Clearing the Dead: Apoptotic Cell Sensing,Recognition, Engulfment,and Digestion

Clearance of apoptotic cells is the final stage of programmed cell death. Uncleared corpses can become secondarily necrotic, promoting inflammation and autoimmunity. Remarkably, even in tissues with high cellular turnover, apoptotic cells are rarely seen because of efficient clearance mechanisms in healthy individuals. Recently, significant progress has been made in understanding the steps involved in prompt cell clearance in vivo. These include the sensing of corpses via “find me” signals, the recognition of corpses via “eat me” signals and their cognate receptors, the signaling pathways that regulate cytoskeletal rearrangement necessary for engulfment, and the responses of the phagocyte that keep cell clearance events “immunologically silent.” This study focuses on our understanding of these steps.Multicellular organisms execute the majority of unwanted cell populations in a regulated fashion via the process of apoptosis (Henson and Hume 2006; Nagata et al. 2010). Examples of unwanted cells include excess cells generated during development, cells infected with intracellular bacteria or viruses, transformed or malignant cells capable of tumorigenesis, and cells irreparably damaged by cytotoxic agents. Swift removal of these cells is necessary for maintenance of overall health and homeostasis and prevention of autoimmunity, pathogen burden, or cancer. Quick removal of dying cells is a key final step, if not the ultimate goal of the apoptotic program.The term “phagocytosis” refers to an internalization process by which larger particles, such as bacteria and dead/dying cells, are engulfed and processed within a membrane-bound vesicle called the phagosome (Ravichandran and Lorenz 2007). A phagocyte is any cell that is capable of engulfment, including “professional” phagocytes such as macrophages, immature dendritic cells, and neutrophils. Metazoa have multiple mechanisms for clearing apoptotic cells, often depending on the tissue and apoptotic cell type (Gregory 2009). Macrophages and immature dendritic cells readily engulf dead or dying cells in tissues such as bone marrow (where a large number of new hematopoietic cells are generated), spleen (during or after an immune response), and the thymus (in young animals during T-lymphocyte development). In other tissues, neighboring “nonprofessional” phagocytes can also mediate the clearance of apoptotic targets. For example, in the mammary epithelium, viable mammary epithelial cells engulf apoptotic mammary epithelial cells after cessation of lactation (Monks et al. 2005, 2008). What distinguishes the phagocytosis of apoptotic cells from the phagocytosis of most bacteria or necrotic cells is the lack of a pro-inflammatory immune response (Henson 2005). This article discusses apoptotic cell engulfment, specifically the recruitment of phagocytes, through “find me” signals, the recognition of apoptotic cells by phagocytes via “eat me” signals, the internalization process and signaling pathways used for cytoskeletal rearrangement, and finally the digestion of apoptotic cells and phagocytic response to this process (Fig. 1).Open in a separate windowFigure 1.The steps of efficient apoptotic cell clearance. First, “find me” signals released by apoptotic cells are recognized via their cognate receptors on the surface of phagocytes. This is the sensing stage and stimulates phagocyte migration to the location of apoptotic cells. Second, phagocytes recognize exposed “eat me” signals on the surface of apoptotic cells via their phagocytic receptors, which leads to downstream signaling events culminating in Rac activation. Finally, further signaling events within the phagocyte regulate the digestion and processing of the apoptotic cell meal and the secretion of anti-inflammatory cytokines.     

Holliday Junction Resolvases

Four-way DNA intermediates, called Holliday junctions (HJs), can form during meiotic and mitotic recombination, and their removal is crucial for chromosome segregation. A group of ubiquitous and highly specialized structure-selective endonucleases catalyze the cleavage of HJs into two disconnected DNA duplexes in a reaction called HJ resolution. These enzymes, called HJ resolvases, have been identified in bacteria and their bacteriophages, archaea, and eukaryotes. In this review, we discuss fundamental aspects of the HJ structure and their interaction with junction-resolving enzymes. This is followed by a brief discussion of the eubacterial RuvABC enzymes, which provide the paradigm for HJ resolvases in other organisms. Finally, we review the biochemical and structural properties of some well-characterized resolvases from archaea, bacteriophage, and eukaryotes.Homologous recombination (HR) is an essential process that promotes genetic diversity during meiosis (see Lam and Keeney 2014; Zickler and Kleckner 2014). However, in somatic cells, HR plays a key role in conserving genetic information by facilitating DNA repair, thereby ensuring faithful genome duplication and limiting the divergence of repetitive DNA sequences (see Mehta and Haber 2014). As shown in Figure 1, HR is initiated by a DNA double-strand break, the ends of which are resected to produce single-stranded (ss) 3′-overhangs (see Symington 2014). Homologous strand invasion by one of the 3′ overhangs (e.g., one catalyzed by Escherichia coli RecA or human RAD51) leads to the formation of a displacement loop (D-loop) (see Morrical 2014). The invading 3′ end of the D-loop can then be extended by a DNA polymerase, which uses the homologous strand as a template for DNA synthesis. Recombination then proceeds in one of several different ways, some of which involve second-end capture, such that the other resected 3′ end anneals to the displaced strand of the D-loop (Szostak et al. 1983). In the resulting recombination intermediate, the two interacting DNAs are linked by nicked Holliday junctions (HJs). Additional DNA synthesis and nick ligation lead to the formation of a double Holliday junction (dHJ) intermediate. In eukaryotes, dHJs are removed primarily by “dissolution” (Fig. 1, bottom left) (see Bizard and Hickson 2014). This pathway involves the combined activities of a DNA helicase and a type IA topoisomerase, which catalyze branch migration and decatenation of the dHJ into noncrossover products (Manthei and Keck 2014). In somatic cells, this is essential for the avoidance of sister-chromatid exchanges (SCEs) and loss of heterozygosity. Alternatively, dHJs can be processed by “resolution” in reactions mediated by canonical or noncanonical mechanisms of endonuclease-mediated cleavage into either crossover or noncrossover products (Fig. 1, bottom middle and right).Open in a separate windowFigure 1.Pathways for the formation and processing of Holliday junctions. Resected DNA double-strand breaks invade homologous duplex DNA to create a joint molecule, or displacement-loop structure. The invading 3′ end then serves as a primer for DNA synthesis, leading to second end capture and the formation of a double Holliday junction. In eukaryotes, these structures are removed by “dissolution” (bottom left panel) or “resolution” (bottom middle and right panels). Canonical Holliday junction resolvases introduce a pair of symmetrical and coordinated nicks across one of the helical axes (bottom middle panel) to generate nicked DNA duplexes that can be directly ligated. Alternatively, noncanonical resolvases cleave Holliday junctions with asymmetric nicks to produce gapped and flapped DNA duplexes that require further processing prior to ligation (bottom right panel). *Mitochondrial Holliday junction resolvase.     

Oligodendrocytes: Myelination and Axonal Support

Myelinated nerve fibers have evolved to enable fast and efficient transduction of electrical signals in the nervous system. To act as an electric insulator, the myelin sheath is formed as a multilamellar membrane structure by the spiral wrapping and subsequent compaction of the oligodendroglial plasma membrane around central nervous system (CNS) axons. Current evidence indicates that the myelin sheath is more than an inert insulating membrane structure. Oligodendrocytes are metabolically active and functionally connected to the subjacent axon via cytoplasmic-rich myelinic channels for movement of macromolecules to and from the internodal periaxonal space under the myelin sheath. This review summarizes our current understanding of how myelin is generated and also the role of oligodendrocytes in supporting the long-term integrity of myelinated axons.When Virchow analyzed the fine structure of the brain in the 1850s, he recognized that there were more cells within the “Nervenkitt” than astrocytes, but because of the imperfect staining methods, they remained obscure and were only named the “third element” (reviewed in Somjen 1988; Rosenbluth 1999). It was decades later that Pío del Río-Hortega (1921) applied a staining method involving silver carbonate, thereby shedding new light on the rest of the interstitial cells. These cells were found to contain numerous short processes and were named oligodendroglia and microglia. Defining features of oligodendrocytes were their small cell bodies filled with nuclei containing large amounts of chromatin, and their cellular extensions that lacked fibers but were filled with cytoplasmic granules (Fig. 1). When the tissue was optimally preserved, the silver impregnation uncovered a tremendous complexity of extensions (Fig. 1). del Río-Hortega was able to distinguish four types of oligodendrocytes (Fig. 1): Type I cells generate many different myelin segments on small diameter axons in diverse orientations; type II cells are similar to type I in size and number, but myelin segments run in parallel to each other; type III oligodendrocytes ensheath fewer axons of larger diameter; and type IV oligodendrocytes have a cell body closely apposed to a single very large axon similar to Schwann cells. From the staining, it became clear that some of the processes run in parallel to axons and appeared to cover the axons with “myelin,” a term that was introduced by Virchow already in 1858, long before its origin and function were known.Open in a separate windowFigure 1.Morphology of oligodendroglia in the cerebellum of a cat. (Top right) Cellular processes and branches follow the orientation of the nerve fibers and form complex wraps as shown in del Río-Hortega (1921). (Top left) White matter of a newborn human brain: A, Elongated interfascicular cells filled with spherical granules of variable size; B and C, round granular cells; D, astrocytes with long processes; and E, nucleus of a microglia as shown in del Río-Hortega (1921). (Bottom left) The four types of oligodendrocytes recognized by del Río-Hortega. (Bottom right) Oligodendrocytes expressing proteolipid protein (PLP)–enhanced green fluorescent protein (EGFP) in transgenic mice. Because of its bulky EGFP tag, most of it is found within the cytoplasmic-rich spaces or myelin, including the myelinic channels.Penfield (1924) reinforced the view that the formation of myelin is likely the main role of oligodendrocytes. However, myelination was not considered their only function. Within the gray matter, a fraction of oligodendrocytes were termed “perineuronal” satellite cells, which did not form myelin, but were in close contact with the cell body of neurons, suggesting an interdependent relation. Some oligodendroglia were found in close association with small vessels and were, therefore, subclassified as “perivascular satellites.”The major findings that supported the role of oligodendrocytes in myelin generation were their high number in white matter tracts, their appearance in development at the time of myelination, and their position close to the myelin sheaths. Curiously, the large number of granules in the cytosol of oligodendrocytes, reminiscent of cells of secretory glands, was also taken as evidence for a function in myelin formation. At that time, myelin was not considered to be an extension of the oligodendroglial plasma membrane, however, but rather a fatty axonal substance secreted into the extracellular space. The importance of the “axon cylinder” for saltatory impulse propagation was well recognized, but it was the seminal work of Betty Ben Geren (1954), using electron microscopy (EM) in the chick peripheral nervous system, which showed that myelin is not axon derived, but rather a continuous membranous extension of Schwann cells. A principally similar relationship, with spiral wrapping of the glial plasma membrane around the axon, was confirmed later for oligodendrocytes in the central nervous system (CNS) (for an excellent early review, see Bunge 1968; Hildebrand et al. 1993). Together, these studies have turned oligodendrocytes and myelin from a putty-amorphous “Nervenkitt” into a fascinating study object of cell biology.     

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).     

Presynaptic Membrane Retrieval and Endosome Biology: Defining Molecularly Heterogeneous Synaptic Vesicles

The release and uptake of neurotransmitters by synaptic vesicles is a tightly controlled process that occurs in response to diverse stimuli at morphologically disparate synapses. To meet these architectural and functional synaptic demands, it follows that there should be diversity in the mechanisms that control their secretion and retrieval and possibly in the composition of synaptic vesicles within the same terminal. Here we pay particular attention to areas where such diversity is generated, such as the variance in exocytosis/endocytosis coupling, SNAREs defining functionally diverse synaptic vesicle populations and the adaptor-dependent sorting machineries capable of generating vesicle diversity. We argue that there are various synaptic vesicle recycling pathways at any given synapse and discuss several lines of evidence that support the role of the endosome in synaptic vesicle recycling.Chemical synapses contain discrete numbers of synaptic vesicles, which are capable of sustaining neurotransmitter release. Sustained neurotransmission occurs despite the secretory demands imposed by persistent and diverse patterns of neuronal electrical activity. Maintaining synaptic vesicle numbers requires local mechanisms to regenerate these vesicles to prevent their exhaustion, preserve plasma membrane surface area, and to maintain the molecularly distinct identity of a vesicle versus plasma membrane. Rizzoli and Betz (2005) eloquently draw a parallel between chemical neurotransmission with synapse chatter saying that some synapses “whisper,” whereas others “shout.” The “louder” the synapse, the more synaptic vesicles are required, extending from a few hundred vesicles (whisperers) to nearly thousands (shouters). This beautiful analogy implies that every synapse has just one “voice” or species of vesicle. Here we will present the case that synapses are more like choirs in which multiple vesicle species or “voices” contribute to the “pianissimo” or “fortissimo” parts of chemical neurotransmission.Synaptic terminals show a range of structural and functional differences in distinct regions of the brain, suggesting that the mechanisms for exocytosis/endocytosis coupling, as well as local vesicle recycling, may also be diverse. On one side, the Calyx of Held nerve terminal participates in fast and sustained synaptic transmission at high frequency (800 Hz), which is crucial for sound localization in the auditory brainstem (Taschenberger and von Gersdorff 2000; Borst and Soria van Hoeve 2012). The Calyx of Held houses ∼70,000 synaptic vesicles with nearly 3000 vesicles docked per Calyx terminal. These docked vesicles are distributed across the ∼500 active zones that exist per Calyx where vesicle fusion occurs (Satzler et al. 2002). On the other hand, hippocampal synapses fire action potentials at ∼0.5 Hz in bursts (Dobrunz and Stevens 1999). This synapse contains ∼200 synaptic vesicles and one active zone with ∼10 vesicles docked (Schikorski and Stevens 1997). With such a wide functional and structural gamut of synapses, it is reasonable to hypothesize that synaptic vesicles may differ in their retrieval mechanisms, not just at the rate at which the process occurs but also in the molecular pathways used.Two synaptic vesicle retrieval mechanisms, namely clathrin/AP-2/dynamin-dependent biogenesis and kiss-and-run, have been summarized in outstanding recent reviews (see, for example, Augustine et al. 2006; Rizzoli and Jahn 2007; Smith et al. 2008; Royle and Lagnado 2010; Ferguson and De Camilli 2012; Saheki and De Camilli 2012). Therefore, here we focus on the coupling of secretion and membrane retrieval, as well as endosome sorting. We will discuss new developments supporting the existence of diverse functional and molecular pools of synaptic vesicles and how endocytosis and endosome retrieval mechanisms may generate these vesicle pools.     

Wnt Signaling in Vertebrate Axis Specification

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

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.     

Synaptic Vesicle Exocytosis

Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for “soluble NSF-attachment protein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for “N-ethylmaleimide sensitive factor”). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSPα, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the “active zone”) that includes Munc13 and RIM proteins as central components.Synaptic vesicles are uniform organelles of ∼40 nm diameter that constitute the central organelle for neurotransmitter release. Each presynaptic nerve terminal contains hundreds of synaptic vesicles that are filled with neurotransmitters. When an action potential depolarizes the presynaptic plasma membrane, Ca²⁺-channels open, and Ca²⁺ flows into the nerve terminal to trigger the exocytosis of synaptic vesicles, thereby releasing their neurotransmitters into the synaptic cleft (Fig. 1). Ca²⁺ triggers exocytosis by binding to synaptotagmin; after exocytosis, vesicles are re-endocytosed, recycled, and refilled with neurotransmitters. Recycling can occur by multiple parallel pathways, either by fast recycling via local reuse of vesicles (“kiss-and-run” and “kiss-and-stay”), or by slower recycling via an endosomal intermediate (Fig. 1).Open in a separate windowFigure 1.The synaptic vesicle cycle. A presynaptic nerve terminal is depicted schematically as it contacts a postsynaptic neuron. The synaptic vesicle cycle consists of exocytosis (red arrows) followed by endocytosis and recycling (yellow arrows). Synaptic vesicles (green circles) are filled with neurotransmitters (NT; red dots) by active transport (neurotransmitter uptake) fueled by an electrochemical gradient established by a proton pump that acidifies the vesicle interior (vesicle acidification; green background). In preparation to synaptic exocytosis, synaptic vesicles are docked at the active zone, and primed by an ATP-dependent process that renders the vesicles competent to respond to a Ca²⁺-signal. When an action potential depolarizes the presynaptic membrane, Ca²⁺-channels open, causing a local increase in intracellular Ca²⁺ at the active zone that triggers completion of the fusion reaction. Released neurotransmitters then bind to receptors associated with the postsynaptic density (PSD). After fusion pore opening, synaptic vesicles probably recycle via three alternative pathways: local refilling with neurotransmitters without undocking (“kiss-and-stay”), local recycling with undocking (“kiss-and-run”), and full recycling of vesicles with passage through an endosomal intermediate. (Adapted from Südhof 2004.)Due to their small size, synaptic vesicles contain a limited complement of proteins that have been described in detail (Südhof 2004; Takamori et al. 2006). Although the functions of several vesicle components remain to be identified, most vesicle components participate in one of three processes: neurotransmitter uptake and storage, vesicle exocytosis, and vesicle endocytosis and recycling. In addition, it is likely that at least some vesicle proteins are involved in the biogenesis of synaptic vesicles and the maintenance of their exquisite uniformity and stability, but little is known about how vesicles are made, and what determines their size.     

Protein Targeting and Transport as a Necessary Consequence of Increased Cellular Complexity

With increasing intracellular complexity, a new cell-biological problem that is the allocation of cytoplasmically synthesized proteins to their final destinations within the cell emerged. A special challenge is thereby the translocation of proteins into or across cellular membranes. The underlying mechanisms are only in parts well understood, but it can be assumed that the course of cellular evolution had a deep impact on the design of the required molecular machines. In this article, we aim to summarize the current knowledge and concepts of the evolutionary development of protein trafficking as a necessary premise and consequence of increased cellular complexity.

The evolution of modern cells is arguably the most challenging and important problem the field of biology has ever faced …—Carl R. Woese(Woese 2002)

Current models may accept that all modern eukaryotic cells arose from a single common ancestor (the cenancestral eukaryote), the nature of which is—owing to the lack of direct living or fossil descendants—still highly under debate (de Duve 2007). The chimeric nature of eukaryotic genomes with eubacterial and archaebacterial shares led to a discussion about the origin of this first “proto-eukaryote.” Several models exist (see Fig. 1), which either place the evolution of the nucleus before or after the emergence of the mitochondrion (outlined in Koonin 2010; Martijn and Ettema 2013). According to the different postulated scenarios (summarized in Embley and Martin 2006), eukaryotes in the latter case might have evolved by endosymbiosis between a hydrogen-producing, oxygen-producing, or sulfur-dependent α-proteobacterium and an archaebacterial host (Fig. 1C). The resulting mitochondriate prokaryote would have evolved the nucleus subsequently. In other scenarios (Fig. 1B), the cenancestral eukaryote emerged by cellular fusion or endosymbiosis of a Gram-negative, maybe hydrogen-producing, eubacterium and a methanogenic archaebacterium or eocyte, leading to a primitive but nucleated amitochondrial (archezoan) cell (Embley and Martin 2006, and references therein). As a third alternative, Cavalier-Smith (2002) suggested a common eubacterial ancestor for eukaryotes and archaebacteria (the Neomuran hypothesis) (Fig. 1A).Open in a separate windowFigure 1.Evolution of the last common ancestor of all eukaryotic cells. A schematic depiction of the early eukaryogenesis. Because of the lack of living and fossil descendants, several opposing models are discussed (A–C). The anticipated order of events is shown as a flow chart. For details, see text. (Derived from Embley and Martin 2006; Koonin 2010.)