The cytoskeleton in plasmodesmata: a role in intercellular transport?

Actin and myosin are components of the plant cell cytoskeleton that extend from cell to cell through plasmodesmata (PD), but it is unclear how they are organized within the cytoplasmic sleeve or how they might behave as regulatory elements. Early work used antibodies to locate actin and myosin to PD, at the electron microscope level, or to pitfields (aggregations of PD in the cell wall), using immunofluorescence techniques. More recently, a green fluorescent protein (GFP)-tagged plant myosin VIII was located specifically at PD-rich pitfields in cell walls. Application of actin or myosin disrupters may modify the conformation of PD and alter rates of cell-cell transport, providing evidence for a role in regulating PD permeability. Intriguingly, there is now evidence of differentiation between types of PD, some of which open in response to both actin and myosin disrupters, and others which are unaffected by actin disrupters or which close in response to myosin inhibitors. Viruses also interact with elements of the cytoskeleton for both intracellular and intercellular transport. The precise function of the cytoskeleton in PD may change during cell development, and may not be identical in all tissue types, or even in all PD within a single cell. Nevertheless, it is likely that actin- and myosin-associated proteins play a key role in regulating cell-cell transport, by interacting with cargo and loading it into PD, and may underlie the capacity for one-way transport across particular cell and tissue boundaries.     

Head-neck domain of <Emphasis Type="Italic">Arabidopsis</Emphasis> myosin XI,MYA2, fused with GFP produces F-actin patterns that coincide with fast organelle streaming in different plant cells

Background  

The cytoskeletal mechanisms that underlie organelle transport in plants are intimately linked to acto-myosin function. This function is mediated by the attachment of myosin heads to F-actin and the binding of cargo to the tails. Acto-myosin also powers vigorous cytoplasmic streaming in plant cells. Class XI myosins exhibit strikingly fast velocities and may have extraordinary roles in cellular motility. Studies of the structural basis of organelle transport have focused on the cargo-binding tails of myosin XI, revealing a close relationship with the transport of peroxisomes, mitochondria, and Golgi-vesicles. Links between myosin heads and F-actin-based motility have been less investigated. To address this function, we performed localization studies using the head-neck domain of AtMYA2, a myosin XI from Arabidopsis.     

Motor proteins: kinesin can replace Myosin

Directional transport of specific cargos is tuned to specific molecular motors and specific cytoskeletal tracks. Myosin V transports its cargo on actin cables, whereas kinesin or dynein transport their cargo on microtubules. A recent study shows that an engineered kinesin can substitute for myosin V and its cargo-specific transport and subsequent cellular functions.     

Identification of Myosin XI Receptors in Arabidopsis Defines a Distinct Class of Transport Vesicles

To characterize the mechanism through which myosin XI-K attaches to its principal endomembrane cargo, a yeast two-hybrid library of Arabidopsis thaliana cDNAs was screened using the myosin cargo binding domain as bait. This screen identified two previously uncharacterized transmembrane proteins (hereinafter myosin binding proteins or MyoB1/2) that share a myosin binding, conserved domain of unknown function 593 (DUF593). Additional screens revealed that MyoB1/2 also bind myosin XI-1, whereas myosin XI-I interacts with the distantly related MyoB7. The in vivo interactions of MyoB1/2 with myosin XI-K were confirmed by immunoprecipitation and colocalization analyses. In epidermal cells, the yellow fluorescent protein–tagged MyoB1/2 localize to vesicles that traffic in a myosin XI–dependent manner. Similar to myosin XI-K, MyoB1/2 accumulate in the tip-growing domain of elongating root hairs. Gene knockout analysis demonstrated that functional cooperation between myosin XI-K and MyoB proteins is required for proper plant development. Unexpectedly, the MyoB1-containing vesicles did not correspond to brefeldin A–sensitive Golgi and post-Golgi or prevacuolar compartments and did not colocalize with known exocytic or endosomal compartments. Phylogenomic analysis suggests that DUF593 emerged in primitive land plants and founded a multigene family that is conserved in all flowering plants. Collectively, these findings indicate that MyoB are membrane-anchored myosin receptors that define a distinct, plant-specific transport vesicle compartment.     

The temporal and spatial expression pattern of myosin Va, Vb and VI in the mouse ovary

There are 16 classes of unconventional myosins. Class V myosins have been shown to be involved in transporting cargo to and from the cell periphery. Class VI myosins have also been shown to transport cargo from the cell periphery, although it seems that these proteins have many roles which include the mediation of cell migration and stereocillia stabilisation. With the requirement of myosin VI for Drosophila oogenesis, the localised expression of Myosin V in the developing egg chamber and recent mounting evidence which links myosin VI to the migration of human ovarian cancer cell lines, we wanted to investigate the expression pattern of these two myosin classes in the normal mouse ovary. Here we show that these myosins are expressed, localised and regulated within the oocyte and granulosa cells of the developing mouse follicle.     

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.     

A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface

We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane‐specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CAR riers of the T GN to the cell S urface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)‐G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic.     

Shaping fission yeast cells by rerouting actin-based transport on microtubules

Kinesins and myosins transport cargos to specific locations along microtubules and actin filaments, respectively. The relative contribution of the two transport systems for cell polarization varies extensively in different cell types, with some cells relying exclusively on actin-based transport while others mainly use microtubules. Using fission yeast, we asked whether one transport system can substitute for the other. In this organism, microtubules and actin cables both contribute to polarized growth by transporting cargos to cell poles, but with distinct roles: microtubules transport landmarks to label cell poles for growth and actin assembly but do not directly contribute to the growth process [1]. Actin cables serve as tracks for myosin V delivery of growth vesicles to cell poles [ [2] , [3] and [4] ]. We engineered a chimera between the motor domain of the kinesin 7 Tea2 and the globular tail of the myosin V Myo52, which we show transports Ypt3, a myosin cargo receptor, to cell poles along microtubules. Remarkably, this chimera restores polarized growth and viability to cells lacking actin cables. It also bypasses the normal microtubule-dependent marking of cell poles for polarized growth, but not for other functions. Thus, a synthetic motor protein successfully redirects cargos along a distinct cytoskeletal route.

Video Abstract

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9.

Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC     

Ashim Rai  Rachit Shrivastava  Duha Vang  Michael Ritt  Fredrik Sadler  Shreyas Bhaban  Murti Salapaka  Sivaraj Sivaramakrishnan 《The Journal of biological chemistry》2022,298(3)

A range of cargo adaptor proteins are known to recruit cytoskeletal motors to distinct subcellular compartments. However, the structural impact of cargo recruitment on motor function is poorly understood. Here, we dissect the multimodal regulation of myosin VI activity through the cargo adaptor GAIP-interacting protein, C terminus (GIPC), whose overexpression with this motor in cancer enhances cell migration. Using a range of biophysical techniques, including motility assays, FRET-based conformational sensors, optical trapping, and DNA origami–based cargo scaffolds to probe the individual and ensemble properties of GIPC–myosin VI motility, we report that the GIPC myosin-interacting region (MIR) releases an autoinhibitory interaction within myosin VI. We show that the resulting conformational changes in the myosin lever arm, including the proximal tail domain, increase the flexibility of the adaptor–motor linkage, and that increased flexibility correlates with faster actomyosin association and dissociation rates. Taken together, the GIPC MIR–myosin VI interaction stimulates a twofold to threefold increase in ensemble cargo speed. Furthermore, the GIPC MIR–myosin VI ensembles yield similar cargo run lengths as forced processive myosin VI dimers. We conclude that the emergent behavior from these individual aspects of myosin regulation is the fast, processive, and smooth cargo transport on cellular actin networks. Our study delineates the multimodal regulation of myosin VI by the cargo adaptor GIPC, while highlighting linkage flexibility as a novel biophysical mechanism for modulating cellular cargo motility.     

10.

Inhibitors of myosin,but not actin,alter transport through <Emphasis Type="Italic">Tradescantia</Emphasis> plasmodesmata     

Radford JE  White RG 《Protoplasma》2011,248(1):205-216

Actin and myosin are components of plasmodesmata, the cytoplasmic channels between plant cells, but their role in regulating these channels is unclear. Here, we investigated the role of myosin in regulating plasmodesmata in a well-studied, simple system comprising single filaments of cells which form stamen hairs in Tradescantia virginiana flowers. Effects of myosin inhibitors were assessed by analysing cell-to-cell movement of fluorescent tracers microinjected into treated cells. Incubation in the myosin inhibitor, 2,3-butanedione monoxime (BDM) or injection of anti-myosin antibodies increased cell–cell transport of fluorescent dextrans, while treatment with the myosin inhibitor N-ethylmaleimide (NEM) decreased cell–cell transport. Pretreatment with the callose synthesis inhibitor, deoxy-d-glucose (DDG), enhanced transport induced by BDM treatment or injection of myosin antibodies but did not relieve NEM-induced reduction in transport. In contrast to the myosin inhibitors, cell-to-cell transport was unaffected by treatment with the actin polymerisation inhibitor, latrunculin B, after controlling for callose synthesis with DDG. Transport was increased following azide treatment, and reduced after injection of ATP, as in previous studies. We propose that myosin detachment from actin, induced by BDM, opens T. virginiana plasmodesmata whereas the firm attachment of myosin to actin, promoted by NEM, closes them.     

11.

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

Sivaraj Sivaramakrishnan  James A. Spudich 《The Journal of cell biology》2009,187(1):53-60

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.     

12.

Dynein anchors its mRNA cargo after apical transport in the Drosophila blastoderm embryo     

Delanoue R  Davis I 《Cell》2005,122(1):97-106

Molecular motors actively transport many types of cargo along the cytoskeleton in a wide range of organisms. One class of cargo is localized mRNAs, which are transported by myosin on actin filaments or by kinesin and dynein on microtubules. How the cargo is kept at its final intracellular destination and whether the motors are recycled after completion of transport are poorly understood. Here, we use a new RNA anchoring assay in living Drosophila blastoderm embryos to show that apical anchoring of mRNA after completion of dynein transport does not depend on actin or on continuous active transport by the motor. Instead, apical anchoring of RNA requires microtubules and involves dynein as a static anchor that remains with the cargo at its final destination. We propose a general principle that could also apply to other dynein cargo and to some other molecular motors, whereby cargo transport and anchoring reside in the same molecule.     

13.

Intracellular transport based on actin polymerization     

S. Yu. Khaitlina 《Biochemistry. Biokhimii?a》2014,79(9):917-927

In addition to the intracellular transport of particles (cargo) along microtubules, there are in the cell two actin-based transport systems. In the actomyosin system the transport is driven by myosin, which moves the cargo along actin microfilaments. This transport requires the hydrolysis of ATP in the myosin molecule motor domain that induces conformational changes in the molecule resulting in the myosin movement along the actin filament. The other actin-based transport system of the cell does not involve myosin or other motor proteins. This system is based on a unidirectional actin polymerization, which depends on ATP hydrolysis in actin polymers and is initiated by proteins bound to the surface of transported particles. Obligatory components of the actin-based transport are proteins of the WASP/Scar family and a complex of Arp2/3 proteins. Moreover, the actin-based systems often contain dynamin and cortactin. It is known that a system of actin filaments formed on the surface of particles, the so-called “comet-like tail”, is responsible for intracellular movements of pathogenic bacteria, micropinocytotic vesicles, clathrin-coated vesicles, and phagosomes. This movement is reproduced in a cell-free system containing extract of Xenopus oocytes. The formation of a comet-like structure capable of transporting vesicles from the plasma membrane into the cell depth has been studied in detail by high performance electron microscopy combined with electron tomography. A similar mechanism provides the movement of vesicles containing membrane rafts enriched with sphingolipids and cholesterol, changes in position of the nuclear spindle at meiosis, and other processes. This review will consider current ideas about actin polymerization and its regulation by actin-binding proteins and show how these mechanisms are realized in the intracellular actin-based vesicular transport system.     

14.

Rapid Actin-Dependent Viral Motility in Live Cells     

Joshua C. Vaughan  Boerries Brandenburg  James M. Hogle 《Biophysical journal》2009,97(6):1647-1656

During the course of an infection, viruses take advantage of a variety of mechanisms to travel in cells, ranging from diffusion within the cytosol to active transport along cytoskeletal filaments. To study viral motility within the intrinsically heterogeneous environment of the cell, we have developed a motility assay that allows for the global and unbiased analysis of tens of thousands of virus trajectories in live cells. Using this assay, we discovered that poliovirus exhibits anomalously rapid intracellular movement that was independent of microtubules, a common track for fast and directed cargo transport. Such rapid motion, with speeds of up to 5 μm/s, allows the virus particles to quickly explore all regions of the cell with the exception of the nucleus. The rapid, microtubule-independent movement of poliovirus was observed in multiple human-derived cell lines, but appeared to be cargo-specific. Other cargo, including a closely related picornavirus, did not exhibit similar motility. Furthermore, the motility is energy-dependent and requires an intact actin cytoskeleton, suggesting an active transport mechanism. The speed of this microtubule-independent but actin-dependent movement is nearly an order of magnitude faster than the fastest speeds reported for actin-dependent transport in animal cells, either by actin polymerization or by myosin motor proteins.     

15.

Motor-cargo interactions: the key to transport specificity     

Karcher RL  Deacon SW  Gelfand VI 《Trends in cell biology》2002,12(1):21-27

Eukaryotic cells organize their cytoplasm by moving different organelles and macromolecular complexes along microtubules and actin filaments. These movements are powered by numerous motor proteins that must recognize their respective cargoes in order to function. Recently, several proteins that interact with motors have been identified by yeast two-hybrid and biochemical analyses, and their roles in transport are now being elucidated. In several cases, analysis of the binding partners helped to identify new transport pathways, new types of cargo, and transport regulated at the level of motor-cargo binding. We discuss here how different motors of the kinesin, dynein and myosin families recognize their cargo and how motor-cargo interactions are regulated.     

16.

Head-to-tail regulation is critical for the in vivo function of myosin V     

Kirk W. Donovan  Anthony Bretscher 《The Journal of cell biology》2015,209(3):359-365

Cell organization requires regulated cargo transport along cytoskeletal elements. Myosin V motors are among the most conserved organelle motors and have been well characterized in both yeast and mammalian systems. Biochemical data for mammalian myosin V suggest that a head-to-tail autoinhibitory interaction is a primary means of regulation, but the in vivo significance of this interaction has not been studied. Here we generated and characterized mutations in the yeast myosin V Myo2p to reveal that it is regulated by a head-to-tail interaction and that loss of regulation renders the myosin V constitutively active. We show that an unregulated motor is very deleterious for growth, resulting in severe defects in Myo2-mediated transport processes, including secretory vesicle transport, mitochondrial inheritance, and nuclear orientation. All of the defects associated with motor misregulation could be rescued by artificially restoring regulation. Thus, spatial and temporal regulation of myosin V in vivo by a head-to-tail interaction is critical for the normal delivery functions of the motor.     

17.

Slac2-a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport   总被引：12，自引：0，他引：12  

Fukuda M  Kuroda TS  Mikoshiba K 《The Journal of biological chemistry》2002,277(14):12432-12436

Myosin Va is a member of the unconventional class V myosin family, and a mutation in the myosin Va gene causes pigment granule transport defects in human Griscelli syndrome and dilute mice. How myosin Va recognizes its cargo (i.e. melanosomes), however, has remained undetermined over the past decade. In this study, we discovered Slac2-a/melanophilin to be the "missing link" between myosin Va and GTP-Rab27A present in the melanosome. Deletion analysis and site-directed mutagenesis showed that the N-terminal Slp (synaptotagmin-like protein) homology domain of Slac2-a specifically binds Rab27A/B isoforms and that the C-terminal half directly binds the globular tail of myosin Va. The tripartite protein complex (Rab27A.Slac2-a.myosin Va) in melanoma cells was further confirmed by immunoprecipitation. The discovery that myosin Va indirectly recognizes its cargo through Slac2-a, a novel Rab27A/B effector, should shed light on molecular recognition of its specific cargo by class V myosin.     

18.

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells          下载免费PDF全文

Au JS  Puri C  Ihrke G  Kendrick-Jones J  Buss F 《The Journal of cell biology》2007,177(1):103-114

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

19.

Unique features of myosin VI: a structural view     

Wei Feng 《生物学前沿》2010,5(3):204-210

Myosin VI is the only known molecular motor for the transportation of cargo vesicles from the plus end to the minus end of actin filaments. Thus, myosin VI possesses several unique features to distinguish it from other myosin family motors, such as the ability to move in a reverse direction, the unusual large walking step size, and the cargo-mediated dimerization. Recent structural studies of myosin VI have provided mechanistic insights into these unique features. On the basis of the resolved structures of myosin VI each domains (i.e., the structures of the N-terminal motor domain, the C-terminal cargo binding domain, and the region in the middle), the unique features of myosin VI will be reviewed here from a structural perspective. The structural studies of myosin VI definitely provide some answers about the unique features of myosin VI, but also raise significant questions on how myosin VI functions as a special motor both for directional cargo transport and for structural anchoring.     

20.

Cargo properties play a critical role in myosin Va-driven cargo transport along actin filaments     

Arthur J. Michalek  M. Yusuf Ali 《Biochemistry and Biophysics Reports》2022

High-resolution experiments revealed that a single myosin-Va motor can transport micron-sized cargo on actin filaments in a stepwise manner. However, intracellular cargo transport is mediated through the dense actin meshwork by a team of myosin Va motors. The mechanism of how motors interact mechanically to bring about efficient cargo transport is still poorly understood. This study describes a stochastic model where a quantitative understanding of the collective behaviors of myosin Va motors is developed based on cargo stiffness. To understand how cargo properties affect the overall cargo transport, we have designed a model in which two myosin Va motors were coupled by wormlike chain tethers with persistence length ranging from 10 to 80 nm and contour length from 100 to 200 nm, and predicted distributions of velocity, run length, and tether force. Our analysis showed that these parameters are sensitive to both the contour and persistence length of cargo. While the velocity of two couple motors is decreased compared to a single motor (from 531 ± 251 nm/s to as low as 318 ± 287 nm/s), the run length (716 ± 563 nm for a single motor) decreased for short, rigid tethers (to as low as 377 ± 187 μm) and increased for long, flexible tethers (to as high as 1.74 ± 1.50 μm). The sensitivity of processive properties to tether rigidity (persistence length) was greatest for short tethers, which caused the motors to exhibit close, yet anti-cooperative coordination. Motors coupled by longer tethers stepped more independently regardless of tether rigidity. Therefore, the properties of the cargo or linkage must play an essential role in motor-motor communication and cargo transport.     

Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC

A range of cargo adaptor proteins are known to recruit cytoskeletal motors to distinct subcellular compartments. However, the structural impact of cargo recruitment on motor function is poorly understood. Here, we dissect the multimodal regulation of myosin VI activity through the cargo adaptor GAIP-interacting protein, C terminus (GIPC), whose overexpression with this motor in cancer enhances cell migration. Using a range of biophysical techniques, including motility assays, FRET-based conformational sensors, optical trapping, and DNA origami–based cargo scaffolds to probe the individual and ensemble properties of GIPC–myosin VI motility, we report that the GIPC myosin-interacting region (MIR) releases an autoinhibitory interaction within myosin VI. We show that the resulting conformational changes in the myosin lever arm, including the proximal tail domain, increase the flexibility of the adaptor–motor linkage, and that increased flexibility correlates with faster actomyosin association and dissociation rates. Taken together, the GIPC MIR–myosin VI interaction stimulates a twofold to threefold increase in ensemble cargo speed. Furthermore, the GIPC MIR–myosin VI ensembles yield similar cargo run lengths as forced processive myosin VI dimers. We conclude that the emergent behavior from these individual aspects of myosin regulation is the fast, processive, and smooth cargo transport on cellular actin networks. Our study delineates the multimodal regulation of myosin VI by the cargo adaptor GIPC, while highlighting linkage flexibility as a novel biophysical mechanism for modulating cellular cargo motility.     

Inhibitors of myosin,but not actin,alter transport through <Emphasis Type="Italic">Tradescantia</Emphasis> plasmodesmata

Actin and myosin are components of plasmodesmata, the cytoplasmic channels between plant cells, but their role in regulating these channels is unclear. Here, we investigated the role of myosin in regulating plasmodesmata in a well-studied, simple system comprising single filaments of cells which form stamen hairs in Tradescantia virginiana flowers. Effects of myosin inhibitors were assessed by analysing cell-to-cell movement of fluorescent tracers microinjected into treated cells. Incubation in the myosin inhibitor, 2,3-butanedione monoxime (BDM) or injection of anti-myosin antibodies increased cell–cell transport of fluorescent dextrans, while treatment with the myosin inhibitor N-ethylmaleimide (NEM) decreased cell–cell transport. Pretreatment with the callose synthesis inhibitor, deoxy-d-glucose (DDG), enhanced transport induced by BDM treatment or injection of myosin antibodies but did not relieve NEM-induced reduction in transport. In contrast to the myosin inhibitors, cell-to-cell transport was unaffected by treatment with the actin polymerisation inhibitor, latrunculin B, after controlling for callose synthesis with DDG. Transport was increased following azide treatment, and reduced after injection of ATP, as in previous studies. We propose that myosin detachment from actin, induced by BDM, opens T. virginiana plasmodesmata whereas the firm attachment of myosin to actin, promoted by NEM, closes them.     

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.     

Dynein anchors its mRNA cargo after apical transport in the Drosophila blastoderm embryo

Molecular motors actively transport many types of cargo along the cytoskeleton in a wide range of organisms. One class of cargo is localized mRNAs, which are transported by myosin on actin filaments or by kinesin and dynein on microtubules. How the cargo is kept at its final intracellular destination and whether the motors are recycled after completion of transport are poorly understood. Here, we use a new RNA anchoring assay in living Drosophila blastoderm embryos to show that apical anchoring of mRNA after completion of dynein transport does not depend on actin or on continuous active transport by the motor. Instead, apical anchoring of RNA requires microtubules and involves dynein as a static anchor that remains with the cargo at its final destination. We propose a general principle that could also apply to other dynein cargo and to some other molecular motors, whereby cargo transport and anchoring reside in the same molecule.     

Intracellular transport based on actin polymerization

In addition to the intracellular transport of particles (cargo) along microtubules, there are in the cell two actin-based transport systems. In the actomyosin system the transport is driven by myosin, which moves the cargo along actin microfilaments. This transport requires the hydrolysis of ATP in the myosin molecule motor domain that induces conformational changes in the molecule resulting in the myosin movement along the actin filament. The other actin-based transport system of the cell does not involve myosin or other motor proteins. This system is based on a unidirectional actin polymerization, which depends on ATP hydrolysis in actin polymers and is initiated by proteins bound to the surface of transported particles. Obligatory components of the actin-based transport are proteins of the WASP/Scar family and a complex of Arp2/3 proteins. Moreover, the actin-based systems often contain dynamin and cortactin. It is known that a system of actin filaments formed on the surface of particles, the so-called “comet-like tail”, is responsible for intracellular movements of pathogenic bacteria, micropinocytotic vesicles, clathrin-coated vesicles, and phagosomes. This movement is reproduced in a cell-free system containing extract of Xenopus oocytes. The formation of a comet-like structure capable of transporting vesicles from the plasma membrane into the cell depth has been studied in detail by high performance electron microscopy combined with electron tomography. A similar mechanism provides the movement of vesicles containing membrane rafts enriched with sphingolipids and cholesterol, changes in position of the nuclear spindle at meiosis, and other processes. This review will consider current ideas about actin polymerization and its regulation by actin-binding proteins and show how these mechanisms are realized in the intracellular actin-based vesicular transport system.     

Rapid Actin-Dependent Viral Motility in Live Cells

During the course of an infection, viruses take advantage of a variety of mechanisms to travel in cells, ranging from diffusion within the cytosol to active transport along cytoskeletal filaments. To study viral motility within the intrinsically heterogeneous environment of the cell, we have developed a motility assay that allows for the global and unbiased analysis of tens of thousands of virus trajectories in live cells. Using this assay, we discovered that poliovirus exhibits anomalously rapid intracellular movement that was independent of microtubules, a common track for fast and directed cargo transport. Such rapid motion, with speeds of up to 5 μm/s, allows the virus particles to quickly explore all regions of the cell with the exception of the nucleus. The rapid, microtubule-independent movement of poliovirus was observed in multiple human-derived cell lines, but appeared to be cargo-specific. Other cargo, including a closely related picornavirus, did not exhibit similar motility. Furthermore, the motility is energy-dependent and requires an intact actin cytoskeleton, suggesting an active transport mechanism. The speed of this microtubule-independent but actin-dependent movement is nearly an order of magnitude faster than the fastest speeds reported for actin-dependent transport in animal cells, either by actin polymerization or by myosin motor proteins.     

Motor-cargo interactions: the key to transport specificity

Eukaryotic cells organize their cytoplasm by moving different organelles and macromolecular complexes along microtubules and actin filaments. These movements are powered by numerous motor proteins that must recognize their respective cargoes in order to function. Recently, several proteins that interact with motors have been identified by yeast two-hybrid and biochemical analyses, and their roles in transport are now being elucidated. In several cases, analysis of the binding partners helped to identify new transport pathways, new types of cargo, and transport regulated at the level of motor-cargo binding. We discuss here how different motors of the kinesin, dynein and myosin families recognize their cargo and how motor-cargo interactions are regulated.     

Head-to-tail regulation is critical for the in vivo function of myosin V

Cell organization requires regulated cargo transport along cytoskeletal elements. Myosin V motors are among the most conserved organelle motors and have been well characterized in both yeast and mammalian systems. Biochemical data for mammalian myosin V suggest that a head-to-tail autoinhibitory interaction is a primary means of regulation, but the in vivo significance of this interaction has not been studied. Here we generated and characterized mutations in the yeast myosin V Myo2p to reveal that it is regulated by a head-to-tail interaction and that loss of regulation renders the myosin V constitutively active. We show that an unregulated motor is very deleterious for growth, resulting in severe defects in Myo2-mediated transport processes, including secretory vesicle transport, mitochondrial inheritance, and nuclear orientation. All of the defects associated with motor misregulation could be rescued by artificially restoring regulation. Thus, spatial and temporal regulation of myosin V in vivo by a head-to-tail interaction is critical for the normal delivery functions of the motor.     

Slac2-a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport

Myosin Va is a member of the unconventional class V myosin family, and a mutation in the myosin Va gene causes pigment granule transport defects in human Griscelli syndrome and dilute mice. How myosin Va recognizes its cargo (i.e. melanosomes), however, has remained undetermined over the past decade. In this study, we discovered Slac2-a/melanophilin to be the "missing link" between myosin Va and GTP-Rab27A present in the melanosome. Deletion analysis and site-directed mutagenesis showed that the N-terminal Slp (synaptotagmin-like protein) homology domain of Slac2-a specifically binds Rab27A/B isoforms and that the C-terminal half directly binds the globular tail of myosin Va. The tripartite protein complex (Rab27A.Slac2-a.myosin Va) in melanoma cells was further confirmed by immunoprecipitation. The discovery that myosin Va indirectly recognizes its cargo through Slac2-a, a novel Rab27A/B effector, should shed light on molecular recognition of its specific cargo by class V myosin.     

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

Unique features of myosin VI: a structural view

Myosin VI is the only known molecular motor for the transportation of cargo vesicles from the plus end to the minus end of actin filaments. Thus, myosin VI possesses several unique features to distinguish it from other myosin family motors, such as the ability to move in a reverse direction, the unusual large walking step size, and the cargo-mediated dimerization. Recent structural studies of myosin VI have provided mechanistic insights into these unique features. On the basis of the resolved structures of myosin VI each domains (i.e., the structures of the N-terminal motor domain, the C-terminal cargo binding domain, and the region in the middle), the unique features of myosin VI will be reviewed here from a structural perspective. The structural studies of myosin VI definitely provide some answers about the unique features of myosin VI, but also raise significant questions on how myosin VI functions as a special motor both for directional cargo transport and for structural anchoring.     

Cargo properties play a critical role in myosin Va-driven cargo transport along actin filaments

High-resolution experiments revealed that a single myosin-Va motor can transport micron-sized cargo on actin filaments in a stepwise manner. However, intracellular cargo transport is mediated through the dense actin meshwork by a team of myosin Va motors. The mechanism of how motors interact mechanically to bring about efficient cargo transport is still poorly understood. This study describes a stochastic model where a quantitative understanding of the collective behaviors of myosin Va motors is developed based on cargo stiffness. To understand how cargo properties affect the overall cargo transport, we have designed a model in which two myosin Va motors were coupled by wormlike chain tethers with persistence length ranging from 10 to 80 nm and contour length from 100 to 200 nm, and predicted distributions of velocity, run length, and tether force. Our analysis showed that these parameters are sensitive to both the contour and persistence length of cargo. While the velocity of two couple motors is decreased compared to a single motor (from 531 ± 251 nm/s to as low as 318 ± 287 nm/s), the run length (716 ± 563 nm for a single motor) decreased for short, rigid tethers (to as low as 377 ± 187 μm) and increased for long, flexible tethers (to as high as 1.74 ± 1.50 μm). The sensitivity of processive properties to tether rigidity (persistence length) was greatest for short tethers, which caused the motors to exhibit close, yet anti-cooperative coordination. Motors coupled by longer tethers stepped more independently regardless of tether rigidity. Therefore, the properties of the cargo or linkage must play an essential role in motor-motor communication and cargo transport.