Replicating Damaged DNA in Eukaryotes

DNA damage is one of many possible perturbations that challenge the mechanisms that preserve genetic stability during the copying of the eukaryotic genome in S phase. This short review provides, in the first part, a general introduction to the topic and an overview of checkpoint responses. In the second part, the mechanisms of error-free tolerance in response to fork-arresting DNA damage will be discussed in some detail.Before eukaryotic cells divide, the successful completion of DNA replication during S phase is essential to preserve genomic integrity from one generation to the next. During this process, the replication apparatus traverses in the form of bidirectionally moving forks to synthesize new daughter strands. Cells use several means to ensure faithful copying of the parental strands—first, by means of regulatory mechanisms a correctly coordinated replication apparatus is established, and second, a high degree of fidelity during DNA synthesis is maintained by replicative polymerases (Kunkel and Bebenek 2000; Reha-Krantz 2010). However, under several stressful circumstances, endogenously or exogenously induced, the replication apparatus can stall (Tourriere and Pasero 2007). Mostly, structural deformations in the form of lesions or special template-specific features arrest the replication process, activate checkpoint pathways and set in motion repair or tolerance mechanisms to counter the stalling (Branzei and Foiani 2009; Zegerman and Diffley 2009). Basic replication mechanism, its regulatory pathways and means to tolerate DNA damage are largely conserved across eukaryotic species (Branzei and Foiani 2010; Yao and O’Donnell 2010). Understanding the mechanisms involved may enable therapeutic intervention to several human conditions arising from an incomplete replication or from the inability to tolerate perturbations (Ciccia et al. 2009; Preston et al. 2010; Abbas et al. 2013). Enhanced replication stress has also been commonly identified in precancerous lesions, and the inactivation of checkpoint responses coping with this presumably oncogene-induced condition is considered necessary to establish the fully malignant phenotype (Bartkova et al. 2005; Negrini et al. 2010).It is not possible to treat this topic in a comprehensive manner in the allotted space; the reader is referred to excellent recent reviews for more details (Branzei and Foiani 2010; Jones and Petermann 2012). We will attempt to provide an overview of the various strategies that a eukaryotic cell invokes to avoid problems caused by replication stress related to DNA damage and, if problems arise, to tolerate damage without endangering the entire process of genome duplication. In this context, we will only give a brief outline of checkpoint responses that are discussed in more detail in Sirbu and Cortez (2013) and Marechal and Zou (2013). Also, a detailed discussion of translesion synthesis can be reviewed in Sale (2013).     

Dormant Origins,the Licensing Checkpoint,and the Response to Replicative Stresses

Only ∼10% of replication origins that are licensed by loading minichromosome maintenance 2-7 (MCM2-7) complexes are normally used, with the majority remaining dormant. If replication fork progression is inhibited, nearby dormant origins initiate to ensure that all of the chromosomal DNA is replicated. At the same time, DNA damage-response kinases are activated, which preferentially suppress the assembly of new replication factories. This diverts initiation events away from completely new areas of the genome toward regions experiencing replicative stress. Mice hypomorphic for MCM2-7, which activate fewer dormant origins in response to replication inhibition, are cancer-prone and are genetically unstable. The licensing checkpoint delays entry into S phase if an insufficient number of origins have been licensed. In contrast, humans with Meier-Gorlin syndrome have mutations in pre-RC proteins and show defects in cell proliferation that may be a consequence of chronic activation of the licensing checkpoint.Replicating the large amount of DNA in eukaryotic cells is a complex task, requiring the activation of hundreds or thousands of origins spread throughout the genome. To maintain genetic stability, it is essential that during S phase genomic DNA is precisely duplicated, with no sections of DNA left unreplicated and no section of DNA replicated more than once. To prevent re-replication, cells divide the process of DNA replication into two non-overlapping phases. Prior to S phase, origins are licensed by the binding of minichromosome maintenance 2-7 (MCM2-7) double hexamers (Gillespie et al. 2001; Blow and Dutta 2005; Arias and Walter 2007). During S phase, these are activated as the core of the CMG (Cdc45-MCM-GINS) replicative helicase (Moyer et al. 2006; Ilves et al. 2010). Prior to the onset of S phase, licensing proteins are down-regulated or inhibited, so that no more origins can be licensed (Wohlschlegel et al. 2000; Tada et al. 2001; Li et al. 2003; Li and Blow 2005). One consequence of using this mechanism for preventing re-replication of DNA is that it is imperative that enough origins are licensed prior to S-phase entry, so that no regions of the genome remain unreplicated, even if some replication forks stall or some origins fail to initiate (Blow et al. 2011). Metazoan cells employ a licensing checkpoint to monitor that sufficient origins are licensed, inhibiting S-phase entry until this is established (Shreeram et al. 2002; Blow and Gillespie 2008).Here we review recent research showing how cells ensure complete genome duplication by licensing more replication origins in G1 than are normally used during S phase. The otherwise dormant replication origins become important for ensuring the completion of DNA replication if replication forks stall or are inhibited during S phase. We also review research showing how the licensing checkpoint ensures that a large enough number of origins are licensed before cells embark on S phase.     

Cadherin-mediated Intercellular Adhesion and Signaling Cascades Involving Small GTPases

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

Mitochondrial Evolution

Viewed through the lens of the genome it contains, the mitochondrion is of unquestioned bacterial ancestry, originating from within the bacterial phylum α-Proteobacteria (Alphaproteobacteria). Accordingly, the endosymbiont hypothesis—the idea that the mitochondrion evolved from a bacterial progenitor via symbiosis within an essentially eukaryotic host cell—has assumed the status of a theory. Yet mitochondrial genome evolution has taken radically different pathways in diverse eukaryotic lineages, and the organelle itself is increasingly viewed as a genetic and functional mosaic, with the bulk of the mitochondrial proteome having an evolutionary origin outside Alphaproteobacteria. New data continue to reshape our views regarding mitochondrial evolution, particularly raising the question of whether the mitochondrion originated after the eukaryotic cell arose, as assumed in the classical endosymbiont hypothesis, or whether this organelle had its beginning at the same time as the cell containing it.In 1970, Lynn Margulis published Origin of Eukaryotic Cells, an influential book that effectively revived the long-standing but mostly moribund idea that mitochondria and plastids (chloroplasts) evolved from free-living bacteria via symbiosis within a eukaryotic host cell (Margulis 1970). The discovery in the 1960s of DNA within these organelles together with the recognition that they contain a translation system distinct from that of the cytosol were two of the observations that Margulis marshaled in support of the endosymbiont hypothesis of organelle origins. Indeed, throughout her career, Margulis forcefully argued that symbiosis is a potent but largely unrecognized and unappreciated force in evolution (Margulis 1981). Technological developments in DNA cloning and sequencing in the 1970s and 1980s opened the way to the detailed characterization of mitochondrial genomes and genes, and the generation of key molecular data that were instrumental in affirming a bacterial origin of the mitochondrial and plastid genomes, allowing researchers to pinpoint the extant bacterial phyla to which these two organelles are most closely related. Over the past several decades, numerous reviews have documented in detail the biochemical and molecular and cell biological data bearing on the endosymbiont hypothesis of organelle origins (Gray 1982, 1983, 1989a,b, 1992, 1993, 1999; Gray and Doolittle 1982; Wallace 1982; Cavalier-Smith 1987b, 1992; Gray and Spencer 1996; Andersson and Kurland 1999; Gray et al. 1999, 2001, 2004; Lang et al. 1999; Andersson et al. 2003; Burger et al. 2003a; Bullerwell and Gray 2004). Various endosymbiotic models proposed over the years have been comprehensively critiqued (Martin et al. 2001), while the debates surrounding the endosymbiont hypothesis have been recounted in an engaging perspective that traces the development of ideas regarding organelle origins (Sapp 1994). Within a historical context, the present article emphasizes more recent data and insights that are relevant to continuing questions regarding how mitochondria originated and have since evolved.     

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).     

Regulating DNA Replication in Plants

Chromosomal DNA replication in plants has requirements and constraints similar to those in other eukaryotes. However, some aspects are plant-specific. Studies of DNA replication control in plants, which have unique developmental strategies, can offer unparalleled opportunities of comparing regulatory processes with yeast and, particularly, metazoa to identify common trends and basic rules. In addition to the comparative molecular and biochemical studies, genomic studies in plants that started with Arabidopsis thaliana in the year 2000 have now expanded to several dozens of species. This, together with the applicability of genomic approaches and the availability of a large collection of mutants, underscores the enormous potential to study DNA replication control in a whole developing organism. Recent advances in this field with particular focus on the DNA replication proteins, the nature of replication origins and their epigenetic landscape, and the control of endoreplication will be reviewed.Faithful genome duplication during the S phase of the cell cycle uses strategies largely conserved in all eukaryotes (DePamphilis and Bell 2011) and is pivotal to preserve genome integrity. Genome duplication in dividing plant cells has the same requirements and constraints than in animal cells, including the strict rule of occurring once and only once every cell cycle. The initial discoveries of several basic biological processes were performed in studies with plant cells, e.g., transposons, telomeres, RNA interference, to cite a few. DNA replication in eukaryotes is not an exception. Pioneering work in the mid-1950s showed the semiconservative nature of chromosomal DNA replication in the common bean Vicia faba (Taylor et al. 1957). Since then, plant DNA replication studies have focused primarily on defining temporal patterns of DNA replication at the chromosomal level along the S phase. DNA fiber autoradiography was first used in plants to determine replicon size at the single molecule level (Nitta and Nagata 1976; Van’t Hof 1976) and genome organization in early and late replicon families (Van’t Hof et al. 1978; Van’t Hof and Bjerknes 1981; reviewed in Bryant 2010; Costas et al. 2011a).In this work, we will focus on: (1) plant DNA replication proteins (see also Supplemental Table 3 online); (2) plant DNA replication origins and their epigenetic landscape; (3) novel licensing mechanisms; (4) the relevance of DNA replication proteins in the control of the endoreplication cycle during plant development, and, finally; (5) a brief overview of duplication of plant DNA viruses.     

Sexual Conflict between Parents: Offspring Desertion and Asymmetrical Parental Care

Parental care is an immensely variable social behavior, and sexual conflict offers a powerful paradigm to understand this diversity. Conflict over care (usually considered as a type of postzygotic sexual conflict) is common, because the evolutionary interests of male and female parents are rarely identical. I investigate how sexual conflict over care may facilitate the emergence and maintenance of diverse parenting strategies and argue that researchers should combine two fundamental concepts in social behavior to understand care patterns: cooperation and conflict. Behavioral evidence of conflict over care is well established, studies have estimated specific fitness implications of conflict for males or females, and experiments have investigated specific components of conflict. However, studies are long overdue to reveal the full implications of conflict for both males and females. Manipulating (or harming) the opposite sex seems less common in postzygotic conflicts than in prezygotic conflicts because by manipulating, coercing, or harming the opposite sex, the reproductive interest of the actor is also reduced. Parental care is a complex trait, although few studies have yet considered the implications of multidimensionality for parental conflict. Future research in parental conflict will benefit from understanding the behavioral interactions between male and female parents (e.g., negotiation, learning, and coercion), the genetic and neurogenomic bases of parental behavior, and the influence of social environment on parental strategies. Empirical studies are needed to put sexual conflict in a population context and reveal feedback between mate choice, pair bonds and parenting strategies, and their demographic consequences for the population such as mortalities and sex ratios. Taken together, sexual conflict offers a fascinating avenue for understanding the causes and consequences of parenting behavior, sex roles, and breeding system evolution.Sexual conflict over care is a type of evolutionary conflict that emerges from the different interests of males and females in regard to parental care (Trivers 1972; Clutton-Brock 1991; Chapman et al. 2003; Arnqvist and Rowe 2005). The conflict arises when the young benefit from the effort of either parent, but each parent pays only the cost of its own effort, so that each parent would have higher fitness if the other parent provides more care (Houston et al. 2005; Lessells 2006; Klug et al. 2012). Conflict refers to the way selection acts on the two sexes that have different optimum values in parental provisioning; between the two optima, sexually antagonistic selection operates (Lessells 2012). Sexual conflict over care can be seen as tug-of-war, because each parent is tempted to pull out of care leaving the other parent to provide more care for the young (Székely et al. 1996; Arnqvist and Rowe 2005; Lessells 2012).Sexual conflict over care seems to be the rule rather than the exception. The conflict may be resolved by one or both parents failing to adopt the optimal parenting for their mate and nonetheless remaining in conflict, or by both parents adopting the optima that suit their mate (i.e., exhibit the maximum provisioning possible). Examples of the latter conflict resolution (whereby the conflict is completely wiped out) are exceedingly rare and seem to be limited to three scenarios. First, conflict over care is not expected in obligate monogamy by both males and females so that the lifetime reproductive successes of both parents are identical. This may occur in semelparous organisms (i.e., both the male and the female put their resources into a single breeding event) or in iteroparous organisms with lifelong exclusive monogamy. Second, males and females might be genetically identical, so even though one or both sexes are polygamous, polygamy would benefit the same genome whether it is in the male or the female phenotype. Third, parental care is cost-free and thus parents provide maximum level of care (P Smiseth, pers. comm.). However, few, if any, organisms fit these restrictive assumptions, and thus conflict-free parenting seems exceedingly rare in nature: (1) some level of polygamy (by males, females, or both sexes) appears to be widespread; (2) the reproduction by genetically identical individuals (clones) as separate sexes (males and females) seems unlikely although not impossible if sex is determined environmentally; and (3) care provisioning, as far as we are aware, does have costs that discourage parents from providing their absolute maxima for a given batch of offspring.Parents may have conflicting interest over caring or deserting the young, the amount of care provided for each young, the number of simultaneous mates, the size and sex ratio of their brood, and the synchronization of birth for a clutch or litter of young (Westneat and Sargent 1996; Houston et al. 2005; Klug et al. 2012; Lessells 2012). Conflict between parents over care is usually labeled as a postzygotic conflict although resources had been already allocated into the gametes before fertilization as part of parental provisioning (Clutton-Brock 1991); other examples of postzygotic conflicts include infanticide and genomic imprinting (Chapman et al. 2003; Tregenza et al. 2006; Lessells 2012; see Palombit 2014).Studies of conflict over care are fascinating for at least four major reasons. First, parental care is diverse. There is great variation both between and within species in the types of care provided, duration of care, and the sex of the care-providing parent (Wilson 1975; Clutton-Brock 1991; McGraw et al. 2010; Royle et al. 2012), and sexual conflict is thought to be one of the main drivers of this diversity. Second, parental care is one of the core themes in breeding systems and sex role evolution, and it is increasingly evident that parental care can only be understood by dissecting the entangled relationships between ecological and life-history settings, and the variety of mating and parenting behavior (Székely et al. 2000; Webb et al. 2002; Wedell et al. 2006; Jennions and Kokko 2010; Klug et al. 2012). Third, parental care was (and is) one of the test beds of evolutionary game theory. Numerous models have been developed to understand how parents interact with each other and with their offspring (Trivers 1972; Maynard Smith 1977; Houston and Davies 1985; Balshine-Earn and Earn 1998; McNamara et al. 1999, 2000; Webb et al. 1999; Johnstone and Hinde 2006; Johnstone et al. 2014). Parental care research is one field in which empiricists are extensively testing the predictions of evolutionary game theoretic models in both the laboratory and wild populations (Székely et al. 1996; Balshine-Earn and Earn 1998; Harrison et al. 2009; Klug et al. 2012; Lessells 2012; van Dijk et al. 2012), although the congruence between theoretical and empirical work is not as tight as often assumed (Houston et al. 2013). Finally, parental care—wherever it occurs—is often a major component of fitness, because whether the offspring are cared for or abandoned has a large impact on their survival, maturation, and reproduction (Smiseth et al. 2012). Therefore, parental care (or the lack of it) may have an impact on population productivity and population growth and influences the resilience of populations to various threats (Bessa-Gomes et al. 2004; Veran and Beissinger 2009; Blumstein 2010). Thus, understanding the behavioral interactions between parents and the fitness implications of these interactions is highly relevant for population dynamics and biodiversity conservation (Alonzo and Sheldon 2010; Blumstein 2010).Sexual conflict over care has been reviewed recently (van Dijk and Székely 2008; Lessells 2012; Houston et al. 2013). Here, I focus on three issues that have not been extensively covered by previous reviews: (1) why sexual conflict over care occurs in some environments, whereas in others parental cooperation appears to dominate; (2) how can one detect sexual conflict over care; and (3) what are the implications of sexual conflict over care for macroevolution. I view causes and implications of parental care primarily from empirical perspectives; there are excellent reviews on the rich theoretical literature (Lessells 2006, 2012; Klug et al. 2012; Houston et al. 2013). My intention is not to be comprehensive; instead, I use selected examples to illustrate salient features of conflict over care. I focus on ecological and evolutionary aspects; for a discussion of the genetic and neuroendocrine bases of parental care, see Adkins-Regan (2005), McGraw et al. (2010), and Champagne and Curley (2012). I prefer to use the term “parental care” instead of “parental investment,” because the latter, as admitted by Trivers (1985), is extremely difficult to estimate empirically and thus may have a limited use in empirical studies (Mock and Parker 1997; McGraw et al. 2010). The term “parental investment” can be deceptive, if used without directly demonstrating the full costs of care. The term “parental care” is less restrictive, because it refers to any form of parental behavior that appears to increase the fitness of an offspring and is likely to have evolved for this function (Clutton-Brock 1991; Smiseth et al. 2012). In this review, I focus on families in the narrow sense (i.e., two parents and their offspring), although in numerous organisms the families are more extensive and may include several generations of offspring living together and/or unrelated individuals that assist the parents rearing the young.     

Retrograde Traffic from the Golgi to the Endoplasmic Reticulum

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

Origin of Microglia: Current Concepts and Past Controversies

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

DNA Replication Origins

The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression.Timely duplication of the genome is an essential step in the reproduction of any cell, and it is not surprising that chromosomal DNA synthesis is tightly regulated by mechanisms that determine precisely where and when new replication forks are assembled. The first model for a DNA synthesis regulatory circuit was described about 50 years ago (Jacob et al. 1963), based on the idea that an early, key step in building new replication forks was the binding of a chromosomally encoded initiator protein to specialized DNA regions, termed replication origins (Fig. 1). The number of replication origins in a genome is, for the most part, dependent on chromosome size. Bacterial and archaeal genomes, which usually consist of a small circular chromosome, frequently have a single replication origin (Barry and Bell 2006; Gao and Zhang 2007). In contrast, eukaryotic genomes contain significantly more origins, ranging from 400 in yeast to 30,000–50,000 in humans (Cvetic and Walter 2005; Méchali 2010), because timely duplication of their larger linear chromosomes requires establishment of replication forks at multiple locations. The interaction of origin DNA and initiator proteins (Fig. 1) ultimately results in the assembly of prereplicative complexes (pre-RCs), whose role is to load and activate the DNA helicases necessary to unwind DNA before replication (Remus and Diffley 2009; Kawakami and Katayama 2010). Following helicase-catalyzed DNA unwinding, replisomal proteins become associated with the single-stranded DNA, and new replication forks proceed bidirectionally along the genome until every region is duplicated (for review, see O’Donnell 2006; Masai et al. 2010).Open in a separate windowFigure 1.Revised versions of the replicon model for all domains of life. For cells of each domain type, trans-acting initiators recognize replication origins to assemble prereplicative complexes required to unwind the DNA and load DNA helicase. Eukaryotic initiators are preassembled into hexameric origin recognition complexes (ORCs) before interacting with DNA. In prokaryotes, single initiators (archaeal Orc1/Cdc6 or bacterial DnaA) bind to recognition sites and assemble into complexes on DNA. In all cases, the DNA helicases (MCMs or DnaB) are recruited to the origin and loaded onto single DNA strands. In bacteria, DNA-bending proteins, such as Fis or IHF, may modulate the assembly of pre-RC by bending the origin DNA. Two activities of DnaA are described in the figure. The larger version binds to recognition sites, and the smaller version represents DnaA required to assist DnaC in loading DnaB helicase on single-stranded DNA.Initiator proteins from all forms of life share structural similarities, including membership in the AAA⁺ family of proteins (ATPases associated with various cellular activities) (Duderstadt and Berger 2008; Wigley 2009) that are activated by ATP binding and inactivated by ATP hydrolysis (Duderstadt and Berger 2008; Duncker et al. 2009; Kawakami and Katayama 2010). Despite these similarities, initiators assemble into prereplicative complexes in two fundamentally different ways (Fig. 2). In prokaryotes, initiator monomers interact with the origin at multiple repeated DNA sequence motifs, and the arrangement of these motifs (see below) can direct assembly of oligomers that mediate strand separation (Erzberger et al. 2006; Rozgaja et al. 2011). In eukaryotes, a hexameric origin recognition complex (ORC) binds to replication origins and then recruit additional factors (as Cdc6 and Cdt1) that will themselves recruit the hexameric MCM2-7 DNA helicase to form a prereplicative complex (for review, see Diffley 2011). This process occurs during mitosis and along G₁ and is called “DNA replication licensing,” a crucial regulation of eukaryotic DNA replication (for review, see Blow and Gillespie 2008). Importantly, this complex is still inactive, and only a subset of these preassembled origins will be activated in S phase. This process is, therefore, fundamentally different from initiation of replication in bacteria. Moreover, because sequence specificity appears more relaxed in large eukaryotic genomes, prokaryotic mechanisms that regulate initiator–DNA site occupation must be replaced by alternative mechanisms, such as structural elements or the use of epigenetic factors.Open in a separate windowFigure 2.Functional elements in some well-studied prokaryotic replication origins. (A) Bacterial oriCs. The DNA elements described in the text are (arrows) DnaA recognition boxes or (boxes) DNA unwinding elements (DUEs). When recognition site affinities are known, colored arrows designate high- (K_d > 100 nm) and low- (K_d < 100 nm) affinity sites. (B) Archaeal oriCs. Arrows and boxes designate DNA elements as in A, but the initiator protein is Orc1/Cdc6 rather than DnaA. (Thick arrows) Long origin recognition boxes (ORBs); (thin arrows) shorter versions (miniORBs). Both ORBs and miniORBs are identified in Pyrococcus. DUEs are not yet well defined for Helicobacter or Sulfolobus genera and are not labeled in this figure.Here, we describe replication origins on prokaryotic and eukaryotic genomes below, with a particular focus on the attributes responsible for orderly initiator interactions and origin selection specificity, as well as on the shift from origin sequence-dependent regulation to epigenetic regulation. You are also referred to other related articles in this collection and several recent reviews covering the topics of DNA replication initiation in more detail (Méchali 2010; Beattie and Bell 2011; Blow et al. 2011; Bryant and Aves 2011; Ding and MacAlpine 2011; Dorn and Cook 2011; Kaguni 2011; Leonard and Grimwade 2011; Sequeira-Mendes and Gomez 2012).     

Postreplicative Mismatch Repair

The mismatch repair (MMR) system detects non-Watson–Crick base pairs and strand misalignments arising during DNA replication and mediates their removal by catalyzing excision of the mispair-containing tract of nascent DNA and its error-free resynthesis. In this way, MMR improves the fidelity of replication by several orders of magnitude. It also addresses mispairs and strand misalignments arising during recombination and prevents synapses between nonidentical DNA sequences. Unsurprisingly, MMR malfunction brings about genomic instability that leads to cancer in mammals. But MMR proteins have recently been implicated also in other processes of DNA metabolism, such as DNA damage signaling, antibody diversification, and repair of interstrand cross-links and oxidative DNA damage, in which their functions remain to be elucidated. This article reviews the progress in our understanding of the mechanism of replication error repair made during the past decade.The mismatch repair (MMR) system is one of the key guardians of genomic integrity. Its malfunction leads to a substantial increase in spontaneous mutagenesis, illegitimate recombination, and cancer in mammals. MMR improves the fidelity of DNA replication by several orders of magnitude by excising sections of the nascent strand containing mispaired nucleotides. It is likely that MMR has evolved to carry out this function in order to ensure that daughter cells inherit an exact replica of the parental genome. But MMR also controls the fidelity of recombination by removing mispairs from heteroduplexes arising between donor and recipient strands and possibly even rejecting synapses between sequences that are too diverged. Indeed, the existence of MMR was first invoked in the 1960s to explain the unanticipated segregation of genetic markers in fungi and bacteria (for a comprehensive overview of the field, see chapter 12 in Friedberg et al. 1995). During the intervening 50 years, our understanding of MMR has made enormous progress; the main protagonists, as well as many “extras” that participate in this complex process, have been identified, initially in a series of genetic and biochemical experiments and later by sequence homology searches that were made possible by the high degree of evolutionary conservation of MMR. Analysis of the primary sequences of these polypeptides then helped to uncover their enzymatic activities that were confirmed by biochemical and structural studies. In vitro MMR assays using cell extracts and recombinant DNA substrates carrying single mismatches at defined positions led to the discovery of criteria required for efficient, strand-directional MMR. Finally, the Escherichia coli and the minimal human MMR systems could be reconstituted from purified recombinant proteins (Dzantiev et al. 2004). Despite this wealth of knowledge, however, we still lack detailed understanding of the molecular transactions that lead to successful repair of replication errors, and our notion of the role(s) of MMR proteins during recombination is highly speculative.Since the discovery of a link between its malfunction and cancer (for recent reviews, see Wimmer and Etzler 2008; Hewish et al. 2010), MMR has attracted a great deal of attention, and recent progress in our understanding of this pathway has been the subject of several reviews (Stojic et al. 2004; Kunkel and Erie 2005; Iyer et al. 2006; Jiricny 2006; Hsieh and Yamane 2008; Li 2008; George and Alani 2012; Peña-Diaz and Jiricny 2012). This article therefore provides only a brief overview of the MMR process and focuses primarily on the most recent insights into this complex pathway of DNA metabolism.     

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.