Molecular Mechanisms of Fibroblast Growth Factor Signaling in Physiology and Pathology

Fibroblast growth factors (FGFs) signal in a paracrine or endocrine fashion to mediate a myriad of biological activities, ranging from issuing developmental cues, maintaining tissue homeostasis, and regulating metabolic processes. FGFs carry out their diverse functions by binding and dimerizing FGF receptors (FGFRs) in a heparan sulfate (HS) cofactor- or Klotho coreceptor-assisted manner. The accumulated wealth of structural and biophysical data in the past decade has transformed our understanding of the mechanism of FGF signaling in human health and development, and has provided novel concepts in receptor tyrosine kinase (RTK) signaling. Among these contributions are the elucidation of HS-assisted receptor dimerization, delineation of the molecular determinants of ligand–receptor specificity, tyrosine kinase regulation, receptor cis-autoinhibition, and tyrosine trans-autophosphorylation. These structural studies have also revealed how disease-associated mutations highjack the physiological mechanisms of FGFR regulation to contribute to human diseases. In this paper, we will discuss the structurally and biophysically derived mechanisms of FGF signaling, and how the insights gained may guide the development of therapies for treatment of a diverse array of human diseases.Fibroblast growth factor (FGF) signaling fulfills essential roles in metazoan development and metabolism. A wealth of literature has documented the requirement for FGF signaling in multiple processes during embryogenesis, including implantation (Feldman et al. 1995), gastrulation (Sun et al. 1999), somitogenesis (Dubrulle and Pourquie 2004; Wahl et al. 2007; Lee et al. 2009; Naiche et al. 2011; Niwa et al. 2011), body plan formation (Martin 1998; Rodriguez Esteban et al. 1999; Tanaka et al. 2005; Mariani et al. 2008), morphogenesis (Metzger et al. 2008; Makarenkova et al. 2009), and organogenesis (Goldfarb 1996; Kato and Sekine 1999; Sekine et al. 1999; Sun et al. 1999; Colvin et al. 2001; Serls et al. 2005; Vega-Hernandez et al. 2011). Recent clinical and biochemical data have uncovered unexpected roles for FGF signaling in metabolic processes, including phosphate/vitamin D homeostasis (Consortium 2000; Razzaque and Lanske 2007; Nakatani et al. 2009; Gattineni et al. 2011; Kir et al. 2011), cholesterol/bile acid homeostasis (Yu et al. 2000a; Holt et al. 2003), and glucose/lipid metabolism (Fu et al. 2004; Moyers et al. 2007). Highlighting its diverse biology, deranged FGF signaling contributes to many human diseases, such as congenital craniosynostosis and dwarfism syndromes (Naski et al. 1996; Wilkie et al. 2002, 2005), Kallmann syndrome (Dode et al. 2003; Pitteloud et al. 2006a), hearing loss (Tekin et al. 2007, 2008), and renal phosphate wasting disorders (Shimada et al. 2001; White et al. 2001), as well as many acquired forms of cancers (Rand et al. 2005; Pollock et al. 2007; Gartside et al. 2009; di Martino et al. 2012). Endocrine FGFs have also been implicated in the progression of acquired metabolic disorders, including chronic kidney disease (Fliser et al. 2007), obesity (Inagaki et al. 2007; Moyers et al. 2007; Reinehr et al. 2012), and insulin resistance (Fu et al. 2004; Chen et al. 2008b; Chateau et al. 2010; Huang et al. 2011), giving rise to many opportunities for drug discovery in the field of FGF biology (Beenken and Mohammadi 2012).Based on sequence homology and phylogeny, the 18 mammalian FGFs are grouped into six subfamilies (Ornitz and Itoh 2001; Popovici et al. 2005; Itoh and Ornitz 2011). Five of these subfamilies act in a paracrine fashion, namely, the FGF1 subfamily (FGF1 and FGF2), the FGF4 subfamily (FGF4, FGF5, and FGF6), the FGF7 subfamily (FGF3, FGF7, FGF10, and FGF22), the FGF8 subfamily (FGF8, FGF17, and FGF18), and the FGF9 subfamily (FGF9, FGF16, and FGF20). In contrast, the FGF19 subfamily (FGF19, FGF21, and FGF23) signals in an endocrine manner (Beenken and Mohammadi 2012). FGFs exert their pleiotropic effects by binding and activating the FGF receptor (FGFR) subfamily of receptor tyrosine kinases that are coded by four genes (FGFR1, FGFR2, FGFR3, and FGFR4) in mammals (Johnson and Williams 1993; Mohammadi et al. 2005b). The extracellular domain of FGFRs consists of three immunoglobulin (Ig)-like domains (D1, D2, and D3), and the intracellular domain harbors the conserved tyrosine kinase domain flanked by the flexible amino-terminal juxtamembrane linker and carboxy-terminal tail (Lee et al. 1989; Dionne et al. 1991; Givol and Yayon 1992). A unique feature of FGFRs is the presence of a contiguous segment of glutamic and aspartic acids in the D1–D2 linker, termed the acid box (AB). The two-membrane proximal D2 and D3 and the intervening D2–D3 linker are necessary and sufficient for ligand binding/specificity (Dionne et al. 1990; Johnson et al. 1990), whereas D1 and the D1–D2 linker are implicated in receptor autoinhibition (Wang et al. 1995; Roghani and Moscatelli 2007; Kalinina et al. 2012). Alternative splicing and translational initiation further diversify both ligands and receptors. The amino-terminal regions of FGF8 and FGF17 can be differentially spliced to yield FGF8a, FGF8b, FGF8e, FGF8f (Gemel et al. 1996; Blunt et al. 1997), and FGF17a and FGF17b isoforms (Xu et al. 1999), whereas cytosine-thymine-guanine (CTG)-mediated translational initiation gives rise to multiple high molecular weight isoforms of FGF2 and FGF3 (Florkiewicz and Sommer 1989; Prats et al. 1989; Acland et al. 1990). The tissue-specific alternative splicing in D3 of FGFR1, FGFR2, and FGFR3 yields “b” and “c” receptor isoforms which, along with their temporal and spatial expression patterns, is the major regulator of FGF–FGFR specificity/promiscuity (Orr-Urtreger et al. 1993; Ornitz et al. 1996; Zhang et al. 2006). A large body of structural data on FGF–FGFR complexes has begun to reveal the intricate mechanisms by which different FGFs and FGFRs combine selectively to generate quantitatively and qualitatively different intracellular signals, culminating in distinct biological responses. In addition, these structural data have unveiled how pathogenic mutations hijack the normal physiological mechanisms of FGFR regulation to lead to pathogenesis. We will discuss the current state of the structural biology of the FGF–FGFR system, lessons learned from studying the mechanism of action of pathogenic mutations, and how the structural data are beginning to shape and advance the translational research.     

Origin and Evolution of the Self-Organizing Cytoskeleton in the Network of Eukaryotic Organelles

The eukaryotic cytoskeleton evolved from prokaryotic cytomotive filaments. Prokaryotic filament systems show bewildering structural and dynamic complexity and, in many aspects, prefigure the self-organizing properties of the eukaryotic cytoskeleton. Here, the dynamic properties of the prokaryotic and eukaryotic cytoskeleton are compared, and how these relate to function and evolution of organellar networks is discussed. The evolution of new aspects of filament dynamics in eukaryotes, including severing and branching, and the advent of molecular motors converted the eukaryotic cytoskeleton into a self-organizing “active gel,” the dynamics of which can only be described with computational models. Advances in modeling and comparative genomics hold promise of a better understanding of the evolution of the self-organizing cytoskeleton in early eukaryotes, and its role in the evolution of novel eukaryotic functions, such as amoeboid motility, mitosis, and ciliary swimming.The eukaryotic cytoskeleton organizes space on the cellular scale and this organization influences almost every process in the cell. Organization depends on the mechanochemical properties of the cytoskeleton that dynamically maintain cell shape, position organelles, and macromolecules by trafficking, and drive locomotion via actin-rich cellular protrusions, ciliary beating, or ciliary gliding. The eukaryotic cytoskeleton is best described as an “active gel,” a cross-linked network of polymers (gel) in which many of the links are active motors that can move the polymers relative to each other (Karsenti et al. 2006). Because prokaryotes have only cytoskeletal polymers but lack motor proteins, this “active gel” property clearly sets the eukaryotic cytoskeleton apart from prokaryotic filament systems.Prokaryotes contain elaborate systems of several cytomotive filaments (Löwe and Amos 2009) that share many structural and dynamic features with eukaryotic actin filaments and microtubules (Löwe and Amos 1998; van den Ent et al. 2001). Prokaryotic cytoskeletal filaments may trace back to the first cells and may have originated as higher-order assemblies of enzymes (Noree et al. 2010; Barry and Gitai 2011). These cytomotive filaments are required for the segregation of low copy number plasmids, cell rigidity and cell-wall synthesis, cell division, and occasionally the organization of membranous organelles (Komeili et al. 2006; Thanbichler and Shapiro 2008; Löwe and Amos 2009). These functions are performed by dynamic filament-forming systems that harness the energy from nucleotide hydrolysis to generate forces either via bending or polymerization (Löwe and Amos 2009; Pilhofer and Jensen 2013). Although the identification of actin and tubulin homologs in prokaryotes is a major breakthrough, we are far from understanding the origin of the structural and dynamic complexity of the eukaryotic cytoskeleton.Advances in genome sequencing and comparative genomics now allow a detailed reconstruction of the cytoskeletal components present in the last common ancestor of eukaryotes. These studies all point to an ancestrally complex cytoskeleton, with several families of motors (Wickstead and Gull 2007; Wickstead et al. 2010) and filament-associated proteins and other regulators in place (Jékely 2003; Richards and Cavalier-Smith 2005; Rivero and Cvrcková 2007; Chalkia et al. 2008; Eme et al. 2009; Fritz-Laylin et al. 2010; Eckert et al. 2011; Hammesfahr and Kollmar 2012). Genomic reconstructions and comparative cell biology of single-celled eukaryotes (Raikov 1994; Cavalier-Smith 2013) allow us to infer the cellular features of the ancestral eukaryote. These analyses indicate that amoeboid motility (Fritz-Laylin et al. 2010; although, see Cavalier-Smith 2013), cilia (Cavalier-Smith 2002; Mitchell 2004; Jékely and Arendt 2006; Satir et al. 2008), centrioles (Carvalho-Santos et al. 2010), phagocytosis (Cavalier-Smith 2002; Jékely 2007; Yutin et al. 2009), a midbody during cell division (Eme et al. 2009), mitosis (Raikov 1994), and meiosis (Ramesh et al. 2005) were all ancestral eukaryotic cellular features. The availability of functional information from organisms other than animals and yeasts (e.g., Chlamydomonas, Tetrahymena, Trypanosoma) also allow more reliable inferences about the ancestral functions of cytoskeletal components (i.e., not only their ancestral presence or absence) and their regulation (Demonchy et al. 2009; Lechtreck et al. 2009; Suryavanshi et al. 2010).The ancestral complexity of the cytoskeleton in eukaryotes leaves a huge gap between prokaryotes and the earliest eukaryote we can reconstruct (provided that our rooting of the tree is correct) (Cavalier-Smith 2013). Nevertheless, we can attempt to infer the series of events that happened along the stem lineage, leading to the last common ancestor of eukaryotes. Meaningful answers will require the use of a combination of gene family history reconstructions (Wickstead and Gull 2007; Wickstead et al. 2010), transition analyses (Cavalier-Smith 2002), and computer simulations relevant to cell evolution (Jékely 2008).     

Origin of Microglia: Current Concepts and Past Controversies

Microglia are the resident macrophages of the central nervous system (CNS), which sit in close proximity to neural structures and are intimately involved in brain homeostasis. The microglial population also plays fundamental roles during neuronal expansion and differentiation, as well as in the perinatal establishment of synaptic circuits. Any change in the normal brain environment results in microglial activation, which can be detrimental if not appropriately regulated. Aberrant microglial function has been linked to the development of several neurological and psychiatric diseases. However, microglia also possess potent immunoregulatory and regenerative capacities, making them attractive targets for therapeutic manipulation. Such rationale manipulations will, however, require in-depth knowledge of their origins and the molecular mechanisms underlying their homeostasis. Here, we discuss the latest advances in our understanding of the origin, differentiation, and homeostasis of microglial cells and their myelomonocytic relatives in the CNS.Microglia are the resident macrophages of the central nervous system (CNS), which are uniformly distributed throughout the brain and spinal cord with increased densities in neuronal nuclei, including the Substantia nigra in the midbrain (Lawson et al. 1990; Perry 1998). They belong to the nonneuronal glial cell compartment and their function is crucial to maintenance of the CNS in both health and disease (Ransohoff and Perry 2009; Perry et al. 2010; Ransohoff and Cardona 2010; Prinz and Priller 2014).Two key functional features define microglia: immune defense and maintenance of CNS homeostasis. As part of the innate immune system, microglia constantly sample their environment, scanning and surveying for signals of external danger (Davalos et al. 2005; Nimmerjahn et al. 2005; Lehnardt 2010), such as those from invading pathogens, or internal danger signals generated locally by damaged or dying cells (Bessis et al. 2007; Hanisch and Kettenmann 2007). Detection of such signals initiates a program of microglial responses that aim to resolve the injury, protect the CNS from the effects of the inflammation, and support tissue repair and remodeling (Minghetti and Levi 1998; Goldmann and Prinz 2013).Microglia are also emerging as crucial contributors to brain homeostasis through control of neuronal proliferation and differentiation, as well as influencing formation of synaptic connections (Lawson et al. 1990; Perry 1998; Hughes 2012; Blank and Prinz 2013). Recent imaging studies revealed dynamic interactions between microglia and synaptic connections in the healthy brain, which contributed to the modification and elimination of synaptic structures (Perry et al. 2010; Tremblay et al. 2010; Bialas and Stevens 2013). In the prenatal brain, microglia regulate the wiring of forebrain circuits, controlling the growth of dopaminergic axons in the forebrain and the laminar positioning of subsets of neocortical interneurons (Squarzoni et al. 2014). In the postnatal brain, microglia-mediated synaptic pruning is similarly required for the remodeling of neural circuits (Paolicelli et al. 2011; Schafer et al. 2012). In summary, microglia occupy a central position in defense and maintenance of the CNS and, as a consequence, are a key target for the treatment of neurological and psychiatric disorders.Although microglia have been studied for decades, a long history of experimental misinterpretation meant that their true origins remained debated until recently. Although we knew that microglial progenitors invaded the brain rudiment at very early stages of embryonic development (Alliot et al. 1999; Ransohoff and Perry 2009), it has now been established that microglia arise from yolk sac (YS)-primitive macrophages, which persist in the CNS into adulthood (Davalos et al. 2005; Nimmerjahn et al. 2005; Ginhoux et al. 2010, 2013; Kierdorf and Prinz 2013; Kierdorf et al. 2013a). Moreover, early embryonic brain colonization by microglia is conserved across vertebrate species, implying that it is essential for early brain development (Herbomel et al. 2001; Bessis et al. 2007; Hanisch and Kettenmann 2007; Verney et al. 2010; Schlegelmilch et al. 2011; Swinnen et al. 2013). In this review, we will present the latest findings in the field of microglial ontogeny, which provide new insights into their roles in health and disease.     

Biology of the TAM Receptors

The TAM receptors—Tyro3, Axl, and Mer—comprise a unique family of receptor tyrosine kinases, in that as a group they play no essential role in embryonic development. Instead, they function as homeostatic regulators in adult tissues and organ systems that are subject to continuous challenge and renewal throughout life. Their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. The TAMs and their ligands—Gas6 and Protein S—are essential for the efficient phagocytosis of apoptotic cells and membranes in these tissues; and in the immune system, they act as pleiotropic inhibitors of the innate inflammatory response to pathogens. Deficiencies in TAM signaling are thought to contribute to chronic inflammatory and autoimmune disease in humans, and aberrantly elevated TAM signaling is strongly associated with cancer progression, metastasis, and resistance to targeted therapies.The name of the TAM family is derived from the first letter of its three constituents—Tyro3, Axl, and Mer (Prasad et al. 2006). As detailed in Figure 1, members of this receptor tyrosine kinase (RTK) family were independently identified by several different groups and appear in the early literature under multiple alternative names. However, Tyro3, Axl, and Mer (officially c-Mer or MerTK for the protein, Mertk for the gene) have now been adopted as the NCBI designations. The TAMs were first grouped into a distinct RTK family (the Tyro3/7/12 cluster) in 1991, through PCR cloning of their kinase domains (Lai and Lemke 1991). The isolation of full-length cDNAs for Axl (O''Bryan et al. 1991), Mer (Graham et al. 1994), and Tyro3 (Lai et al. 1994) confirmed their segregation into a structurally distinctive family of orphan RTKs (Manning et al. 2002b). The two ligands that bind and activate the TAMs—Gas6 and Protein S (Pros1)—were identified shortly thereafter (Ohashi et al. 1995; Stitt et al. 1995; Mark et al. 1996; Nagata et al. 1996).Open in a separate windowFigure 1.TAM receptors and ligands. The TAM receptors (red) are Tyro3 (Lai and Lemke 1991; Lai et al. 1994)—also designated Brt (Fujimoto and Yamamoto 1994), Dtk (Crosier et al. 1994), Rse (Mark et al. 1994), Sky (Ohashi et al. 1994), and Tif (Dai et al. 1994); Axl (O''Bryan et al. 1991)—also designated Ark (Rescigno et al. 1991), Tyro7 (Lai and Lemke 1991), and Ufo (Janssen et al. 1991); and Mer (Graham et al. 1994)—also designated Eyk (Jia and Hanafusa 1994), Nyk (Ling and Kung 1995), and Tyro12 (Lai and Lemke 1991). The TAMs are widely expressed by cells of the mature immune, nervous, vascular, and reproductive systems. The TAM ligands (blue) are Gas6 and Protein S (Pros1). The carboxy-terminal SHBG domains of the ligands bind to the immunoglobulin (Ig) domains of the receptors, induce dimerization, and activate the TAM tyrosine kinases. When γ-carboxylated in a vitamin-K-dependent reaction, the amino-terminal Gla domains of the dimeric ligands bind to the phospholipid phosphatidylserine expressed on the surface on an apposed apoptotic cell or enveloped virus. See text for details. (From Lemke and Burstyn-Cohen 2010; adapted, with permission, from the authors.)Subsequent progress on elucidating the biological roles of the TAM receptors was considerably slower and ultimately required the derivation of mouse loss-of-function mutants (Camenisch et al. 1999; Lu et al. 1999). The fact that Tyro3^−/−, Axl^−/−, and Mer^−/− mice are all viable and fertile permitted the generation of a complete TAM mutant series that included all possible double mutants and even triple mutants that lack all three receptors (Lu et al. 1999). Remarkably, these Tyro3^−/−Axl^−/−Mer^−/− triple knockouts (TAM TKOs) are viable, and for the first 2–3 wk after birth, superficially indistinguishable from their wild-type counterparts (Lu et al. 1999). Because many RTKs play essential roles in embryonic development, even single loss-of-function mutations in RTK genes often result in an embryonic-lethal phenotype (Gassmann et al. 1995; Lee et al. 1995; Soriano 1997; Arman et al. 1998). The postnatal viability of mice in which an entire RTK family is ablated completely—the TAM TKOs can survive for more than a year (Lu et al. 1999)—is therefore highly unusual. Their viability notwithstanding, the TAM mutants go on to develop a plethora of phenotypes, some of them debilitating (Camenisch et al. 1999; Lu et al. 1999; Lu and Lemke 2001; Scott et al. 2001; Duncan et al. 2003; Prasad et al. 2006). Almost without exception, these phenotypes are degenerative in nature and reflect the loss of TAM signaling activities in adult tissues that are subject to regular challenge, renewal, and remodeling. These activities are the subject of this review.     

Molecular Bases of Cell–Cell Junctions Stability and Dynamics

Epithelial cell–cell junctions are formed by apical adherens junctions (AJs), which are composed of cadherin adhesion molecules interacting in a dynamic way with the cortical actin cytoskeleton. Regulation of cell–cell junction stability and dynamics is crucial to maintain tissue integrity and allow tissue remodeling throughout development. Actin filament turnover and organization are tightly controlled together with myosin-II activity to produce mechanical forces that drive the assembly, maintenance, and remodeling of AJs. In this review, we will discuss these three distinct stages in the lifespan of cell–cell junctions, using several developmental contexts, which illustrate how mechanical forces are generated and transmitted at junctions, and how they impact on the integrity and the remodeling of cell–cell junctions.Cell–cell junction formation and remodeling occur repeatedly throughout development. Epithelial cells are linked by apical adherens junctions (AJs) that rely on the cadherin-catenin-actin module. Cadherins, of which epithelial E-cadherin (E-cad) is the most studied, are Ca²⁺-dependent transmembrane adhesion proteins forming homophilic and heterophilic bonds in trans between adjacent cells. Cadherins and the actin cytoskeleton are mutually interdependent (Jaffe et al. 1990; Matsuzaki et al. 1990; Hirano et al. 1992; Oyama et al. 1994; Angres et al. 1996; Orsulic and Peifer 1996; Adams et al. 1998; Zhang et al. 2005; Pilot et al. 2006). This has long been attributed to direct physical interaction of E-cad with β-catenin (β-cat) and of α-catenin (α-cat) with actin filaments (for reviews, see Gumbiner 2005; Leckband and Prakasam 2006; Pokutta and Weis 2007). Recently, biochemical and protein dynamics analyses have shown that such a link may not exist and that instead, a constant shuttling of α-cat between cadherin/β-cat complexes and actin may be key to explain the dynamic aspect of cell–cell adhesion (Drees et al. 2005; Yamada et al. 2005). Regardless of the exact nature of this link, several studies show that AJs are indeed physically attached to actin and that cadherins transmit cortical forces exerted by junctional acto-myosin networks (Costa et al. 1998; Sako et al. 1998; Pettitt et al. 2003; Dawes-Hoang et al. 2005; Cavey et al. 2008; Martin et al. 2008; Rauzi et al. 2008). In addition, physical association depends in part on α-cat (Cavey et al. 2008) and additional intermediates have been proposed to represent alternative missing links (Abe and Takeichi 2008) (reviewed in Gates and Peifer 2005; Weis and Nelson 2006). Although further work is needed to address the molecular nature of cadherin/actin dynamic interactions, association with actin is crucial all throughout the lifespan of AJs. In this article, we will review our current understanding of the molecular mechanisms at work during three different developmental stages of AJs biology: assembly, stabilization, and remodeling, with special emphasis on the mechanical forces controlling AJs integrity and development.     

Glial Cell Development and Function in Zebrafish

The zebrafish is a premier vertebrate model system that offers many experimental advantages for in vivo imaging and genetic studies. This review provides an overview of glial cell types in the central and peripheral nervous system of zebrafish. We highlight some recent work that exploited the strengths of the zebrafish system to increase the understanding of the role of Gpr126 in Schwann cell myelination and illuminate the mechanisms controlling oligodendrocyte development and myelination. We also summarize similarities and differences between zebrafish radial glia and mammalian astrocytes and consider the possibility that their distinct characteristics may represent extremes in a continuum of cell identity. Finally, we focus on the emergence of zebrafish as a model for elucidating the development and function of microglia. These recent studies have highlighted the power of the zebrafish system for analyzing important aspects of glial development and function.Following the pioneering work of George Streisinger in the early 1980s, the zebrafish has emerged as a premier vertebrate model system (Streisinger et al. 1981). A key strength of the zebrafish is that the embryos and early larvae are transparent, allowing exquisite cellular analysis of many dynamic processes, including cell migration, axonal pathfinding, and myelination, among many others (e.g., Gilmour et al. 2002; Lyons et al. 2005; Czopka et al. 2013). The zebrafish also has many advantages for large-scale genetic studies, including relatively small size and rapid development, high fecundity, and the ability to manipulate the ploidy of gametes and early embryos (Kimmel 1989). Through the 1980s and early 1990s, insightful studies of several interesting mutations elegantly exploited these experimental advantages (e.g., Kimmel et al. 1989; Ho and Kane 1990; Hatta et al. 1991; Grunwald and Eisen 2002), attracting many researchers from other fields to the zebrafish system. Following the explosion of interest in the zebrafish in the 1990s, advances in many areas have added to the strengths of the system, including large-scale screens that identified thousands of new mutations (Driever et al. 1996; Haffter et al. 1996), rapid transgenesis (Kawakami et al. 2004), new methods for imaging and tracking all cells during development (Huisken 2012), genetic mapping and sequencing to identify genes and mutated loci (Postlethwait et al. 1994; Howe et al. 2013), optogenetic methods to control neural activity (Portugues et al. 2013), the advent of targeted nucleases to create mutations in genes of interest (Huang et al. 2011; Sander et al. 2011; Bedell et al. 2012; Chang et al. 2013; Hwang et al. 2013), and small molecule screening approaches to isolate compounds with novel biological activities in vivo (Peterson and Fishman 2011).Many fundamental similarities in physiology and body plan unite the zebrafish and other vertebrates (Kimmel 1989). In addition, analysis of genes and genomes has revealed that sequence, expression, and function of many genes are conserved among zebrafish and other vertebrates (Postlethwait and Talbot 1997; Howe et al. 2013). Thus, insights from studies in zebrafish will apply broadly to other vertebrates, including humans. On the other hand, there are important genetic, genomic, and physiological differences among vertebrates. It is, therefore, important to keep possible differences in mind and to recognize that analyzing the diversity among different species may enhance overall understanding of important processes. For example, zebrafish and other teleosts have a much more extensive regenerative ability than mammals, so that studies of fin, heart, and spinal cord regeneration in zebrafish may suggest avenues toward new therapeutic approaches in humans (Gemberling et al. 2013; Becker and Becker 2014).In this review, we provide an overview of different types of glia in the zebrafish, with a focus on some recent studies that highlight the power of the zebrafish system to analyze different aspects of glial development and function.     

Retrograde Traffic from the Golgi to the Endoplasmic Reticulum

Proteins to be secreted are transported from the endoplasmic reticulum (ER) to the Golgi apparatus. The transport of these proteins requires the localization and activity of proteins that create ER exit sites, coat proteins to collect cargo and to reshape the membrane into a transport container, and address labels—SNARE proteins—to target the vesicles specifically to the Golgi apparatus. In addition some proteins may need export chaperones or export receptors to enable their exit into transport vesicles. ER export factors, SNAREs, and misfolded Golgi-resident proteins must all be retrieved from the Golgi to the ER again. This retrieval is also part of the organellar homeostasis pathway essential to maintaining the identity of the ER and of the Golgi apparatus. In this review, I will discuss the different processes in retrograde transport from the Golgi to the ER and highlight the mechanistic insights we have obtained in the last couple of years.Proteins that are exposed at the plasma membrane or populate a membrane-bounded organelle are synthesized into the endoplasmic reticulum (ER). In the ER, the folding of these proteins takes place and posttranslational modifications such as N-glycosylation and disulfide bridge formation occur. Upon adopting a suitable, often correct, conformation, proteins destined to locations beyond the ER are concentrated at so-called ER exit sites (ERES) and incorporated into nascent COPII-coated vesicles. These COPII vesicles eventually bud off the ER membrane and are transported to the Golgi (in yeast, Drosophila, and C. elegans) or the ER-Golgi intermediate compartment (in mammalian cells) (Schweizer et al. 1990; Kondylis and Rabouille 2003; Spang 2009; Witte et al. 2011).It is assumed that the vesicle coat is at least partially destabilized through the hydrolysis of GTP by the small GTPase Sar1 (Oka and Nakano 1994; Springer et al. 1999). However, some of the destabilized coat components have to stay on the vesicle until it has reached the Golgi apparatus because coat components participate in the recognition and the tethering process (Barlowe 1997; Cai et al. 2007; Lord et al. 2011; Zong et al. 2012). Subsequently, SNARE proteins on the vesicles (v-SNAREs) zipper up with cognate SNAREs on the Golgi (target SNAREs, t-SNAREs) to drive membrane fusion (Hay et al. 1998; Cao and Barlowe 2000; Parlati et al. 2002). The content of the ER-derived COPII vesicles is thereby released into the lumen of the cis-cisterna of the Golgi apparatus. Most proteins will continue their journey through the Golgi apparatus and encounter further modifications such as extension of the glycosylation tree or lipidation. However, some proteins, especially those involved in the fusion process, i.e., the v-SNAREs or proteins that act as export factors of the ER, such as Vma21, which is essential for export of the correctly folded and assembled V0 sector of the V-ATPase, need to be recycled back to the ER for another round of transport (Ballensiefen et al. 1998; Malkus et al. 2004). Moreover, cis-Golgi proteins are returned to the ER for quality/functional control (Todorow et al. 2000; Sato et al. 2004; Valkova et al. 2011). Finally, some ER-resident proteins, such as the ER Hsp70 chaperone BiP/Kar2, can escape the ER, but are captured at the cis-Golgi by the H/KDEL receptor Erd2 and returned to the ER (Lewis et al. 1990; Semenza et al. 1990; Aoe et al. 1997).Unfortunately, the retrograde transport route is also hijacked by toxins. For example, endocytosed cholera toxin subunit A contains a KDEL sequence and can thereby exploit the system to access the ER (Majoul et al. 1996, 1998). From there, it is retro-translocated into the cytoplasm where it can exert its detrimental function.     

Functional Differentiation of Adult-Born Neurons along the Septotemporal Axis of the Dentate Gyrus

Over the past several decades, the proliferation and integration of adult-born neurons into existing hippocampal circuitry has been implicated in a wide range of behaviors, including novelty recognition, pattern separation, spatial learning, anxiety behaviors, and antidepressant response. In this review, we suggest that the diversity in behavioral requirements for new neurons may be partly caused by separate functional roles of individual neurogenic niches. Growing evidence shows that the hippocampal formation can be compartmentalized not only along the classic trisynaptic circuit, but also along a longitudinal septotemporal axis. We suggest that subpopulations of hippocampal adult-born neurons may be specialized for distinct mnemonic- or mood-related behavioral tasks. We will examine the literature supporting a functional and anatomical dissociation of the hippocampus along the longitudinal axis and discuss techniques to functionally dissect the roles of adult-born hippocampal neurons in these distinct subregions.Since the presence of dividing cells in the mostly postmitotic adult brain was first described (Altman and Das 1965), the generation of new neurons in adulthood has been proposed to be involved in a variety of behaviors (Doetsch and Hen 2005; Becker and Wojtowicz 2007; Sahay and Hen 2007; Deng et al. 2010; Ming and Song 2011; Miller and Hen 2014). Adult neurogenesis in the healthy mammalian brain is consistently seen in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). Recent studies have implicated hippocampal neurogenesis in learning- and memory-related tasks, such as contextual discrimination and spatial navigation and, specifically, in behavioral pattern separation (Clelland et al. 2009; Sahay et al. 2011; Nakashiba et al. 2012; Niibori et al. 2012; see also reviews in Deng et al. 2010; Ming and Song 2011; Marin-Burgin and Schinder 2012), but also in some behavioral effects of antidepressants (Santarelli et al. 2003; see also reviews in Sahay and Hen 2007; Kheirbek et al. 2012; Tanti and Belzung 2013). However, the exact role of adult hippocampal neurogenesis in some of these behaviors has been debated as some studies have shown no effects of altering adult neurogenesis on spatial navigation or antidepressant response. Proposed explanations have included differences in the behavioral tasks used to measure cognition or emotion, motivational state of subjects, species differences, or in how neurogenesis is defined, either as proliferation, survival, or differentiation (see reviews in Zhao et al. 2008; Aimone et al. 2011; Petrik et al. 2012b; Miller and Hen 2014).It must also be noted, however, that these hippocampal neurons are not born into a singular structure. Work in the past several decades has shown that the hippocampus can be divided, not only along the classic trisynaptic loop, but also longitudinally along a septotemporal axis. The septal (dorsal in rodents; posterior in primates) and temporal (ventral in rodents; anterior in primates) poles, as well as potential intermediate zones of the hippocampus, have different anatomic connections and electrophysiological properties, express a gradient of molecular markers, and play different functional roles, such as performance in spatial learning tasks and stress responses (see reviews in Moser and Moser 1998; Fanselow and Dong 2010). Consequently, adult-born neurons in the hippocampal DG may also be segregated along this longitudinal axis, and conflicting functional roles for neurogenesis may be a result of attempting to examine hippocampal neurogenesis as a unitary phenomenon. It is possible that there are intrinsic, cell-autonomous differences in adult-born neurons generated at opposite poles of the DG. An alternative, although not mutually exclusive, hypothesis is that progenitor cells are initially identical, but differentiate in a dissimilar manner as a result of integration into distinct network circuitry. We will, therefore, first discuss heterogeneity of the hippocampus along its longitudinal axis before reviewing differences in neurogenesis between the septal and temporal poles of the DG. As these topics have been reviewed extensively elsewhere (Moser and Moser 1998; Deng et al. 2010; Fanselow and Dong 2010; Koehl and Abrous 2011; Samuels and Hen 2011; Kheirbek et al. 2012; Petrik et al. 2012b), we will not try to exhaustively cover all the current literature. Rather, we attempt to gather key studies examining a septotemporal gradient of the hippocampus and hippocampal neurogenesis. We will then suggest possible approaches to examine neurogenesis in specific subregions of the hippocampal DG. Finally, a short section will examine segregation of the DG along its transverse axis.     

Cadherin-mediated Intercellular Adhesion and Signaling Cascades Involving Small GTPases

Epithelia form physical barriers that separate the internal milieu of the body from its external environment. The biogenesis of functional epithelia requires the precise coordination of many cellular processes. One of the key events in epithelial biogenesis is the establishment of cadherin-dependent cell–cell contacts, which initiate morphological changes and the formation of other adhesive structures. Cadherin-mediated adhesions generate intracellular signals that control cytoskeletal reorganization, polarity, and vesicle trafficking. Among such signaling pathways, those involving small GTPases play critical roles in epithelial biogenesis. Assembly of E-cadherin activates several small GTPases and, in turn, the activated small GTPases control the effects of E-cadherin-mediated adhesions on epithelial biogenesis. Here, we focus on small GTPase signaling at E-cadherin-mediated epithelial junctions.Cell–cell adhesions are involved in a diverse range of physiological processes, including morphological changes during tissue development, cell scattering, wound healing, and synaptogenesis (Adams and Nelson 1998; Gumbiner 2000; Halbleib and Nelson 2006; Takeichi 1995; Tepass et al. 2000). In epithelial cells, cell–cell adhesions are classified into three kinds of adhesions: adherens junction, tight junction, and desmosome (for more details, see Meng and Takeichi 2009, Furuse 2009, and Delva et al. 2009, respectively). A key event in epithelial polarization and biogenesis is the establishment of cadherin-dependent cell–cell contacts. Cadherins belong to a large family of adhesion molecules that require Ca²⁺ for their homophilic interactions (Adams and Nelson 1998; Blanpain and Fuchs 2009; Gumbiner 2000; Hartsock and Nelson 2008; Takeichi 1995; Tepass et al. 2000). Cadherins form transinteraction on the surface of neighboring cells (for details, see Shapiro and Weis 2009). For the development of strong and rigid adhesions, cadherins are clustered concomitantly with changes in the organization of the actin cytoskeleton (Tsukita et al. 1992). Classical cadherins are required, but not sufficient, to initiate cell–cell contacts, and other adhesion protein complexes subsequently assemble (for details, see Green et al. 2009). These complexes include the tight junction, which controls paracellular permeability, and desmosomes, which support the structural continuum of epithelial cells. A fundamental problem is to understand how these diverse cellular processes are regulated and coordinated. Intracellular signals, generated when cells attach with one another, mediate these complicated processes.Several signaling pathways upstream or downstream of cadherin-mediated cell–cell adhesions have been identified (Perez-Moreno et al. 2003) (see also McCrea et al. 2009). Among these pathways, small GTPases including the Rho and Ras family GTPases play critical roles in epithelial biogenesis and have been studied extensively. Many key morphological and functional changes are induced when these small GTPases act at epithelial junctions, where they mediate an interplay between cell–cell adhesion molecules and fundamental cellular processes including cytoskeletal activity, polarity, and vesicle trafficking. In addition to these small GTPases, Ca²⁺ signaling and phosphorylation of cadherin complexes also play pivotal roles in the formation and maintenance of cadherin-mediated adhesions. Here, we focus on signaling pathways involving the small GTPases in E-cadherin-mediated cell–cell adhesions. Other signaling pathways are described in recent reviews (Braga 2002; Fukata and Kaibuchi 2001; Goldstein and Macara 2007; McLachlan et al. 2007; Tsukita et al. 2008; Yap and Kovacs 2003; see also McCrea et al. 2009).     

Mediation of T-Cell Activation by Actin Meshworks

Although the actin cytoskeleton and T-cell receptor (TCR) signaling complexes are seemingly distinct molecular structures, they are tightly integrated in T cells. The signaling pathways initiated by TCRs binding to peptide MHC complexes are extensively influenced by the actin cytoskeletal activities of the motile phase before TCR signaling, the signalosome scaffolding function of the cytoskeleton, and the translocation of signaling clusters that precedes the termination of signaling at these complexes. As these three successive phases constitute essentially all the steps consequent to immune synapse formation, it has become clear that the substantial physical forces and signaling interactions generated by the actin cytoskeleton dominate the signaling life cycle of TCR signalosomes. We discuss the contributions of the actin cytoskeleton to TCR signaling phases and model some remaining questions about how specific cytoskeletal factors regulate TCR signaling outcomes.The activation of T cells is controlled primarily by T-cell receptors (TCRs) interacting with peptide-loaded major histocompatibility complexes (pMHCs) as T cells scan the surface of antigen presenting cells (APCs). Because T cells are continuously motile cells that transit through lymph nodes in their surveillance, it is clear that TCR triggering must occur within the context of physical forces that might rapidly separate TCRs from agonist pMHCs. Moreover, crawling T cells do not truly come to rest at the surfaces of APCs following TCR engagement. Instead, they continuously extend protrusions over APCs and move along the surface of their partner (Gunzer et al. 2000). In their initial encounters with antigen-bearing dendritic cells (DCs), T cells also often rapidly couple and uncouple on the order of minutes, rather than dwelling for extended periods of time on single DCs (Gunzer et al. 2000; Mempel et al. 2004). This dynamic coupling allows T cells to quickly sample a large proportion of the total APC membrane pool in search of their cognate antigen. Still, these transient contacts are productive—they induce calcium fluxes and the expression of markers of activated T cells—indicating that TCR signalosome outputs can be initiated in mere minutes and survive the dissolution of contacts, even under the mechanical stress of cytoskeletal remodeling.TCR signaling requires the dynamic recruitment of a macromolecular complex of kinases, scaffolding molecules, and other signaling effectors to a triggered TCR. Assembly of this macromolecular signaling complex must be very sensitive and occur rapidly, or there is a risk that the TCR will release the pMHC ligand, and the T cell will fail to register the antigen hit. Conversely, the signalosome assembly mechanism needs to discriminate against TCRs interacting transiently with a vast array of pMHCs presenting nonagonist peptides. Viewed in this manner, a scheme that rapidly dissociates TCRs from MHCs loaded with endogenous peptide, freeing them to rebind and test other MHCs, is desirable. It is notable that several TCR signaling factors carry binding sites for actin binding proteins or actin itself (Rozdzial et al. 1995; Zhang et al. 1999; Zeng et al. 2003; Phee et al. 2005; Gomez et al. 2006). Through these actin-associated factors, agonist-triggered TCRs rapidly assemble stabilized signaling platforms that survive mechanical disruption.In concert with adhesive integrin interactions and costimulatory receptor signaling, TCRs orchestrate a reorganization of the T-cell plasma membrane that may begin with a handful of receptors and eventually encompasses the entire contact face with the APC (some 50–100 µm²). TCRs first aggregate into micron scale clusters of TCRs, then flow to the center of the contact face, generating the central supramolecular activating complex (cSMAC) of the immune synapse (Monks et al. 1998; Grakoui et al. 1999; Krummel et al. 2000). Underscoring the importance of the cytoskeleton, the actin depolymerizing toxins latrunculin A and cytochalasin D are potent inhibitors of T-cell activation and block both TCR microcluster formation and cSMAC coalescence (Wulfing et al. 1998; Grakoui et al. 1999; Krummel et al. 2000; Varma et al. 2006). Ultimately, it is the coordination of the local interactions between receptors and effectors with the cell morphological level rearrangements that determines the nature and magnitude of T-cell responses to pathogens. Regulation of TCR signaling lifecycles and T-cell responses, therefore, falls squarely on the actin cytoskeleton.     

What Was the Real Contribution of Endosymbionts to the Eukaryotic Nucleus? Insights from Photosynthetic Eukaryotes

Eukaryotic genomes are composed of genes of different evolutionary origins. This is especially true in the case of photosynthetic eukaryotes, which, in addition to typical eukaryotic genes and genes of mitochondrial origin, also contain genes coming from the primary plastids and, in the case of secondary photosynthetic eukaryotes, many genes provided by the nuclei of red or green algal endosymbionts. Phylogenomic analyses have been applied to detect those genes and, in some cases, have led to proposing the existence of cryptic, no longer visible endosymbionts. However, detecting them is a very difficult task because, most often, those genes were acquired a long time ago and their phylogenetic signal has been heavily erased. We revisit here two examples, the putative cryptic endosymbiosis of green algae in diatoms and chromerids and of Chlamydiae in the first photosynthetic eukaryotes. We show that the evidence sustaining them has been largely overestimated, and we insist on the necessity of careful, accurate phylogenetic analyses to obtain reliable results.Today it is widely accepted that photosynthesis originated in eukaryotes by the endosymbiosis of a cyanobacterium within a heterotrophic eukaryotic host. This occurred in a lineage that subsequently diversified to give rise to the three contemporary groups of primary photosynthetic eukaryotes: Viridiplantae (including green algae and land plants), Rhodophyta and Glaucophyta, grouped collectively within a unique eukaryotic superphylum called Archaeplastida (Adl et al. 2005) or Plantae (Cavalier-Smith 1982). Recently, a second case of primary endosymbioses has been unveiled thanks to the characterization of Paulinella chromatophora, a filose amoeba that hosts a cyanobacterium with a reduced genome that has been described as “a plastid in the making” (Marin et al. 2005; Keeling and Archibald 2008; Nowack et al. 2008). Primary endosymbioses resulted in the establishment of plastids with two membranes. However, a vast variety of eukaryotes possess plastids with three or more membranes. They derive from the endosymbioses of primary photosynthetic eukaryotes within other eukaryotic cells (Delwiche 1999; Keeling 2013). Such secondary endosymbioses have spread photosynthesis across the eukaryotic tree, either by the endosymbiosis of red or of green algae. Whereas it is almost certain that secondary endosymbioses of green algae occurred twice (in euglenids and chlorarachniophytes), secondary red algal plastids are found in a variety of alveolates, stramenopiles, cryptophytes, and haptophytes, and the number of red algal endosymbioses at the origin of these groups has been matter of intense debate (Baurain et al. 2010; Keeling 2010, 2013; Burki et al. 2012b). Moreover, the existence of tertiary endosymbioses (namely, the symbiosis of a secondary photosynthetic eukaryote within another eukaryotic cell) and of plastid replacements makes the picture of plastid evolution in eukaryotes even more complex. Dinoflagellates, some of which have replaced their ancestral red algal plastids by green algae, diatoms, haptophytes, or cryptophytes, are paradigmatic examples of such complex situations (Keeling 2013).The evolution of plastids has been studied using genes from the plastid genome as well as typical eukaryotic nuclear genes, which allow inferring the phylogenies of both the plastids and their hosts. The use of those markers has led to interesting discoveries, such as the monophyly of the Archaeplastida (Moreira et al. 2000; Rodríguez-Ezpeleta et al. 2005) or the difficulties in reconciling the plastid and host histories in eukaryotes with red algal plastids (Baurain et al. 2010; Burki et al. 2012b). However, a third class of genes can also provide useful complementary information: the genes of plastid origin retrieved within the nuclear genome of the host. In fact, contemporary plastids have small genomes, which is due to the fact that most of the original cyanobacterial symbiont genes were lost or transferred to the host nucleus (by a process called endosymbiotic gene transfer, EGT) during the evolution of plastids (Weeden 1981; Martin et al. 1998). These transfer events are not restricted to plastid endosymbioses—the same phenomenon occurred during the endosymbiosis that gave rise to the mitochondria (Gray et al. 1999; Burger et al. 2003).EGT genes may serve to study the evolutionary history of plastids and, in particular, the presence of cryptic endosymbioses. In fact, species that had a plastid in the past but lost photosynthesis may have conserved genes of plastid origin in their nuclear genomes. This has been shown for a variety of nonphotosynthetic eukaryotes, such as, for example, apicomplexan parasites (Fast et al. 2001; Roos et al. 2002; Williams and Keeling 2003; Huang et al. 2004), perkinsids (Stelter et al. 2007; Matsuzaki et al. 2008; Fernández Robledo et al. 2011) or nonphotosynthetic dinoflagellates (Sanchez-Puerta et al. 2007; Slamovits and Keeling 2008), and green algae (de Koning and Keeling 2004). Although much more controversial, potential EGTs have also been used to propose a photosynthetic ancestry for ciliates (Reyes-Prieto et al. 2008) or that algae with secondary plastids of red algal origin, such as diatoms and chromerids, may have contained green algal endosymbionts in their past (Moustafa et al. 2009; Woehle et al. 2011). Likewise, several dozens of potential EGTs have been detected in algae and plants that appear to have been acquired from Chlamydiae, a group of parasitic bacteria (Huang and Gogarten 2007; Becker et al. 2008; Moustafa et al. 2008), which led to proposing that cryptic chlamydial endosymbionts may have helped to establish the first plastids, in particular, by providing essential functions for plastid activity (Greub and Raoult 2003; Ball et al. 2013; Baum 2013).We revise here some of these cases of cryptic endosymbiosis, with special attention on the difficulties in accurately detecting EGT and the importance of proper phylogenetic analysis and of an adequate taxonomic sampling to achieve that task.     

Not Just Fat: The Structure and Function of the Lipid Droplet

Lipid droplets (LDs) are independent organelles that are composed of a lipid ester core and a surface phospholipid monolayer. Recent studies have revealed many new proteins, functions, and phenomena associated with LDs. In addition, a number of diseases related to LDs are beginning to be understood at the molecular level. It is now clear that LDs are not an inert store of excess lipids but are dynamically engaged in various cellular functions, some of which are not directly related to lipid metabolism. Compared to conventional membrane organelles, there are still many uncertainties concerning the molecular architecture of LDs and how each function is placed in a structural context. Recent findings and remaining questions are discussed.Lipid droplets (LDs) were recognized as a mere deposit of lipid esters for many years. Recently, LDs have been redefined as authentic organelles that are actively engaged in multiple functions, and these have been reviewed in many recent articles (Murphy 2001; Martin et al. 2006; Ducharme et al. 2008; Fujimoto et al. 2008; Goodman 2008; Olofsson et al. 2008; Thiele et al. 2008; Murphy et al. 2009; Walther et al. 2009; Beller et al. 2010). The name, lipid droplets, implies the opposite: a static and inert nature. Alternative names have been proposed, but lipid droplet is now a generally agreed nomenclature (Martin et al. 2006).LDs are intimately related to many aspects of lipid metabolism. The lipid storage function of LDs is most conspicuous in white adipocytes, which have a gigantic unilocular LD. The regulation of the process of lipid storage and utilization has been the focus of many studies because of the prevalence of obesity, type II diabetes, and metabolic syndrome in the modern world.Besides the canonical lipid-related function, various new functions have been ascribed to LDs more recently, including some that do not appear to be directly related to lipid metabolism. However, both the canonical and noncanonical functions of LDs, are rooted in the unique architecture of LDs. In contrast to other organelles that have aqueous content within a phospholipid bilayer membrane, the basic structure of LDs is thought to be a mass of lipid esters covered by a phospholipid monolayer (Murphy et al. 1999; Tauchi-Sato et al. 2002). In this article, we first review what is known about LD structure and then discuss various functions. We review this information by considering the extent to which the functions can be understood based on the current structural paradigm.     

Cell Adhesion, the Backbone of the Synapse: ��Vertebrate�� and ��Invertebrate�� Perspectives

Synapses are asymmetric intercellular junctions that mediate neuronal communication. The number, type, and connectivity patterns of synapses determine the formation, maintenance, and function of neural circuitries. The complexity and specificity of synaptogenesis relies upon modulation of adhesive properties, which regulate contact initiation, synapse formation, maturation, and functional plasticity. Disruption of adhesion may result in structural and functional imbalance that may lead to neurodevelopmental diseases, such as autism, or neurodegeneration, such as Alzheimer''s disease. Therefore, understanding the roles of different adhesion protein families in synapse formation is crucial for unraveling the biology of neuronal circuit formation, as well as the pathogenesis of some brain disorders. The present review summarizes some of the knowledge that has been acquired in vertebrate and invertebrate genetic model organisms.Synapses are asymmetric, intercellular junctions that are the basic structural units of neuronal transmission. The correct development of synaptic specializations and the establishment of appropriate connectivity patterns are crucial for the assembly of functional neuronal circuits. Improper synapse formation and function may cause neurodevelopmental disorders, such as mental retardation (MsR) and autism spectrum disorders (ASD) (McAllister 2007; Sudhof 2008), and likely play a role in neurodegenerative disorders, such as Alzheimer''s disease (AD) (Haass and Selkoe 2007).At chemical synapses (reviewed in Sudhof 2004; Zhai and Bellen 2004; Waites et al. 2005; McAllister 2007; Jin and Garner 2008), the presynaptic compartment contains synaptic vesicles (SV), organized in functionally distinct subcellular pools. A subset of SVs docks to the presynaptic membrane around protein-dense release sites, named active zones (AZ). Upon the arrival of an action potential at the terminal, the docked and “primed” SVs fuse with the plasma membrane and release neurotransmitter molecules into the synaptic cleft. Depending on the type of synapse (i.e., excitatory vs. inhibitory synapses), neurotransmitters ultimately activate an appropriate set of postsynaptic receptors that are accurately apposed to the AZ.Synapse formation occurs in several steps (Fig. 1) (reviewed in Eaton and Davis 2003; Goda and Davis 2003; Waites et al. 2005; Garner et al. 2006; Gerrow and El-Husseini 2006; McAllister 2007). Spatiotemporal signals guide axons through heterogeneous cellular environments to contact appropriate postsynaptic targets. At their destination, axonal growth cones initiate synaptogenesis through adhesive interactions with target cells. In the mammalian central nervous system (CNS), immature postsynaptic dendritic spines initially protrude as thin, actin-rich filopodia on the surface of dendrites. Similarly, at the Drosophila neuromuscular junction (NMJ), myopodia develop from the muscles (Ritzenthaler et al. 2000). The stabilization of intercellular contacts and their elaboration into mature, functional synapses involves cytoskeletal arrangements and recruitment of pre- and postsynaptic components to contact sites in spines and boutons. Conversely, retraction of contacts results in synaptic elimination. Both stabilization and retraction sculpt a functional neuronal circuitry.Open in a separate windowFigure 1.(A–C) Different stages of synapse formation. (A) Target selection, (B) Synapse assembly, (C) Synapse maturation and stabilization. (D–F) The role of cell adhesion molecules in synapse formation is exemplified by the paradigm of N-cadherin and catenins in regulation of the morphology and strength of dendritic spine heads. (D) At an early stage the dendritic spines are elongated from motile structures “seeking” their synaptic partners. (E) The contacts between the presynaptic and postsynaptic compartments are stabilized by recruitment of additional cell adhesion molecules. Adhesional interactions activate downstream pathways that remodel the cytoskeleton and organize pre- and postsynaptic apparatuses. (F) Cell adhesion complexes, stabilized by increased synaptic activity, promote the expansion of the dendritic spine head and the maturation/ stabilization of the synapse. Retraction and expansion is dependent on synaptic plasticity.In addition to the plastic nature of synapse formation, the vast heterogeneity of synapses (in terms of target selection, morphology, and type of neurotransmitter released) greatly enhances the complexity of synaptogenesis (reviewed in Craig and Boudin 2001; Craig et al. 2006; Gerrow and El-Husseini 2006). The complexity and specificity of synaptogenesis relies upon the modulation of adhesion between the pre- and postsynaptic components (reviewed in Craig et al. 2006; Gerrow and El-Husseini 2006; Piechotta et al. 2006; Dalva et al. 2007; Shapiro et al. 2007; Yamada and Nelson 2007; Gottmann 2008). Cell adhesive interactions enable cell–cell recognition via extracellular domains and also mediate intracellular signaling cascades that affect synapse morphology and organize scaffolding complexes. Thus, cell adhesion molecules (CAMs) coordinate multiple synaptogenic steps.However, in vitro and in vivo studies of vertebrate CAMs are often at odds with each other. Indeed, there are no examples of mutants for synaptic CAMs that exhibit prominent defects in synapse formation. This apparent “resilience” of synapses is probably caused by functional redundancy or compensatory effects among different CAMs (Piechotta et al. 2006). Hence, studies using simpler organisms less riddled by redundancy, such as Caenorhabditis elegans and Drosophila, have aided in our understanding of the role that these molecules play in organizing synapses.In this survey, we discuss the roles of the best characterized CAM families of proteins involved in synaptogenesis. Our focus is to highlight the complex principles that govern the molecular basis of synapse formation and function from a comparative perspective. We will present results from cell culture studies as well as in vivo analyses in vertebrate systems and refer to invertebrate studies, mainly performed in Drosophila and C. elegans, when they have provided important insights into the role of particular CAM protein families. However, we do not discuss secreted factors, for which we refer the reader to numerous excellent reviews (as for example Washbourne et al. 2004; Salinas 2005; Piechotta et al. 2006; Shapiro et al. 2006; Dalva 2007; Yamada and Nelson 2007; Biederer and Stagi 2008; Salinas and Zou 2008).     

Diversity of Neural Precursors in the Adult Mammalian Brain

Aided by advances in technology, recent studies of neural precursor identity and regulation have revealed various cell types as contributors to ongoing cell genesis in the adult mammalian brain. Here, we use stem-cell biology as a framework to highlight the diversity of adult neural precursor populations and emphasize their hierarchy, organization, and plasticity under physiological and pathological conditions.The adult mammalian brain displays remarkable structural plasticity by generating and incorporating new neural cell types into an already formed brain (Kempermann and Gage 1999). Largely restricted within the subventricular zone (SVZ) along the lateral ventricle and the subgranular zone (SGZ) in the dentate gyrus (DG), neural genesis is thought to arise from neural stem cells (NSCs) (Ming and Song 2011). Stem cells are defined by hallmark functions: capacity to self-renew, maintenance of an immature state over a long duration, and ability to generate specialized cell types (Fig. 1). These features distinguish stem cells from committed progenitor cells that more readily differentiate into specialized cell types (Fig. 1). Stem and progenitor cells (collectively called precursors) are additionally characterized by their lineage capacity. For example, multipotential neural precursors generate neurons and glia, whereas unipotential cells produce only one cell type, such as neurons (Gage 2000; Ma et al. 2009). The classical NSC definition is based on cell culture experiments in which a single cell can self-renew and generate neurons, astrocytes, and oligodendrocytes (Gage 2000; Ma et al. 2009). Yet, reprogramming studies have raised the question of whether cultured lineage-restricted neural progenitors acquire additional potential not evident in vivo (Palmer et al. 1999; Kondo and Raff 2000; Gabay et al. 2003). As a result, various lineage models have been proposed to explain cell generation in the adult brain (Fig. 1) (Ming and Song 2011). In one model, bona fide adult stem cells generate multiple lineages at the individual cell level. In another, cell genesis represents a collective property from a mixed population of unipotent progenitors. Importantly, these models are not mutually exclusive as evidence for the coexistence of multiple precursors has been observed in several adult somatic tissues, in which one population preferentially maintains homeostasis and another serves as a cellular reserve (Li and Clevers 2010; Mascre et al. 2012). Recent technical advances, including single-cell lineage tracing (Kretzschmar and Watt 2012), have made it possible to dissect basic cellular and behavioral processes of neural precursors in vivo (Fig. 4) (Bonaguidi et al. 2012). In this work, we review our current knowledge of precursor cell identity, hierarchical organization, and regulation to examine the diverse origins of cell genesis in the adult mammalian brain.Open in a separate windowFigure 1.Models of generating cell diversity in the adult tissues. (A,B) Definitions of stem and progenitor cells. In A, quiescent stem cells (S_q) become active stem cells (S_a) that proliferate to generate different types of specialized cells (C₁, C₂, C₃) and new stem cells (S). The active stem cell can return to quiescence and remain quiescent over long periods of time. In B, lineage-restricted progenitor cells lacking self-renewal capacity (P₁, P₂, P₃) each give rise to distinct populations of specialized cells (C₁, C₂, C₃). (C) Generation of specialized cells in a tissue could be explained by three models. (1) The stem-cell model, in which multipotent stem cells give rise to all the specialized cells in the tissue. (2) The progenitor cell model, in which diverse, lineage-restricted progenitor cells give rise to different cell types in the tissue. (3) A hybrid model, in which a mixture of stem cells and lineage-restricted progenitor cells generate specialized cells of the adult tissue.

Table 1.

Comparison of different methods used to study the generation of new cells in the adult mammalian nervous system

Quality Control of Mitochondrial Proteostasis

A decline in mitochondrial activity has been associated with aging and is a hallmark of many neurological diseases. Surveillance mechanisms acting at the molecular, organellar, and cellular level monitor mitochondrial integrity and ensure the maintenance of mitochondrial proteostasis. Here we will review the central role of mitochondrial chaperones and proteases, the cytosolic ubiquitin-proteasome system, and the mitochondrial unfolded response in this interconnected quality control network, highlighting the dual function of some proteases in protein quality control within the organelle and for the regulation of mitochondrial fusion and mitophagy.In all cellular compartments, correct protein folding is critical to maintain cellular homeostasis. In cases where proteins become misfolded or damaged, it is imperative that they are turned over and removed to prevent the formation of toxic folding intermediates or the accumulation of aggregates to levels that can be deleterious for the cell. Several neurodegenerative diseases share a common pathogenic mechanism, which involves the formation of fibrillar aggregates of a particular protein that can accumulate in the cytosol, the nucleus, or the mitochondria. Examples of this include accumulation of the amyloid-β peptide in Alzheimer’s disease (Kayed et al. 2003; Tanzi and Bertram 2005), accumulation of α-synuclein in Parkinson’s disease (Spillantini et al. 1997; Zarranz et al. 2004), and aggregation of a mutant form of the huntingtin protein caused by extended polyglutamine stretches in Huntington’s disease (DiFiglia et al. 1997). Although the exact mechanism of pathogenesis for these diseases remains unresolved, mitochondrial dysfunction is implicated in their progression, which may in turn be responsible for the loss of neurological cell populations because of their sensitivity and requirement for functional mitochondria (Rodolfo et al. 2010).The evolution of mitochondria began approximately 1.5 billion years ago after an α-proteobacterium was engulfed by a preeukaryotic cell (Gray et al. 1999). Since that time, mitochondria have retained two phospholipid bilayers that segregate two aqueous compartments, the mitochondrial intermembrane space (IMS) and the mitochondrial matrix (Palade 1953). Mitochondria are found in essentially all eukaryotic cells and play integral roles in a number of the cell''s metabolic pathways. For example, mitochondria are the key players in cellular ATP production through an elaborate respiratory chain network found in the organelles inner membrane (IM) (Mitchell 1961; Leonard and Schapira 2000). Mitochondria are also required for the β-oxidation of fatty acids, Fe-S biosynthesis, and Ca²⁺ homeostasis (Pinton et al. 1998; Rizzuto et al. 2000; Lill 2009; Modre-Osprian et al. 2009). Moreover, mitochondria are key regulators of programmed cell death and they participate in developmental processes as well as aging (Singh 2004; Green 2005).In contrast to early depictions of mitochondria as singular kidney bean shaped entities, it is now well established that mitochondria form elaborate, reticular networks in many tissues (Bereiter-Hahn 1990). The ability of mitochondria to form such networks arises from two major factors: (1) Specialized machineries in the mitochondrial outer membrane (OM) and the IM allow mitochondria to fuse and divide and (2) mitochondria are able to be shuttled along cytoskeletal elements (Anesti and Scorrano 2006; Hoppins et al. 2007). This plasticity of mitochondria ensures that they are able to respond to different cellular cues, which is potentially important for their numerous functions. In different cell types, mitochondria adopt varying morphologies (Kuznetsov et al. 2009). For example, in cultured fibroblasts mitochondria form extensive reticular networks, whereas in neuronal cells, mitochondria can be found enriched at areas of high-energy demand, including presynaptic termini, axon initial segments, and growth cones. Furthermore, in muscle cells, mitochondria adopt a very uniform intermyofibrillar conformation (Vendelin et al. 2005). The dynamic nature of mitochondria provides an explanation as to how they adopt varying organizations in different cell populations. The importance of mitochondrial networks is highlighted by the fact that mutations in components involved in maintaining mitochondrial dynamics results in neurodegenerative diseases (Chan 2006; Olichon et al. 2006; Knott et al. 2008; Martinelli and Rugarli 2010; Winklhofer and Haass 2010).     

Unconventional Functions for Clathrin,ESCRTs, and Other Endocytic Regulators in the Cytoskeleton,Cell Cycle,Nucleus, and Beyond: Links to Human Disease

The roles of clathrin, its regulators, and the ESCRT (endosomal sorting complex required for transport) proteins are well defined in endocytosis. These proteins can also participate in intracellular pathways that are independent of endocytosis and even independent of the membrane trafficking function of these proteins. These nonendocytic functions involve unconventional biochemical interactions for some endocytic regulators, but can also exploit known interactions for nonendocytic functions. The molecular basis for the involvement of endocytic regulators in unconventional functions that influence the cytoskeleton, cell cycle, signaling, and gene regulation are described here. Through these additional functions, endocytic regulators participate in pathways that affect infection, glucose metabolism, development, and cellular transformation, expanding their significance in human health and disease.The discovery and characterization of clathrin (Pearse 1975) initiated molecular definition of the many endocytosis regulators described in this collection, which mediate the clathrin-dependent and -independent pathways for membrane internalization (see Kirchhausen et al. 2014; Mayor et al. 2014; Merrifield and Kaksonen 2014). In accompanying reviews, we have seen how these endocytic pathways influence nutrition and metabolism (see Antonescu et al. 2014), signal transduction (see Bökel and Brand 2014; Di Fiore and von Zastrow 2014), neuronal function (see Morgan et al. 2013; Cosker and Segal 2014), infection and immunity (see ten Broeke et al. 2013; Cossart and Helenius 2014), tissue polarity and development (see Eaton and Martin-Belmonte 2014; Gonzalez-Gaitan and Jülicher 2014), and migration and metastasis (see Mellman and Yarden 2013). Recently, it has been established that some endocytic regulators have molecular properties that expand their functions beyond endocytosis. These include molecular interactions that affect the microtubule and actin cytoskeletons, nuclear translocation that influences gene regulation, and the formation of membrane-associated scaffolds that serve as signaling and sorting platforms. Through these diverse nonendocytic functions, endocytosis regulators play additional roles in cell division, pathogen infection, cell adhesion, and oncogenesis. In this article, we review the nonconventional behavior of endocytic regulators, first discussing the molecular properties that enable their moonlighting functions and then discussing the cellular processes and disease states that are influenced by these functions.