Sorting of cargos between axons and dendrites: modelling of differences in cargo transport in these two types of neurites

Explaining how intracellular cargos are sorted between axons and dendrites is important for a mechanistic understanding of what happens in many neurodegenerative disorders. A simple model of cargo sorting relies on differences in microtubule (MT) orientation between axons and dendrites: in mammalian neurons all MTs in axons have their plus ends directed outward while in proximal regions of dendrites the MT polarity is mixed. It can therefore be assumed that cargos that need to be driven into axons associate with kinesin motors while cargos that need to be driven into dendrites associate with dynein motors. This paper develops equations of cargo transport in axons and dendrites based on the above assumptions. Propagation of a pulse of radiolabelled cargos entering an axon and dendrite is simulated. The model equations are solved utilising the Laplace transform method. Differences in cargo transport between axons and dendrites are discussed.     

Microtubules have opposite orientation in axons and dendrites of Drosophila neurons

In vertebrate neurons, axons have a uniform arrangement of microtubules with plus ends distal to the cell body (plus-end-out), and dendrites have equal numbers of plus- and minus-end-out microtubules. To determine whether microtubule orientation is a conserved feature of axons and dendrites, we analyzed microtubule orientation in invertebrate neurons. Using microtubule plus end dynamics, we mapped microtubule orientation in Drosophila sensory neurons, interneurons, and motor neurons. As expected, all axonal microtubules have plus-end-out orientation. However, in proximal dendrites of all classes of neuron, approximately 90% of dendritic microtubules were oriented with minus ends distal to the cell body. This result suggests that minus-end-out, rather than mixed orientation, microtubules are the signature of the dendritic microtubule cytoskeleton. Surprisingly, our map of microtubule orientation predicts that there are no tracks for direct cargo transport between the cell body and dendrites in unipolar neurons. We confirm this prediction, and validate the completeness of our map, by imaging endosome movements in motor neurons. As predicted by our map, endosomes travel smoothly between the cell body and axon, but they cannot move directly between the cell body and dendrites.     

Growing dendrites and axons differ in their reliance on the secretory pathway

Little is known about how the distinct architectures of dendrites and axons are established. From a genetic screen, we isolated dendritic arbor reduction (dar) mutants with reduced dendritic arbors but normal axons of Drosophila neurons. We identified dar2, dar3, and dar6 genes as the homologs of Sec23, Sar1, and Rab1 of the secretory pathway. In both Drosophila and rodent neurons, defects in Sar1 expression preferentially affected dendritic growth, revealing evolutionarily conserved difference between dendritic and axonal development in the sensitivity to limiting membrane supply from the secretory pathway. Whereas limiting ER-to-Golgi transport resulted in decreased membrane supply from soma to dendrites, membrane supply to axons remained sustained. We also show that dendritic growth is contributed by Golgi outposts, which are found predominantly in dendrites. The distinct dependence between dendritic and axonal growth on the secretory pathway helps to establish different morphology of dendrites and axons.     

Paradoxical relationships between active transport and global protein distributions in neurons

Neural function depends on continual synthesis and targeted trafficking of intracellular components, including ion channel proteins. Many kinds of ion channels are trafficked over long distances to specific cellular compartments. This raises the question of whether cargo is directed with high specificity during transit or whether cargo is distributed widely and sequestered at specific sites. We addressed this question by experimentally measuring transport and expression densities of Kv4.2, a voltage-gated transient potassium channel that exhibits a specific dendritic expression that increases with distance from the soma and little or no functional expression in axons. In over 500 h of quantitative live imaging, we found substantially higher densities of actively transported Kv4.2 subunits in axons as opposed to dendrites. This paradoxical relationship between functional expression and traffic density supports a model—commonly known as the sushi belt model—in which trafficking specificity is relatively low and active sequestration occurs in compartments where cargo is expressed. In further support of this model, we find that kinetics of active transport differs qualitatively between axons and dendrites, with axons exhibiting strong superdiffusivity, whereas dendritic transport resembles a weakly directed random walk, promoting mixing and opportunity for sequestration. Finally, we use our data to constrain a compartmental reaction-diffusion model that can recapitulate the known Kv4.2 density profile. Together, our results show how nontrivial expression patterns can be maintained over long distances with a relatively simple trafficking mechanism and how the hallmarks of a global trafficking mechanism can be revealed in the kinetics and density of cargo.     

Molecular motors and mechanisms of directional transport in neurons

Intracellular transport is fundamental for neuronal morphogenesis, function and survival. Many proteins are selectively transported to either axons or dendrites. In addition, some specific mRNAs are transported to dendrites for local translation. Proteins of the kinesin superfamily participate in selective transport by using adaptor or scaffolding proteins to recognize and bind cargoes. The molecular components of RNA-transporting granules have been identified, and it is becoming clear how cargoes are directed to axons and dendrites by kinesin superfamily proteins. Here we discuss the molecular mechanisms of directional axonal and dendritic transport with specific emphasis on the role of motor proteins and their mechanisms of cargo recognition.     

Wnt/PCP proteins regulate stereotyped axon branch extension in Drosophila

Branching morphology is a hallmark feature of axons and dendrites and is essential for neuronal connectivity. To understand how this develops, I analyzed the stereotyped pattern of Drosophila mushroom body (MB) neurons, which have single axons branches that extend dorsally and medially. I found that components of the Wnt/Planar Cell Polarity (PCP) pathway control MB axon branching. frizzled mutant animals showed a predominant loss of dorsal branch extension, whereas strabismus (also known as Van Gogh) mutants preferentially lost medial branches. Further results suggest that Frizzled and Strabismus act independently. Nonetheless, branching fates are determined by complex Wnt/PCP interactions, including interactions with Dishevelled and Prickle that function in a context-dependent manner. Branching decisions are MB-autonomous but non-cell-autonomous as mutant and non-mutant neurons regulate these decisions collectively. I found that Wnt/PCP components do not need to be asymmetrically localized to distinct branches to execute branching functions. However, Prickle axonal localization depends on Frizzled and Strabismus.     

Monastrol, a selective inhibitor of the mitotic kinesin Eg5, induces a distinctive growth profile of dendrites and axons in primary cortical neuron cultures

Various factors including some motor proteins regulate microtubule (MT) transport and influence the formation of neuronal processes. Eg5, a slow and non-processive (+)-end directed motor molecule, is expressed in developing and differentiated neurons. However, how Eg5 works in neurons is still elusive. Thus, we treated primary rat cortical neuron cultures with monastrol, a specific inhibitor of Eg5, to investigate its role in neurons. Immature neurons treated with monastrol extended longer processes than control within a few hours. After 3 days, immature neurons treated with monastrol had longer dendrites but slightly shorter axons than control. This difference in growth between dendrites and axons became more prominent as the cells differentiated until 5 days. Interestingly, MT distributions in the cell bodies of monastrol-treated neurons appeared somewhat circular surrounding the nucleus, while MTs in the cell bodies of control neurons were primarily distributed in the MT organizing center (MTOC) just beside the nucleus. In mature neurons, monastrol treatment induced the axonal clusters of tubulins, grossly not affecting dendrites. Taken together, we conclude that Eg5 acts distinctively on dendrites and axons in neurons and suggest a putative model of how Eg5 works distinctively on dendrites and axons.     

Are dendrites in Drosophila homologous to vertebrate dendrites?

Dendrites represent arborising neurites in both vertebrates and invertebrates. However, in vertebrates, dendrites develop on neuronal cell bodies, whereas in higher invertebrates, they arise from very different neuronal structures, the primary neurites, which also form the axons. Is this anatomical difference paralleled by principal developmental and/or physiological differences? We address this question by focussing on one cellular model, motorneurons of Drosophila and characterise the compartmentalisation of these cells. We find that motorneuronal dendrites of Drosophila share with typical vertebrate dendrites that they lack presynaptic but harbour postsynaptic proteins, display calcium elevation upon excitation, have distinct cytoskeletal features, develop later than axons and are preceded by restricted localisation of Par6-complex proteins. Furthermore, we demonstrate in situ and culture that Drosophila dendrites can be shifted from the primary neurite to their soma, i.e. into vertebrate-like positions. Integrating these different lines of argumentation, we propose that dendrites in vertebrates and higher invertebrates have a common origin, and differences in dendrite location can be explained through translocation of neuronal cell bodies introduced during the evolutionary process by which arthropods and vertebrates diverged from a common urbilaterian ancestor. Implications of these findings for studies of dendrite development, neuronal polarity, transport and evolution are discussed.     

A role for kinesin heavy chain in controlling vesicle transport into dendrites in Drosophila

The unique architecture of neurons requires the establishment and maintenance of polarity, which relies in part on microtubule-based transport to deliver essential cargo into dendrites. To test different models of differential motor protein regulation and to understand how different compartments in neurons are supplied with necessary functional proteins, we studied mechanisms of dendritic transport, using Drosophila as a model system. Our data suggest that dendritic targeting systems in Drosophila and mammals are evolutionarily conserved, since mammalian cargoes are moved into appropriate domains in Drosophila. In a genetic screen for mutants that mislocalize the dendritic marker human transferrin receptor (hTfR), we found that kinesin heavy chain (KHC) may function as a dendritic motor. Our analysis of dendritic and axonal phenotypes of KHC loss-of-function clones revealed a role for KHC in maintaining polarity of neurons, as well as ensuring proper axonal outgrowth. In addition we identified adenomatous polyposis coli 1 (APC1) as an interaction partner of KHC in controlling directed transport and modulating kinesin function in neurons.     

Neuronal polarity and trafficking

Among the most morphologically complex cells, neurons are masters of membrane specialization. Nowhere is this more striking than in the division of cellular labor between the axon and the dendrites. In morphology, signaling properties, cytoskeletal organization, and physiological function, axons and dendrites (or more properly, the somatodendritic compartment) are radically different. Such polarization of neurons into domains specialized for either receiving (dendrites) or transmitting (axons) cellular signals provides the underpinning for all neural circuitry. The initial specification of axonal and dendritic identity occurs early in neuronal life, persists for decades, and is manifested by the presence of very different sets of cell surface proteins. Yet, how neuronal polarity is established, how distinct axonal and somatodendritic domains are maintained, and how integral membrane proteins are directed to dendrites or accumulate in axons remain enduring and formidable questions in neuronal cell biology.     

Molecular mechanisms for Drosophila neuronetwork formation

Understanding the establishment of functional neuronetworks is one of the frontiers of developmental neurobiology. The use of axons and dendrites from Drosophila nervous system as a model allows the identification of molecular mechanisms which give neurons the ability to guide their processes en route to connect precisely with their partners. By focusing on selected Drosophila model systems, we discuss the recent advances in our understanding of the molecular mechanisms regulating guidance, branching and targeting of axons and dendrites required for the establishment of a functional neuronetwork.     

Microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) guides kinesin‐3‐mediated cargo transport to dendrites

In neurons, the polarized distribution of vesicles and other cellular materials is established through molecular motors that steer selective transport between axons and dendrites. It is currently unclear whether interactions between kinesin motors and microtubule‐binding proteins can steer polarized transport. By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin‐3 (KIF1) and kinesin‐4 (KIF21) subfamily that can also target dendrites. We found that microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1‐dependent dense‐core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule‐binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport.     

The role of selective transport in neuronal polarization

Neurons are functionally and morphologically polarized and possess two distinct types of neurites: axons and dendrites. Key molecules for axon formation are transported along microtubules and accumulated at the distal end of the nascent axons. In this review, we summarize recent advances in the understanding of the mechanisms involved in selective transport in neurons. In addition, we focus on motor proteins, cargo, cargo adaptors, and the loading and unloading of cargo.     

Vesicular trafficking of semaphorin 3A is activity-dependent and differs between axons and dendrites

Secreted semaphorins act as guidance cues in the developing nervous system and may have additional functions in mature neurons. How semaphorins are transported and secreted by neurons is poorly understood. We find that endogenous semaphorin 3A (Sema3A) displays a punctate distribution in axons and dendrites of cultured cortical neurons. GFP-Sema3A shows a similar distribution and co-localizes with secretory vesicle cargo proteins. Live-cell imaging reveals highly dynamic trafficking of GFP-Sema3A vesicles with distinct properties in axons and dendrites regarding directionality, velocity, mobility and pausing time. In axons, most GFP-Sema3A vesicles move fast without interruption, almost exclusively in the anterograde direction, while in dendrites many GFP-Sema3A vesicles are stationary and move equally frequent in both directions. Disruption of microtubules, but not of actin filaments, significantly impairs GFP-Sema3A transport. Interestingly, depolarization induces a reversible arrest of axonal transport of GFP-Sema3A vesicles but has little effect on dendritic transport. Conversely, action potential blockade using tetrodotoxin (TTX) accelerates axonal transport, but not dendritic transport. These data indicate that axons and dendrites regulate trafficking of Sema3A and probably other secretory vesicles in distinct ways, with axons specializing in fast, uninterrupted, anterograde transport. Furthermore, neuronal activity regulates secretory vesicle trafficking in axons by a depolarization-evoked trafficking arrest.     

Modelling active transport in Drosophila unipolar motor neurons

This paper develops a model for simulating organelle transport in Drosophila unipolar motor neurons. The paper is motivated by a recent experimental investigation by Stone et al. (Microtubules have opposite orientation in axons and dendrites of Drosophila neurons. Mol Biol Cell.19:4122-4129) who proposed a map of microtubule (MT) orientation in Drosophila neurons, and explained why dynein mutations selectively impede dendritic growth without having much effect on axonal growth. Two different approaches to modelling the effect of dynein mutations are utilised: one through assuming a reduced average velocity of a dynein mutant motor and the other through assuming its decreased processivity (an increased detachment rate from MTs). Modified Smith-Simmons equations are used for developing a continuum model of the process. Distributions of organelle concentrations as well as distributions of diffusion, motor-driven and total organelle fluxes are simulated.     

Organelle distribution in neurons: Logistics behind polarized transport

Highly polarized neurons need to carefully regulate the distribution of organelles and other cargoes into their two morphologically and functionally distinct domains, the somatodendritic and axonal compartments, to maintain proper neuron homeostasis. An outstanding question in the field is how organelles reach their correct destination. Long-range transport along microtubules, driven by motors, ensures a fast and controlled availability of organelles in axons and dendrites, but it remains largely unclear what rules govern their transport into the correct compartment. Here, we review the emerging concepts of polarized cargo trafficking in neurons, highlighting the role of microtubule organization, microtubule-associated proteins, and motor proteins and discuss compartment-specific inclusion and exclusion mechanisms as well as the regulation of correct coupling of cargoes to motor proteins.     

Modelling active transport in Drosophila unipolar motor neurons

This paper develops a model for simulating organelle transport in Drosophila unipolar motor neurons. The paper is motivated by a recent experimental investigation by Stone et al. (Microtubules have opposite orientation in axons and dendrites of Drosophila neurons. Mol Biol Cell.19:4122-4129) who proposed a map of microtubule (MT) orientation in Drosophila neurons, and explained why dynein mutations selectively impede dendritic growth without having much effect on axonal growth. Two different approaches to modelling the effect of dynein mutations are utilised: one through assuming a reduced average velocity of a dynein mutant motor and the other through assuming its decreased processivity (an increased detachment rate from MTs). Modified Smith–Simmons equations are used for developing a continuum model of the process. Distributions of organelle concentrations as well as distributions of diffusion, motor-driven and total organelle fluxes are simulated.     

Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast.

The mushroom bodies (MBs) are prominent structures in the Drosophila brain that are essential for olfactory learning and memory. Characterization of the development and projection patterns of individual MB neurons will be important for elucidating their functions. Using mosaic analysis with a repressible cell marker (Lee, T. and Luo, L. (1999) Neuron 22, 451-461), we have positively marked the axons and dendrites of multicellular and single-cell mushroom body clones at specific developmental stages. Systematic clonal analysis demonstrates that a single mushroom body neuroblast sequentially generates at least three types of morphologically distinct neurons. Neurons projecting into the (gamma) lobe of the adult MB are born first, prior to the mid-3rd instar larval stage. Neurons projecting into the alpha' and beta' lobes are born between the mid-3rd instar larval stage and puparium formation. Finally, neurons projecting into the alpha and beta lobes are born after puparium formation. Visualization of individual MB neurons has also revealed how different neurons acquire their characteristic axon projections. During the larval stage, axons of all MB neurons bifurcate into both the dorsal and medial lobes. Shortly after puparium formation, larval MB neurons are selectively pruned according to birthdays. Degeneration of axon branches makes early-born gamma neurons retain only their main processes in the peduncle, which then project into the adult gamma lobe without bifurcation. In contrast, the basic axon projections of the later-born (alpha'/beta') larval neurons are preserved during metamorphosis. This study illustrates the cellular organization of mushroom bodies and the development of different MB neurons at the single cell level. It allows for future studies on the molecular mechanisms of mushroom body development.     

Polarized secretory trafficking directs cargo for asymmetric dendrite growth and morphogenesis

Proper growth of dendrites is critical to the formation of neuronal circuits, but the cellular machinery that directs the addition of membrane components to generate dendritic architecture remains obscure. Here, we demonstrate that post-Golgi membrane trafficking is polarized toward longer dendrites of hippocampal pyramidal neurons in vitro and toward apical dendrites in vivo. Small Golgi outposts partition selectively into longer dendrites and are excluded from axons. In dendrites, Golgi outposts concentrate at branchpoints where they engage in post-Golgi trafficking. Within the cell body, the Golgi apparatus orients toward the longest dendrite, and this Golgi polarity precedes asymmetric dendrite growth. Manipulations that selectively block post-Golgi trafficking halt dendrite growth in developing neurons and cause a shrinkage of dendrites in mature pyramidal neurons. Further, disruption of Golgi polarity produces neurons with symmetric dendritic arbors lacking a single longest principal dendrite. These results define a novel polarized organization of neuronal secretory trafficking and demonstrate a mechanistic link between directed membrane trafficking and asymmetric dendrite growth.     

Dendrites of distinct classes of Drosophila sensory neurons show different capacities for homotypic repulsion

BACKGROUND: Understanding how dendrites establish their territory is central to elucidating how neuronal circuits are built. Signaling between dendrites is thought to be important for defining their territories; however, the strategies by which different types of dendrites communicate are poorly understood. We have shown previously that two classes of Drosophila peripheral da sensory neurons, the class III and class IV neurons, provide complete and independent tiling of the body wall. By contrast, dendrites of class I and class II neurons do not completely tile the body wall, but they nevertheless occupy nonoverlapping territories. RESULTS: By developing reagents to permit high-resolution studies of dendritic tiling in living animals, we demonstrate that isoneuronal and heteroneuronal class IV dendrites engage in persistent repulsive interactions. In contrast to the extensive dendritic exclusion shown by class IV neurons, duplicated class III neurons showed repulsion only at their dendritic terminals. Supernumerary class I and class II neurons innervated completely overlapping regions of the body wall, and this finding suggests a lack of like-repels-like behavior. CONCLUSIONS: These data suggest that repulsive interactions operate between morphologically alike dendritic arbors in Drosophila. Further, Drosophila da sensory neurons appear to exhibit at least three different types of class-specific dendrite-dendrite interactions: persistent repulsion by all branches, repulsion only by terminal dendrites, and no repulsion.