has both MyTH4 and FERM domains, and has a potential to function in vesicle transport or anchoring between membrane and actin filaments. Subclass 2 comprised of 4 myosins has RCC1 (regulator of chromosome condensation 1) domains, which are found only in some protists, and may have unconventional features. Subclass 3 is comprised of one myosin, which has a long coiled-coil domain like class II myosin. In addition, phylogenetic analysis on the basis of motor domains showed that T. thermophila myosins are separated into two clusters: one consists of subclasses 1 and 2, and the other consists of subclass 3.     

Endocytic transit inDictyostelium discoideum

Summary The cells ofDictyostelium discoideum are soil amoebae with a simple endocytic pathway: Particles or fluid are taken up at the plasma membrane in a process dependent on the actin cytoskeleton. After rapid acidification and subsequent neutralisation of the food vacuoles during which breakdown of the contents occurs, indigestible remnants are exocytosed. This tight coupling between endocytosis and exocytosis is thought to maintain membrane homeostasis. In spite of the apparent overall difference between the endocytic pathways of mammalian cells andD. discoideum, conserved proteins are involved in individual steps of endocytic transport, possibly indicating that in mammalian cells it is only the routing of marker that has evolved from a simple transit to a complex, branched pathway.     

Left-right asymmetry: class I myosins show the direction

Myosins are actin-based molecular motors that are found in almost all eukaryotes. Phylogenetic analysis allows the discrimination of 37 different types of myosins, most with unknown functions. Recent work in Drosophila has revealed a crucial role for type ID unconventional myosin in left-right asymmetry. Mutations in Myosin ID completely reverse the left-right axis (situs inversus), a phenotype that is dependent on an intact actin cytoskeleton. How this myosin might orient the left-right axis has began to be elucidated by showing that it interacts directly with beta-catenin, suggesting that myosin ID interacts with the adherens junction to control the direction of organ looping. This is the first demonstration of a role of a myosin in body patterning.     

Unconventional myosins at the crossroad of signal transduction and cytoskeleton remodeling

The cytoplasm of eukaryotic cells is a complex milieu and unraveling how its unique cytoarchitecture is achieved and maintained is a central theme in modern cell biology. The actin cytoskeleton is essential for the maintenance of cell shape and locomotion, and also provides tracks for active intracellular transport. Myosins, the actin-dependent motor proteins form a superfamily of at least 15 structural classes and have been identified in a wide variety of organisms, making the presence of actin and myosins a hallmark feature of eukaryotes. Direct connections of myosins to a variety of cellular tasks are now emerging, such as in cytokinesis, phagocytosis, endocytosis, polarized secretion and exocytosis, axonal transport. Recent studies reveal that myosins also play an essential role in many aspects of signal transduction and neurosensation.     

A class VII unconventional myosin is required for phagocytosis

BACKGROUND: Phagocytosis, the process by which cells internalize particles, is essential for the defense of multicellular organisms against invading pathogens and is the major means by which many unicellular organisms obtain nutrients. The actin cytoskeleton plays a critical role in phagocytosis and the observation that a significant amount of force (10-20 nN) is generated during internalization, suggests that a myosin participates in the process. Although more than 15 distinct classes of myosin have been identified, their roles in phagocytosis are unknown. RESULTS: The identification of a class VII unconventional myosin (DdMVII) in the Dictyostelium discoideum amoeba, which is a model for phagocytosis, is reported here. Mutant cells lacking DdMVII exhibited an 80% decrease in the uptake of particles whereas all other actin-based behaviors that were tested, including pinocytosis, exocytosis, cytokinesis and morphogenesis, proceeded normally. The defect in phagocytosis was neither because of altered particle binding nor inability to form actin-filled phagocytic cups. CONCLUSIONS: Molecular genetic analysis of Dictyostelium myosin VII reveals that this motor protein plays a specific and significant role in phagocytosis.     

Unconventional myosins at the crossroad of signal transduction and cytoskeleton remodeling

Summary The cytoplasm of eukaryotic cells is a complex milieu and unraveling how its unique cytoarchitecture is achieved and maintained is a central theme in modern cell biology. The actin cytoskeleton is essential for the maintenance of cell shape and locomotion, and also provides tracks for active intracellular transport. Myosins, the actin-dependent motor proteins form a superfamily of at least 15 structural classes and have been identified in a wide variety of organisms, making the presence of actin and myosins a hallmark feature of eukaryotes. Direct connections of myosins to a variety of cellular tasks are now emerging, such as in cytokinesis, phagocytosis, endocytosis, polarized secretion and exocytosis, axonal transport. Recent studies reveal that myosins also play an essential role in many aspects of signal transduction and neurosensation.