Wnt Signaling in Vertebrate Axis Specification

The Wnt pathway is a major embryonic signaling pathway that controls cell proliferation, cell fate, and body-axis determination in vertebrate embryos. Soon after egg fertilization, Wnt pathway components play a role in microtubule-dependent dorsoventral axis specification. Later in embryogenesis, another conserved function of the pathway is to specify the anteroposterior axis. The dual role of Wnt signaling in Xenopus and zebrafish embryos is regulated at different developmental stages by distinct sets of Wnt target genes. This review highlights recent progress in the discrimination of different signaling branches and the identification of specific pathway targets during vertebrate axial development.Wnt pathways play major roles in cell-fate specification, proliferation and differentiation, cell polarity, and morphogenesis (Clevers 2006; van Amerongen and Nusse 2009). Signaling is initiated in the responding cell by the interaction of Wnt ligands with different receptors and coreceptors, including Frizzled, LRP5/6, ROR1/2, RYK, PTK7, and proteoglycans (Angers and Moon 2009; Kikuchi et al. 2009; MacDonald et al. 2009). Receptor activation is accompanied by the phosphorylation of Dishev-elled (Yanagawa et al. 1995), which appears to transduce the signal to both the cell membrane and the nucleus (Cliffe et al. 2003; Itoh et al. 2005; Bilic et al. 2007). Another common pathway component is β-catenin, an abundant component of adherens junctions (Nelson and Nusse 2004; Grigoryan et al. 2008). In response to signaling, β-catenin associates with T-cell factors (TCFs) and translocates to the nucleus to stimulate Wnt target gene expression (Behrens et al. 1996; Huber et al. 1996; Molenaar et al. 1996).This β-catenin-dependent activation of specific genes is often referred to as the “canonical” pathway. In the absence of Wnt signaling, β-catenin is destroyed by the protein complex that includes Axin, GSK3, and the tumor suppressor APC (Clevers 2006; MacDonald et al. 2009). Wnt proteins, such as Wnt1, Wnt3, and Wnt8, stimulate Frizzled and LRP5/6 receptors to inactivate this β-catenin destruction complex, and, at the same time, trigger the phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2 (HIPK2) (Hikasa et al. 2010; Hikasa and Sokol 2011). Both β-catenin stabilization and the regulation of TCF protein function by phosphorylation appear to represent general strategies that are conserved in multiple systems (Sokol 2011). Thus, the signaling pathway consists of two branches that together regulate target gene expression (Fig. 1).Open in a separate windowFigure 1.Conserved Wnt pathway branches and components. In the absence of Wnt signals, glycogen synthase kinase 3 (GSK3) binds Axin and APC to form the β-catenin destruction complex. Some Wnt proteins, such as Wnt8 and Wnt3a, stimulate Frizzled and LRP5/6 receptors to inhibit GSK3 activity and stabilize β-catenin (β-cat). Stabilized β-cat forms a complex with T-cell factors (e.g., TCF1/LEF1) to activate target genes. Moreover, GSK3 inhibition leads to target gene derepression by promoting TCF3 phosphorylation by homeodomain-interacting protein kinase 2 (HIPK2) through an unknown mechanism, for which β-catenin is required as a scaffold. This phosphorylation results in TCF3 removal from target promoters and gene activation. Other Wnt proteins, such as Wnt5a and Wnt11, use distinct receptors such as ROR2 and RYK, in addition to Frizzled, to control the the cytoskeletal organization through core planar cell polarity (PCP) proteins, small GTPases (Rho/Rac/Cdc42), and c-Jun amino-terminal kinase (JNK).Other Wnt proteins, such as Wnt5a or Wnt11, strongly affect the cytoskeletal organization and morphogenesis without stabilizing β-catenin (Torres et al. 1996; Angers and Moon 2009; Wu and Mlodzik 2009). These “noncanonical” ligands do not influence TCF3 phosphorylation (Hikasa and Sokol 2011), but may use distinct receptors such as ROR1/2 and RYK instead of or in addition to Frizzled (Hikasa et al. 2002; Lu et al. 2004; Mikels and Nusse 2006; Nishita et al. 2006, 2010; Schambony and Wedlich 2007; Grumolato et al. 2010; Lin et al. 2010; Gao et al. 2011). In such cases, signaling mechanisms are likely to include planar cell polarity (PCP) components, such as Vangl2, Flamingo, Prickle, Diversin, Rho GTPases, and c-Jun amino-terminal kinases (JNKs), which do not directly affect β-catenin stability (Fig. 1) (Sokol 2000; Schwarz-Romond et al. 2002; Schambony and Wedlich 2007; Komiya and Habas 2008; Axelrod 2009; Itoh et al. 2009; Tada and Kai 2009; Sato et al. 2010; Gao et al. 2011). This simplistic dichotomy of the Wnt pathway does not preclude some Wnt ligands from using both β-catenin-dependent and -independent routes in a context-specific manner.Despite the existence of many pathway branches, only the β-catenin-dependent branch has been implicated in body-axis specification. Recent experiments in lower vertebrates have identified additional pathway components and targets and provided new insights into the underlying mechanisms.     

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

Gap Junctions

Gap junctions are aggregates of intercellular channels that permit direct cell–cell transfer of ions and small molecules. Initially described as low-resistance ion pathways joining excitable cells (nerve and muscle), gap junctions are found joining virtually all cells in solid tissues. Their long evolutionary history has permitted adaptation of gap-junctional intercellular communication to a variety of functions, with multiple regulatory mechanisms. Gap-junctional channels are composed of hexamers of medium-sized families of integral proteins: connexins in chordates and innexins in precordates. The functions of gap junctions have been explored by studying mutations in flies, worms, and humans, and targeted gene disruption in mice. These studies have revealed a wide diversity of function in tissue and organ biology.Gap junctions are clusters of intercellular channels that allow direct diffusion of ions and small molecules between adjacent cells. The intercellular channels are formed by head-to-head docking of hexameric assemblies (connexons) of tetraspan integral membrane proteins, the connexins (Cx) (Goodenough et al. 1996). These channels cluster into polymorphic maculae or plaques containing a few to thousands of units (Fig. 1). The close membrane apposition required to allow the docking between connexons sterically excludes most other membrane proteins, leaving a narrow ∼2 nm extracellular “gap” for which the junction is named (Fig. 2). Gap junctions in prechordates are composed of innexins (Phelan et al. 1998; Phelan 2005). In chordates, connexins arose by convergent evolution (Alexopoulos et al. 2004), to expand by gene duplication (Cruciani and Mikalsen 2007) into a 21-member gene family. Three innexin-related proteins, called pannexins, have persisted in vertebrates, although it is not clear if they form intercellular channels (Panchin et al. 2000; Bruzzone et al. 2003). 7Å-resolution electron crystallographic structures of intercellular channels composed of either a carboxy-terminal truncation of Cx43 (Unger et al. 1999; Yeager and Harris 2007) or an M34A mutant of Cx26 (Oshima et al. 2007) are available. The overall pore morphologies are similar with the exception of a “plug” in the Cx26 channel pore. The density of this plug is substantively decreased by deletion of amino acids 2–7, suggesting that the amino-terminus contributes to this structure (Oshima et al. 2008). A 3.5-Å X-ray crystallographic structure has visualized the amino-terminus of Cx26 folded into the mouth of the channel without forming a plug, thought to be an image of the open channel conformation (Maeda et al. 2009). The amino-terminus has been physiologically implicated in voltage-gating of the Cx26 and Cx32 channels (Purnick et al. 2000; Oh et al. 2004), lending support to a role for the amino-terminus as a gating structure. However, Cx43 also shows voltage-gating, and its lack of any structure resembling a plug remains unresolved. A comparison of a 1985 intercellular channel structure (Makowski 1985) with the 2009 3.5Å structure (Maeda et al. 2009) summarizes a quarter-century of X-ray progress (Fig. 3).Open in a separate windowFigure 1.A diagram showing the multiple levels of gap junction structure. Individual connexins assemble intracellularly into hexamers, called connexons, which then traffic to the cell surface. There, they dock with connexons in an adjacent cell, assembling an axial channel spanning two plasma membranes and a narrow extracellular “gap.”Open in a separate windowFigure 2.Electron microscopy of gap junctions joining adjacent hepatocytes in the mouse. The gap junction (GJ) is seen as an area of close plasma membrane apposition, clearly distinct from the tight junction (TJ) joining these cells. (Inset A) A high magnification view of the gap junction revealing the 2–3 nm “gap” (white arrows) separating the plasma membranes. (Inset B) A freeze-fracture replica of a gap junction showing the characteristic particles on the protoplasmic (P) fracture face and pits on the ectoplasmic (E) fracture face. The particles and pits show considerable disorder in their packing with an average 9-nm center-to-center spacing.Open in a separate windowFigure 3.A comparison of axial sections through gap-junction structures deduced from X-ray diffraction. The 1985 data (Makowski 1985) were acquired from gap junctions isolated biochemically from mouse liver containing mixtures of Cx32 and Cx26. The intercellular channel (CHANNEL) is blocked at the two cytoplasmic surfaces by electron density at the channel mouths along the sixfold symmetry axis. The 2009 data (Maeda et al. 2009), acquired from three-dimensional crystals of recombinant Cx26, resolve this density at the channel opening as the amino-termini of the connexin proteins, the 2009 model possibly showing an open channel structure.Most cells express multiple connexins. These may co-oligomerize into the same (homomeric) or mixed (heteromeric) connexons, although only certain combinations are permitted (Falk et al. 1997; Segretain and Falk 2004). A connexon may dock with an identical connexon to form a homotypic intercellular channel or with a connexon containing different connexins to form a heterotypic channel (Dedek et al. 2006). Although only some assembly combinations are permitted (White et al. 1994), the number of possible different intercellular channels formed by this 21-member family is astonishingly large. This diversity has significance because intercellular channels composed of different connexins have different physiological properties, including single-channel conductances and multiple conductance states (Takens-Kwak and Jongsma 1992), as well as permeabilities to experimental tracers (Elfgang et al. 1995) and to biologically relevant permeants (Gaunt and Subak-Sharpe 1979; Veenstra et al. 1995; Bevans et al. 1998; Gong and Nicholson 2001; Goldberg et al. 2002; Ayad et al. 2006; Harris 2007).Opening of extrajunctional connexons in the plasma membrane, described as “hemichannel” activity, can be experimentally induced in a variety of cell types. Because first observations of hemichannel activity were in an oocyte expression system (Paul et al. 1991) and dissociated retinal horizontal cells (DeVries and Schwartz 1992), the possible functions of hemichannels composed of connexins and pannexins has enjoyed vigorous investigation (Goodenough and Paul 2003; Bennett et al. 2003; Locovei et al. 2006; Evans et al. 2006; Srinivas et al. 2007; Schenk et al. 2008; Thompson and MacVicar 2008; Anselmi et al. 2008; Goodenough and Paul 2003). Hemichannels have been implicated in various forms of paracrine signaling, for example in providing a pathway for extracellular release of ATP (Cotrina et al. 1998; Kang et al. 2008), glutamate (Ye et al. 2003), NAD⁺ (Bruzzone et al. 2000), and prostaglandins (Jiang and Cherian 2003).     

The Desmosome

Desmosomes are intercellular junctions that tether intermediate filaments to the plasma membrane. Desmogleins and desmocollins, members of the cadherin superfamily, mediate adhesion at desmosomes. Cytoplasmic components of the desmosome associate with the desmosomal cadherin tails through a series of protein interactions, which serve to recruit intermediate filaments to sites of desmosome assembly. These desmosomal plaque components include plakoglobin and the plakophilins, members of the armadillo gene family. Linkage to the cytoskeleton is mediated by the intermediate filament binding protein, desmoplakin, which associates with both plakoglobin and plakophilins. Although desmosomes are critical for maintaining stable cell–cell adhesion, emerging evidence indicates that they are also dynamic structures that contribute to cellular processes beyond that of cell adhesion. This article outlines the structure and function of the major desmosomal proteins, and explores the contributions of this protein complex to tissue architecture and morphogenesis.The desmosome is an adhesive intercellular junction that is crucial to tissues that experience mechanical stress, such as the myocardium, bladder, gastrointestinal mucosa, and skin (Getsios et al. 2004b; Holthofer et al. 2007). The desmosome was first observed in the spinous layer of epidermis by the Italian pathologist Giulio Bizzozero (1846–1901). Bizzozero''s observations of these small dense nodules, subsequently named “nodes of Bizzozero,” led him to the insightful interpretation of these structures as adhesive cell–cell contact points. The term desmosome was later coined by Josef Schaffer in 1920 and is derived from the Greek words “desmo,” meaning bond or fastening, and “soma,” meaning body (Wells 2005; Calkins and Setzer 2007). The introduction of electron microscopy yielded a series of advances by Porter, Odland, and Kelly in the 1950s and 1960s, which revealed desmosome organization at the ultrastructural level. These studies and others indicated that the desmosome can be divided into three morphologically identifiable zones: the extracellular core region (desmoglea), the outer dense plaque (ODP), and the inner dense plaque (IDP) (Fig. 1A) (Kowalczyk et al. 1994; Schmidt et al. 1994; Green and Jones 1996; North et al. 1999; Garrod and Chidgey 2008).Open in a separate windowFigure 1.A model for the structure of desmosomes. (A) Electron micrograph of a desmosome. (B) Schematic of desmosomal proteins and relative distance from the plasma membrane (PM). The desmosomal cadherins, the desmogleins and desmocollins, extend into extracellular core and outer dense plaque (ODP) to establish contact and adhere to neighboring cells in a Ca²⁺-dependent manner. The cadherin cytoplasmic tails associate linker proteins, plakoglobin (PG), the plakophilins (PKP), and desmoplakin (DP). DP binds to keratin intermediate filaments (KIF) within the inner dense plaque (IDP), serving to tether the intermediate filaments to the plasma membrane. (Adapted with permission from Kottke et al. 2006.)In the mid 1970s, Skerrow and Matoltsy (Skerrow and Matoltsy 1974a; Skerrow and Matoltsy 1974b) advanced the field by isolating desmosomes using biochemical approaches (Bass-Zubek and Green 2007).These landmark studies provided a foundation for the Franke and Steinberg laboratories to characterize the transmembrane glycoproteins and cytoplasmic plaque proteins that linked the structure to the intermediate filament cytoskeleton, and to develop immunological tools for localizing specific components (Franke et al. 1981; Kapprell et al. 1985; Steinberg et al. 1987). Collectively, these and other studies shaped our current view of how desmosomal components are organized.The transmembrane glycoproteins, termed desmogleins and desmocollins (Garrod and Chidgey 2008), represent separate subfamilies of the cadherin superfamily of calcium dependent adhesion molecules. The extracellular domains of the desmogleins and desmocollins mediate adhesion, whereas the cytoplasmic tails of these cadherins associate with the desmosomal plaque proteins. The outer dense plaque consists of the cytoplasmic tails of the desmosomal cadherins, which bind to members of the armadillo and plakin family of linker proteins (Kowalczyk et al. 1994; Getsios et al. 2004b; Garrod and Chidgey 2008). Plakoglobin, a member of the armadillo family, binds directly to the cytoplasmic tails of both the desmogleins and the desmocollins (Wahl et al. 1996; Witcher et al. 1996). Desmoplakin, a member of the plakin family, interacts with both plakoglobin and another subgroup of armadillo family proteins, the plakophilins (Cowin and Burke 1996). Finally, the interaction between desmoplakin and the keratin filaments forms the inner dense plaque, tethering the cytoskeletal network to the adhesion complex (Fig. 1B) (Kowalczyk et al. 1994; Getsios et al. 2004b; Garrod and Chidgey 2008).The following sections of this article describe the structural and functional characteristics of the major desmosomal proteins. In addition, we discuss differences in tissue expression patterns of desmosomal proteins and the role of desmosomes in human disease. A comprehensive review of additional proteins found to regulate or associate with desmosomes is provided elsewhere (Holthofer et al. 2007) and discussion of desmosome dynamics is provided in Green et al. 2009.     

Central Role of RET in Thyroid Cancer

RET (rearranged during transfection) is a receptor tyrosine kinase involved in the development of neural crest derived cell lineages, kidney, and male germ cells. Different human cancers, including papillary and medullary thyroid carcinomas, lung adenocarcinomas, and myeloproliferative disorders display gain-of-function mutations in RET. Accordingly, RET protein has become a promising molecular target for cancer treatment.The human RET (rearranged during transfection) gene maps on 10q11.2 and is composed of 21 exons spanning a region of 55,000 bp. It encodes a single-pass trans-membrane protein, RET, that belongs to the receptor tyrosine kinase (RTK) family (Pasini et al. 1995). The RET extracellular segment contains four cadherin-like domains, followed by a domain containing cysteine residues involved in the formation of intramolecular disulfide bonds (Fig. 1A) (Anders et al. 2001; Airaksinen and Saarma 2002). RET protein is highly glycosylated and N-glycosylation is necessary for its transport to the cell surface. Only the fully mature glycosylated 170 kDa RET protein isoform is exposed to the extracellular compartment, whereas the mannose-rich 150 kDa isoform is confined to the Golgi (Takahashi et al. 1993; Carlomagno et al. 1996). The transmembrane segment is composed of 22 amino acids, among which S649 and S653 mediate self-association and dimerization of RET, possibly via formation of inter-molecular hydrogen bonding (Kjaer et al. 2006). The intracellular portion of RET contains the tyrosine kinase domain split into two subdomains by the insertion of 27 amino acids. The RET COOH-terminal tail varies in length as a result of alternative splicing of the 3′ end (carboxy terminal with respect to glycine 1063), generating three different isoforms that contain 9 (RET9), 43 (RET43), or 51 (RET51) amino acids (Myers et al. 1995). RET9 and RET51 are the most abundant isoforms, and they activate similar signaling pathways through interaction with diverse protein complexes, and may exert a differential role in development (Fig. 1A) (de Graaff et al. 2001).Open in a separate windowFigure 1.Illustration of the mechanisms of activation of wild-type (wt) RET and RET-derived oncoproteins. (A) Wild-type RET activation is mediated by ligand (GFL)-induced dimerization; ligand binding to RET is not direct and mediated by GFR-α coreceptors (not shown); major RET autophosphorylation sites and downstream signaling pathways are indicated. RET extracellular cadherin-like domains are represented in red. The split intracellular RET tyrosine kinase domain, as well as the three alternative carboxy-terminal RET tails, are also depicted. (B) RET/PTC activation is mediated by coiled-coil-induced dimerization (left); activation of RET cysteine mutants associated with MEN2A or FMTC is mediated by disulfide bonds-mediated dimerization (right).RET shows several autophosphorylation sites (Fig. 1A) (Liu et al. 1996; Kawamoto et al. 2004). RET tyrosine 1062 (Y1062) functions as a multidocking site for signaling molecules containing a phosphotyrosine-binding (PTB) domain (Asai et al. 1996). Phospho-Y1062 binding proteins include SHC, N-SHC (RAI), FRS2, IRS1/2, DOK1, and DOK4/5 that, in turn, contribute to the activation of RAS-MAPK (mitogen-activated protein kinases) and PI3K (phosphatidyl inositol 3 kinase)-AKT pathways. Y1096, specific to the RET51 splicing variant, couples to the PI3K-AKT and RAS-MAPK pathways, as well. These signaling cascades mediate RET-dependent cell survival, proliferation, and motility (Alberti et al. 1998; Murakami et al. 1999; Segouffin-Cariou and Billaud 2000; Melillo et al. 2001a,b; Schuetz et al. 2004). Y905 is located in the activation loop of the RET kinase and its phosphorylation is associated with RET kinase activation (Knowles et al. 2006). Finally, Y981 and Y1015 have been shown to be coupled to important signaling molecules such as SRC and PLC-γ, respectively (Borrello et al. 1996; Encinas et al. 2004).RET is the receptor for a group of neurotrophic growth factors that belong to the glial cell line-derived neurotrophic factor (GDNF) family (GFLs, GDNF family ligands), namely, GDNF, Neurturin (NRT), Artemin (ART), and Persephin (PSF) (Airaksinen and Saarma 2002). GFLs mediate RET protein dimerization and activation (Fig. 1A). GFLs are presented to RET by GPI (glycosylphosphatidylinositol)-anchored coreceptors, called GFR-α (GDNF family receptor α 1-4). Differential tissue expression dictates the specificity of action displayed by alternative GLF-GFR-α pairs during development and adult life (Baloh et al. 2000; Airaksinen and Saarma 2002).Together with other membrane (DCC and p75NTR) or nuclear (androgen receptor, AR) receptors, RET belongs to the family of so-called “dependence” receptors (Mehlen and Bredesen 2011). In the absence of ligand, RET exerts a proapoptotic activity, that is blocked on ligand stimulation (Bordeaux et al. 2000). Such pro-apoptotic activity is RET kinase-independent and mediated by cleavage of RET cytosolic portion by caspase-3, which, in turn, releases a carboxy-terminal RET peptide that is able to induce cell death (Bordeaux et al. 2000). It is feasible that such activity is important for RET developmental function, because it may control migration of RET-expressing cells by limiting survival of cells that move beyond ligand availability (Bordeaux et al. 2000; Cañibano et al. 2007). Whether modulation of this function is also important for RET-associated diseases is still unknown. However, it is interesting to note that a cancer-associated RET mutant (RET-C634R, see below) does not exert cleavage-dependent proapoptotic effects, whereas RET mutants associated with defective development (Hirschsprung disease, see below) exert strong proapoptotic activity that is refractory to modulation by ligand (Bordeaux et al. 2000).RET is expressed in enteric ganglia, adrenal medulla chromaffin cells, thyroid C cells, sensory and autonomic ganglia of the peripheral nervous system, a subset of central nervous system nuclei, developing kidney and testis germ cells (Manié et al. 2001; de Graaff et al. 2001). RET null mice display impaired development of superior cervical ganglia and enteric nervous system, kidney agenesia, reduction of thyroid C cells, and impaired spermatogenesis (Manié et al. 2001). Accordingly, individuals with germline loss-of-function mutations of RET are affected by intestinal aganglionosis causing congenital megacolon (Hirschsprung disease) (Brooks et al. 2005). RET loss-of-function mutations have also been identified in congenital anomalies of kidney and urinary tract (CAKUT), either isolated or in combination with Hirschsprung disease (Jain 2009).Several genetic alterations convert RET into a dominantly transforming oncogene. This review will describe RET-derived oncogenes that are associated with different types of human neoplasia (Fig. 1B).     

Poles Apart: Prokaryotic Polar Organelles and Their Spatial Regulation

While polar organelles hold the key to understanding the fundamentals of cell polarity and cell biological principles in general, they have served in the past merely for taxonomical purposes. Here, we highlight recent efforts in unraveling the molecular basis of polar organelle positioning in bacterial cells. Specifically, we detail the role of members of the Ras-like GTPase superfamily and coiled-coil-rich scaffolding proteins in modulating bacterial cell polarity and in recruiting effector proteins to polar sites. Such roles are well established for eukaryotic cells, but not for bacterial cells that are generally considered diffusion-limited. Studies on spatial regulation of protein positioning in bacterial cells, though still in their infancy, will undoubtedly experience a surge of interest, as comprehensive localization screens have yielded an extensive list of (polarly) localized proteins, potentially reflecting subcellular sites of functional specialization predicted for organelles.Since the first electron micrographs that revealed flagella at the cell poles of bacteria, we have known that bacterial cells are polarized and that they are able to decode the underlying positional information to confine the assembly of an extracellular organelle to a polar cellular site (Fig. 1). Foraging into this unknown territory has been challenging, but recent efforts that exploit the power of bacterial genetics along with modern imaging methods to visualize proteins in the minute bacterial cells has yielded several enticing entry points to dissect polarity-based mechanisms and explore potentially contributing subdiffusive characteristics (Golding and Cox 2006).Open in a separate windowFigure 1.Transmission electron micrograph (taken by Jeff Skerker) of a Caulobacter crescentus swarmer cell showing the polar pili (empty arrowheads), the polar flagellum with the flagellar filament (filled arrowheads), and the hook (white arrow) (see Fig. 2A).While polar organelles are a visual manifestation of polarity, it is important to point out that polarity can also be inherent to cells, at least in molecular terms, even in the absence of discernible polar structures. In other words, molecular anatomy can reveal that a bacterial cell, such as an Escherichia coli cell, features specialized protein complexes at or near the poles, despite a perfectly symmetrical morphology (Maddock and Shapiro 1993; Lindner et al. 2008). Such systemic polarization in bacteria, likely stemming from the distinctive division history of each pole, has the potential to be widespread and to be exploited for positioning of polar organelles and protein complexes. As excellent reviews have been published detailing the interplay between cell polarity and protein localization (Dworkin 2009; Shapiro et al. 2009; Kaiser et al. 2010; Rudner and Losick 2010), here we focus on recent progress in understanding the function and localization of spatial regulators of polar organelles. Considering that the ever-growing list of polar protein complexes emerging from systematic and comprehensive localization studies (Kitagawa et al. 2005; Russell and Keiler 2008; Werner et al. 2009; Hughes et al. 2010) is suggestive of multiple polarly confined (organelle-like) functions, understanding their spatial regulation is also of critical relevance in the realm of medical bacteriology, as many virulence determinants also underlie polarity (Goldberg et al. 1993; Scott et al. 2001; Judd et al. 2005; Jain et al. 2006; Jaumouille et al. 2008; Carlsson et al. 2009). Below, we highlight a few prominent examples of overtly polar organelles and the proteins known to date that regulate their polar positioning.     

Base Excision Repair

Base excision repair (BER) corrects DNA damage from oxidation, deamination and alkylation. Such base lesions cause little distortion to the DNA helix structure. BER is initiated by a DNA glycosylase that recognizes and removes the damaged base, leaving an abasic site that is further processed by short-patch repair or long-patch repair that largely uses different proteins to complete BER. At least 11 distinct mammalian DNA glycosylases are known, each recognizing a few related lesions, frequently with some overlap in specificities. Impressively, the damaged bases are rapidly identified in a vast excess of normal bases, without a supply of energy. BER protects against cancer, aging, and neurodegeneration and takes place both in nuclei and mitochondria. More recently, an important role of uracil-DNA glycosylase UNG2 in adaptive immunity was revealed. Furthermore, other DNA glycosylases may have important roles in epigenetics, thus expanding the repertoire of BER proteins.Base excision repair (BER) corrects small base lesions that do not significantly distort the DNA helix structure. Such damage typically results from deamination, oxidation, or methylation (Fig. 1). Much of the damage is the result of spontaneous decay of DNA (Lindahl 1993), although similar damage may also be caused by environmental chemicals, radiation, or treatment with cytostatic drugs. BER takes place in nuclei, as well as in mitochondria, largely using different isoforms of proteins or genetically distant proteins. The identification of Escherichia coli uracil-DNA glycosylase (Ung) in 1974 by Tomas Lindahl marks the discovery of BER. Lindahl searched for an enzyme activity that would act on genomic uracil resulting from cytosine deamination. Such an activity was found, but rather unexpectedly, it was not a nuclease. Instead, Lindahl identified an enzyme that cleaved the bond between uracil and deoxyribose. The resulting abasic site (AP-site) was suggested to be further processed by an AP-endonuclease, an exonuclease, a DNA polymerase, and a ligase. Thus, the fundamental steps in the BER pathway were outlined already in the very first paper (Lindahl 1974). Enzymes that cleave the bond between deoxyribose and a modified or mismatched DNA base are now called DNA glycosylases. Collectively these enzymes initiate base excision repair of a large number of base lesions, each recognized by one or a few DNA glycosylases with overlapping specificities.Open in a separate windowFigure 1.Chemistry of common base lesions and abasic sites.This relatively brief review focuses on recent advances in the mechanism and function of BER with a focus on mammalian proteins. The current view is that BER is important in relation to cancer, neurodegeneration, and aging (Jeppesen et al. 2011; Wallace et al. 2012). Because of limited space, we have referred to reviews for the majority of results published more than 6–7 years ago. Also, for more detailed analyses of different aspects of BER, the reader is referred to excellent reviews on BER proteins and pathways published in Huffman et al. (2005), Beard and Wilson (2006), Berti and McCann (2006), Cortázar et al. (2007), Kavli et al. (2007), Sousa et al. (2007), Tubbs et al. (2007), Berger et al. (2008), Robertson et al. (2009), Friedman and Stivers (2010), Wilson et al. (2010), Svilar et al. (2011), and Jacobs and Schar (2012).     

P-Bodies and Stress Granules: Possible Roles in the Control of Translation and mRNA Degradation

The control of translation and mRNA degradation is important in the regulation of eukaryotic gene expression. In general, translation and steps in the major pathway of mRNA decay are in competition with each other. mRNAs that are not engaged in translation can aggregate into cytoplasmic mRNP granules referred to as processing bodies (P-bodies) and stress granules, which are related to mRNP particles that control translation in early development and neurons. Analyses of P-bodies and stress granules suggest a dynamic process, referred to as the mRNA Cycle, wherein mRNPs can move between polysomes, P-bodies and stress granules although the functional roles of mRNP assembly into higher order structures remain poorly understood. In this article, we review what is known about the coupling of translation and mRNA degradation, the properties of P-bodies and stress granules, and how assembly of mRNPs into larger structures might influence cellular function.The translation and decay of mRNAs play key roles in the control of eukaryotic gene expression. The determination of eukaryotic mRNA decay pathways has allowed insight into how translation and mRNA degradation are coupled. Degradation of eukaryotic mRNAs is generally initiated by shortening of the 3′ poly (A) tail (Fig. 1A) (reviewed in Parker and Song 2004; Garneau et al. 2007) by the major mRNA deadenylase, the Ccr4/Pop2/Not complex (Daugeron et al. 2001; Tucker et al. 2001; Thore et al. 2003). Following deadenylation, mRNAs can be degraded 3′ to 5′ by the exosome (Anderson and Parker 1998; Wang and Kiledjian 2001). However, more commonly, mRNAs are decapped by the Dcp1/Dcp2 decapping enzyme and then degraded 5′ to 3′ by the exonuclease, Xrn1 (Decker and Parker 1993; Hsu and Stevens 1993; Muhlrad et al. 1994, 1995; Dunckley and Parker 1999; van Dijk et al. 2002; Steiger et al. 2003). In metazoans, a second decapping enzyme, Nudt16, also contributes to mRNA turnover (Song et al. 2010).Open in a separate windowFigure 1.Eukaryotic mRNA decay pathways. (A) General mRNA decay pathways. (B) Specialized decay pathways that degrade translationally aberrant mRNAs.The processes of mRNA decay and translation are interconnected in eukaryotic cells in many ways. For example, quality control mechanisms exist to detect aberrancies in translation, which then lead to mRNAs being degraded by specialized mRNA decay pathways (Fig. 1B). Nonsense-mediated decay (NMD) is one such mRNA quality control system that degrades mRNAs that terminate translation aberrantly. In yeast, aberrant translation termination leads to deadenylation-independent decapping (Muhlrad and Parker 1994), whereas in metazoan cells NMD substrates can be both decapped and endonucleolytically cleaved and degraded (reviewed in Isken and Maquat 2007). A second quality control system for mRNA translation is referred to as no-go decay (NGD) and leads to endonucleolytic cleavage of mRNAs with strong stalls in translation elongation (Doma and Parker 2006; reviewed in Harigaya and Parker 2010). Another mechanism of mRNA quality control is the rapid 3′ to 5′ degradation of mRNAs that do not contain translation termination codons, which is referred to as non-stop decay (NSD) (Frischmeyer et al. 2002; van Hoof et al. 2002). The available evidence suggests these specialized mechanisms function primarily on aberrant mRNAs that are produced by defects in splicing, 3′ end formation, or damage to RNAs.The main pathway of mRNA degradation is also in competition with translation initiation. Competition between the two processes was first suggested by the observation that removal of the poly (A) tail and the cap structure, both of which stimulate translation initiation, were the key steps in mRNA degradation. In addition, inhibition of translation initiation by strong secondary structures in the 5′UTR, translation initiation inhibitors, a poor AUG context, or mutations in initiation factors increases the rates of deadenylation and decapping (Muhlrad et al. 1995; Muckenthaler et al. 1997; Lagrandeur and Parker 1999; Schwartz and Parker 1999). Moreover, the cap binding protein eIF4E, known to stimulate translation initiation, inhibits the decapping enzyme, Dcp1/Dcp2, both in vivo and in vitro (Schwartz and Parker 1999; Schwartz and Parker 2000). Finally, many mRNA specific regulatory factors, (e.g., miRNAs or PUF proteins), both repress translation and accelerate deadenylation and decapping (reviewed in Wickens et al. 2002; Behm-Ansmant et al. 2006; Franks and Lykke-Anderson 2008; Shyu et al. 2008).In the simplest model, the competition between translation and mRNA degradation can be understood through changes in the proteins bound to the cap and poly (A) tail that then influence the accessibility of these structures to deadenylases and decapping enzymes. For example, given that the Ccr4/Pop2/Not deadenylase complex is inhibited by poly (A)-binding protein (Pab1) (Tucker et al. 2002), the effects of translation on deadenylation are most likely through dynamic changes in the association of Pab1 binding with the poly (A) tail. One possibility is that defects in translation initiation either directly or indirectly decrease Pab1 association with the poly (A) tail. Deadenylation is also affected by aspects of translation termination. For instance, premature translation termination in yeast accelerates poly (A) shortening as part of the process of NMD (Cao and Parker 2003; Mitchell and Tollervey 2003). The coupling of translation termination to deadenylation has been suggested to occur through direct interactions of the translation termination factor eRF3 with Pab1 (Cosson et al. 2002), which may lead to Pab1 transiently dissociating from the poly (A) tail. Interestingly, in yeast, once the poly (A) tail reaches an oligo (A) length of 10–12 residues, a length that reduces the affinity of Pab1, the mRNA can become a substrate for decapping and for binding of the Pat1/Lsm1-7 complex (Tharun and Parker 2001; Chowdhury et al. 2007), which enhances the rate of decapping. This exchange of the Pab1 protein for the Pat1/Lsm1-7 complex is part of the mechanism that allows decapping to be promoted following deadenylation.A similar mRNP dynamic is also likely to occur on the cap structure. Specifically, the competition between translation initiation and decapping suggests that prior to decapping, translation initiation factors are exchanged for decapping factors, thereby assembling a distinct “decapping” mRNP that is no longer capable of translation initiation (Tharun and Parker 2001). This idea is supported by the observation that some decapping activators also function as translational repressors (Coller and Parker 2005; Pilkington and Parker 2008; Nissan et al. 2010). Thus, mRNA decapping appears to occur in two steps, first inhibition of translation initiation and exchange of translation factors for the general repression/degradation machinery, and a second step whereby the mRNA is actually degraded. Thus, by understanding the changes in mRNP states between actively translating mRNAs and mRNAs that are translationally repressed and possibly stored or ultimately degraded we will better understand how the fate of mRNAs is controlled in the cytoplasm.     

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.     

Receptor Tyrosine Kinases: Legacy of the First Two Decades

Receptor tyrosine kinases (RTKs) and their cellular signaling pathways play important roles in normal development and homeostasis. Aberrations in their activation or signaling leads to many pathologies, especially cancers, motivating the development of a variety of drugs that block RTK signaling that have been successfully applied for the treatment of many cancers. As the current field of RTKs and their signaling pathways are covered by a very large amount of literature, spread over half a century, I am focusing the scope of this review on seminal discoveries made before tyrosine phosphorylation was discovered, and on the early days of research into RTKs and their cellular signaling pathways. I review the history of the early days of research in the field of RTKs. I emphasize key early findings, which provided conceptual frameworks for addressing the questions of how RTKs are activated and how they regulate intracellular signaling pathways.The family of cell-surface receptors designated receptor tyrosine kinases (RTK) received their name more that a decade after the same molecules were already known as the cell-surface receptors for insulin (insulin receptor), epidermal growth factor (EGFR), and many other growth factor receptors. Following the pioneering discoveries of nerve growth factor and epidermal growth factor (EGF; Levi-Montalcini and Booker 1960; Cohen 1962) and the establishment of the important roles of these two growth factors in the control of neuronal differentiation and cell proliferation in vivo and in vitro, it became clear that these cytokines bind specifically to cell-surface receptors. Insulin had already been discovered by this time, and had been applied successfully to treat diabetes patients since the early twentieth century. The resulting homogenous preparations of pure insulin enabled the quantitative characterization of insulin binding to its receptor on intact cells or to solubilized insulin receptor preparations using radiolabeled insulin (De Meyts et al. 1973). These studies greatly advanced understanding of the ligand binding characteristics of insulin receptor and, later on EGFR (Carpenter et al. 1975), including the establishment of negative cooperativity in insulin binding to its receptor expressed on the surface of living cells (De Meyts et al. 1973). Moreover, these studies shed important light on the dynamic nature of the cellular behavior of these receptors. The capacities of insulin receptor and EGFR to undergo ligand-dependent down-regulation and desensitization through receptor-mediated internalization and degradation (Carpenter and Cohen 1976; Gordon et al. 1978; Schlessinger et al. 1978a,b; Carpentier et al. 1979; Haigler et al. 1979) were also established well before the realization that growth factors receptors are endowed with intrinsic protein tyrosine kinase activities (Fig. 1).Open in a separate windowFigure 1.A time line of key findings during the history of RTKs, with emphasis on findings and discoveries that produced the conceptual framework in the development of the RTK field and its application for cancer therapy. References for the key findings are also presented in the text (Lee et al. 1985; Libermann et al. 1985; Margolis et al. 1990; Bottaro et al. 1991; Bae et al. 2009).Progress was also made in elucidating the role of growth factors in normal embryonic development, wound healing, and pathological conditions such as cancer. Early studies in the 1960s and 1970s showed that growth factors play an important role in oncogenesis induced by retroviruses and in the proliferation of tumor-derived cancer cells. Pioneering studies performed by Howard Temin (1966, 1967) showed that cancer cells need less insulin and serum growth factors for cell proliferation compared with normal cells, suggesting that cancer cells produce and use their own growth factors and/or use cellular processes that in normal cells are regulated by exogenously supplied growth factors; both predictions were subsequently confirmed. A variety of new polypeptide growth factors that stimulate cell proliferation by binding to receptors at the cell surface were subsequently discovered. Those include a growth factor isolated from human platelets designated platelet-derived growth factor (PDGF; Antoniades et al. 1979; Heldin et al. 1979), a growth factor isolated from bovine brain designated fibroblast growth factor (FGF; Gospodarowicz et al. 1978), a growth factor isolated from rat platelets that stimulates the proliferation of mature hepatocytes, designated hepatocyte growth factor (HGF; Nakamura et al. 1986). In addition to EGF, another growth factor that binds selectively to cells expressing EGFR was isolated from virally and chemically transformed cells, suggesting that this growth factor—designated transforming growth factor α—may play a role in oncogenesis by an autocrine mechanism (Roberts et al. 1980, 1982). This discovery provided further support to the earlier finding that transformation by murine and feline sarcoma viruses selectively interferes with EGF binding to EGFR in transformed cells (Todaro et al. 1976). Together with many other studies published since the 1980s, this work showed that growth factors and their receptors play numerous important roles during development and in many normal cellular processes as well as in pathologies such as cancer, diabetes, atherosclerosis, severe bone disorders, and tumor angiogenesis.Visualization of dynamic cellular redistribution of ligand/receptor complexes, and rapid receptor-mediated internalization of growth factors such as insulin or EGF, led to the proposal that cell-surface receptors for these ligands may play a passive role in delivering them to intracellular compartments in which internalized EGF or insulin molecules exert their actions (Vigneri et al. 1978; Podlecki et al. 1986; Jiang and Schindler 1990). In other words, according to this hypothesis, the biological signals induced by insulin or EGF were thought to be mediated by binding of the ligands themselves to intracellular target(s) in the cytoplasm or nucleus, with the role of the cell-surface receptor being to act as a “carrier” that delivers them directly to these targets. An alternative hypothesis was that insulin or EGF activates their cognate receptors at the cell surface, which in turn stimulate the production of an intracellular second messenger molecule analogous to cAMP in signaling by the G-protein-activating β-adrenergic receptor. Indeed, several potential second messengers that are generated in cells on stimulation with insulin or other growth factors were proposed before (and even after) it became clear that insulin receptor, EGFR, and other RTKs are endowed with intrinsic tyrosine kinase activity (Larner et al. 1979; Das 1980; Saltiel and Cuatrecasas 1986).A demonstration that anti-insulin receptor antibodies from the serum of certain diabetic patients could mimic cellular responses of insulin (Flier et al. 1977; Van Obberghen et al. 1979) provided the first conclusive answer to the question of whether the biological activity of growth factors is mediated directly or indirectly through their membrane receptors. This experiment ruled out the possibility that insulin receptor functions as a passive carrier that delivers insulin to an intracellular target to induce cellular responses. Studies showing that intact, bivalent antibodies against the insulin receptor can activate its signaling, whereas monovalent Fab fragments of the same antibodies cannot further argued that ligand-induced receptor dimerization or stimulation of a particular arrangement between two receptor molecules in a dimer can activate the insulin receptor (Kahn et al. 1978).A similar conclusion was reached using certain monoclonal antibodies that bind to the extracellular region of EGFR and block ligand binding (Schreiber et al. 1981). Whereas intact antibodies were able to mimic EGF in stimulating a variety of EGF-like responses including cell proliferation, monovalent Fab fragments of the same monoclonal EGFR antibodies failed to do so—and acted instead as EGFR antagonists (Schreiber et al. 1981, 1983). These experiments provided strong evidence both that EGFR plays a crucial role in mediating EGF-induced cellular responses and that EGFR is activated by ligand-induced receptor dimerization (Schreiber 1981, 1983).     

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).     

Modular Design of Immunological Synapses and Kinapses

The concept of an immunological synapse goes back to the early 1980s with the discovery of the relationship between T-cell antigen receptor mediated Ca²⁺ signaling, adhesion, and directed secretion. However, this concept did not gain traction until images were published starting in 1998 that revealed a specific molecular pattern in the interface between T cells and model antigen-presenting cells or supported planar bilayers. The dominant pattern, a ring of adhesion molecules surrounding a central cluster of antigen receptors, was observed in both model systems. Analysis of the origins of this pattern over the past 10 years has presented a solution for a difficult problem in lymphocyte biology—how a highly motile cell can suddenly stop when it encounters a signal delivered by just a few antigenic ligands on the surface of another cell without disabling the sensory machinery of the motile cell. The T lymphocyte actively assembles the immunological synapse pattern following a modular design with roots in actin–myosin‐based motility.The immune system provides an outstanding model for study of both dynamic and stable cell–cell adhesion (Dustin and Springer 1989; Lawrence and Springer 1991; Miller et al. 2002; Mempel et al. 2004). Early studies on molecules important for the function of cytotoxic lymphocytes, cells that kill virally infected cells and contribute to destruction of transplanted organs and tumors, identified two families of adhesion molecules (Davignon et al. 1981) (Fig. 1). Monoclonal antibodies that blocked the activity of cytotoxic lymphocytes identified an integrin, still widely referred to as LFA-1, and two immunoglobulin superfamily members, LFA-2 and LFA-3, now known more commonly as CD2 and CD58, respectively (Sanchez-Madrid et al. 1982). CD2 and CD58 were defined as a receptor ligand pair making it the first clearly defined heterophilic cell–cell adhesion system (Dustin et al. 1987a; Selvaraj et al. 1987). The identification of a ligand for LFA-1, ICAM-1, revealed that it too was a transmembrane member of the immunoglobulin superfamily providing a mechanism for involvement of integrins in cell–cell junction formation (Marlin and Springer 1987). These studies were contemporaneous with evidence for adhesion via direct ligand binding as a mechanism of matrix binding integrins, and homophilic interactions of NCAM and cadherins (Rutishauser et al. 1982; Peyrieras et al. 1983; Gardner and Hynes 1985; Horwitz et al. 1985; Pytela et al. 1985; Wright and Meyer 1985; Cunningham et al. 1987; Nagafuchi et al. 1987). In some respects, the complexity of the role of oligosaccharides in NCAM-mediated adhesion and studying homophilic systems has resulted in clearer results coming from the heterophilic immune cell adhesion systems in the early to mid 1980s. It was speculated that the CD2–CD58 interaction might have evolved from an ancestral homophilic system like NCAM and subsequently it has been determined that CD58 is located in a rapidly evolving gene cluster including several important homophilic adhesion molecules on chromosome 1 (Wong et al. 1990). The number of receptor ligand interactions that are involved in immune cell interactions has grown significantly since these early studies, but the LFA-1/ICAM-1 and CD2 family interactions still appear to be major contributors in cell adhesion in many functional settings. In this article, I review the role of LFA-1/ICAM-1, CD2/CD58, and CD2 family homophilic adhesion molecules, like SLAM, in immune cell interactions. I describe the supported planar bilayer model in some detail because this has played an important role in the characterization of immune-cell adhesion systems, but also discuss recent studies using in vivo imaging that have also provided insight into the unique demands of in situ immune-cell interactions leading to specific molecular requirements.Open in a separate windowFigure 1.Cytotoxic T lymphocyte (CTL) life cycle. Naïve cells hunt for evidence of infection on the surface of self dendritic cells in lymphoid tissue. One antigen, if found on the primary immunological synapse, leads to activation, proliferation, and development of a CTL. This CTL exits the lymph node and uses the blood to reach a tissue. In the tissue, the CTL migrates to find its target and then uses a secondary synapse to kill the target.     

Membrane Domains Based on Ankyrin and Spectrin Associated with Cell–Cell Interactions

Nodes of Ranvier and axon initial segments of myelinated nerves, sites of cell–cell contact in early embryos and epithelial cells, and neuromuscular junctions of skeletal muscle all perform physiological functions that depend on clustering of functionally related but structurally diverse ion transporters and cell adhesion molecules within microdomains of the plasma membrane. These specialized cell surface domains appeared at different times in metazoan evolution, involve a variety of cell types, and are populated by distinct membrane-spanning proteins. Nevertheless, recent work has shown that these domains all share on their cytoplasmic surfaces a membrane skeleton comprised of members of the ankyrin and spectrin families. This review will summarize basic features of ankyrins and spectrins, and will discuss emerging evidence that these proteins are key players in a conserved mechanism responsible for assembly and maintenance of physiologically important domains on the surfaces of diverse cells.Spectrins are flexible rods 0.2 microns in length with actin-binding sites at each end (Shotton et al. 1979; Bennett et al. 1982) (Fig. 1A). Spectrins are assembled from α and β subunits, each comprised primarily of multiple copies of a 106-amino acid repeat (Speicher and Marchesi 1984). In addition to the canonical 106-residue repeat, β spectrins also have a carboxy-terminal pleckstrin homology domain (Zhang et al. 1995; Macias et al. 1994) and tandem amino-terminal calponin homology domains (Bañuelos et al. 1998), whereas α spectrins contain an Src homology domain 3 (SH3) site (Musacchio et al. 1992), a calmodulin-binding site (Simonovic et al. 2006), and EF hands (Travé et al. 1995) (Fig. 1A). Spectrin α and β subunits are assembled antiparallel and side-to-side into heterodimers, which in turn are associated head-to-head to form tetramers (Clarke 1971; Shotton et al. 1979; Davis and Bennett 1983) (Fig. 1A). In human erythrocytes, in which spectrin was first characterized (Marchesi and Steers 1968; Clarke 1971), actin oligomers containing 10–14 monomers are each linked to five to six spectrin tetramers by accessory proteins to form a geodesic domelike structure that has been resolved by electron microscopy (Byers and Branton 1985). The principal proteins at the spectrin–actin junction are protein 4.1, adducin, tropomyosin, tropomodulin, and dematin (Bennett and Baines 2001) (Open in a separate windowFigure 1.Domain structure and variants of spectrin and ankyrin proteins. (A) Molecular domains of spectrins: Two α spectrins and five β spectrins are shown. Spectrins are comprised of modular units called spectrin repeats (yellow). Other domains such as the ankyrin binding domain (purple), Src-homology domain 3 (SH3, blue), EF-hand domain (red), and calmodulin-binding domain (green) promote interactions with binding targets important for spectrin function. The pleckstrin homology domain (black) promotes association with the plasma membrane and the actin binding domain (grey) tethers the spectrin-based membrane skeleton to the underlying actin cytoskeleton. (B) The spectrin tetramer, the fundamental unit of the spectrin-based membrane skeleton. The spectrin repeat domains of α and β spectrin associate end-to-end to form heterodimers. Heterodimers associate laterally in an antiparallel fashion to form tetramers. The tetramers can then associate end-to-end to form extended macromolecules that link into a geodesic dome shape directly underneath the plasma membrane. (C) Molecular domains present in canonical ankyrins. The membrane binding domain of ankyrin isoforms (orange) is comprised of 24 ANK repeats. The spectrin binding domain (green-blue) allows ankyrins to coordinate integral membrane proteins to the membrane skeleton. The death domain (pink) is the most highly conserved domain. The regulatory domain (brown) is the most variable region of ankyrins. The regulatory domain interacts intramolecularly with the membrane binding domain to modulate ankyrin’s affinity for other binding partners. All ankyrins and spectrins are subject to alternative splicing, which further increases their functional diversity.

Table 1.

Binding partners of spectrin and ankyrins