Myosin-dependent transport in neurons

Axonal transport in neurons has been shown to be microtubule dependent, driven by the molecular motor proteins kinesin and dynein. However, organelles undergoing fast transport can often pause or rapidly change directions without apparent dissociation from their transport tracks. Cytoskeletal polymers such as neurofilaments and microtubules have also been shown to make infrequent but rapid movements in axons indicating that their transport is likely to involve molecular motors. In addition, neurons have multiple compartments that are devoid of microtubules where transport of organelles is still seen to occur. These areas are rich in other cytoskeletal polymers such as actin filaments. Transported organelles have been shown to associate with multiple motor proteins including myosins. This suggests that nonmicrotubule-based transport may be myosin driven. In this review we will focus our attention on myosin motors known to be present in neurons and evaluate the evidence that they contribute to transport or other functions in the different compartments of the neuron.     

Molecular motors in axonal transport

Neurons require a large amount of intracellular transport. Cytoplasmic polypeptides and membrane-bounded organelles move from the perikaryon, down the length of the axon, and to the synaptic terminals. This movement occurs at distinct rates and is termed axonal transport. Axonal transport is divided into the slow transport of cytoplasmic proteins including glycolytic enzymes and cytoskeletal structures and the fast transport of membrane-bounded organelles along linear arrays of microtubules. The polypeptide compositions of the rate classes of axonal transport have been well characterized, but the underlying molecular mechanisms of this movement are less clear. Progress has been particularly slow toward understanding force-generation in slow transport, but recent developments have provided insight into the molecular motors involved in fast axonal transport. Recent advances in the cellular and molecular biology of one fast axonal transport motor, kinesin, have provided a clearer understanding of organelle movement along microtubules. The availability of cellular and molecular probes for kinesin and other putative axonal transport motors have led to a reevaluation of our understanding of intracellular motility.     

Cytoskeletal elements and intracellular transport

Recent advances in the understanding of the functions of various components of the cytoskeleton indicate that, besides serving a structural role, the cytoskeletal elements may regulate the transport of several proteins in the cell. Studies reveal that there are co-operative interactions between the actin and microtubule cytoskeletons including functional overlap in the transport influenced by different motor families. Multiple motors are probably involved in the control of the dynamics of many proteins and intriguing hints about how these motors are co-ordinated are appearing. It has been shown that some of the intermediate elements also participate in selected intracellular transport mechanisms. In view of the author's preoccupation with the steroid receptor systems, special attention has been given to the role of the cytoskeletal elements, particularly actin, in the intracellular transport of steroid receptors and receptor-related proteins.     

Dynamics of nonmembranous cell components: Role of active transport along microtubules

Here we discuss some common mechanisms of microtubule-dependent active transport of nonmembranous components in animal cells. We summarize data about mRNA, cytoskeletal elements, structural proteins, and signaling complexes transport. We also characterize the series of molecular interactions that connect nonmembranous cargoes and microtubules and describe the regulatory pathways for these interactions.     

The molecular mechanism of targeted vesicle transport in cytokinesis

Recent studies have demonstrated that vesicle transport to cleavage furrow is indispensable for cytokinesis. Some animal and plant cells form distinct structures during cell division known as central spindle and phragmoplast, respectively. Several essential factors involved in the vesicle transport have been isolated so far. SNARE proteins and molecular motors play a central role in this process. For future research of cytokinesis, it is important to investigate these factors as well as cytoskeletal components of the contractile ring in detail. This review focuses on the molecular mechanism of targeted vesicle transport in cytokinesis.     

Characterization of a messenger RNA transport protein

A cytoplasmic protein which facilitates the energy-dependent transport of mRNA from isolated nuclei to a specified medium has been further characterized, since it could have relevance to the mechanism of mRNA nucleo-cytoplasmic transport in vivo. This protein is now shown, by cDNA hybridization analysis using appropriate recombinant probes, to be obligatory for the transport of alpha 2u-globulin and albumin mRNA from male rat liver nuclei. It is concentrated in the cytoplasm. When isolated under conditions where they retain nuclear proteins, the nuclei contain less than 2% of the total mRNA transport activity. Approx. 20% is recovered in the cytosol, while the rest (80%) copurifies with the messenger ribonucleoproteins in the polyribosome fraction. The protein is eluted from the poly A-messenger ribonucleoproteins between 0.25 and 0.50 M NaCl. The activities of the cytosolic- and messenger ribonucleoprotein-derived transport proteins were mutually additive below saturation of the transport system. Further, the activities of both fractions were increased when they were fortified with the catalytic subunit of the cAMP-dependent protein kinase in the presence of ATP. On the other hand, protein kinase-induced thiophosphorylation of the protein with ATP[S] decreased transport activity. The molecular weight of the transport protein from either cell compartment as judged by molecular sieving is approx. 35,000. It has now been purified 2000-fold and requires manganese ions and serum albumin for stabilization of activity. The highly purified transport factor from the cytosol is tentatively assigned a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis.     

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.     

New horizons in cytoskeletal dynamics: transport of intermediate filaments along microtubule tracks

Until recently, the dynamic properties of intermediate filaments (IF) were attributed primarily to the exchange of subunits between a disassembled pool and polymerized 10nm filaments. During interphase, this subunit exchange process was thought to produce local modifications in IF structure. During cell division, shifts in the equilibrium between subunits and polymers were thought to lead to either the global or regional disassembly of IF networks, thereby facilitating their distribution into daughter cells. Recently, novel structural forms of IF that undergo rapid and directed transport in several cell types were revealed. Time-lapse observations of motile IF structures in different cell systems have also revealed novel insights into the mechanisms underlying the transport of cytoskeletal components throughout the cytoplasm and the molecular basis of the 'crosstalk' between different cytoskeletal systems.     

Cargo-carrying motor vehicles on the neuronal highway: transport pathways and neurodegenerative disease

Within axons vital cargoes must be transported over great distances along microtubule tracks to maintain neuronal viability. Essential to this system are the molecular motors, kinesin and dynein, which transport a variety of neuronal cargoes. Elucidating the transport pathways, the identity of the cargoes transported, and the regulation of motor-cargo complexes are areas of intense investigation. Evidence suggests that essential components, including signaling proteins, neuroprotective and repair molecules, and vesicular and cytoskeletal components are all transported. In addition newly emerging data indicate that defects in axonal transport pathways may contribute to the initiation or progression of chronic neuronal dysfunction. In this review we concentrate on microtubule-based motor proteins, their linkers, and cargoes and discuss how factors in the axonal transport pathway contribute to disease states. As additional cargo complexes and transport pathways are identified, an understanding of the role these pathways play in the development of human disease will hopefully lead to new diagnostic and treatment strategies.     

Inhibition of protein phosphatases induces transport deficits and axonopathy

The activity of protein phosphatase (PP)-2A and PP-1 decreased in the brains of Alzheimer's disease and inhibition of the phosphatases led to spatial memory deficit in rats. However, the molecular basis underlying memory impairment of the phosphatase inhibition is elusive. In the present study, we observed a selective inhibition of PP-2A and PP-1 with Calyculin A (CA) not only caused hyperphosphorylation of cytoskeletal proteins, but also impaired the transport of pEGFP-labeled neurofilament-M subunit in the axon-like processes of neuroblastoma N2a cells and resulted in accumulation of neurofilament in the cell bodies. To analyze the morphological alteration of the cells during inhibition of the phosphatases, we established a cell model showing steady outgrowth of axon-like cell processes and employed a stereological system to analyze the retraction of the processes. We found CA treatment inhibited outgrowth of the cell processes and prolonged treatment with CA caused retraction of the processes and meanwhile, the early neurodegenerative varicosities were also obvious in the CA-treated cells. We conclude suppression of PP-2A and PP-1 by CA not only damages intracellular transport but also leads to cell degeneration, which may serve as the functional and structural elements for the memory deficits induced by suppression of the phosphatases.     

The Emerging Role of Cytoskeletal Proteins as Reliable Biomarkers

Cytoskeletal proteins are essential building blocks of cells. More than 100 cytoskeletal and cytoskeleton‐associated proteins are known and for some, their function and regulation are understood in great detail. Apart from cell shape and support, they facilitate many processes such as intracellular signaling and transport, and cancer related processes such as proliferation, migration, and invasion. During the last decade, comparative proteomic studies have identified cytoskeletal proteins as in vitro markers for tumor progression and metastasis. Here, these results are summarized and a number of unrelated studies are highlighted, identifying the same cytoskeletal proteins as potential biomarkers. These findings might indicate that the abundance of these potential markers of tumor progression is associated with the biological outcome and are independent of the cancer origin. This correlates well with recently published results from the Cancer Genome Atlas, indicating that cancers show remarkable similarities in their analyzed molecular information, independent of their organ of origin. It is postulated that the quantification of cytoskeletal proteins in healthy tissues, tumors, in adjacent tissues, and in stroma, is a great source of molecular information, which might not only be used to classify tumors, but more importantly to predict patients’ outcome or even best treatment choices.     

Axonal transport and neurodegenerative disease

Neurons have extensive processes and communication between those processes and the cell body is crucial to neuronal function and survival. Thus, neurons are uniquely dependent on microtubule based transport. Growing evidence supports the idea that deficits in axonal transport contribute to pathogenesis in multiple neurodegenerative diseases. We describe the motor, cytoskeletal, and adaptor proteins involved in axonal transport and their interactions. Data linking disruption of axonal transport to diseases such as ALS are discussed. Finally, we explore the pathways that may cause neuronal dysfunction and death.     

Formation of AP-3 transport intermediates requires Vps41 function

Transport of a subset of membrane proteins to the yeast vacuole requires the function of the AP-3 adaptor protein complex. To define the molecular requirements of vesicular transport in this pathway, we used a biochemical approach to analyse the formation and content of the AP-3 transport intermediate. A vam3tsf (vacuolar t-SNARE) mutant blocks vesicle docking and fusion with the vacuole and causes the accumulation of 50-130-nanometre membrane vesicles, which we isolated and showed by biochemical analysis and immunocytochemistry to contain both AP-3 adaptors and alkaline phosphatase (ALP) pathway cargoes. Inactivation of AP-3 or the protein Vps41 blocks formation of this vesicular intermediate. Vps41 binds to the AP-3 delta-adaptin subunit, suggesting that they function together in the formation of ALP pathway transport intermediates at the late Golgi.     

Protein transport in intact, purified pea etioplasts

We have developed a method to isolate intact, purified pea etioplasts. These etioplasts were capable of recognizing, transporting, and processing the precursor form of the small subunit of the ribulose-1,5-bisphosphate carboxylase, a protein which is not detectable at this developmental stage. Transport of proteins was completely dependent on ATP and could not be substituted for or stimulated by light. The transported precursor protein was processed to its proper molecular weight. The mature form of the small subunit was assembled with the large subunit of the ribulose-1,5-bisphosphate carboxylase already present at this stage to form an oligomer. Protein transport was completely abolished using the phosphatase inhibitor sodium fluoride. This is the first time protein transport has been demonstrated in isolated, purified etioplasts.     

Stochastic simulation of neurofilament transport in axons: the "stop-and-go" hypothesis

According to the "stop-and-go" hypothesis of slow axonal transport, cytoskeletal and cytosolic proteins are transported along axons at fast rates but the average velocity is slow because the movements are infrequent and bidirectional. To test whether this hypothesis can explain the kinetics of slow axonal transport in vivo, we have developed a stochastic model of neurofilament transport in axons. We propose that neurofilaments move in both anterograde and retrograde directions along cytoskeletal tracks, alternating between short bouts of rapid movement and short "on-track" pauses, and that they can also temporarily disengage from these tracks, resulting in more prolonged "off-track" pauses. We derive the kinetic parameters of the model from a detailed analysis of the moving and pausing behavior of single neurofilaments in axons of cultured neurons. We show that the model can match the shape, velocity, and spreading of the neurofilament transport waves obtained by radioisotopic pulse labeling in vivo. The model predicts that axonal neurofilaments spend approximately 8% of their time on track and approximately 97% of their time pausing during their journey along the axon.     

The molecular control of transport vesicle fusion

The fusion of transport vesicles with the appropriate target membrane in constitutive transport is a complex and well-controlled process. Many of the molecular details of the reactions that result in this control are being revealed through the use of cell-free assays of protein transport as well as by the study of the molecular genetics of secretion in yeast. Kinetic analyses have indicated that several structural intermediates are formed after transport vesicles attach to their destination, but before they fuse with the appropriate membrane. Proteins that mediate the formation and processing of these intermediates have been identified. Included among these are small molecular weight GTP-binding proteins. This intricate set of reactions may ensure the fidelity of transport and guard the integrity of the organelles along the transport pathway.     

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).     

Axonal transport: beyond kinesin and cytoplasmic dynein.

In vitro and in vivo studies of specific neuronal fast and slow transport components are presently reshaping our understanding of how the processes of vesicular and cytoskeletal transport are regulated in axons and dendrites. Evidence suggests that vesicles possess an inherent directionality, possibly the result of their motor receptor proteins responding to intracellular cues, which then allows movement with either kinesin or cytoplasmic dynein.     

Separation and reconstitution of sodium-dependent glucose transport activity from renal brush-border membranes using gel-filtration chromatography

Pig kidney brush-border membrane vesicles were solubilized using a final concentration of 1% Triton X-100, found optimal for quantitative reconstitution of d-glucose transport into liposomes. Using reconstituted proteoliposomes, selective permeability towards d-glucose compared to other sugars tested was shown as well as the main features of d-glucose transport in native membranes, namely sodium dependence and phlorizin inhibition of d-glucose accumulation. After removal of Triton X-100 from the detergent extract, some membrane proteins (about 40%), which are insoluble in the absence of detergent, were isolated. Among these proteins resolubilized by 1% Triton X-100, the component catalyzing the d-glucose transport was located by gel-filtration chromatography separation, using reconstitution of transport as the assay. The active fraction displayed a molecular size of 50 Å; when analyzed on SDS polyacrylamide gel electrophoresis, it contained one major protein subunit with an apparent molecular weight close to 65 000. We conclude that this protein fraction is involved in d-glucose transport by renal brush borders.     

Low molecular weight GTP-binding proteins are associated with neuronal organelles involved in rapid axonal transport and exocytosis

Recent evidence suggests that low molecular weight GTP-binding proteins may play important roles in a variety of membrane transport processes. In order to address the question of whether these proteins are involved in transport processes in the nerve axon, we have assessed their presence in rapid transport membranes from rabbit optic nerve. We report the characterization of a group of low molecular weight GTP-binding proteins which are constituents of rapid transport vesicles. Although these proteins are components of rapid transport vesicles, they are apparently not major rapidly transported species. They are localized in cytosolic as well as in membrane fractions of axons, and the membrane-associated form behaves as an integral membrane protein(s). These proteins are also found in association with a variety of vesicular and organellar components of neurons including coated vesicles, synaptic vesicles, synaptic plasma membranes, and mitochondria. We discuss the possible roles of these proteins in rapid axonal transport and exocytosis.