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).     

Synchronization of Chromosome Dynamics and Cell Division in Bacteria

Bacterial cells have evolved a variety of regulatory circuits that tightly synchronize their chromosome replication and cell division cycles, thereby ensuring faithful transmission of genetic information to their offspring. Complex multicomponent signaling cascades are used to monitor the progress of cytokinesis and couple replication initiation to the separation of the two daughter cells. Moreover, the cell-division apparatus actively participates in chromosome partitioning and, particularly, in the resolution of topological problems that impede the segregation process, thus coordinating chromosome dynamics with cell constriction. Finally, bacteria have developed mechanisms that harness the cell-cycle-dependent positioning of individual chromosomal loci or the nucleoid to define the cell-division site and control the timing of divisome assembly. Each of these systems manages to integrate a complex set of spatial and temporal cues to regulate and execute critical steps in the bacterial cell cycle.In recent years, considerable progress has been made in understanding the cell biology of bacteria and, in particular, the organization and dynamics of their chromosomes (Shih and Rothfield 2006; Graumann 2007; Morris and Jensen 2008; Reyes-Lamothe et al. 2008b; Thanbichler and Shapiro 2008). It has emerged that bacterial cells have evolved a variety of mechanisms to closely coordinate replication and segregation of chromosomal DNA with cell division, thus ensuring that genetic information is passed on faithfully. In the absence of these regulatory circuits, premature formation of a division septum can lead to dissection of the nucleoid and generation of anucleate cells. Conversely, untimely origin firing before the end of cell division may result in the accumulation of supernumerary chromosomes, thereby interfering with proper cell cycle and gene regulation, and loading the cell with a substantial metabolic burden. This article first summarizes our current knowledge on checkpoints that couple replication initiation and the last stages of chromosome segregation to the progression of cell constriction. Subsequently, it discusses effects of chromosome dynamics on the temporal and spatial control of divisome assembly.     

Self-avoidance and Tiling: Mechanisms of Dendrite and Axon Spacing

The spatial pattern of branches within axonal or dendritic arbors and the relative arrangement of neighboring arbors with respect to one another impact a neuron''s potential connectivity. Although arbors can adopt diverse branching patterns to suit their functions, evenly spread branches that avoid clumping or overlap are a common feature of many axonal and dendritic arbors. The degree of overlap between neighboring arbors innervating a surface is also characteristic within particular neuron types. The arbors of some populations of neurons innervate a target with a comprehensive and nonoverlapping “tiled” arrangement, whereas those of others show substantial territory overlap. This review focuses on cellular and molecular studies that have provided insight into the regulation of spatial arrangements of neurite branches within and between arbors. These studies have revealed principles that govern arbor arrangements in dendrites and axons in both vertebrates and invertebrates. Diverse molecular mechanisms controlling the spatial patterning of sister branches and neighboring arbors have begun to be elucidated.Axonal and dendritic arbors adopt complex and morphologically diverse shapes that influence neural connectivity and information processing. In this article we review anatomical and molecular studies that elucidate how the arrangements of branches within neuronal arbors are established during development (isoneuronal spacing) and how the relative spacing of arbors is determined when multiple neurons together innervate a defined territory (heteroneuronal spacing). Together these mechanisms ensure that arbors achieve functionally appropriate coverage of input or output territories.Isoneuronal and heteroneuronal processes display a variety of spacing arrangements, suggesting a diversity of underlying molecular mechanisms. Self-avoidance can occur between branches that arise from a single soma (Yau 1976; Kramer and Kuwada 1983; Kramer and Stent 1985), implying that neurons are able to discriminate “self,” which they avoid, from “nonself” arbors, with which they coexist (Kramer and Kuwada 1983). Similarly, arbors from different cells that share the same function and together innervate a defined territory can create a pattern of minimally overlapping neighboring dendritic or axonal fields, known as tiling. Such spacing mechanisms ensure that arbors maximize their spread across a territory while minimizing the redundancy with which the territory is innervated. In contrast, adhesive interactions between arbors can operate to maintain coherence of dendrites at specific targets (Zhu and Luo 2004), or to bundle functionally similar processes and possibly coordinate their activity (Campbell et al. 2009). Understanding how processes are patterned relative to one another can help to uncover the functional logic of neural circuit organization.Here we focus primarily on mechanisms of isoneuronal and heteroneuronal avoidance that result in complete and nonredundant innervation of sensory or synaptic space. Such mechanisms have been studied extensively in systems where neuronal arbors innervate a two-dimensional plane, such as the retina or body wall (Wassle et al. 1981; Perry and Linden 1982; Hitchcock 1989; Lin and Masland 2004; Fuerst et al. 2009; Kramer and Stent 1985; Grueber et al. 2003; Sugimura et al. 2003; Sagasti et al. 2005). However, the principles regulating process spacing in these regions likely also apply in three dimensions, most prominently where processes are segregated into nonoverlapping domains or columns (Huckfeldt et al. 2009). It is also notable that nonneuronal cell types might similarly engage in self-avoidance and form tiling arrangements, including leech comb cells (Jellies and Kristan 1991) and mammalian astrocytes (Bushong et al. 2002; Ogata and Kosaka 2002; Livet et al. 2007). Elucidating the mechanisms of process spacing during development is therefore relevant for understanding principles of tissue organization inside and outside of the nervous system.     

Architecture of the Mammalian Golgi

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

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).     

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.     

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.     

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).     

Metabolic Stress in Autophagy and Cell Death Pathways

Growth factors and oncogenic kinases play important roles in stimulating cell growth during development and transformation. These processes have significant energetic and synthetic requirements and it is apparent that a central function of growth signals is to promote glucose metabolism to support these demands. Because metabolic pathways represent a fundamental aspect of cell proliferation and survival, there is considerable interest in targeting metabolism as a means to eliminate cancer. A challenge, however, is that molecular links between metabolic stress and cell death are poorly understood. Here we review current literature on how cells cope with metabolic stress and how autophagy, apoptosis, and necrosis are tightly linked to cell metabolism. Ultimately, understanding of the interplay between nutrients, autophagy, and cell death will be a key component in development of new treatment strategies to exploit the altered metabolism of cancer cells.Although single-celled organisms grow and proliferate based on nutrient availability, metazoan cells rely on growth factor input to promote nutrient uptake, regulate growth and proliferation, and survive (Raff 1992; Rathmell et al. 2000). Access and competition for these signals are critical in developmental patterning and to maintain homeostasis of mature tissues. Cells that do not receive proper growth factor signals typically atrophy, lose the ability to uptake and use extracellular nutrients, and instead induce the self-digestive process of autophagy as an intracellular energy source before ultimately undergoing programmed cell death. Cancer cells, in contrast, often become independent of extracellular growth signals by gaining mutations or expressing oncogenic kinases to drive intrinsic growth signals that mimic growth factor input, which can be the source of oncogene addiction. Growth factor input or oncogenic signals often drive highly elevated glucose uptake and metabolism (Rathmell et al. 2000; DeBerardinis et al. 2008; Michalek and Rathmell 2010). First described in cancer by Warburg in the 1920s, this highly glycolytic metabolic program is termed aerobic glycolysis and is a general feature of many nontransformed proliferative cells (Warburg 1956; DeBerardinis et al. 2008).Nutrient uptake and aerobic glycolysis induced by growth signals play key roles in cell survival (Vander Heiden et al. 2001). Manipulating cell metabolism as a means to promote the death of inappropriately dividing cells, therefore, is a promising new avenue to treat disease. Targeting the altered metabolism of cancer cells in particular is of great interest. It is still unclear at the molecular level, however, how inhibiting or modulating cell metabolism leads to apoptosis, and how these pathways may best be exploited (Dang et al. 2009; Wise and Thompson 2010).Growth factor or oncogenic kinases promote multiple metabolic pathways that are essential to prevent metabolic stress and may be targets in efforts to link metabolism and cell death (Vander Heiden et al. 2001). Decreased glucose metabolism on loss of growth signals leads to decreased ATP generation as well as loss in generation of many biosynthetic precursor molecules, including nucleic acids, fatty acids, and acetyl-CoA for acetylation (Zhao et al. 2007; Wellen et al. 2009; Coloff et al. 2011). Glucose is also important as a precursor for the hexosamine pathway, to allow proper glycosylation and protein folding in the endoplasmic reticulum (Dennis et al. 2009; Kaufman et al. 2010). If glucose metabolism remains insufficient or disrupted, the cells can switch to rely on mitochondrial oxidation of fatty acids and amino acids, which are energy rich but do not readily support cell growth and can lead to potentially dangerous levels of reactive oxygen species (Wellen and Thompson 2010). Amino acid deficiency can directly inhibit components of the signaling pathways downstream from growth factors and activate autophagy (Lynch 2001; Beugnet et al. 2003; Byfield et al. 2005; Nobukuni et al. 2005). Finally, hypoxia induces a specific pathway to increase nutrient uptake and metabolism via the hypoxia-inducible factor (HIF1/2α) that promotes adaptation to anaerobic conditions, but may lead to apoptosis if hypoxia is severe (Saikumar et al. 1998; Suzuki et al. 2001; Fulda and Debatin 2007).Typically a combination of metabolic stresses rather than loss of a single nutrient input occur at a given time (Degenhardt et al. 2006) and autophagy is activated to mitigate damage and provide nutrients for short-term survival (Bernales et al. 2006; Tracy et al. 2007; Altman et al. 2011; Guo et al. 2011). Autophagy is a cellular process of bulk cytoplasmic and organelle degradation common to nearly all eukaryotes. Unique double-membraned vesicles known as autophagosomes engulf cellular material and fuse with lysosomes to promote degradation of the contents (Kelekar 2005). Described in greater detail below, autophagy can reduce sources of stress, such as protein aggregates and damaged or dysfunctional intracellular organelles, and provide nutrients during times of transient and acute nutrient withdrawal.Despite the protective effects of autophagy, cells deprived of growth signals, nutrients, or oxygen for prolonged times will eventually succumb to cell death. Apoptosis is the initial death response on metabolic stress and is regulated by Bcl-2 family proteins. In healthy cells, antiapoptotic Bcl-2 family proteins, such as Bcl-2, Bcl-xl, and Mcl-1, bind and inhibit the multidomain proapoptotic proteins Bax and Bak (van Delft and Huang 2006; Walensky 2006; Chipuk et al. 2010). In metabolic stress, proapoptotic “BH3-only” proteins of the Bcl-2 family are induced or activated and bind to and inhibit the antiapoptotic Bcl-2 family proteins to allow activation of the proapoptotic Bax and Bak (Galonek and Hardwick 2006). The BH3-only proteins Bim, Bid, and Puma can also directly bind and activate Bax and Bak (Letai et al. 2002; Ren et al. 2010). Active Bax and Bak disrupt the outer mitochondrial membrane (termed mitochondrial outer-membrane permeabilization, or MOMP) and release several proapoptotic factors including cytochrome-C that activate the apoptosome that in turn activates effector caspases to cleave a variety of cellular proteins and drive apoptosis (Schafer and Kornbluth 2006). In cases in which these apoptotic pathways are suppressed, metabolic stress can instead lead to necrotic cell death (Jin et al. 2007).     

The Biochemistry of Mitosis

In this article, we will discuss the biochemistry of mitosis in eukaryotic cells. We will focus on conserved principles that, importantly, are adapted to the biology of the organism. It is vital to bear in mind that the structural requirements for division in a rapidly dividing syncytial Drosophila embryo, for example, are markedly different from those in a unicellular yeast cell. Nevertheless, division in both systems is driven by conserved modules of antagonistic protein kinases and phosphatases, underpinned by ubiquitin-mediated proteolysis, which create molecular switches to drive each stage of division forward. These conserved control modules combine with the self-organizing properties of the subcellular architecture to meet the specific needs of the cell. Our discussion will draw on discoveries in several model systems that have been important in the long history of research on mitosis, and we will try to point out those principles that appear to apply to all cells, compared with those in which the biochemistry has been specifically adapted in a particular organism.The aim of mitosis is to separate the genome and ensure that the two daughter cells inherit an equal and identical complement of chromosomes (Yanagida 2014). To achieve this, eukaryotic cells completely reorganize their microtubules to build a mitotic spindle that pulls apart the sister chromatids after the cohesin complexes are cut (see Cheeseman 2014; Hirano 2015; Reber and Hyman 2015; Westhorpe and Straight 2015) and, subsequently, use the actin cytoskeleton to divide the cell into two (cytokinesis) (see D’Avino et al. 2015). In some cells, such as in budding and fission yeasts, the spindle is built within the nucleus (closed mitosis), whereas in others, the nuclear envelope breaks down and the condensed chromosomes are captured by microtubules in the cytoplasm (open mitosis). This difference in the spatial organization of the mitotic cell has ramifications for the machinery controlling mitosis. In particular, the breakdown of the nuclear compartment disrupts the guanosine triphosphate (GTP)–guanosine diphosphate (GDP) gradient of the small GTPase called Ran. Ran usually controls nuclear-cytoplasmic transport through the importin chaperones; Ran-GDP in the cytoplasm promotes binding to nuclear transport substrates, whereas Ran-GTP in the nucleus promotes their dissociation (Güttler and Görlich 2011). As a result of nuclear envelope breakdown (NEBD), another Ran-GTP gradient is generated around the chromosomes, to which the RCC1 GTP-exchange factor binds (Clarke 2008). This Ran-GTP gradient is important for the interaction between microtubules and chromosomes because the high Ran-GTP levels around chromosomes promote the dissociation between the importin β chaperone and its binding partners, several of which help to stabilize or nucleate microtubules (Carazo-Salas et al. 1999; Kalab et al. 1999; Gruss et al. 2001; Wilde et al. 2001; Yokoyama et al. 2008).The dramatic reorganization of the cell at mitosis must be coordinated in both time and space. There are several key temporal events: entry to mitosis, sister chromatid separation, and mitotic exit, and these are effectively made unidirectional by the biochemical machinery. We will discuss the biochemistry behind each of these temporal events, in turn, but it is important to emphasize that the control mechanisms are also spatially organized. Our understanding of this spatial organization has improved dramatically with advances in the technology to detect gradients of activity in cells, and this has revealed the importance of local gradients of antagonistic protein kinases and phosphatases, GTP-binding protein regulators, and ubiquitin ligases and deubiquitylases, to name only a few of the more prominent examples (reviewed in Pines and Hagan 2011).     

Protein Solubility and Protein Homeostasis: A Generic View of Protein Misfolding Disorders

According to the “generic view” of protein aggregation, the ability to self-assemble into stable and highly organized structures such as amyloid fibrils is not an unusual feature exhibited by a small group of peptides and proteins with special sequence or structural properties, but rather a property shared by most proteins. At the same time, through a wide variety of techniques, many of which were originally devised for applications in other disciplines, it has also been established that the maintenance of proteins in a soluble state is a fundamental aspect of protein homeostasis. Taken together, these advances offer a unified framework for understanding the molecular basis of protein aggregation and for the rational development of therapeutic strategies based on the biological and chemical regulation of protein solubility.Virtually every complex biochemical process taking place in living cells depends on the ability of the molecules involved to self-assemble into functional structures (Dobson 2003; Robinson et al. 2007; Russel et al. 2009), and a sophisticated quality control system is responsible for regulating the reactions leading to this organization within the cellular environment (Dobson 2003; Balch et al. 2008; Hartl and Hayer-Hartl 2009; Powers et al. 2009; Vendruscolo and Dobson 2009). Proteins are the molecules that are essential for enabling, regulating, and controlling almost all the tasks necessary to maintain such a balance. To function, the majority of our proteins need to fold into specific three-dimensional structures following their biosynthesis in the ribosome (Hartl and Hayer-Hartl 2002). The wide variety of highly specific structures that results from protein folding, and which serve to bring key functional groups into close proximity, has enabled living systems to develop an astonishing diversity and selectivity in their underlying chemical processes by using a common set of just 20 basic molecular components, the amino acids (Dobson 2003). Given the central importance of protein folding, it is not surprising that the failure of proteins to fold correctly, or to remain correctly folded, is at the origin of a wide variety of pathological conditions, including late-onset diabetes, cystic fibrosis, and Alzheimer’s and Parkinson’s diseases (Dobson 2003; Chiti and Dobson 2006; Haass and Selkoe 2007). In many of these disorders proteins self-assemble in an aberrant manner into large molecular aggregates, notably amyloid fibrils (Chiti and Dobson 2006; Ramirez-Alvarado et al. 2010).