Neuronal polarization: the cytoskeleton leads the way

The morphology of cells is key to their function. Neurons extend a long axon and several shorter dendrites to transmit signals in the nervous system. This process of neuronal polarization is driven by the cytoskeleton. The first and decisive event during neuronal polarization is the specification of the axon. Distinct cytoskeletal dynamics and organization of the cytoskeleton determine the future axon while the other neurites become dendrites. Here, we will review how the cytoskeleton and its effectors drive axon specification and neuronal polarization. First, the role of the actin cytoskeleton and microtubules in axon specification will be presented. Then, we will discuss the role of the centrosome in axon determination as well as how microtubules are generated in axons and dendrites. Finally, we will discuss potential mechanisms leading to axon specification, such as positive feedback loops that could be a coordinated interaction between actin and microtubules. Together, this review will present the recent advances on the role of the microtubules and the actin cytoskeleton during neuronal polarization. We will pinpoint the upcoming challenges to gain a better understanding of neuronal polarization on a fundamental intracellular level. Finally, we will outline how reactivation of the intrinsic polarization program may help to induce axon regeneration after CNS injury.     

Interaction of nonreceptor tyrosine-kinase Fer and p120 catenin is involved in neuronal polarization

The neuronal cytoskeleton is essential for establishment of neuronal polarity, but mechanisms controlling generation of polarity in the cytoskeleton are poorly understood. The nonreceptor tyrosine kinase, Fer, has been shown to bind to microtubules and to interact with several actin-regulatory proteins. Furthermore, Fer binds p120 catenin and has been shown to regulate cadherin function by modulating cadherin-beta-catenin interaction. Here we show involvement of Fer in neuronal polarization and neurite development. Fer is concentrated in growth cones together with cadherin, beta-catenin, and cortactin in stage 2 hippocampal neurons. Inhibition of Fer-p120 catenin interaction with a cell-permeable inhibitory peptide (FerP) increases neurite branching. In addition, the peptide significantly delays conversion of one of several dendrites into an axon in early stage hippocampal neurons. FerP-treated growth cones also exhibit modified localization of the microtubule and actin cytoskeleton. Together, this indicates that the Fer-p120 interaction is required for normal neuronal polarization and neurite development.     

APC nuclear membrane association and microtubule polarity.

BACKGROUND INFORMATION: Directional cell migration is a fundamental feature of embryonic development, the inflammatory response and the metastatic spread of cancer. Migrating cells have a polarized morphology with an asymmetric distribution of signalling molecules and of the actin and microtubule cytoskeletons. The dynamic reorganization of the actin cytoskeleton provides the major driving force for migration in all mammalian cell types, but microtubules also play an important role in many cells, most notably neuronal precursors. RESULTS: We previously showed, using primary fibroblasts and astrocytes in in vitro scratch-induced migration assays, that the accumulation of APC (adenomatous polyposis coli; the APC tumour suppressor protein) at microtubule plus-ends promotes their association with the plasma membrane at the leading edge. This is required for polarization of the microtubule cytoskeleton during directional migration. Here, we have examined the organization of microtubules in the soma of migrating neurons and fibroblasts. CONCLUSIONS: We find that APC, through a direct interaction with the NPC (nuclear pore complex) protein Nup153 (nucleoporin 153), promotes the association of microtubules with the nuclear membrane.     

Rho GTPases in neuronal morphogenesis

The Rho family of small GTPases act as intracellular molecular switches that transduce signals from extracellular stimuli to the actin cytoskeleton and the nucleus. Recent evidence implicates Rho GTPases in the regulation of neuronal morphogenesis, including migration, polarity, axon growth and guidance, dendrite elaboration and plasticity, and synapse formation. Signalling pathways from membrane receptors to Rho GTPases and from Rho GTPases to the actin cytoskeleton are beginning to be discovered. Mutations in these signalling pathways have been reported in human neurological diseases, which underscores their importance in the development and function of the nervous system.     

The role of the cytoskeleton during neuronal polarization

The formation of an axon and dendrites, neuronal polarization, is a prerequisite for neurons to integrate and propagate information within the brain. During the past years progress has been made toward understanding the initial stage of neuronal polarization, axon formation. First, the physiological role of some candidate regulators of neuronal polarity has been affirmed, including Sad kinases, the Rho-GTPase Cdc42, and the actin regulators Ena/VASP proteins. Second, recent studies have revealed microtubule stabilization as a mechanism complementary to actin dynamics underlying neuronal polarization. Moreover, stable microtubules in the axon may form a landmark to confer identity to the axon. This review highlights the recent advances in understanding the intracellular mechanisms underlying neuronal polarization and discusses them in the context of putative cytoskeletal effectors.     

APC nuclear membrane association and microtubule polarity

Background information. Directional cell migration is a fundamental feature of embryonic development, the inflammatory response and the metastatic spread of cancer. Migrating cells have a polarized morphology with an asymmetric distribution of signalling molecules and of the actin and microtubule cytoskeletons. The dynamic reorganization of the actin cytoskeleton provides the major driving force for migration in all mammalian cell types, but microtubules also play an important role in many cells, most notably neuronal precursors. Results. We previously showed, using primary fibroblasts and astrocytes in in vitro scratch‐induced migration assays, that the accumulation of APC (adenomatous polyposis coli; the APC tumour suppressor protein) at microtubule plus‐ends promotes their association with the plasma membrane at the leading edge. This is required for polarization of the microtubule cytoskeleton during directional migration. Here, we have examined the organization of microtubules in the soma of migrating neurons and fibroblasts. Conclusions. We find that APC, through a direct interaction with the NPC (nuclear pore complex) protein Nup153 (nucleoporin 153), promotes the association of microtubules with the nuclear membrane.     

Establishing cell polarity in development

Polarity is a common feature of many different cell types, including the Caenorhabditis elegans zygote, the Drosophila oocyte and mammalian epithelial cells. The initial establishment of cell polarity depends on asymmetric cues that lead to reorganization of the cytoskeleton and polarized localization of several cortical proteins that act downstream of the polarization cues. The past year revealed that homologs of the C. elegans par (partitioning defective) genes are also essential for establishing polarity in Drosophila and vertebrate cells. There is growing evidence that the proteins encoded by these genes interact with key regulators of both the actin and the microtubule cytoskeletons.     

Neuronal polarization and the cytoskeleton

Neuronal polarization, the formation of one long axon and several short dendrites, is an obligatory process to integrate and propagate information within the brain. Axon formation is the key event during neuronal polarization and is based on tightly regulated rearrangements of the cytoskeleton. Here, we discuss how the cytoskeleton drives neuronal polarization. First, we convey the role of the actin cytoskeleton and microtubules during axon formation. Second, we discuss different cytoskeletal binding and regulating proteins, which are essential to specify the axon. Finally, we outline plus end tracking proteins (+TIPs) as important regulators for neuronal polarization by mediating the interaction between the actin cytoskeleton and microtubules and compare this function to other polarity processes.     

Microtubules guide root hair tip growth

The ability to establish cell polarity is crucial to form and function of an individual cell. Polarity underlies critical processes during cell development, such as cell growth, cell division, cell differentiation and cell signalling. Interphase cytoplasmic microtubules in tip-growing fission yeast cells have been shown to play a particularly important role in regulating cell polarity. By placing proteins that serve as spatial cues in the cell cortex of the expanding tip, microtubules determine the site where exocytosis, and therefore growth, takes place. Transport and the targeting of exocytotic vesicles to the very tip depend on the actin cytoskeleton. Recently, endoplasmic microtubules have been identified in tip-growing root hairs, which are an experimental system for plant cell growth. Here, we review the data that demonstrate involvement of microtubules in hair elongation and polarity of the model plants Medicago truncatula and Arabidopsis thaliana. Differences and similarities between the microtubule organization and function in these two species are discussed and we compare the observations in root hairs with the microtubule-based polarity mechanism in fission yeast.     

Crosstalk of cell polarity signaling pathways

Cell polarity, the asymmetric organization of cellular components along one or multiple axes, is present in most cells. From budding yeast cell polarization induced by pheromone signaling, oocyte polarization at fertilization to polarized epithelia and neuronal cells in multicellular organisms, similar mechanisms are used to determine cell polarity. Crucial role in this process is played by signaling lipid molecules, small Rho family GTPases and Par proteins. All these signaling circuits finally govern the cytoskeleton, which is responsible for oriented cell migration, cell shape changes, and polarized membrane and organelle trafficking. Thus, typically in the process of cell polarization, most cellular constituents become polarized, including plasma membrane lipid composition, ion concentrations, membrane receptors, and proteins in general, mRNA, vesicle trafficking, or intracellular organelles. This review gives a brief overview how these systems talk to each other both during initial symmetry breaking and within the signaling feedback loop mechanisms used to preserve the polarized state.     

Microtubules and growth cone function

It has been recognized for a long time that the neuronal cytoskeleton plays an important part in neurite growth and growth cone pathfinding, the mechanism by which growing axons find an appropriate route through the developing embryo to their target cells. In the growth cone, many intracellular signaling pathways that are activated by guidance cues converge on the growth cone cytoskeleton and regulate its dynamics. Most of the research effort in this area has focussed on the actin, microfilament cytoskeleton of the growth cone, principally because it underlies growth cone motility, the extension and retraction of filopodia and lamellipodia, and these structures are the first to encounter guidance cues during growth cone advance. However, more recently, it has become apparent that the microtubule cytoskeleton also has a role in growth cone pathfinding and is also regulated by guidance cues operating through intracellular signaling pathways via engagement with cell membrane receptors. Furthermore, recent work has revealed an interaction between these two components of the growth cone cytoskeleton that is probably essential for growth cone turning, a fundamental growth cone behavior during pathfinding. In this short review I discuss recent experiments that uncover the function of microtubules in growth cones, how their behavior is regulated, and how they interact with the actin filaments.     

Cell polarity: ROPing the ends together

Cell polarity plays an important role in plant development, but the mechanisms that first establish polarity cues remain obscure. By contrast, a flurry of information has recently emerged on the elaboration of cell shape from such unknown initial cell-polarity cues. Recent studies suggest that Rho-related GTPases in plants (ROPs), and their effector targets among the ROP-interactive CRIB motif-containing proteins (RICs), mediate two antagonistic pathways that have opposing action on cell polarization. ROP proteins appear to interact directly with upstream regulators of the ARP2/3 complex, which are conserved modulators of the actin cytoskeleton. ROP function is dependent on the class 1 ADP-ribosylation factors (ARFs), which are core components of the vesicle transport machinery that are also involved in the polar localization of PIN-FORMED (PIN) family auxin efflux facilitators.     

Neuronal Polarity

The assembly of functional neuronal networks in the developing animal relies on the polarization of neurons, i.e., the formation of a single axon and multiple dendrites. Breaking the symmetry of neurons depends on cytoskeletal rearrangements. In particular, axon specification requires local dynamic instability of actin and stabilization of microtubules. The polarized cytoskeleton also provides the basis for selective trafficking and retention of cellular components in the future somatodendritic or axonal compartments. Hence, these mechanisms are not only essential to achieve neuronal polarization, but also to maintain it. Different extracellular and intracellular signals converge on the regulation of the cytoskeleton. Most notably, Rho GTPases, PI3K, Ena/VASP, cofilin and SAD kinases are major intracellular regulators of neuronal polarity. Analyzing polarity signals under physiological conditions will provide a better understanding of how neurons can be induced to repolarize under pathological conditions, i.e., to regenerate their axons after central nervous system (CNS) injury.One ambitious aim in cellular biology is to unravel the molecular mechanisms driving cellular asymmetry and polarization. The polarity of neurons is particularly dramatic as neurons undergo complex morphological rearrangements to assemble into neuronal circuits and propagate signals. They start as round neuronal spheres, gradually adopting a complex morphology by forming one long axon and several shorter dendrites to eventually connect to other neurons via synapses. Neuronal compartments segregate into molecularly and functionally distinct zones. For example, signal input takes place at the postsynaptic densities where a chemical signal elicits electric postsynaptic potentials. These potentials are integrated along the dendritic tree and cell body to trigger an action potential arising at the axon hillock and propagating further along the axon. At their terminals, the electrical signal is reconverted into a chemical signal by the release of synaptic vesicles containing neurotransmitter.Neurons maintain their polarity throughout life by different intracellular mechanisms and molecular signals. During the last decade, cell biological and molecular approaches helped to discover many of the molecules and signaling mechanisms regulating neuronal polarity (Yoshimura et al. 2006; Arimura and Kaibuchi 2007; Witte and Bradke 2008). The aim of this article is to summarize the current knowledge and principles of breaking neuronal symmetry to generate functional neurons, and to discuss the future challenges in the field. The article covers two different topics: intrinsic mechanisms that govern symmetry breaking in the absence of external cues (in vitro systems) and the role of extracellular signaling in the establishment of neuronal polarity in vivo.     

The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.     

Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis

The Drosophila melanogaster body axes are defined by the precise localization and the restriction of molecular determinants in the oocyte. Polarization of the oocyte during oogenesis is vital for this process. The directed traffic of membranes and proteins is a crucial component of polarity establishment in various cell types and organisms. Here, we investigate the role of the small GTPase Rab6 in the organization of the egg chamber and in asymmetric determinant localization during oogenesis. We show that exocytosis is affected in rab6-null egg chambers, which display a loss of nurse cell plasma membranes. Rab6 is also required for the polarization of the oocyte microtubule cytoskeleton and for the posterior localization of oskar mRNA. We show that, in vivo, Rab6 is found in a complex with Bicaudal-D, and that Rab6 and Bicaudal-D cooperate in oskar mRNA localization. Thus, during Drosophila oogenesis, Rab6-dependent membrane trafficking is doubly required; first, for the general organization and growth of the egg chamber, and second, more specifically, for the polarization of the microtubule cytoskeleton and localization of oskar mRNA. These findings highlight the central role of vesicular trafficking in the establishment of polarity and in determinant localization in Drosophila.     

A role of local signalling in the establishment and maintenance of the asymmetrical architecture of a neuron

Significant progress has been made in the identification of intrinsic and extrinsic factors involved in the development of nervous system. It is remarkable that the establishment and maintenance of the asymmetrical architecture of a neuron is coordinated by a limited repertoire of signalling machineries. However, the details of signalling mechanisms responsible for creating specificity and diversity required for proper development of the nervous system remain largely to be investigated. An emerging body of evidence suggests that specificity and diversity can be achieved by differential regulation of signalling components at distinct subcellular localizations. Many aspects of neuronal polarization and morphogenesis are attributed to localized signalling. Further diversity and specificity of receptor signalling can be achieved by the regulation of molecules outside the cell. Recent evidence suggests that extracellular matrix molecules are essential extrinsic cues that function to foster the growth of neurons. Therefore, it is important to understand where the signalling machineries are activated and how they are combined with other factors in order to understand the molecular mechanism underlying neuronal development.     

Cdc42: an important regulator of neuronal morphology

Regulation of neuronal morphology and activity-dependent synaptic modifications involves reorganization of the actin cytoskeleton. Dynamic changes of the actin cytoskeleton in many cell types are controlled by small GTPases of the Rho family, such as RhoA, Rac1 and Cdc42. As key regulators of both actin and microtubule cytoskeleton, Rho GTPases have also emerged as important regulators of dendrite and spine structural plasticity. Multiple studies suggest that Rac1 and Cdc42 are positive regulators promoting neurite outgrowth and growth cone protrusion, while the activation of RhoA induces stress fiber formation, leading to growth cone collapse and neurite retraction. This review focuses on recent advances in our understanding of the molecular mechanisms underlying physiological and pathological functions of Cdc42 in the nervous system. We also discuss application of different FRET-based biosensors as a powerful approach to examine the dynamics of Cdc42 activity in living cells.     

The Actin Cytoskeleton and Signaling Network during Pollen Tube Tip Growth

The organization and dynamics of the actin cytoskeleton play key roles in many aspects of plant cell development. The actin cytoskeleton responds to internal developmental cues and environmental signals and is involved in cell division, subcellular organelle movement, cell polarity and polar cell growth. The tipgrowing pollen tubes provide an ideal model system to investigate fundamental mechanisms of underlying polarized cell growth. In this system, most signaling cascades required for tip growth...     

Rearrangement of microtubule polarity orientation during conversion of dendrites to axons in cultured pyramidal neurons

Axons and dendrites of neurons differ in the polarity orientation of their microtubules. Whereas the polarity orientation of microtubules in axons is uniform, with all plus ends distal, that in dendrites is nonuniform. The mechanisms responsible for establishment and maintenance of microtubule polarity orientation in neuronal processes remain unclear, however. We previously described a culture system in which dendrites of rat cortical neurons convert to axons. In the present study, we examined changes in microtubule polarity orientation in such dendrites. With the use of the hooking procedure and electron microscopy, we found that microtubule polarity orientation changed from nonuniform to uniform, with a plus end-distal arrangement, in dendrites that gave rise to axons during culture of neurons for 24 h. Microtubule polarity orientation remained nonuniform in dendrites that did not elongate. Axon regeneration at the dendritic tip thus triggered the disappearance of minus end-distal microtubules from dendrites. These minus end-distal microtubules also disappeared from dendrites during axon regeneration in the presence of inhibitors of actin polymerization, suggesting that actin-dependent transport of microtubules is not required for this process and implicating a previously unidentified mechanism in the establishment and maintenance of microtubule polarity orientation in neuronal processes.