Molecular motors and the Golgi complex: Staying put and moving through

The Golgi apparatus is a highly dynamic organelle through which nascent proteins released from the endoplasmic reticulum (ER) are trafficked. Proteins are post-translationally modified within the Golgi and subsequently packaged into carriers for transport to a variety of cellular destinations. This transit of proteins, as well as the maintenance of Golgi structure and position, is highly dependent upon the actin and microtubule cytoskeletons and their associated molecular motors. Here we review how motors contribute to the correct functioning of the Golgi in higher eukaryotes and discuss the secretory pathway as a model system for studying cooperation between motor proteins.     

In vitro assays demonstrate that pollen tube organelles use kinesin-related motor proteins to move along microtubules

The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed their ability to move along in vitro-polymerized microtubules under different experimental conditions. Regardless of their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules. Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity, and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear evidence that the movement of pollen tube organelles is not just actin based; rather, they show a microtubule-based motion as well. This unexpected finding suggests new insights into the use of pollen tube microtubules, which could be used for short-range transport, as actin filaments are in animal cells.     

Actin dynamics at intracellular membranes: the Spir/formin nucleator complex

The assembly of actin monomers into filaments is a highly regulated basic cellular function. The structural organization of a cell, morphological changes or cell motility is dependent on actin filament dynamics. While within the last decade substantial knowledge has been acquired about actin dynamics at the cell membrane, today only little is known about the actin cytoskeleton and its functions at intracellular endosomal and organelle membranes. The Spir actin nucleators are specifically targeted towards endosomal membranes by a FYVE zinc finger membrane localization domain, and provide an important link to study the role of actin dynamics in the regulation of intracellular membrane transport. Spir proteins are the founding members of a novel class of actin nucleation factors, which initiate actin polymerization by binding of actin monomers to one or multiple Wiskott-Aldrich syndrome protein (WASp) homology 2 (WH2) domains. Although Spir proteins can nucleate actin polymerization in vitro by themselves, they form a regulatory complex with the distinct actin nucleators of the formin subgroup (Fmn) of formins. A cooperative mechanism in actin nucleation has been proposed. Ongoing studies on the function and regulation of the Spir proteins in vesicle transport processes will reveal important insights into actin dynamics at intracellular membranes and how this regulates the highly directed and controlled routes of intracellular membrane trafficking.     

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.     

Regulation of melanosome movement in the cell cycle by reversible association with myosin V.

Previously, we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner, and that myosin V is bound to purified melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. Biol. 8:161-164). In the present study, we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport. To examine whether myosin V was regulated in a cell cycle-dependent manner, purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motility of organelles treated with mitotic extract was found to decrease dramatically, as compared with untreated or interphase extract-treated melanosomes. This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes, but the activity of soluble motor remained unaffected. Furthermore, we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts. We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles, and that this binding is likely regulated by phosphorylation of myosin V during mitosis.     

Organelle motility: running on unleadened

The study of fish and amphibian melanocytes has yielded a wealth of information on the regulation of microtubule- and actin-based motor proteins involved in organelle transport. A new zebrafish mutant provides further insight into how the actions of these motors are coordinated in vivo.     

GTP gamma S inhibits organelle transport along axonal microtubules

Movements of membrane-bounded organelles through cytoplasm frequently occur along microtubules, as in the neuron-specific case of fast axonal transport. To shed light on how microtubule-based organelle motility is regulated, pharmacological probes for GTP-binding proteins, or protein kinases or phosphatases were perfused into axoplasm extruded from squid (Loligo pealei) giant axons, and effects on fast axonal transport were monitored by quantitative video-enhanced light microscopy. GTP gamma S caused concentration-dependent and time-dependent declines in organelle transport velocities. GDP beta S was a less potent inhibitor. Excess GTP, but not GDP, masked the effects of coperfused GTP gamma S. The effects of GTP gamma S on transport were not mimicked by broad spectrum inhibitors of protein kinases (K-252a) or phosphatases (microcystin LR and okadaic acid), or as shown earlier, by ATP gamma S. Therefore, suppression of organelle motility by GTP gamma S was guanine nucleotide- specific and evidently did not involve irreversible transfer of thiophosphate groups to protein. Instead, the data imply that organelle transport in the axon is modulated by cycles of GTP hydrolysis and nucleotide exchange by one or more GTP-binding proteins. Fast axonal transport was not perturbed by AlF4-, indicating that the GTP gamma S- sensitive factors do not include heterotrimeric G-proteins. Potential axoplasmic targets of GTP gamma S include dynamin and multiple small GTP-binding proteins, which were shown to be present in squid axoplasm. These collective findings suggest a novel strategy for regulating microtubule-based organelle transport and a new role for GTP-binding proteins.     

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.     

Motoring down the highways of the cell

All eukaryotic cells contain large numbers of motor proteins (kinesins, dyneins and myosins), each of which appears to carry out a specialized force-generating function within the cell. They are known to have roles in muscle contraction, ciliary movement, organelle and vesicle transport, mitosis and cytokinesis. These motor proteins operate on different cytoskeletal filaments; myosins move along actin filaments, and kinesins and dyneins along microtubules. Recently published crystal structures of the motor domains of two members of the kinesin superfamily reveal that they share the same overall fold that is also found at the core of the larger myosin motor. This suggests that they may share a common mechanism as well as a common ancestry.     

Orchestrating organelle inheritance in Saccharomyces cerevisiae

The biochemical functions of eukaryotic cells are often compartmentalized into membrane-bound organelles to increase their overall efficiency. Although some organelles can be formed anew, cells have evolved elaborate mechanisms to ensure the faithful inheritance of their organelles. In contrast to cells that divide by fission, the budding yeast Saccharomyces cerevisiae must actively and vectorially deliver half of its organelles to the growing bud. To achieve this, proteins called formins are strategically localized to the bud, where they assemble an array of actin cables that radiate deep into the mother cell. Class V myosin motors use these cables as tracks to transport various organelles, including peroxisomes, a portion of the vacuole and elements of the endoplasmic reticulum and Golgi complex. By contrast, mitochondria do not engage a myosin motor for their movement but instead use Arp2/3-nucleated actin polymerization for their bud-directed motility. The translocation machineries work cooperatively with molecular devices that retain organelles within both mother cell and bud to ensure an equitable division of organelles between them. While organelle inheritance requires specific proteins tailored for the inheritance of each type of organelle, it is becoming apparent that a set of fundamental rules underlies the inheritance of all organelles.     

Regulatory Control of Both Microtubule‐ and Actin‐dependent Fish Melanosome Movement

In fish melanophores, melanosomes can either aggregate around the cell centre or disperse uniformly throughout the cell. This organelle transport involves microtubule‐ and actin‐dependent motors and is regulated by extracellular stimuli that modulate levels of intracellular cyclic adenosine 3‐phosphate (cAMP). We analysed melanosome dynamics in Atlantic cod melanophores under different experimental conditions in order to increase the understanding of the regulation and relative contribution of the transport systems involved. By inhibiting dynein function via injection of inhibitory antidynein IgGs, and modulating cAMP levels using forskolin, we present cellular evidence that dynein is inactivated by increased cAMP during dispersion and that the kinesin‐related motor is inactivated by low cAMP levels during aggregation. Inhibition of dynein further resulted in hyperdispersed melanosomes, which subsequently reversed movement towards a more normal dispersed state, pointing towards a peripheral feedback regulation in maintaining the evenly dispersed state. This reversal was blocked by noradrenaline. Analysis of actin‐mediated melanosome movements shows that actin suppresses aggregation and dispersion, and indicates the possibility of down‐regulating actin‐dependent melanosome movement by noradrenaline. Data from immuno‐electron microscopy indicate that myosinV is associated with fish melanosomes. Taken together, our study presents evidence that points towards a model where both microtubule‐ and actin‐mediated melanosome transport are synchronously regulated during aggregation and dispersion, and this provides a cell physiological explanation behind the exceptionally fast rate of background adaptation in fish.     

Regulation of kinesin-directed movements

Bidirectional organelle transport along microtubules is most likely mediated by the opposing forces generated by two microtubule-based motors: kinesin and cytoplasmic dynein. Because the direction and timing of organelle movements are controlled by the cell, the activity of one or both of these motor molecules must be regulated. Recent studies demonstrate that kinesin, kinesin-like proteins and kinesin-associated proteins can be phosphorylated, and suggest that changes in their phosphorylation state may modulate kinesin's ability to interact with either microtubules or organelles. Thus, it is possible that phosphorylation regulates kinesin-driven movements.     

Molecular mechanisms of pigment transport in melanophores.

We present an overview of the research on intracellular transport in pigment cells, with emphasis on the most recent discoveries. Pigment cells of lower vertebrates have been traditionally used as a model for studies of intracellular transport mechanisms, because these cells transport pigment organelles to the center or to the periphery of the cell in a highly co-ordinated fashion. It is now well established that both aggregation and dispersion of pigment in melanophores require two elements of the cytoskeleton: microtubules and actin filaments. Melanosomes are moved along these cytoskeletal tracks by motor proteins. Recent studies have identified the motors responsible for pigment dispersion and aggregation in melanophores. We propose a model for the possible roles of the two cytoskeletal transport systems and how they might interact. We also discuss the putative mechanisms of regulation of pigment transport, especially phosphorylation. Last, we suggest areas of research that will receive attention in the future in order to elucidate the mechanisms of organelle transport.     

Molecular Mechanisms of Pigment Transport in Melanophores

We present an overview of the research on intracellular transport in pigment cells, with emphasis on the most recent discoveries. Pigment cells of lower vertebrates have been traditionally used as a model for studies of intracellular transport mechanisms, because these cells transport pigment organelles to the center or to the periphery of the cell in a highly co-ordinated fashion. It is now well established that both aggregation and dispersion of pigment in melanophores require two elements of the cytoskeleton: microtubules and actin filaments. Melanosomes are moved along these cytoskeletal tracks by motor proteins. Recent studies have identified the motors responsible for pigment dispersion and aggregation in melanophores. We propose a model for the possible roles of the two cytoskeletal transport systems and how they might interact. We also discuss the putative mechanisms of regulation of pigment transport, especially phosphorylation. Last, we suggest areas of research that will receive attention in the future in order to elucidate the mechanisms of organelle transport.     

How peptide hormone vesicles are transported to the secretion site for exocytosis

Post-Golgi transport of peptide hormone-containing vesicles from the site of genesis at the trans-Golgi network to the release site at the plasma membrane is essential for activity-dependent hormone secretion to mediate various endocrinological functions. It is known that these vesicles are transported on microtubules to the proximity of the release site, and they are then loaded onto an actin/myosin system for distal transport through the actin cortex to just below the plasma membrane. The vesicles are then tethered to the plasma membrane, and a subpopulation of them are docked and primed to become the readily releasable pool. Cytoplasmic tails of vesicular transmembrane proteins, as well as many cytosolic proteins including adaptor proteins, motor proteins, and guanosine triphosphatases, are involved in vesicle budding, the anchoring of the vesicles, and the facilitation of movement along the transport systems. In addition, a set of cytosolic proteins is also necessary for tethering/docking of the vesicles to the plasma membrane. Many of these proteins have been identified from different types of (neuro)endocrine cells. Here, we summarize the proteins known to be involved in the mechanisms of sorting various cargo proteins into regulated secretory pathway hormone-containing vesicles, movement of these vesicles along microtubules and actin filaments, and their eventual tethering/docking to the plasma membrane for hormone secretion.     

The yeast class V myosins, Myo2p and Myo4p, are nonprocessive actin-based motors

The motor properties of the two yeast class V myosins, Myo2p and Myo4p, were examined using in vitro motility assays. Both myosins are active motors with maximum velocities of 4.5 microm/s for Myo2p and 1.1 microm/s for Myo4p. Myo2p motility is Ca(2+) insensitive. Both myosins have properties of a nonprocessive motor, unlike chick myosin-Va (M5a), which behaves as a processive motor when assayed under identical conditions. Additional support for the idea that Myo2p is a nonprocessive motor comes from actin cosedimentation assays, which show that Myo2p has a low affinity for F-actin in the presence of ATP and Ca(2+), unlike chick brain M5a. These studies suggest that if Myo2p functions in organelle transport, at least five molecules of Myo2p must be present per organelle to promote directed movement.     

Making sense of melanosome dynamics in mouse melanocytes

Molecular motors drive most if not all organelle movements in Eukaryotic cells. These proteins are thought to bind to the organelle surface and, through the action of their mechanochemical domains, to translocate the organelle along a cytoskeletal track. In the case of the myosin family of molecular motors, the cytoskeletal track is filamentous actin. Microtubules serve as the cytoskeletal track for the kinesins and dyneins. While a considerable amount is known about the motors and tracks responsible for the bi-directional movement of pigment granules in fish and frog melanophores, relatively little is known about how melanosomes in mammalian melanocytes are transported out the cells dendritic arbor, accumulated at the ends of these dendrites, and transferred to keratinocytes. In this short review, we focus on the use of video microscopy to address these questions in mouse melanocytes, and we describe how an analysis of melanosome dynamics within wild type and dilute melanocytes shaped our thinking regarding the role of an unconventional myosin in melanosome transport and distribution.     

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).     

Microtubule motors and pollen tube growth—still an open question

The growth of pollen tubes is supported by the continuous supply of secretory vesicles in the tip area. Movement and accumulation of vesicles is driven by the dynamic interplay between the actin cytoskeleton and motor proteins of the myosin family. A combination of the two protein systems is also responsible for the bidirectional movement of larger organelle classes. In contrast, the role of microtubules and microtubule-based motors is less clear and often ambiguous. Nevertheless, there is evidence which shows that the pollen tube contains a number of microtubule-based motors of the kinesin family. These motor proteins are likely to be associated with pollen tube organelles and, consequently, they have been hypothesized to participate in the distribution of organelles during pollen tube growth. Whether microtubule motor proteins take part in either the transport or positioning of organelles is not known for sure, but there is evidence for this second possibility. This review will discuss the current knowledge of microtubule-based motor proteins (including kinesins and hypothetical dyneins) and will make some hypothesis about their role in the pollen tube.