Special issue on ‘Cytoskeleton-dependent regulation of neuronal network formation’

How does a single neuron develop its prominent polar morphology, how do multiple neurons connect with each other and how is communication between these neurons established, controlled and maintained? These are fundamental questions one needs to ask when trying to understand the complex structure and function of the brain. As in all other eukaryotic cell types, cellular morphogenesis and the interaction between cells is firmly orchestrated by the underlying cell cytoskeleton. In this Special Issue, experts in the field of cytoskeleton research discuss key structural proteins, their regulators and signaling pathways that are instrumental in establishing neuronal polarity, driving neurite outgrowth and establishing and maintaining synaptic connections between neurons.

The reviews featured in this issue deal with the structural characteristics and dynamics of the three major filament systems that comprise the cell cytoskeleton: [1] actin or microfilament system, [2] microtubules and [3] intermediate filaments. Although the major focus of this issue is to provide an overview of the current knowledge in the field, it also includes reports on some new findings on the regulation of the cytoskeleton in neurons. This comprises an analysis of the spatiotemporal expression of tubulin isoforms in the embryonic cortex (Breuss et al., 2017), the identification of the ENA/VASP family member Mena as a binding partner for myosin VI together with a role of myosin VI in recruiting secretory granules to the cortical actin in PC12 cells (Tomatis et al., 2017) and a role for the actin-associated protein tropomyosins Tpm3.1 and Tpm4.2 in bulk endocytosis (Gormal et al., 2017). The following section will provide brief synopses of the articles, presented in this Special Issue.

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  The direction of nerve growth cones is defined by attractive and repulsive guidance cues that act via the actin cytoskeleton to control motility. In their review, James Zheng and colleagues focus on the recent findings in this field (Omotade et al., 2017). After describing growth cone structure, they briefly introduce actin-binding proteins, such as Arp2/3, formin, ADF/cofilin, profilin and capping proteins known to be crucial for actin dynamics. The authors discuss the ‘molecular clutch’ model, the mechanical coupling between F-actin and the extracellular matrix mediated by cell adhesion complexes responsible for the forward movement of the growth cone. A particular focus of the review is a discussion on different pools of G-actin, newly synthesized, cytosolic and recycling in different sub-cellular regions of the growth cone. The regulation of these pools plays a critical role during growth cone dynamics and neurite growth.

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  After reviewing the role of the actin cytoskeleton in driving the outgrowth of neurites, Max Schelski and colleagues discuss positive and negative feedback loops that are underpinning the differentiation of one of these neurites to become an axon (Schelski et al., 2017). This addresses a central question of how neurons establish polarity interrogating the role of polarity effectors and signaling pathways that feed into these feedback loops. Central to their proposed model is the concept of ‘Local Activation – Global Inhibition’ in which the accumulation of growth-promoting factors promotes local signaling feedback loops driving axonal specification while global inhibition occurs in distant neurites through an antagonistic role of cAMP and cGMP. Next, the interplay and integration of neurite length-dependent feedback loops with cellular adhesion-induced signaling is discussed where axonal specification can be accelerated by adhesion-induced signaling. The review then draws the attention to kinesin-mediated anterograde transport of polarity effectors and the role of microtubules in the transport machinery, which is determined by changes in the organization and function (number, orientation and posttranslational modifications) of microtubules. The second part of the review then comprises a detailed discussion on how polarity effectors lead to changes in the organization of the actin cytoskeleton and microtubules and linking dynamic changes in actin cytoskeleton organization with microtubule dynamics in subdomains of growth cones.

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  Leading on from the discussion of various feedback loops and signaling pathways that regulate neurite growth and polarization, the review by Gasperini et al. moves Ca2+ signaling to the center stage. The authors examine what is known, and perhaps more importantly what is unknown about how calcium regulates the cytoskeleton in navigating growth cones during axon guidance. The review focuses on calcium regulation of phosphorylation, describing several of the well described phosphorylation pathways. What is clear from this work, is that the complete repertoire of calcium-dependent phosphorylation signaling to the cytoskeleton is still to be deciphered. One of the more complete calcium-phosphorylation signaling stories in growth cones is the elegant work of Kamiguchi and colleagues who have done much to describe how membrane endocytosis and exocytosis is regulated during growth cone navigation. The latter part of the review moves to explore new models of calcium-cytoskeletal regulation, examining potential direct links between store operated calcium entry and the cytoskeleton. Most of these ideas are still to be demonstrated in growth cones, but when they are, they will have profound effects on our understanding of how calcium regulates growth cone motility and hence axon guidance.

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  In order to form a complex network of communicating neurons, the primary neurites that are formed need to undergo arborization (or branching). The review by Weingart and Gallo addresses the question of how axons form collateral branches that can then innervate multiple target sites (Weingart and Gallo, 2017). As for the process of polarization and neurite extension, the interplay of microtubules and axons is critical to drive branching. In this context, local protein synthesis, signaling pathways and organelle function in axons are discussed. In the first part of their review, the authors discuss the molecular players involved in actin cytoskeleton remodeling. Their focus is in axonal filopodia, small axonal protrusions that emerge and elongate along the axonal shaft. These filopodia are then invaded by individual microtubules which facilitates the formation of a branch. A detailed discussion on the regulation of microtubule dynamics and their interaction with the actin cytoskeleton then follows. The second part of the review is dedicated to the discussion of key signaling pathways involved in axonal branching. These pathways include neurotrophin and GSK3 serine/threonine kinase, 3′-5′-cyclic adenosine monophosphate (cAMP), small GTPase and calcium signaling. In the last part, a brief overview on the link between intra-axonal protein synthesis and mitochondrial function with the regulation of actin cytoskeleton dynamics is provided.

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  As highlighted in the first reviews of this Special Issue, the actin cytoskeleton underlies the control of many regulatory and effector proteins. Two of the key effector proteins are the actin filament pointed end-capping protein tropomodulin and the actin-associated proteins tropomyosin. The interaction of these proteins and their function in regulating actin filament dynamics in neurons is discussed in detail by Gray and colleagues (Gray et al., 2017). The review furthermore provides a comprehensive reference for the expression of these two classes of proteins in the nervous system and their role in neurite outgrowth. More recent work also suggests a role for these actin-associated proteins in synaptic plasticity and function, which is the focus of current research in the field. The discussion of the individual and synergistic effect of tropomodulins and tropomyosins in neuronal function is followed by a brief overview on the role of these proteins in the diseased nervous system.

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  Breuss et al. (2017) review the role of the microtubule cytoskeleton in brain development, setting out to remind readers that “tubulins are not just controls on your average western blot”. They focus on the essential role of microtubules in neurogenesis, neuronal migration and neuronal differentiation. The authors examine the tubulin family which comprise 8 alpha- and 9 beta-tubulin genes that encode human microtubules. Disorders that result from mutations in selected members of the tubulin family are collectively called tubulinopathies. The authors describe how the tubulinopathies have aided in deciphering the different roles of specific tubulin genes. For example, a mutation in the tubulin gene TUBA4A is identified as a causative mutation in amyotrophic lateral sclerosis, while other tubulin gene mutations have overlapping phenotypes. The authors suggest that specific tubulin expression patterns might provide clues to the functional roles of those isoforms. They present some new data demonstrating the spatiotemporal expression of tubulin isoforms Tubb2b, Tubb3 and Tubb5in the embryonic cortex, illustrating how the expression of some tubulin isoforms changes with development. The authors also discuss other factors that contribute to the diversity of tubulin genes including how mRNA might be targeted by microRNAs to regulate translation efficiency or stability and the growing array of post-translational modifications that are now known to regulate tubulin function.

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  In addition to actin filaments and microtubules, intermediate filaments are another class of filamentous proteins that compose the cellular cytoskeleton and help to maintain axons, the longest cellular appendages. In their review Matthew Kirkcaldie and Samuel Dwyer describe neuronal intermediate filaments as a third wave of neuron structural maturation (Kirkcaldie and Dwyer, 2017). After short introduction the authors review structure, roles and localizations of different types of neuronal intermediate filaments. This section is followed by the section with thorough description of developmental regulation and expression of neuronal intermediate filaments in different regions of nervous system.

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  Next, Bertling & Hotulainen give more detailed characteristic of actin cytoskeleton in dendritic spines in their review (Bertling and Hotulainen, 2017). The introduction describes modern view of the role of spines in brain function. Mature dendritic spines have mushroom-like shape with distinct head and neck regions. Actin filament orientation has more heterogeneous pattern in the head, and F-actin rings were shown in necks. The section on dendritic spine morphogenesis is divided to several subsections discussing processes of filopodia initiation, elongation, spine head enlargement, actin dynamics and remodeling. The authors give detailed description how spine density is maintained by controlled filopodia initiation (classified by the authors into two categories, activity-dependent and activity-independent) and inhibition that can be promoted by such proteins as MIM/Mtss1, Ephexin5 and ARHGAP44. Different mechanisms of the initiation/inhibition are discussed and summarized in the figure. Peculiarities of actin cytoskeleton of dendritic filopodia and filopodia in axon growth cones are described as well as roles of mDia2 and Arp2/3 in filopodia elongation. It still needs to be learned what signals cause turning filopodia to spines. In the section about actin modifications, the authors review recent data on control of actin cytoskeleton in dendritic spines via actin phosphorylation.

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  The story about actin cytoskeleton then is continued in the review written by Tomoaki Shirao and colleagues (Koganezawa et al., 2017). The role of drebrin, an actin-binding protein, in dendritic spines formation and synaptic plasticity is discussed. Actin filaments in spines can be in two pools, the stable F-actin localized to the base core of the spine and the dynamic F-actin observed in the spine tip. In the section describing actin cytoskeleton in dendritic spines the authors propose a stochastic autocatalytic branching model of F-actin in the dynamic pool. Two different isoforms, embryonic drebrin E and adult drebrin A, are actin-binding proteins that similarly to tropomyosins bind along actin filaments, stabilize F-actin and prevent its depolymerization. The authors show data obtained by super resolution microscopy to demonstrate preferential localization of drebrin to the center of dendritic spines. Effects of overexpression and inhibition of expression of drebrin A on spine morphology is discussed from the point of spine maturation. Reviewing studies from the Shirao lab the authors explain why distinct roles of the isoforms in the brain function and isoform conversion from E to A are critical for long-term potentiation.

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  Besides neurite growth and spine plasticity, another important function of the actin cytoskeleton is to keep plasma membrane lipids and proteins in specific compartments. Andreas Papadopulos review literature on modulation of plasma membrane properties by actomyosin cortex and the role of cortical actin network in exocytosis (Papadopoulos, 2017). The author discusses proteins involved in the fusion pore formation when cellular and vesicle membranes are merged. The author suggests that future studies could be directed at understanding connection between actin reorganization and membrane compartmentalization / tension with the help of modern methods, such as single molecule and high-speed microscopy, FRET and electrophysiological measurements.

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  The story about the role of the actin cytoskeleton in exocytosis continues in the paper of Frederic Meunier and colleagues (Tomatis et al., 2017). They identified Mena, a protein from the ENA/VASP family of actin-binding proteins, as a binding partner for myosin VI. The authors describe the potential confounding effects of examining Mena function, when they are built in redundancy with other members of the ENA/VASP family. To account for extraneous ENA/VASP interactions, they exploit “knock-sideways approach”, where all ENA/VASP proteins are bound to the mitochondrial membrane, although importantly for this study, myosin VI is unaffected and remains in the cytoplasm. The authors then demonstrated that this unconventional myosin isoform participates in recruitment of secretory granules to the cortical actin network. ENA/VASP proteins target myosin VI to secretory granules and consequently regulate their exocytosis. Of three Mena isoforms that co-precipitate with myosin VI, the 80 kDa isoform suggested to be responsible for targeting myosin VI to secretory granules, tethering the granules to the cortical actin network. This work provides important new information on the cellular mechanisms that underpin exocytosis.

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  A key role of the cytoskeleton is to regulate membrane endo- and exocytosis. Gormal et al. (2017), recognizing that neurosecretory cells can be studied as a model of synaptic vesicle release, have exploited the neurosecretory cells to decipher the cell signals that underpin vesicle endocytosis. The authors focus on activity dependent bulk endocytosis, a mechanism of membrane retrieval, employed during periods of high activity to replenish vesicle pools. The authors examine the actin-based mechanisms that regulate the activity dependent bulk endocytosis, describing the actin rings that form around newly forming bulk endosomes. These actin rings facilitate the fission process in neurosecretory cells and form during major re-organization of the cortical actin network. It is likely that myosin II is important in maintaining actin ring stability and possibly constriction of the actin rings. The authors then extend their analysis to the tropomyosins, a group of proteins thought to be important regulators of actin-myosin function. They provide novel data to demonstrate that tropomyosin (Tpm) 3.1 and 4.2 are present in the actin ring complexes after activity dependent bulk endocytosis and that activity stimulates Tpm3.1 recruitment to actin ring complexes possibly to act in recruiting myosin II and stabilizing the ring complex. They go on to examine the function of signaling lipids, dynamin and interacting calcium signals in activity dependent bulk endocytosis concluding that the regulation of actin function in endo- and exocytosis is vital, but remains to be completely deciphered.

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  To this stage, the structure and function of the cytoskeletal filament systems in the nervous system have been discussed. The review by Hoffman and colleagues shifts its focus on the class of serine/threonine phosphatases as important regulators of microtubules, the actin cytoskeleton and intermediate filaments (Hoffman et al., 2017). Controlling the phosphorylation status of signaling molecules, effector proteins and cytoskeletal building blocks is major regulatory mechanisms for driving changes and maintaining homeostasis of the cell architecture. In this review, the major serine/threonine phosphatases PP2A, PP1, calcineurin are discussed in the context of post-translational regulation of cytoskeletal components. A general discussion of the structure and function of these phosphatases is followed by a discussion on their interaction with microtubules and microtubule-associated proteins. A major aspect of this discussion is how the control of the phosphorylation level of tubulin and microtubule-associated proteins regulates microtubule dynamics. This detailed discussion on the regulation of the microtubule cytoskeleton by PP2A, PP1 and calcineurin is then complemented by a discourse of the effect of these phosphatases on intermediate filaments and the actin cytoskeleton. An important implication of the deregulation of these regulatory mechanisms is the association of altered phosphorylation of cytoskeletal proteins with several neurological diseases, which is discussed in the last part of this review. This aspect is further elaborated on in the following review by Roland Brandt and colleagues, discussing the role of the microtubule-associated protein tau in the nervous system and its deregulation in disease.

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  The microtubule-associated neuronal protein, Tau is the focus of the review by Brandt et al. (2017). The authors set out to examine the question of whether Tau is simply a regulator of microtubule assembly in axons, or plays more complex roles in neuronal network function, with systemic effects on synaptic plasticity and neuronal transmission. The hyperphosphorylation state of Tau in Alzheimer’s disease has led to the term “tauopathies”, and is associated with a number of neurodegenerative conditions. Tau is promiscuous in its binding and so the question of how it actually functions in neurodegeneration is unclear. The authors examine a number of studies that evaluate motor behavior, learning and memory in several models of Tau knockout mice. The authors suggest that Tau function may change during tauopathies, by potential toxic gain of function mechanisms. They examine the evidence that Tau is important in synaptic long-term potentiation and depression, concluding that in addition to the canonical role of microtubule regulation, Tau may have systemic and possibly microtubule-independent functions in neuronal connectivity that underscore its role in neurodegenerative disease.