Endocytosis of Viruses and Bacteria

Of the many pathogens that infect humans and animals, a large number use cells of the host organism as protected sites for replication. To reach the relevant intracellular compartments, they take advantage of the endocytosis machinery and exploit the network of endocytic organelles for penetration into the cytosol or as sites of replication. In this review, we discuss the endocytic entry processes used by viruses and bacteria and compare the strategies used by these dissimilar classes of pathogens.Many of the most widespread and devastating diseases in humans and livestock are caused by viruses and bacteria that enter cells for replication. Being obligate intracellular parasites, viruses have no choice. They must transport their genome to the cytosol or nucleus of infected cells to multiply and generate progeny. Bacteria and eukaryotic parasites do have other options; most of them can replicate on their own. However, some have evolved to take advantage of the protected environment in the cytosol or in cytoplasmic vacuoles of animal cells as a niche favorable for growth and multiplication. In both cases (viruses and intracellular bacteria), the outcome is often destructive for the host cell and host organism. The mortality and morbidity caused by infectious diseases worldwide provide a strong rationale for research into pathogen–host cell interactions and for pursuing the detailed mechanisms of transmission and dissemination. The study of viruses and bacteria can, moreover, provide invaluable insights into fundamental aspects of cell biology.Here, we focus on the mechanisms by which viral and bacterial pathogens exploit the endocytosis machinery for host cell entry and replication. Among recent reviews on this topic, dedicated uniquely to either mammalian viruses or bacterial pathogens, we recommend the following: Cossart and Sansonetti (2004); Pizarro-Cerda and Cossart (2006); Kumar and Valdivia (2009); Cossart and Roy (2010); Mercer et al. (2010b); Grove and Marsh (2011); Kubo et al. (2012); Vazquez-Calvo et al. (2012a); Sun et al. (2013).The term “endocytosis” is used herein in its widest sense, that is, to cover all processes whereby fluid, solutes, ligands, and components of the plasma membrane as well as particles (including pathogenic agents) are internalized by cells through the invagination of the plasma membrane and the scission of membrane vesicles or vacuoles. This differs from current practice in the bacterial pathogenesis field, where the term “endocytosis” is generally reserved for the internalization of molecules or small objects, whereas the uptake of bacteria into nonprofessional phagocytes is called “internalization” or “bacterial-induced phagocytosis.” In addition, the term “phagocytosis” is reserved for internalization of bacteria by professional phagocytes (macrophages, polymorphonuclear leucocytes, dendritic cells, and amoebae), a process that generally but not always leads to the destruction of the ingested bacteria (Swanson et al. 1999; May and Machesky 2001; Henry et al. 2004; Zhang et al. 2010). With a few exceptions, we will not discuss phagocytosis of bacteria or the endocytosis of protozoan parasites such as Toxoplasma and Plasmodium (Robibaro et al. 2001).     

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.     

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).     

Fibronectins,Their Fibrillogenesis,and In Vivo Functions

Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN–FN and cell–FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.As a ubiquitous component of the extracellular matrix (ECM), fibronectin (FN) provides essential connections to cells through integrins and other receptors and regulates cell adhesion, migration, and differentiation. FN is secreted as a large dimeric glycoprotein with subunits that range in size from 230 kDa to 270 kDa (Mosher 1989; Hynes 1990). Variation in subunit size depends primarily on alternative splicing. FN was first isolated from blood more than 60 years ago (Edsall 1978), and this form is called plasma FN. The other major form, called cellular FN, is abundant in the fibrillar matrices of most tissues. Although FN is probably best known for promoting attachment of cells to surfaces, this multidomain protein has many interesting structural features and functional roles beyond cell adhesion.FN is composed of three different types of modules termed type I, II, and III repeats (Fig. 1) (Petersen et al. 1983; Hynes 1990). These repeats have distinct structures. Although the conformations of type I and type II repeats are maintained by pairs of intramodule disulfide bonds, the type III repeat is a 7-stranded β-barrel structure that lacks disulfide bonds (Main et al. 1992; Leahy et al. 1996, 1992) and, therefore, can undergo conformational changes. FN type III repeats are widely distributed among animal, bacterial, and plant proteins and are found in both extracellular and intracellular proteins (Bork and Doolittle 1992; Tsyguelnaia and Doolittle 1998).Open in a separate windowFigure 1.FN domain organization and isoforms. Each FN monomer has a modular structure consisting of 12 type I repeats (cylinders), 2 type II repeats (diamonds), and 15 constitutive type III repeats (hexagons). Two additional type III repeats (EIIIA and EIIIB, green) are included or omitted by alternative splicing. The third region of alternative splicing, the V region (green box), is included (V120), excluded (V0), or partially included (V95, V64, V89). Sets of modules comprise domains for binding to other extracellular molecules as indicated. Domains required for fibrillogenesis are in red: the assembly domain (repeats I_1-5) binds FN, III_9-10 contains the RGD and synergy sequences for integrin binding, and the carboxy-terminal cysteines form the disulfide-bonded FN dimer (‖). The III_1-2 domain (light red) has two FN binding sites that are important for fibrillogenesis. The amino-terminal 70-kDa fragment contains assembly and gelatin-binding domains and is routinely used in FN binding and matrix assembly studies.Sets of adjacent modules form binding domains for a variety of proteins and carbohydrates (Fig. 1). ECM proteins, including FN, bind to cells via integrin receptors, αβ heterodimers with two transmembrane subunits (Hynes 2002). FN-binding integrins have specificity for one of the two cell-binding sites within FN, either the RGD-dependent cell-binding domain in III₁₀ (Pierschbacher and Ruoslahti 1984) or the CS1 segment of the alternatively spliced V region (IIICS) (Wayner et al. 1989; Guan and Hynes 1990). Some integrins require a synergy sequence in repeat III₉ for maximal interactions with FN (Aota et al. 1994; Bowditch et al. 1994). Another family of cell surface receptors is the syndecans, single-chain transmembrane proteoglycans (Couchman 2010). Syndecans use their glycosaminoglycan (GAG) chains to interact with FN at its carboxy-terminal heparin-binding (HepII) domain (Fig. 1) (Saunders and Bernfield 1988; Woods et al. 2000), which binds to heparin, heparan sulfate, and chondroitin sulfate GAGs (Hynes 1990; Barkalow and Schwarzbauer 1994). Syndecan binding to the HepII domain enhances integrin-mediated cell spreading and intracellular signaling, suggesting that syndecans act as coreceptors with integrins in cell–FN binding (Woods and Couchman 1998; Morgan et al. 2007).A major site for FN self-association is within the amino-terminal assembly domain spanning the first five type I repeats (I_1-5) (Fig. 1) (McKeown-Longo and Mosher 1985; McDonald et al. 1987; Schwarzbauer 1991b; Sottile et al. 1991). This domain plays an essential role in FN fibrillogenesis. As a major blood protein, FN interacts with fibrin during blood coagulation, also using the I_1-5 domain (Mosher 1989; Hynes 1990). As fibrin polymerizes, factor XIII transglutaminase covalently cross-links glutamine residues near the amino terminus of FN to fibrin α chains (Mosher 1975; Corbett et al. 1997). The amino-terminal domain has multiple binding partners in addition to FN and fibrin; these include heparin, S. aureus, and other bacteria, thrombospondin-1, and tenascin-C (Hynes 1990; Ingham et al. 2004; Schwarz-Linek et al. 2006). Adjacent to this domain is the gelatin/collagen-binding domain composed of type I and type II modules (Ingham et al. 1988). This domain also binds to tissue transglutaminase (Radek et al. 1993) and fibrillin-1 (Sabatier et al. 2009). Within the 15 type III repeats reside several FN binding sites that interact with the amino-terminal assembly domain as well as three sites of alternative splicing that generate multiple isoforms. At the carboxyl terminus is a pair of cysteine residues that form the FN dimer through antiparallel disulfide bonds (Hynes 1990). This dimerization may be facilitated by disulfide isomerase activity located in the last set of type I repeats (Langenbach and Sottile 1999).The diverse set of binding domains provides FN with the ability to interact simultaneously with other FN molecules, other ECM components (e.g., collagens and proteoglycans), cell surface receptors, and extracellular enzymes (Pankov and Yamada 2002; Fogelgren et al. 2005; Hynes 2009; Singh et al. 2010). Multitasking by FN probably underlies its essential role during embryogenesis (George et al. 1993). Furthermore, FN''s interactions can be modulated by exposure or sequestration of its binding sites within matrix fibrils, through the presence of ECM proteins that bind to FN, or through variation in structure by alternative splicing.     

Autophagy and Neuronal Cell Death in Neurological Disorders

Autophagy is implicated in the pathogenesis of major neurodegenerative disorders although concepts about how it influences these diseases are still evolving. Once proposed to be mainly an alternative cell death pathway, autophagy is now widely viewed as both a vital homeostatic mechanism in healthy cells and as an important cytoprotective response mobilized in the face of aging- and disease-related metabolic challenges. In Alzheimer’s, Parkinson’s, Huntington’s, amyotrophic lateral sclerosis, and other diseases, impairment at different stages of autophagy leads to the buildup of pathogenic proteins and damaged organelles, while defeating autophagy’s crucial prosurvival and antiapoptotic effects on neurons. The differences in the location of defects within the autophagy pathway and their molecular basis influence the pattern and pace of neuronal cell death in the various neurological disorders. Future therapeutic strategies for these disorders will be guided in part by understanding the manifold impact of autophagy disruption on neurodegenerative diseases.Soon after the discovery of lysosomes by de Duve in the 1950s, electron microscopists recognized the presence of cytoplasmic organelles within membrane-limited vacuoles (Clark 1957) and observed what appeared to be the progressive breakdown of these contents (Ashford and Porter 1962). Proposing that “prelysosomes” containing sequestered cytoplasm matured to autolysosomes by fusion with primary lysosomes, de Duve and colleagues (de Duve 1963; de Duve and Wattiaux 1966) named this process “autophagy” (self-eating). Neurons, as cells particularly rich in acid phosphatase-positive lysosomes, were a preferred model in the initial investigations of autophagy. Early studies of pathologic states such as neuronal chromatolysis (Holtzman and Novikoff 1965; Holtzman et al. 1967) linked neurodegenerative phenomena to robust proliferation of autophagic vacuoles (AVs) and lysosomes. Although de Duve appreciated the importance of lysosomes for maintaining cell homeostasis, he was especially intrigued with their potential as “suicide bags” capable of triggering cell death by releasing proteases into the cytoplasm. Despite some support for this notion (Brunk and Brun 1972), the concept was not significantly embraced until many decades later. Instead, for many years, lysosomes and autophagy were mainly considered to perform cellular housekeeping and to scavenge and clean up debris during neurodegeneration in preparation for regenerative processes. The connection between autophagy and neuronal cell death reemerged in the 1970s from observations of Clarke and colleagues, who presented evidence that the developing brain deployed autophagy as a form of programmed neuronal cell death during which autophagy was massively up-regulated to eliminate cytoplasmic components, at once killing the neuron and reducing its cell mass for easy removal. Self-degradation was suggested as a more efficient elimination mechanism than apoptosis, which requires a large population of phagocytic cells and access of these cells to the dying region (Baehrecke 2005). Indeed, the best evidence for this process is in the context of massive cell death, as in metamorphosis and involutional states (Das et al. 2012).Clarke proposed that autophagic cell death (ACD)—type 2 programmed cell death (PCD)—could be a relatively common alternative route to death distinct from apoptosis—type 1 PCD (Clarke 1990)—or caspase-independent cell death—type 3 PCD (Fig. 1). The distinguishing features of ACD are marked proliferation of AVs and progressive disappearance of organelles but relative preservation of cytoskeletal and nuclear integrity until late in the process (Schweichel and Merker 1973; Hornung et al. 1989). In this original concept of ACD or type 2 PCD, death is achieved by autophagic digestion of organelles and essential regulatory molecules and elimination of death inhibitory factors (Baehrecke 2005). With the advent of the molecular era of autophagy research in the 1990s, it became possible to verify the most important implication of ACD, namely, that the death could be prevented by inhibiting autophagy genetically or pharmacologically. Meanwhile, reports of prominent lysosomal/autophagic pathology in Alzheimer’s disease (AD) (Cataldo et al. 1997; Nixon et al. 2000, 2005) and other neuropathic states (Anglade et al. 1997; Rubinsztein et al. 2005) raised important questions about whether autophagy pathology signifies a prodeath program or an attempt to maintain survival—a critical question for any potential therapy based on autophagy modulation. In this article, we will examine evidence for the various neuroprotective roles of autophagy and review our current understanding of how specific stages of autophagy may become disrupted and influence the neurodegenerative pattern seen in major adult-onset neurological diseases. We will particularly focus on how neurons regulate the balance between prosurvival autophagy and well-established cell death mechanisms in making life or death decisions.Open in a separate windowFigure 1.Neuronal cell death: three general morphological types of dying cells in the developing nervous system, as initially classified by Schweichel and Merker (1973) and later Clarke (1990). (A,B) Type 1 (“apoptotic”) cell death: (A) A neuron, from the brain of a postnatal day 6 mouse pup, in the middle of apoptotic degeneration showing cell shrinkage, cytoplasmic condensation, ruffled plasma membrane, and a highly electron-dense nucleus. Endoplasmic reticulum (ER) is still recognizable and some are dilated. A small number of autophagic vacuoles (AVs) can be seen (arrows). (B) A late-stage apoptotic neuron displaying electron-dense chromatin balls (CB), each surrounded by a small amount of highly condensed cytoplasm. (Panel from Yang et al. 2008; reprinted, with permission, from the American Association of Pathologists and Bacteriologists.) (C) Type 2 (“autophagic”) cell death: a deafferented isthmo-optic neuron in developing chick brain after uptake of horseradish peroxidase to highlight (electron dense) endocytic and autophagic compartments. The cell death pattern features pyknosis, abundant AVs, and sometimes dilated ER and mitochondria. (Panel from Hornung et al. 1989; reproduced, with permission, from John Wiley & Sons) (D) Type 3 (“cytoplasmic, nonlysosomal”) cell death: a motoneuron displaying markedly dilated rough ER, Golgi, and nuclear envelope, late vacuolization, and increased chromatin granularity. (Panel from Chu-Wang and Oppenheim 1978; reproduced, with permission, from John Wiley & Sons) Scale bars, 1 µm (A,B); 2 µm (C,D).     

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).     

Architecture of the Mammalian Golgi

Since its first visualization in 1898, the Golgi has been a topic of intense morphological research. A typical mammalian Golgi consists of a pile of stapled cisternae, the Golgi stack, which is a key station for modification of newly synthesized proteins and lipids. Distinct stacks are interconnected by tubules to form the Golgi ribbon. At the entrance site of the Golgi, the cis-Golgi, vesicular tubular clusters (VTCs) form the intermediate between the endoplasmic reticulum and the Golgi stack. At the exit site of the Golgi, the trans-Golgi, the trans-Golgi network (TGN) is the major site of sorting proteins to distinct cellular locations. Golgi functioning can only be understood in light of its complex architecture, as was revealed by a range of distinct electron microscopy (EM) approaches. In this article, a general concept of mammalian Golgi architecture, including VTCs and the TGN, is described.In 1898 Camillo Golgi was the first to visualize, describe, and ultimately name the Golgi complex. Using a histochemical impregnation method causing the reduction and deposition of silver, he defined the Golgi in neuronal cells as a reticular apparatus stained by the “black reaction” (Golgi 1898). In the 1950s, the first ultrastructural images of the Golgi were revealed using the then newly developed electron microscope (EM) (Dalton 1954; Farquhar and Rinehart 1954; Sjostrand and Hanzon 1954; Dalton and Felix 1956), reviewed by Farquhar and Palade (1981). In 1961, the thiamine pyrophosphatase reaction developed by Novikoff and Goldfischer allowed cytochemical labeling of Golgi membranes, which revealed the ubiquitous cellular distribution of this organelle (Novikoff and Goldfischer 1961). In the many years of ultrastructural research that have followed, the visualization of the Golgi has gone hand-in-hand with the developing EM techniques.The intriguing structural complexity of the Golgi has made it one of the most photographed organelles in the cell. However, a full understanding of Golgi architecture is hard to deduce from the ultrathin (70–100 nm) sections used in standard transmission EM preparations. Rambourg and Clermont (1974) were the first to investigate the Golgi in three dimensions (3D), using stereoscopy (Rambourg 1974). In this approach a “thick” (150–200 nm), EM section is photographed at two distinct angles, after which the pairs of photographs are viewed with a stereoscope. Over the years, stereoscopy was applied to a variety of cells and has greatly contributed to our current understanding of Golgi architecture (Lindsey and Ellisman 1985; Rambourg and Clermont 1990; Clermont et al. 1994; Clermont et al. 1995). An alternative approach to study 3D structure is serial sectioning, by which a series of adjacent (serial) thin sections are collected. The Golgi can be followed throughout these sections and be constructed into a 3D model (Beams and Kessel 1968; Dylewski et al. 1984; Rambourg and Clermont 1990). In the nineties, 3D-EM was boosted by the introduction of high-voltage, dual axis 3D electron tomography (Ladinsky et al. 1999; Koster and Klumperman 2003; Marsh 2005; Marsh 2007; Noske et al. 2008), which allows the analysis of sections of up to 3–4 µm with a 4–6 nm resolution in the z-axis. The sections are photographed in a tilt series of different angles, which are reconstructed into a 3D tomogram that allows one to “look beyond” a given structure and reveals how it relates to other cellular compartments.Membranes with a similar appearance can differ in protein content and function. These differences are revealed by protein localization techniques. Therefore, in addition to the “classical” EM techniques providing ultrastructural details, EM methods that determine protein localization within the context of the cellular morphology have been crucial to further our understanding on the functional organization of the Golgi. For example, by enzyme-activity-based cytochemical staining the cis-to-trans-polarity in the distribution of Golgi glycosylation enzymes was discovered, reviewed by Farquhar and Palade (1981), which was key to understanding the functional organization of the Golgi stack in protein and lipid glycosylation. With the development of immunoEM methods, using antibodies, the need for enzyme activity for protein localization was overcome. This paved the way for the localization of a wide variety of proteins, such as the cytoplasmic coat complexes associated with the Golgi (Rabouille and Klumperman 2005).A logical next step in EM-based imaging of the Golgi would be to combine protein localization with 3D imaging, but this is technically challenging. A number of protocols enabling protein localization in 3D have recently been described (Trucco et al. 2004; Grabenbauer et al. 2005; Gaietta et al. 2006; Zeuschner et al. 2006; Meiblitzer-Ruppitsch et al. 2008), but these have only been applied in a limited manner to Golgi studies. Another approach that holds great potential for Golgi research is correlative microscopy (CLEM). Live cell imaging of fluorescent proteins has revolutionized cell biology by the real time visualization of dynamic events. However, live cell imaging does not reveal membrane complexity. By CLEM, live cells are first viewed by light microscopy and then prepared for EM (Mironov et al. 2008; van Rijnsoever et al. 2008). When coupled with the recent introduction of super resolution light microscopy techniques for real time imaging, the combination with EM for direct correlation with ultrastructural resolution has great potential (Hell 2009; Lippincott-Schwartz and Manley 2009).The 100th anniversary of the discovery of the Golgi, in 1998, triggered a wave of reviews on this organelle, including those focusing on Golgi architecture (Rambourg 1997; Farquhar and Palade 1998). More recent reviews that describe Golgi structure in great detail are provided by Marsh (2005) and Hua (2009). In this article, the most recent insights in mammalian Golgi architecture as revealed by distinct EM approaches are integrated into a general concept.     

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).     

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).     

The EGFR Family: Not So Prototypical Receptor Tyrosine Kinases

The epidermal growth factor receptor (EGFR) was among the first receptor tyrosine kinases (RTKs) for which ligand binding was studied and for which the importance of ligand-induced dimerization was established. As a result, EGFR and its relatives have frequently been termed “prototypical” RTKs. Many years of mechanistic studies, however, have revealed that—far from being prototypical—the EGFR family is quite unique. As we discuss in this review, the EGFR family uses a distinctive “receptor-mediated” dimerization mechanism, with ligand binding inducing a dramatic conformational change that exposes a dimerization arm. Intracellular kinase domain regulation in this family is also unique, being driven by allosteric changes induced by asymmetric dimer formation rather than the more typical activation-loop phosphorylation. EGFR family members also distinguish themselves from other RTKs in having an intracellular juxtamembrane (JM) domain that activates (rather than autoinhibits) the receptor and a very large carboxy-terminal tail that contains autophosphorylation sites and serves an autoregulatory function. We discuss recent advances in mechanistic aspects of all of these components of EGFR family members, attempting to integrate them into a view of how RTKs in this important class are regulated at the cell surface.The epidermal growth factor receptor (EGFR) is often considered the “prototypical” receptor tyrosine kinase (RTK) and has been intensively studied. It is one of a family of four RTKs in humans, the others being ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4 (Fig. 1). EGFR and its relatives are known oncogenic drivers in cancers such as lung cancer (Mok 2011), breast cancer (Arteaga et al. 2011), and glioblastoma (Libermann et al. 1985; Lee et al. 2006a; Vivanco et al. 2012), and inhibitors of these receptors have been among the most successful examples of targeted cancer therapies to date (Arteaga 2003; Moasser 2007; Zhang et al. 2007), including antibody therapeutics (e.g., trastuzumab and cetuximab) and small-molecule tyrosine kinase inhibitors (e.g., erlotinib, gefitinib, lapatinib).Open in a separate windowFigure 1.Schematic representation of EGFR/ErbB family receptors and their ligands. (A) The domain composition of human EGFR is shown. The extracellular region contains four domains: Domain I (amino acids 1–165), domain II (amino acids 165–310), domain III (amino acids 310–480), and domain IV (amino acids 480–620). Domains I and III are closely related in sequence, as are domains II and IV. Shown are representations of the structures of domains I and IV. Domain IV contains two types of disulfide-bonded module (C1 and C2). In C1 domains, a single disulfide constrains an intervening bow-like loop. In C2 modules, two disulfides link four successive cysteines in the patterns C1–C3 and C2–C4 to give a knot-like structure. A short extracellular juxtamembrane (eJM) region separates the extracellular region from the ∼23-amino-acid transmembrane (TM) domain. Within the cell, a short intracellular juxtamembrane (iJM) region separates the tyrosine kinase domain (TKD) from the membrane. A representative EGFR TKD structure is shown. The TKD is followed by a carboxy-terminal largely unstructured tail (amino acids 953–1186) that contains at least five tyrosine autophosphorylation sites. (B) EGFR is one of four members of the EGFR/ErbB family in humans. The other members are ErbB2/HER2, for which no soluble activating ligand is shown; ErbB3/HER3, which has a significantly impaired kinase domain (Jura et al. 2009b; Shi et al. 2010); and ErbB4/HER4. The primary active moiety of the ligands for these receptors is the EGF-like domain, shown as a cartoon structure (top right). EGFR is activated by the EGFR agonists: EGF itself, TGF-α (transforming growth factor α), ARG (amphiregulin), and EGN (epigen). The bispecific ligands regulate both EGFR and ErbB4: HB-EGF (heparin-binding EGF-like growth factor), EPR (epiregulin), and BTC (betacellulin). Neuregulins (NRGs) 1 and 2 regulate ErbB3 and ErbB4, whereas NRG3 and NRG4 appear to be specific for ErbB4 (Wilson et al. 2009).Far from being prototypical, however, it is now clear that regulation of EGFR family members is unique among RTKs (Ferguson 2008; Lemmon 2009; Lemmon and Schlessinger 2010). Structural studies have revealed how the ∼620-amino-acid isolated extracellular region is induced to dimerize after growth factor binding (Burgess et al. 2003) and how the isolated intracellular tyrosine kinase domain (TKD) becomes allosterically activated after forming an asymmetric dimer (Zhang et al. 2006; Jura et al. 2009a; Red Brewer et al. 2009). These findings have typically been interpreted in the context of a model in which EGF family receptors are regulated through ligand-induced receptor homodimerization or heterodimerization, with growth factor binding converting the receptor from an inactive monomeric configuration to an active dimeric conformation (Yarden and Schlessinger 1987; Schlessinger 1988, 2014; Ullrich and Schlessinger 1990). Although this original model has stood the test of time and initiated a whole field of studies of ligand-induced RTK dimerization, recent work has provided highly sophisticated views of a unique mode of allosteric regulation used by EGFR.     

Cellular Mechanisms Controlling Caspase Activation and Function

Caspases are the primary drivers of apoptotic cell death, cleaving cellular proteins that are critical for dismantling the dying cell. Initially translated as inactive zymogenic precursors, caspases are activated in response to a variety of cell death stimuli. In addition to factors required for their direct activation (e.g., dimerizing adaptor proteins in the case of initiator caspases that lie at the apex of apoptotic signaling cascades), caspases are regulated by a variety of cellular factors in a myriad of physiological and pathological settings. For example, caspases may be modified posttranslationally (e.g., by phosphorylation or ubiquitylation) or through interaction of modulatory factors with either the zymogenic or active form of a caspase, altering its activation and/or activity. These regulatory events may inhibit or enhance enzymatic activity or may affect activity toward particular cellular substrates. Finally, there is emerging literature to suggest that caspases can participate in a variety of cellular processes unrelated to apoptotic cell death. In these settings, it is particularly important that caspases are maintained under stringent control to avoid inadvertent cell death. It is likely that continued examination of these processes will reveal new mechanisms of caspase regulation with implications well beyond control of apoptotic cell death.Apoptosis is a form of programmed cell death that eliminates individual cells within an organism while preserving the overall structure of surrounding tissue. Many of the prominent morphological features of apoptosis were first described in 1972 by Kerr, Wyllie, and Currie (Kerr et al. 1972). However, it was not until the mid-1990s that apoptosis was linked to the activation of the cysteine-dependent aspartate-driven proteases (caspases), which cleave key intracellular substrates to promote cell death (Cerretti et al. 1992; Nicholson et al. 1995; Alnemri et al. 1996; Liu et al. 1996; Thornberry and Lazebnik 1998). Given the critical role that caspases play in dismantling the cell during apoptosis, their activation and subsequent activity are highly regulated. Failure of a cell to properly modulate caspase activity can cause aberrant or untimely apoptotic cell death, potentially leading to carcinogenesis, autoimmunity, neurodegeneration, and immunodeficiency (Thompson 1995; Hanahan and Weinberg 2000; Yuan and Yankner 2000; Li and Yuan 2008).Caspases are synthesized within the cell as inactive zymogens that lack significant protease activity. Thus, caspases are, in essence, regulated from the moment of protein synthesis in that they are not activated until receipt of specific death stimuli (Earnshaw et al. 1999). The primary structure of a caspase is an amino-terminal prodomain and a carboxy-terminal protease domain, which contains the key catalytic cysteine residue. Caspases are categorized as initiator or effector caspases, based on their position in apoptotic signaling cascades. The initiator caspases (caspase-2, -8, -9, and -10) act apically in cell death pathways and all share long, structurally similar prodomains. This group of enzymes is activated through “induced proximity” when adaptor proteins interact with the prodomains and promote caspase dimerization (Boatright et al. 2003; Baliga et al. 2004; Pop et al. 2006; Riedl and Salvesen 2007; Wachmann et al. 2010). In contrast, the effector caspases (caspase-3, -6, and -7) have shorter prodomains and exist in the cell as preformed, but inactive, homodimers. Following cleavage mediated by an initiator caspase, effector caspases act directly on specific cellular substrates to dismantle the cell. Although many individual caspase substrates have been implicated in specific aspects of cellular destruction (e.g., lamin cleavage is required for the efficient packaging of nuclei into small membrane-bound vesicles), recent proteomic approaches have greatly expanded the known repertoire of proteolytic products generated during apoptosis (Van Damme et al. 2005; Dix et al. 2008; Mahrus et al. 2008). Further work will be needed to confirm these findings and to determine how (or if) all of these substrates participate in the apoptotic process (see Poreba et al. 2013), especially as new details emerge on the relationship between posttranslational modifications, like phosphorylation, and caspase cleavage (Dix et al. 2012).     

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.     

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.     

Morphogen Gradient Formation

How morphogen gradients are formed in target tissues is a key question for understanding the mechanisms of morphological patterning. Here, we review different mechanisms of morphogen gradient formation from theoretical and experimental points of view. First, a simple, comprehensive overview of the underlying biophysical principles of several mechanisms of gradient formation is provided. We then discuss the advantages and limitations of different experimental approaches to gradient formation analysis.How a multicellular organism develops from a single fertilized cell has fascinated people throughout history. By looking at chick embryos of different developmental stages, Aristotle first noted that development is characterized by growing complexity and organization of the embryo (Balme 2002). During the 19th century, two events were recognized as key in development: cell proliferation and differentiation. Driesch first noted that to form organisms with correct morphological pattern and size, these processes must be controlled at the level of the whole organism. When he separated two sea urchin blastomeres, they produced two half-sized blastula, showing that cells are potentially independent, but function together to form a whole organism (Driesch 1891, 1908). Morgan noted the polarity of organisms and that regeneration in worms occurs with different rates at different positions. This led him to postulate that regeneration phenomena are influenced by gradients of “formative substances” (Morgan 1901).The idea that organisms are patterned by gradients of form-providing substances was explored by Boveri and Hörstadius to explain the patterning of the sea urchin embryo (Boveri 1901; Hörstadius 1935). The discovery of the Spemann organizer, i.e., a group of dorsal cells that when grafted onto the opposite ventral pole of a host gastrula induce a secondary body axis (Spemann and Mangold 1924), suggested that morphogenesis results from the action of signals that are released from localized groups of cells (“organizing centers”) to induce the differentiation of the cells around them (De Robertis 2006). Child proposed that these patterning “signals” represent metabolic gradients (Child 1941), but the mechanisms of their formation, regulation, and translation into pattern remained elusive.In 1952, Turing showed that chemical substances, which he called morphogens (to convey the idea of “form producers”), could self-organize into spatial patterns, starting from homogenous distributions (Turing 1952). Turing’s reaction–diffusion model shows that two or more morphogens with slightly different diffusion properties that react by auto- and cross-catalyzing or inhibiting their production, can generate spatial patterns of morphogen concentration. The reaction–diffusion formalism was used to model regeneration in hydra (Turing 1952), pigmentation of fish (Kondo and Asai 1995; Kondo 2002), and snails (Meinhardt 2003).At the same time that Turing showed that pattern can self-organize from the production, diffusion, and reaction of morphogens in all cells, the idea that morphogens are released from localized sources (“organizers” à la Spemann) and form concentration gradients was still explored. This idea was formalized by Wolpert with the French flag model for generation of positional information (Wolpert 1969). According to this model, morphogen is secreted from a group of source cells and forms a gradient of concentration in the target tissue. Different target genes are expressed above distinct concentration thresholds, i.e., at different distances to the source, hence generating a spatial pattern of gene expression (Fig. 1C).Open in a separate windowFigure 1.Tissue geometry and simplifications. (A) Gradients in epithelia (left) and mesenchymal tissues (right). Because of symmetry considerations, one row of cells (red outline) is representative for the whole gradient. (B) Magnified view of the red row of cells shown in A. Cells with differently colored nuclei (brown, orange, and blue) express different target genes. (C) A continuum model in which individual cells are ignored and the concentration is a function of the positions x. The morphogen activates different target genes above different concentration thresholds (brown and orange).Experiments in the 1970s and later confirmed that tissues are patterned by morphogen gradients. Sander showed that a morphogen released from the posterior cytoplasm specifies anterioposterior position in the insect egg (Sander 1976). Chick wing bud development was explained by a morphogen gradient emanating from the zone of polarizing activity to specify digit positions (Saunders 1972; Tickle, et al. 1975; Tickle 1999). The most definitive example of a morphogen was provided with the identification of Bicoid function in the Drosophila embryo (Nüsslein-Volhard and Wieschaus 1980; Frohnhöfer and Nüsslein-Volhard 1986; Nüsslein-Volhard et al. 1987) and the visualization of its gradient by antibody staining (Driever and Nüsslein-Volhard 1988b, 1988a; reviewed in Ephrussi and St Johnston 2004). Since then, many examples of morphogen gradients acting in different organs and species have been found.In an attempt to understand pattern formation in more depth, quantitative models of gradient formation have been developed. An early model by Crick shows that freely diffusing morphogen produced in a source cell and destroyed in a “sink” cell at a distance would produce a linear gradient in developmentally relevant timescales (Crick 1970). Today, it is known that a localized “sink” is not necessary for gradient formation: Gradients can form if all cells act as sinks and degrade morphogen, or even if morphogen is not degraded at all. Here, we review different mechanisms of gradient formation, the properties of these gradients, and the implications for patterning. We discuss the theory behind these mechanisms and the supporting experimental data.     

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

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.