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.     

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).     

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.     

The melanosome as a model to study organelle motility in mammals

Melanosomes are lysosome-related organelles within which melanin pigment is synthesized. The molecular motors that allow these organelles to move within melanocytes have been the subject of intense study in several organisms. In mammals, melanosomes travel bi-directionally along microtubule tracks. The anterograde movement, i.e., towards microtubule plus-ends at the periphery, is accomplished by proteins of the kinesin superfamily, whereas the retrograde movement, i.e., towards microtubule minus-ends at the cell center, is achieved by dynein and dynein-associated proteins. At the periphery, melanosomes interact with the actin cytoskeleton via a tripartite complex formed by the small GTPase Rab27a, melanophilin and myosin Va, an actin-based motor. This interaction is essential for the maintenance of a dispersed state of the melanosomes, as shown by the perinuclear clustering of organelles in mutants in any of the referred proteins. In the retinal pigment epithelium, a similar complex formed by Rab27a, a melanophilin homolog called MyRIP and myosin VIIa is probably responsible for the tethering of melanosomes to the actin cytoskeleton. The coordination of motor activities is still poorly characterized, although some models have emerged in recent years and are discussed here. Unraveling regulatory mechanisms responsible for melanosome motility in pigmented cells will provide general insights into organelles dynamics within eukaryotic cells.     

Mechanical properties of organelles driven by microtubule-dependent molecular motors in living cells

The organization of the cytoplasm is regulated by molecular motors which transport organelles and other cargoes along cytoskeleton tracks. Melanophores have pigment organelles or melanosomes that move along microtubules toward their minus and plus end by the action of cytoplasmic dynein and kinesin-2, respectively. In this work, we used single particle tracking to characterize the mechanical properties of motor-driven organelles during transport along microtubules. We tracked organelles with high temporal and spatial resolutions and characterized their dynamics perpendicular to the cytoskeleton track. The quantitative analysis of these data showed that the dynamics is due to a spring-like interaction between melanosomes and microtubules in a viscoelastic microenvironment. A model based on a generalized Langevin equation explained these observations and predicted that the stiffness measured for the motor complex acting as a linker between organelles and microtubules is ～ one order smaller than that determined for motor proteins in vitro. This result suggests that other biomolecules involved in the interaction between motors and organelles contribute to the mechanical properties of the motor complex. We hypothesise that the high flexibility observed for the motor linker may be required to improve the efficiency of the transport driven by multiple copies of motor molecules.     

Biogenesis of melanosomes - the chessboard of pigmentation

Melanosomes are lysosome-related organelles in retinal pigment epithelial cells and epidermal melanocytes in which melanin pigments are synthesized and stored. Melanosomes are generated by multistep processes in which an immature unpigmented organelle forms and then subsequently matures. Such maturation requires inter-organellar transport of protein cargos required for pigment synthesis but also recruitment of effector proteins necessary for the correct transport of melanosomes to the cell periphery. Several studies have started to unravel the main pathways and mechanisms exploited by melanosomal proteins involved in melanosome structure and melanin synthesis. A major unexpected finding seen early in melanosome biogenesis showed the similarities between the fibrillar sheets of premelanosomes and amyloid fibrils. Late steps of melanosome formation are dependent on pathways regulated by proteins encoded by genes mutated in genetic diseases such as the Hermansky-Pudlak Syndrom (HPS) and different types of albinism. Altogether the findings from the past recent years have started to unravel how specialized cells integrate unique and ubiquitous molecular mechanisms in subverting the endosomal system to generate cell-type specific structures and their associated functions. Further dissection of the melanosomal system will likely shed light not only on the biogenesis of lysosome-related organelles but also on general aspects of vesicular transport in the endosomal system.     

Molecular motors and their role in pigmentation.

Skin pigmentation is orchestrated through a series of complementary processes. After migration of melanoblasts out of the neural crest to epidermis and hair follicle, these cells mature into melanocytes. Differentiated melanocytes produce melanin in specialized organelles, the melanosomes. Moreover, the cytoplasm of melanocytes branches into extensions, the dendrites. Via the tips of these dendrites they donate their mature melanosomes to the keratinocytes resulting in skin pigmentation. Thus, one essential part of the process of pigmentation is the translocation of melanosomes from their site of origin in the perinuclear cytoplasm towards the dendrite tips. Motor proteins are molecules which use the energy derived from ATP hydrolysis to move along cytoskeletal elements, either actin filaments or microtubules, to transport their cargo, which can be organelles, vesicles or chromosomes. This review describes the different classes of microtubule-based and actin-based motor proteins with their characteristics and functional importance in cell biology and organelle transport. Some of them will be highlighted and several recent studies in mammalian pigment cells indicating their role in pigment granule transport will be discussed. As a result of these data and previous suggestions, a model will be proposed for the possible cooperation of both systems in melanosome movement.     

Organelle-localized potassium transport systems in plants

Some intracellular organelles found in eukaryotes such as plants have arisen through the endocytotic engulfment of prokaryotic cells. This accounts for the presence of plant membrane intrinsic proteins that have homologs in prokaryotic cells. Other organelles, such as those of the endomembrane system, are thought to have evolved through infolding of the plasma membrane. Acquisition of intracellular components (organelles) in the cells supplied additional functions for survival in various natural environments. The organelles are surrounded by biological membranes, which contain membrane-embedded K⁺ transport systems allowing K⁺ to move across the membrane. K⁺ transport systems in plant organelles act coordinately with the plasma membrane intrinsic K⁺ transport systems to maintain cytosolic K⁺ concentrations. Since it is sometimes difficult to perform direct studies of organellar membrane proteins in plant cells, heterologous expression in yeast and Escherichia coli has been used to elucidate the function of plant vacuole K⁺ channels and other membrane transporters. The vacuole is the largest organelle in plant cells; it has an important task in the K⁺ homeostasis of the cytoplasm. The initial electrophysiological measurements of K⁺ transport have categorized three classes of plant vacuolar cation channels, and since then molecular cloning approaches have led to the isolation of genes for a number of K⁺ transport systems. Plants contain chloroplasts, derived from photoautotrophic cyanobacteria. A novel K⁺ transport system has been isolated from cyanobacteria, which may add to our understanding of K⁺ flux across the thylakoid membrane and the inner membrane of the chloroplast. This chapter will provide an overview of recent findings regarding plant organellar K⁺ transport proteins.     

Kinesin superfamily proteins and their various functions and dynamics

Kinesin superfamily proteins (KIFs) are motor proteins that transport membranous organelles and macromolecules fundamental for cellular functions along microtubules. Their roles in transport in axons and dendrites have been studied extensively, but KIFs are also used in intracellular transport in general. Recent findings have revealed that in many cases, the specific interaction of cargoes and motors is mediated via adaptor/scaffolding proteins. Cargoes are sorted to precise destinations, such as axons or dendrites. KIFs also participate in polarized transport in epithelial cells as shown in the apical transport of annexin XIIIb-containing vesicles by KIFC3. KIFs play important roles in higher order neuronal activity; transgenic mice overexpressing KIF17, which transports N-methyl-d-asp (NMDA) receptors to dendrites, show enhanced memory and learning. KIFs also play significant roles in neuronal development and brain wiring: KIF2A suppresses elongation of axon collaterals by its unique microtubule-depolymerizing activity. X-ray crystallography has revealed the structural uniqueness of KIF2 underlying the microtubule-depolymerizing activity. In addition, single molecule biophysics and optical trapping have shown that the motility of monomeric KIF1A is caused by biased Brownian movement, and X-ray crystallography has shown how the conformational changes occur for KIF1A to move during ATP hydrolysis. These multiple approaches in analyzing KIF functions will illuminate many basic mechanisms underlying intracellular events and will be a very promising and fruitful area for future studies.     

The mechanochemistry of integrated motor protein complexes

The assembly of molecular motor proteins into multi-unit protein complexes plays an important role in determining the intracellular transport and trafficking properties of many subcellular commodities. Yet, it is not known how proteins within these complexes interact and function collectively. Considering the established ties between motor transport and diseases, it has become increasingly important to investigate the functional properties of these essential transport ‘motifs’. Doing so requires that the composite motile and force-generating properties of multi-unit motor assemblies are characterized. However, such analyses are typically confounded by a lack of understanding of the links between the structural and mechanical properties of many motor complexes. New experimental challenges also emerge when one examines motor cooperation. Distributions in the mechanical microstates available to motor ensembles must be examined in order to fully understand the transport behavior of multi-motor complexes. Furthermore, mechanisms by which motors communicate must be explored to determine whether motor groups can move cargo together in a truly cooperative fashion. Resolving these issues requires the development of experimental methods that allow the dynamics of complex systems of transport proteins to be monitored with the same precision available to single-molecule biophysical assays. Herein, we discuss key fundamental principles governing the function of motor complexes and their relation to mechanisms that regulate intracellular cargo transport. We also outline new experimental strategies to resolve these essential features of intracellular transport.     

Non-vesicular sterol transport in cells

Sterols such as cholesterol are important components of cellular membranes. They are not uniformly distributed among organelles and maintaining the proper distribution of sterols is critical for many cellular functions. Both vesicular and non-vesicular pathways move sterols between membranes and into and out of cells. There is growing evidence that a number of non-vesicular transport pathways operate in cells and, in the past few years, a number of proteins have been proposed to facilitate this transfer. Some are soluble sterol transfer proteins that may move sterol between membranes. Others are integral membranes proteins that mediate sterol efflux, uptake from cells, and perhaps intracellular sterol transfer as well. In most cases, the mechanisms and regulation of these proteins remains poorly understood. This review summarizes our current knowledge of these proteins and how they could contribute to intracellular sterol trafficking and distribution.     

Putting On The Breaks: Regulating Organelle Movements in Plant Cells†

A striking characteristic of plant cells is that their organelles can move rapidly through the cell. This movement, commonly referred to as cytoplasmic streaming, has been observed for over 200 years, but we are only now beginning to decipher the mechanisms responsible for it. The identification of the myosin motor proteins responsible for these movements allows us to probe the regulatory events that coordinate organelle displacement with normal cell physiology. This review will highlight several recent developments that have provided new insight into the regulation of organelle movement, both at the cellular level and at the molecular level.     

Regulation of dynein‐dynactin‐driven vesicular transport

Most of the long‐range intracellular movements of vesicles, organelles and other cargoes are driven by microtubule (MT)‐based molecular motors. Cytoplasmic dynein, a multisubunit protein complex, with the aid of dynactin, drives transport of a wide variety of cargoes towards the minus end of MTs. In this article, I review our current understanding of the mechanisms underlying spatiotemporal regulation of dynein‐dynactin‐driven vesicular transport with a special emphasis on the many steps of directional movement along MT tracks. These include the recruitment of dynein to MT plus ends, the activation and processivity of dynein, and cargo recognition and release by the motor complex at the target membrane. Furthermore, I summarize the most recent findings about the fine control mechanisms for intracellular transport via the interaction between the dynein‐dynactin motor complex and its vesicular cargoes.      

Signaling pathways in melanosome biogenesis and pathology

Melanosomes are the specialized intracellular organelles of pigment cells devoted to the synthesis, storage and transport of melanin pigments, which are responsible for most visible pigmentation in mammals and other vertebrates. As a direct consequence, any genetic mutation resulting in alteration of melanosomal function, either because affecting pigment cell survival, migration and differentiation, or because interfering with melanosome biogenesis, transport and transfer to keratinocytes, is immediately translated into color variations of skin, fur, hair or eyes. Thus, over 100 genes and proteins have been identified as pigmentary determinants in mammals, providing us with a deep understanding of this biological system, which functions by using mechanisms and processes that have parallels in other tissues and organs. In particular, many genes implicated in melanosome biogenesis have been characterized, so that melanosomes represent an incredible source of information and a model for organelles belonging to the secretory pathway. Furthermore, the function of melanosomes can be associated with common physiological phenotypes, such as variation of pigmentation among individuals, and with rare pathological conditions, such as albinism, characterized by severe visual defects. Among the most relevant mechanisms operating in melanosome biogenesis are the signal transduction pathways mediated by two peculiar G protein-coupled receptors: the melanocortin-1 receptor (MC1R), involved in the fair skin/red hair phenotype and skin cancer; and OA1 (GPR143), whose loss-of-function results in X-linked ocular albinism. This review will focus on the most recent novelties regarding the functioning of these two receptors, by highlighting emerging signaling mechanisms and general implications for cell biology and pathology.     

Transporters for amino acids in plant cells: some functions and many unknowns

Membrane proteins are essential to move amino acids in or out of plant cells as well as between organelles. While many putative amino acid transporters have been identified, function in nitrogen movement in plants has only been shown for a few proteins. Those studies demonstrate that import systems are fundamental in partitioning of amino acids at cellular and whole plant level. Physiological data further suggest that amino acid transporters are key-regulators in plant metabolism and that their activities affect growth and development. By contrast, knowledge on the molecular mechanisms of cellular export processes as well as on intracellular transport of amino acids is scarce. Similarly, little is known about the regulation of amino acid transporter function and involvement of the transporters in amino acid signaling. Future studies need to identify the missing components to elucidate the importance of amino acid transport processes for whole plant physiology and productivity.     

Linkers, packages and pathways: new concepts in axonal transport

The molecular mechanisms that generate efficient and directed transport of proteins and organelles in axons remain poorly understood. In the past year, many studies have identified specific transmembrane or scaffold proteins that might link motor proteins to their cargoes. These studies have also identified previously unsuspected pathways and raised the intriguing possibility that pre-packaged groups of functionally related proteins are transported together in the axon. Evidence suggests that fast molecular motor proteins have a role in slow axonal transport, and the axonal transport machinery has been implicated in the genesis of neurodegenerative diseases.     

Movement of organelles along filaments dissociated from the axoplasm of the squid giant axon

Cytoplasmic filaments, separated from the axoplasm of the squid giant axon and visualized by video-enhanced differential interference contrast microscopy, support the directed movement of organelles in the presence of ATP. All organelles, regardless of size, move continuously along isolated transport filaments at 2.2 +/- 0.2 micron/sec. In the intact axoplasm, however, movements of the larger organelles are slow and saltatory. These movements may reflect a resistance to movement imposed by the intact axoplasm. The uniform rate of all organelles along isolated transport filaments suggests that a single type of molecular motor powers fast axonal transport. Organelles can attach to and move along more than one filament at a time, suggesting that organelles have multiple binding sites for this motor.     

Kinesin proteins: A phylum of motors for microtubule-based motility

The cellular processes of transport, division and, possibly, early development all involve microtubule-based motors. Recent work shows that, unexpectedly, many of these cellular functions are carried out by different types of kinesin and kinesin-related motor proteins. The kinesin proteins are a large and rapidly growing family of microtubule-motor proteins that share a 340-amino-acid motor domain. Phylogenetic analysis of the conserved motor domains groups the kinesin proteins into a number of subfamilies, the members of which exhibit a common molecular organization and related functions. The kinesin proteins that belong to different subfamilies differ in their rates and polarity of movement along microtubules, and probably in the particles/organelles that they transport. The kinesins arose early in eukaryotic evolution and gene duplication has allowed functional specialization to occur, resulting in a surprisingly large number of different classes of these proteins adapted for intracellular transport of vesicles and organelles, and for assembly and force generation in the meiotic and mitotic spindles.     

Mechanisms and regulation of autophagosome formation

Autophagy is an intracellular pathway for the bulk degradation of cytoplasmic substances such as cytosol, protein aggregates and organelles. Autophagy is characterized by the formation of double-membrane bound vesicles called autophagosomes, which engulf the cargo and transport it to the vacuole/lysosome for breakdown and recycling. Even though several proteins in this pathway have been identified, little is known about the mechanism of action of these proteins during autophagosome biogenesis. In this review we briefly discuss recent findings on the molecular players and mechanisms involved in autophagosome formation. In particular, we will focus on the mechanisms regulating membrane recruitment as well as membrane remodeling during autophagosome formation.