Cell Biology of Mitotic Recombination

Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.Homologous recombination (HR) is defined as the homology-directed exchange of genetic information between two DNA molecules (Fig. 1). Mitotic recombination is often initiated by single-stranded DNA (ssDNA), which can arise by several avenues (Mehta and Haber 2014). They include the processing of DNA double-strand breaks by 5′ to 3′ resection, during replication of damaged DNA, or during excision repair (Symington 2014). The ssDNA is bound by replication protein A (RPA) to control its accessibility to the Rad51 recombinase (Sung 1994, 1997a; Sugiyama et al. 1997; Morrical 2014). The barrier to Rad51-catalyzed recombination imposed by RPA can be overcome by a number of mediators, such as BRCA2 and Rad52, which serve to replace RPA with Rad51 on ssDNA, and the Rad51 paralogs Rad55-Rad57 (RAD51B-RAD51C-XRCC2-XRCC3) and the Psy3-Csm2-Shu1-Shu2 complex (SHU) (RAD51D-XRCC2-SWS1), which stabilize Rad51 filaments on ssDNA (see Sung 1997b; Sigurdsson et al. 2001; Martin et al. 2006; Bernstein et al. 2011; Liu et al. 2011; Qing et al. 2011; Amunugama et al. 2013; Zelensky et al. 2014). The Rad51 nucleoprotein filament catalyzes the invasion into a homologous duplex to produce a displacement loop (D-loop) (Fig. 1). At this stage, additional antirecombination functions are exerted by Srs2 (FBH1, PARI), which dissociates Rad51 filaments from ssDNA, and Mph1 (FANCM), which disassembles D-loops (see Daley et al. 2014). Upon Rad51-catalyzed strand invasion, the ATP-dependent DNA translocase Rad54 enables the invading 3′ end to be extended by DNA polymerases to copy genetic information from the intact duplex (Li and Heyer 2009). Ligation of the products often leads to joint molecules (JMs), such as single- or double-Holliday junctions (s/dHJs) or hemicatenanes (HCs), which must be processed to allow separation of the sister chromatids during mitosis. JMs can be dissolved by the Sgs1-Top3-Rmi1 complex (STR) (BTR, BLM-TOP3α-RMI1-RMI2) (see Bizard and Hickson 2014) or resolved by structure-selective nucleases, such as Mus81-Mms4 (MUS81-EME1), Slx1-Slx4, and Yen1 (GEN1) (see Wyatt and West 2014). Mitotic cells favor recombination events that lead to noncrossover events likely to avoid potentially detrimental consequences of loss of heterozygosity and translocations.Open in a separate windowFigure 1.Primary pathways for homology-dependent double-strand break (DSB) repair. Recombinational repair of a DSB is initiated by 5′ to 3′ resection of the DNA end(s). The resulting 3′ single-stranded end(s) invade an intact homologous duplex (in red) to prime DNA synthesis. For DSBs that are repaired by the classical double-strand break repair (DSBR) model, the displaced strand from the donor duplex pairs with the 3′ single-stranded DNA (ssDNA) tail at the other side of the break, which primes a second round of DNA synthesis. After ligation of the newly synthesized DNA to the resected 5′ strands, a double-Holliday junction (dHJ) intermediate is generated. The dHJ can be either dissolved by branch migration (indicated by arrows) into a hemicatenane (HC) leading to noncrossover (NCO) products or resolved by endonucleolytic cleavage (indicated by triangles) to produce NCO (positions 1, 2, 3, and 4) or CO (positions 1, 2, 5, and 6) products. Alternatively to the double-strand break repair (DSBR) pathway, the invading strand is often displaced after limited synthesis and the nascent complementary strand anneals with the 3′ single-stranded tail of the other end of the DSB. After fill-in synthesis and ligation, this pathway generates NCO products and is referred to as synthesis-dependent strand annealing (SDSA).

Table 1.

Evolutionary conservation of homologous recombination proteins between Saccharomyces cerevisiae and Homo sapiens

Archaeology of Eukaryotic DNA Replication

Recent advances in the characterization of the archaeal DNA replication system together with comparative genomic analysis have led to the identification of several previously uncharacterized archaeal proteins involved in replication and currently reveal a nearly complete correspondence between the components of the archaeal and eukaryotic replication machineries. It can be inferred that the archaeal ancestor of eukaryotes and even the last common ancestor of all extant archaea possessed replication machineries that were comparable in complexity to the eukaryotic replication system. The eukaryotic replication system encompasses multiple paralogs of ancestral components such that heteromeric complexes in eukaryotes replace archaeal homomeric complexes, apparently along with subfunctionalization of the eukaryotic complex subunits. In the archaea, parallel, lineage-specific duplications of many genes encoding replication machinery components are detectable as well; most of these archaeal paralogs remain to be functionally characterized. The archaeal replication system shows remarkable plasticity whereby even some essential components such as DNA polymerase and single-stranded DNA-binding protein are displaced by unrelated proteins with analogous activities in some lineages.Double-stranded DNA is the molecule that carries genetic information in all cellular life-forms; thus, replication of this genetic material is a fundamental physiological process that requires high accuracy and efficiency (Kornberg and Baker 2005). The general mechanism and principles of DNA replication are common in all three domains of life—archaea, bacteria, and eukaryotes—and include recognition of defined origins, melting DNA with the aid of dedicated helicases, RNA priming by the dedicated primase, recruitment of DNA polymerases and processivity factors, replication fork formation, and simultaneous replication of leading and lagging strands, the latter via Okazaki fragments (Kornberg and Baker 2005; Barry and Bell 2006; Hamdan and Richardson 2009; Hamdan and van Oijen 2010). Thus, it was a major surprise when it became clear that the protein machineries responsible for this complex process are drastically different, especially in bacteria compared with archaea and eukarya. The core components of the bacterial replication systems, such as DNA polymerase, primase, and replication helicase, are unrelated or only distantly related to their counterparts in the archaeal/eukaryotic replication apparatus (Edgell 1997; Leipe et al. 1999).The existence of two distinct molecular machines for genome replication has raised obvious questions on the nature of the replication system in the last universal common ancestor (LUCA) of all extant cellular life-forms, and three groups of hypotheses have been proposed (Leipe et al. 1999; Forterre 2002; Koonin 2005, 2006, 2009; Glansdorff 2008; McGeoch and Bell 2008): (1) The replication systems in Bacteria and in the archaeo–eukaryotic lineage originated independently from an RNA-genome LUCA or from a noncellular ancestral state that encompassed a mix of genetic elements with diverse replication strategies and molecular machineries. (2) The LUCA was a typical cellular life-form that possessed either the archaeal or the bacterial replication apparatus in which several key components have been replaced in the other major cellular lineage. (3) The LUCA was a complex cellular life-form that possessed both replication systems, so that the differentiation of the bacterial and the archaeo–eukaryotic replication machineries occurred as a result of genome streamlining in both lines of descent that was accompanied by differential loss of components. With regard to the possible substitution of replication systems, a plausible mechanism could be replicon takeover (Forterre 2006; McGeoch and Bell 2008). Under the replicon takeover hypothesis, mobile elements introduce into cells a new replication system or its components, which can displace the original replication system through one or several instances of integration of the given element into the host genome accompanied by inactivation of the host replication genes and/or origins of replication. This scenario is compatible with the experimental results showing that DNA replication DNA in Escherichia coli with an inactivated DnaAgene or origin of replication can be rescued by the replication apparatus of R1 or F1 plasmids integrated into the bacterial chromosome (Bernander et al. 1991; Koppes 1992). Furthermore, genome analysis suggests frequent replicon fusion in archaea and bacteria (McGeoch and Bell 2008); in particular, such events are implied by the observation that in archaeal genomes, genes encoding multiple paralogs of the replication helicase MCM and origins of replication are associated with mobile elements (Robinson and Bell 2007; Krupovic et al. 2010). Replicon fusion also is a plausible path from a single origin of replication that is typical of bacteria to multiple origins present in archaea and eukaryotes. However, all the evidence in support of frequent replicon fusion and the plausibility of replicon takeover notwithstanding, there is no evidence of displacement of the bacterial replication apparatus with the archaeal version introduced by mobile elements, or vice versa, displacement of the archaeal machinery with the bacterial version, despite the rapid accumulation of diverse bacterial and archaeal genome sequences. Thus, the displacement scenarios of DNA replication machinery evolution are so far not supported by comparative genomic data.Regardless of the nature of the DNA replication system (if any) in the LUCA and the underlying causes of the archaeo–bacterial dichotomy of replication machineries, the similarity between the archaeal and eukaryotic replication systems is striking (Leipe et al. 1999; Bell and Dutta 2002; Bohlke et al. 2002; Kelman and White 2005; Barry and Bell 2006). Thus, the archaeal replication system appears to be an ancestral version of the eukaryotic system and hence a good model for functional and structural studies aimed at gaining mechanistic insights into eukaryotic replication.

Table 1.

The relationship between archaeal and eukaryotic replication systems

Noncanonical Sites of Adult Neurogenesis in the Mammalian Brain

Two decades after the discovery that neural stem cells (NSCs) populate some regions of the mammalian central nervous system (CNS), deep knowledge has been accumulated on their capacity to generate new neurons in the adult brain. This constitutive adult neurogenesis occurs throughout life primarily within remnants of the embryonic germinal layers known as “neurogenic sites.” Nevertheless, some processes of neurogliogenesis also occur in the CNS parenchyma commonly considered as “nonneurogenic.” This “noncanonical” cell genesis has been the object of many claims, some of which turned out to be not true. Indeed, it is often an “incomplete” process as to its final outcome, heterogeneous by several measures, including regional location, progenitor identity, and fate of the progeny. These aspects also strictly depend on the animal species, suggesting that persistent neurogenic processes have uniquely adapted to the brain anatomy of different mammals. Whereas some examples of noncanonical neurogenesis are strictly parenchymal, others also show stem cell niche-like features and a strong link with the ventricular cavities. This work will review results obtained in a research field that expanded from classic neurogenesis studies involving a variety of areas of the CNS outside of the subventricular zone (SVZ) and subgranular zone (SGZ). It will be highlighted how knowledge concerning noncanonical neurogenic areas is still incomplete owing to its regional and species-specific heterogeneity, and to objective difficulties still hampering its full identification and characterization.The central nervous system (CNS) of adult mammals is assembled during developmental neurogenesis, and its architectural specificity is maintained through a vast cohort of membrane-bound and extracellular matrix molecules (Gumbiner 1996; Bonfanti 2006). Although CNS structure is sculpted by experience-dependent synaptic plasticity at different postnatal developmental stages (critical periods) (see Sale et al. 2009) and, to a lesser extent, during adulthood (Holtmaat and Svoboda 2009), the neural networks are rather stabilized in the “mature” nervous tissue (Spolidoro et al. 2009). The differentiated cellular elements forming adult neural circuitries remain substantially unchanged in terms of their number and types, because cell renewal/addition in the CNS is very low. This situation is intuitive because connectional, neurochemical, and functional specificities are fundamental features of the mature CNS in highly complex brains, allowing specific cell types to be connected and to act in a relatively invariant way (Frotscher 1992).Since the discovery of neural stem cells (NSCs) (Reynolds and Weiss 1992), we realized that the aforementioned rules of CNS stability have a main exception in two brain regions: the forebrain subventricular zone (SVZ) (Lois and Alvarez-Buylla 1994) and the hippocampal subgranular zone (SGZ) (Gage 2000). These “adult neurogenic sites” are remnants of the embryonic germinal layers (although indirectly for the SGZ, which forms ectopically from the embryonic germinative matrix), which retain stem/progenitor cells within a special microenvironment, a “niche,” allowing and regulating NSC activity (Kriegstein and Alvarez-Buylla 2009). In addition, the areas of destination (olfactory bulb and dentate gyrus) reached by neuroblasts generated within these neurogenic sites harbor specific, not fully identified yet, environmental signals allowing the integration of young, newborn neurons. These two “canonical” sites of adult neurogenesis have been found in all animal species studied so far, including humans (reviewed in Lindsey and Tropepe 2006; Bonfanti and Ponti 2008; Kempermann 2012; Grandel and Brand 2013). Although in several classes of vertebrates including fish, amphibians, and reptiles, adult neurogenesis is widespread in many areas of the CNS (Zupanc 2006; Chapouton et al. 2007; Grandel and Brand 2013), in mammals, the vast majority of the brain and spinal cord regions out of the germinal-layer-derived neurogenic sites are commonly referred to as “nonneurogenic parenchyma” (Sohur et al. 2006; Bonfanti and Peretto 2011; Bonfanti and Nacher 2012). However, this viewpoint has changed during the last few years. New examples of cell genesis, involving both neurogenesis and gliogenesis, have been shown to occur in the so-called nonneurogenic regions of the mammalian CNS (Horner et al. 2000; Dayer et al. 2005; Kokoeva et al. 2005; Luzzati et al. 2006; Ponti et al. 2008; reviewed in Butt et al. 2005; Nishiyama et al. 2009; Migaud et al. 2010; Bonfanti and Peretto 2011), suggesting that structural plasticity involving de novo neural cell genesis could be more widespread than previously thought. Apart from their temporal persistence (some of them represent examples of delayed developmental neurogenesis, which persist postnatally; see below), neurogliogenic processes vary as to their regional localization, origin, and final outcome. In this review, “noncanonical” neurogenic processes occurring in adult mammals will be reviewed by underlining their heterogeneity across the species and their differences in intensity and outcome with respect to canonical neurogenic sites.

Table 1.

Main sites of noncanonical neurogenesis in the mammalian brain

Visualization of Ca2+ Signaling During Embryonic Skeletal Muscle Formation in Vertebrates

Dynamic changes in cytosolic and nuclear Ca²⁺ concentration are reported to play a critical regulatory role in different aspects of skeletal muscle development and differentiation. Here we review our current knowledge of the spatial dynamics of Ca²⁺ signals generated during muscle development in mouse, rat, and Xenopus myocytes in culture, in the exposed myotome of dissected Xenopus embryos, and in intact normally developing zebrafish. It is becoming clear that subcellular domains, either membrane-bound or otherwise, may have their own Ca²⁺ signaling signatures. Thus, to understand the roles played by myogenic Ca²⁺ signaling, we must consider: (1) the triggers and targets within these signaling domains; (2) interdomain signaling, and (3) how these Ca²⁺ signals integrate with other signaling networks involved in myogenesis. Imaging techniques that are currently available to provide direct visualization of these Ca²⁺ signals are also described.The recognition of Ca²⁺ as a key regulator of muscle contraction dates back to Sydney Ringer''s seminal observations in the latter part of the 19th Century (Ringer 1883; Ringer 1886; Ringer and Buxton 1887; see reviews by Martonosi 2000; Szent-Györgyi 2004). More recently, evidence is steadily accumulating to support the proposition that Ca²⁺ also plays a necessary and essential role in regulating embryonic muscle development and differentiation (Flucher and Andrews 1993; Ferrari et al. 1996; Lorenzon et al. 1997; Ferrari and Spitzer 1998, 1999; Wu et al. 2000; Powell et al. 2001; Jaimovich and Carrasco 2002; Li et al. 2004; Brennan et al. 2005; Harris et al. 2005; Campbell et al. 2006; Terry et al. 2006; Fujita et al. 2007; and see reviews by Berchtold et al. 2000; Ferrari et al. 2006; Al-Shanti and Stewart 2009). What is currently lacking, however, is extensive direct visualization of the spatial dynamics of the Ca²⁺ signals generated by developing and differentiating muscle cells. This is especially so concerning in situ studies. The object of this article, therefore, is to review and report the current state of our understanding concerning the spatial nature of Ca²⁺ signaling during embryonic muscle development, especially from an in vivo perspective, and to suggest possible directions for future research. The focus of our article is embryonic skeletal muscle development because of this being an area of significant current interest. Several of the basic observations reported, however, may also be common to cardiac muscle development and in some cases to smooth muscle development. What the recent development of reliable imaging techniques has most certainly done, is to add an extra dimension of complexity to understanding the roles played by Ca²⁺ signaling in skeletal muscle development. For example, it is clear that membrane-bound subcellular compartments, such as the nucleus (Jaimovich and Carrasco 2002), may have endogenous Ca²⁺ signaling activities, as do specific cytoplasmic domains, such as the subsarcolemmal space (Campbell et al. 2006). How these Ca²⁺ signals interact with specific down-stream targets within their particular domain, and how they might serve to communicate information among domains, will most certainly be one of the future challenges in elucidating the Ca²⁺-mediated regulation of muscle development.Any methodology used to study the properties of biological molecules and how they interact during development should ideally provide spatial information, because researchers increasingly need to integrate data about the interactions that underlie a biological process (such as differentiation) with information regarding the precise location within cells or an embryo where these interactions take place. Current Ca²⁺ imaging techniques are beginning to provide us with this spatial information, and are thus opening up exciting new avenues of investigation in our quest to understand the signaling pathways that regulate muscle development (

Endocytosis: Past,Present, and Future

Endocytosis may have been a driving force behind the evolution of eukaryotic cells. It plays critical roles in cell biology (e.g., signal transduction) and in organismal physiology (e.g., tissue morphogenesis).Endocytosis, the process of cellular ingestion, may have been the driving force behind evolution of the eucaryotic cell (de Duve 2007). Acquiring the ability to internalize macromolecules and digest them intracellularly would have allowed primordial cells to move out from their food sources and pursue a predatory existence; one that might have led to the development of endosymbiotic relationships with mitochondria and plastids. Thus, it is fitting that endocytosis was first discovered and named as the processes of cell “eating” and “drinking.” In 1883, the developmental biologist Ilya Metchnikoff coined the term phagocytosis, from the Greek “phagos” (to eat) and “cyte” (cell), after observing motile cells in transparent starfish larva surround and engulf small splinters that he had inserted (Tauber 2003). Decades later, in 1931, Warren H. Lewis, one of the earliest cell “cinematographers” coined the term pinocytosis, from the Greek “pinean” (to drink), after observing the uptake of surrounding media into large vesicles in cultured macrophages, sarcoma cells, and fibroblasts by time-lapse imaging (Lewis 1931; Corner 1967).Importantly, these pioneering studies also revealed that the function of endocytosis goes well beyond eating and drinking. Indeed, Metchnikoff, considered one of the founders of modern immunology, realized that the phagocytic behavior of the mesodermal amoeboid cells he had observed under the microscope could serve as a general defense system against invasive parasites, in the larva as in man. This revolutionary concept, termed the phagocytic theory, earned Metchnikoff the 1908 Nobel Prize in Physiology or Medicine for his work on phagocytic immunity, which he shared with Paul Ehrlich who discovered the complementary mechanisms of humoral immunity that led to the identification of antibodies (Vaughan 1965; Tauber 2003; Schmalstieg and Goldman 2008). The phagocytic theory was a milestone in immunology and cell biology, and formally gave birth to the field of endocytosis.Key discoveries over the intervening years, aided in large part by the advent of electron microscopy, revealed multiple pathways for endocytosis in mammalian cells that fulfill multiple critical cellular functions (Fig. 1). These mechanistically and morphologically distinct pathways, and their discoverers, include clathrin-mediated endocytosis (Roth and Porter 1964), caveolae uptake (Palade 1953; Yamada 1955), cholesterol-sensitive clathrin- and caveolae-independent pathways (Moya et al. 1985; Hansen et al. 1991; Lamaze et al. 2001), and, more recently, the large capacity CLIC/GEEC pathway (Kirkham et al. 2005). In place of Metchnikoff’s splinters, many of these discoveries resulted from the detection and tracking of internalized HRP-, ferritin-, or gold-conjugated ligands by electron microscopy. These electron-dense tracers allowed researchers to identify cellular structures associated with the uptake and intracellular sorting of receptor-bound ligands. A particularly striking example is the pioneering work of Roth and Porter, who in 1964 observed the uptake of yolk proteins into mosquito oocytes. To synchronize uptake, they killed female mosquitos at timed intervals after a blood feed and observed the sequential appearance of electron-dense yolk granules in coated pits, coated and uncoated vesicles, and progressively larger vesicles. Their remarkable observations accurately described coated vesicle budding, uncoating, homo- and heterotypic fusion events, as well as the emergence of tubules from early endosomes (Fig. 2), all of which are now known hallmarks of the early endocytic trafficking events.Open in a separate windowFigure 1.Time line for discoveries of endocytic pathways and their discoverers. Boxes are color-coded by pathway. *, Nobel laureate. HRP, horseradish peroxidase; CCVs, clathrin-coated vesicles; CCPs, clathrin-coated pits; EGFR, epidermal growth factor receptor; PM, plasma membrane; ER, endoplasmic reticulum; CLIC/GEEC, clathrin-independent carriers/GPI-enriched endocytic compartments.Open in a separate windowFigure 2.Fiftieth anniversary of the discovery of clathrin-mediated endocytosis by Roth and Porter (1964). The image is the hand-drawn summary of observations made by electron microscopic examination of the trafficking of yolk proteins in a mosquito oocyte. Note the many details, later confirmed and mechanistically studied over the intervening 50 years. These include the growth, invagination, and pinching off of coated pits (1,2), which concentrate cargo taken up by coated vesicles (3), the rapid uncoating of nascent-coated vesicles (4), homotypic fusion of nascent endocytic vesicles in the cell periphery (5), the formation of tubules from early endosomes (7), and changes in the intraluminal properties of larger endosomes (6). Finally, yolk proteins are stored in granules as crystalline bodies (8). (From Roth and Porter 1964; reprinted, with express permission, from Rockefeller University Press © 1964, The Journal of Cell Biology 20: 313–332, doi: 10.1083/jcb.20.2.313.)Another milestone in the field of endocytosis was the discovery of the lysosome by Christian de Duve (Appelmans et al. 1955). Whereas the finding of phagocytosis and other endocytic pathways was possible through microscopy, the discovery of lysosomes originated from a biochemical approach (Courtoy 2007), which benefited from the invention of the ultracentrifuge. de Duve and coworkers observed that preparations of acid phosphatase obtained by subcellular fractionation had an unusual behavior: contrary to most enzymatic activities, the activity of acid phosphatase increased rather than decreased with time, freezing–thawing of the fractions and the presence of detergents. Interestingly, the same was true for other hydrolases, which depended on acidic pH for their optimal activity. This led him to postulate that the acid hydrolases were contained in acidified membrane-bound vesicles. In collaboration with Alex Novikoff, he visualized these vesicles, the lysosomes, by electron microscopy (Beaufay et al. 1956) and later showed their content of acid phosphatase (Farquhar et al. 1972). In 1974, de Duve was awarded the Nobel Prize for Physiology or Medicine for his seminal finding of the lysosomes and peroxisomes. He shared it with Albert Claude and George E. Palade “for their discoveries concerning the structural and functional organization of the cell.” The importance of this work lies also in the significant therapeutic applications that followed. The discovery by Elizabeth Neufeld and collaborators of uptake of lysosomal enzymes by cells provided the foundation for enzyme replacement therapy for lysosomal storage disorders (Neufeld 2011).In the 1970s, research in endocytosis entered the molecular era. Using de Duve and Albert Claude-like methods of subcellular fractionation, Barbara M. Pearse purified clathrin-coated vesicles from pig brain (Pearse 1975). A year later, she isolated a major protein species of 180 kDa, which she named clathrin “to indicate the lattice-like structures which it forms” (Pearse 1976). It was a breakthrough that inaugurated the molecular dissection of clathrin-mediated endocytosis.Over the intervening years, the continued application of microscopy (which now spans from electron cryotomography to live cell, high-resolution fluorescence microscopy), genetics (in particular, in yeast, Caenorhabditis elegans and Drosophila melanogaster), biochemistry (including cell-free reconstitution of endocytic membrane trafficking events), as well as molecular and structural biology have revealed a great deal about the cellular machineries and mechanisms that govern trafficking along the endocytic pathway. A partial, and because of space limitations, necessarily incomplete list of milestones (YearMechanistic milestonesDiscoverers1973Identification of shibirets (dynamin) mutant in DrosophilaD. Suzuki and C. Poodry1974–1976Zipper mechanism for phagocytosisS. Silverstein1975–1976Isolation of CCVs, purification of clathrinB. Pearse1982–1984Phosphomannose, M6PR, and lysosomal targetingW. Sly, S. Kornfeld, E. Neufeld, G. Sahagian1983–1984Isolation of clathrin adapters/localization to distinct membranesJ. Keen, B. Pearse, M. Robinson1986Isolation of endocytosis mutants (End) in yeastH. Riezman1986–1987Isolation of vacuolar protein sorting mutants in yeastS. Emr, T. Stevens1986Endosome fusion in vitroJ. Gruenberg and K. Howell1986EGF and insulin receptor signaling from endosomesJ. Bergeron and B. Posner1986Macropinocytosis induced in stimulated cellsD. Bar-Sagi and J. Feramisco1987Endocytic sorting motifs (FxNPxY, YxxF)M. Brown and J. Goldstein, I. Trowbridge, T. McGraw1987–1989Cloning of CHC, CLC, AP2T. Kirchhausen, M. Robinson1988Isolation of biochemically distinct early and late endosomesS. Schmid and I. Mellman1989–1991Clathrin-mediated endocytosis reconstituted in vitroE. Smythe, G. Warren, S. Schmid1990Localization of endosomal Rab5 and Rab7P. Chavrier, R. Parton, M. Zerial1991Endosome to trans-Golgi network (TGN) transport reconstituted in vitroS. Pfeffer1992Rab5 and Rab4 as early endocytic regulators in vivoM. Zerial, R. Parton, I. Mellman1992–1995Caveolin/VIP21 identified as caveolar coat proteinR. Anderson, T. Kurzchalia, R. Parton, K. Simons1992Vacuolar fusion reconstituted in vitroW. Wickner1992–1994Trigger mechanism for phagocytosis of bacteriaS. Falkow, J. Galán, J. Swanson1993Actin’s role in endocytosis in yeastH. Riezman1993Isolation of autophagy mutants (Atg) in yeastY. Ohsumi1993PI3 kinase activity (PI3P) and endosome functionS. Emr1993Dynamin’s role in clathrin-mediated endocytosisR. Vallee, S. Schmid1995Dynamin assembles into ringsS. Schmid, P. De Camilli1996Clathrin-mediated endocytosis requirement for signalingS. Schmid1996Long distance retrograde transport of signaling endosomes in neuronsW. Mobley1996PI5 phosphatase activity (PI(4,5)P₂) and clathrin-mediated endocytosisP. De Camilli1996Ubiquitin-dependent sorting in endocytosisR. Haguenauer-Tsapis; L. Hicke and H. Riezman1997AP3 and endosomal/lysosomal sortingJ. Bonifacino, S. Robinson1998FYVE fingers bind to PI3PH. Stenmark1998LBPA in MVB biogenesisT. Kobayashi, R. Parton, J. Gruenberg1997–1998Sorting nexinsG. Gill, S. Emr1998Structural basis for Y-based sorting signal recognitionD. Owen1998Retromer coat and endosome to TGN sortingS. Emr1998β-Propeller structure of clathrin heavy chain terminal domainT. Kirchhausen and S. Harrison1998Cargo-specific subpopulations of clathrin-coated pitsM. von Zastrow1999Structure of the clathrin coat protein superhelical motifsJ. Ybe and F. Brodsky1999Imaging green fluorescent protein–clathrin in living cellsJ. Keen1999Biochemical purification of Rab5 effectorsS. Christoforidis and M. Zerial1999Genetic screen for endocytosis mutants in C. elegansB. Grant2000Role of endocytosis in establishing morphogenic gradientsM. Gonzalez-Gaitan, S.M. Cohen2000Identification of GGA coats and lysosomal sortingJ. Bonifacino, S. Kornfeld, M. Robinson2000Identification of endosomal sorting complex required for transport (ESCRT) machinery for multivesicular body (MVB) formationS. Emr2001Ubiquitin-dependent sorting into MVBsR. Piper, S. Emr, H. Pelham2002Structure of the AP2 coreD. Owen2003Lipid conjugation of LC3/Atg8Y. Ohsumi2003–2004siRNA studies of endocytic componentsS. Robinson, E. Ungewickell, A. Sorkin2004BAR domains and membrane curvature generationH. McMahon, P. De Camilli20048-Å structure of a complete clathrin coatT. Kirchhausen and S. Harrison2005Modular design of yeast endocytosis machineryD. Drubin and M. Kaksonen2005Kinome-wide RNAi analysis of clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE)M. Zerial and L. Pelkmans2006–2008Reconstitution of dynamin-mediated membrane fissionA. Roux, P. De Camilli, S. Schmid, J. Zimmerberg, V. Frolov2007Glycosphingolipid-induced endocytosisL. Johannes2009Reconstitution of Rab- and SNARE-dependent vacuolar and endosome fusion from purified componentsW. Wickner, M. Zerial2010Cavins as major caveolae coat componentsR. Parton; B. Nichols2010Reconstitution of ESCRT-dependent internal vesicle formationT. Wollert and J. Hurley2012Reconstitution of CCV formation from minimal componentsE. UngewickellOpen in a separate window     

Heparan Sulfate Proteoglycans

Heparan sulfate proteoglycans are found at the cell surface and in the extracellular matrix, where they interact with a plethora of ligands. Over the last decade, new insights have emerged regarding the mechanism and biological significance of these interactions. Here, we discuss changing views on the specificity of protein–heparan sulfate binding and the activity of HSPGs as receptors and coreceptors. Although few in number, heparan sulfate proteoglycans have profound effects at the cellular, tissue, and organismal level.Heparan sulfate proteoglycans (HSPGs) are glycoproteins, with the common characteristic of containing one or more covalently attached heparan sulfate (HS) chains, a type of glycosaminoglycan (GAG) (Esko et al. 2009). Cells elaborate a relatively small set of HSPGs (∼17) that fall into three groups according to their location: membrane HSPGs, such as syndecans and glycosylphosphatidylinositol-anchored proteoglycans (glypicans), the secreted extracellular matrix HSPGs (agrin, perlecan, type XVIII collagen), and the secretory vesicle proteoglycan, serglycin (Esko et al. 1985), which allowed functional studies in the context of a cell culture model (Zhang et al. 2006). A decade later, the first HSPG mutants in a model organism (Drosophila melanogaster) were identified (Rogalski et al. 1993; Nakato et al. 1995; Häcker et al. 1997; Bellaiche et al. 1998; Lin et al. 1999), which was followed by identification of mutants in nematodes, tree frogs, zebrafish, and mice (​and3).3). HS is evolutionarily ancient and its composition has remained relatively constant from Hydra to humans (Yamada et al. 2007; Lawrence et al. 2008).

Table 1.

Heparan sulfate proteoglycans

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

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

Table 1.

Binding partners of spectrin and ankyrins

Mechanisms and Evidence of Genital Coevolution: The Roles of Natural Selection,Mate Choice,and Sexual Conflict

Genital coevolution between the sexes is expected to be common because of the direct interaction between male and female genitalia during copulation. Here we review the diverse mechanisms of genital coevolution that include natural selection, female mate choice, male–male competition, and how their interactions generate sexual conflict that can lead to sexually antagonistic coevolution. Natural selection on genital morphology will result in size coevolution to allow for copulation to be mechanically possible, even as other features of genitalia may reflect the action of other mechanisms of selection. Genital coevolution is explicitly predicted by at least three mechanisms of genital evolution: lock and key to prevent hybridization, female choice, and sexual conflict. Although some good examples exist in support of each of these mechanisms, more data on quantitative female genital variation and studies of functional morphology during copulation are needed to understand more general patterns. A combination of different approaches is required to continue to advance our understanding of genital coevolution. Knowledge of the ecology and behavior of the studied species combined with functional morphology, quantitative morphological tools, experimental manipulation, and experimental evolution have been provided in the best-studied species, all of which are invertebrates. Therefore, attention to vertebrates in any of these areas is badly needed.Of all the evolutionary interactions between the sexes, the mechanical interaction of genitalia during copulation in species with internal fertilization is perhaps the most direct. For this reason alone, coevolution between genital morphologies of males and females is expected. Morphological and genetic components of male and female genitalia have been shown to covary in many taxa (Sota and Kubota 1998; Ilango and Lane 2000; Arnqvist and Rowe 2002; Brennan et al. 2007; Rönn et al. 2007; Kuntner et al. 2009; Tatarnic and Cassis 2010; Cayetano et al. 2011; Evans et al. 2011, 2013; Simmons and García-González 2011; Yassin and Orgogozo 2013; and see examples in TaxaMale structuresFemale structuresEvidenceLikely mechanismReferencesMollusks Land snails (Xerocrassa)Spermatophore-producing organsSpermatophore-receiving organsComparative among speciesSAC or female choiceSauder and Hausdorf 2009 SatsumaPenis lengthVagina lengthCharacter displacementLock and keyKameda et al. 2009Arthropods Arachnids (Nephilid spiders)MultipleMultipleComparative among speciesSACKuntner et al. 2009 Pholcidae spidersCheliceral apophysisEpigynal pocketsComparative (no phylogenetic analysis)Female choiceHuber 1999 Harvestmen (Opiliones)Hardened penes and loss of nuptial giftsSclerotized pregenital barriersComparative among speciesSACBurns et al. 2013Millipedes Parafontaria tonomineaGonopod sizeGenital segment sizeComparative in species complexMechanical incompatibility resulting from Intersexual selectionSota and Tanabe 2010 Antichiropus variabilisGonopod shape and sizeAccesory lobe of the vulva and distal projectionFunctional copulatory morphologyLock and keyWojcieszek and Simmons 2012Crustacean Fiddler crabs, UcaGonopodeVulva, vagina, and spermathecaTwo-species comparison, shape correspondenceNatural selection against fluid loss, lock and key, and sexual selectionLautenschlager et al. 2010Hexapodes OdonatesClasping appendagesAbdominal shape and sensory hairsFunctional morphology, comparative among speciesLock and key via female sensory systemRobertson and Paterson 1982; McPeek et al. 2009Insects Coleoptera: seed beetlesSpiny aedagusThickened walls of copulatory ductComparative among speciesSACRönn et al. 2007 Callosobruchus: Callosobruchus maculatusDamage inflictedSusceptibility to damageFull sib/half sib mating experimentsSACGay et al. 2011Reduced spinesNo correlated responseExperimental evolutionSACCayetano et al. 2011 Carabid beetles (Ohomopterus)Apophysis of the endophallusVaginal appendix (pocket attached to the vaginal apophysis)Cross-species matingsLock and keySota and Kubota 1998; Sasabi et al. 2010 Dung beetle: Onthophagus taurusShape of the parameres in the aedagusSize and location of genital pitsExperimental evolutionFemale choiceSimmons and García-González 2011 Diptera: Drosophila santomea and D. yakubaSclerotized spikes on the aedagusCavities with sclerotized plateletsCross-species matingsSACKamimura 2012 Drosophila melanogaster species complexEpandrial posterior lobes
Oviscapt pouchesComparative among speciesSAC or female choiceYassin and Orgogozo 2013Phallic spikesOviscapt furrowsCercal teeth, phallic hook, and spinesUterine, vulval, and vaginal shields D. mauritiana and D. secheliaPosterior lobe of the genital archWounding of the female abdomenMating with introgressed linesSACMasly and Kamimura 2014 Stalk-eyed flies (Diopsidae)Genital processCommon spermathecal ductComparative among species and morphologicalFemale choiceKotrba et al. 2014 Tse-tse flies: Glossina pallidipesCercal teethFemale-sensing structuresExperimental copulatory functionFemale choiceBriceño and Eberhard 2009a,b Phelebotomine: sand fliesAedagal filaments, aedagal sheathsSpermathecal ducts length, base of the ductComparative among speciesNone specifiedIlango and Lane 2000 Heteroptera: Bed bugs (Cimiciidae)Piercing genitaliaSpermalege (thickened exosqueleton)Comparative among speciesSACCarayon 1966; Morrow and Arnqvist 2003 Plant bugs (Coridromius)Changes in male genital shapeExternal female paragenitaliaComparative among speciesSACTatarnic and Cassis 2010 Waterstriders (Gerris sp.)Grasping appendagesAntigrasping appendagesComparative among speciesSACArnqvist and Rowe 2002 Gerris incognitusGrasping appendagesAntigrasping appendagesComparative among populationsSACPerry and Rowe 2012 Bee assassins (Apiomerus)AedagusBursa copulatrixComparative among speciesNoneForero et al. 2013 Cave insects (Psocodea), NeotroglaMale genital chamberPenis-like gynosomeComparative among speciesFemale competition (role reversal), coevolution SACYoshizawa et al. 2014 Butterflies (Heliconiinae)Thickness of spermatophore wallSigna: Sclerotized structure to break spermatophoresComparative among speciesSACSánchez and Cordero 2014Fish Basking shark: Cetorhinus maximusClasper clawThick vaginal padsMorphological observationNoneMatthews 1950 GambusiaGonopodial tipsGenital papillae within openingsComparative among speciesStrong character displacementLangerhans 2011 Poecilia reticulataGonopodium tip shapeFemale gonopore shapeComparative among populationsSACEvans et al. 2011Reptiles AnolesHemipene shapeVagina shapeShape correspondence, two speciesSexual selectionKöhler et al. 2012 Several speciesHemipene shapeVagina shapeShape correspondenceLock and key, female choice, and SACPope 1941; Böhme and Ziegler 2009; King et al. 2009 Asiatic pit vipersSpininess in hemipenesThickness of vagina wallTwo-species comparisonNonePope 1941 Garter snake: Thamnophis sirtalisBasal hempene spineVaginal muscular controlExperimental manipulationSACFriesen et al. 2014Birds WaterfowlPenis lengthVaginal elaborationComparative among speciesSACBrennan et al. 2007 TinamousPenis length/presenceVaginal elaborationComparative among speciesFemale choice/natural selectionPLR Brennan, K Zyscowski, and RO Prum, unpubl.Mammals MarsupialsBifid penisTwo lateral vaginaeShape correspondenceNoneRenfree 1987 EquidnaBifid penis with four rosettesSingle vagina splits into two uteriShape correspondenceNoneAugee et al. 2006; Johnston et al. 2007 Insectivores: Short-tailed shrew: Blarina brevicaudaS-shaped curve of the erect penisCoincident curve in the vaginaShape correspondenceNoneBedford et al. 2004 Common tenrec: Tenrec caudatusFiliform penis (up to 70% of the male’s body length)Internal circular folds in the vaginaLength correspondenceNoneBedford et al. 2004 Rodents: Cape dune mole: Bathyergus suillusPenis and baculum lengthVaginal lengthAllometric relationships within speciesNoneKinahan et al. 2007 Australian hopping mice (Notomys)Spiny penisDerived distal region in the vaginaMorphological observation and two-species comparisonCopulatory lockBreed et al. 2013 Pig: Sus domesticusFiliform penis endCervical ridgesArtificial inseminationFemale choiceBonet et al. 2013 Primates: Macaca arctoidesLong and filamentous glansVestibular colliculus (fleshy fold) that partially obstructs the entrance to the vaginaShape correspondence and comparison with close relativesNoneFooden 1967Open in a separate windowThe likely mechanism is that suggested by the authors, and it includes sexually antagonistic coevolution (SAC), natural selection, sexual selection, female choice, or none specified. The evidence provided by the studies can be comparative among species or among populations, experimental evolution, cross-species matings, full-sibling (sib)/half-sib matings, shape, and length correspondence. Shape correspondence is often taken as evidence of coevolution, although it is not as conclusive as other approaches.Male genitalia are among the most variable structures in nature (Eberhard 1985). In contrast, female genitalia have typically been found not to be as interspecifically variable as male genitalia in several studies that specifically examined and described them (Eberhard 1985, 2010a,b). Female genitalia are not studied as often as male genitalia, perhaps because of a male-biased view of evolutionary processes by researchers (Ah-King et al. 2014). However, studying female genitalia is undeniably challenging. Male genitalia are generally kept inside of the body cavity, but are everted before, or during copulation, so their functional morphology can be more easily studied than the internal genitalia of females. Female genitalia also tend to be softer than male genitalia and thus their morphology may be more difficult to describe, and can more easily be distorted on dissection and preservation. Female adaptations to sense or oppose features of male genitalia can be subtle, requiring careful study. Female genital tracts are under multiple sources of selection: not just mating, but also storing sperm, egg laying, birthing, and often interfacing with the terminal portion of the digestive tract. Therefore, selection balancing multiple functions may further constrain morphological evolution in female genitalia. However, even small morphological changes in female genitalia, for example, increases in vaginal muscle, may change a female’s ability to choose or reject a male during mating, or to manage the costs of mating. Thus, the functional consequences to male and female genital morphology are hard to predict unless one knows how genitalia function during intromission. Despite these challenges, recent studies have examined variation of female genitalia and evidence is accumulating that features of female genitalia are variable enough to support coevolutionary processes (Polihronakis 2006; Puniamoorthy et al. 2010; Siegel et al. 2011; Showalter et al. 2013; and see additional references in Ah-King et al. 2014).In this article, we will discuss different hypotheses of genital evolution that predict coevolution; however, this is not a review of that entire subject (but see Eberhard et al. 2010b; Simmons 2013). Rather, we discuss the various mechanisms of genital coevolution differentiating the potentially independent or overlapping roles of natural selection, female choice, and male–male competition (Fig. 1). This classification allows us to distinguish specifically those mechanisms of genital coevolution that involve sexual conflict (i.e., when the evolutionary interests of individuals of different sexes, particularly over mating, are different). We then highlight examples in different taxa organisms with particular emphasis on those that provide evidence of sexual conflict.Open in a separate windowFigure 1.Graphical classification of mechanisms of genital evolution and coevolution. Three circles depict the independent and co-occurring actions of natural selection, female choice, and male–male competition. Different specific versions of genital coevolution can occur depending on which of the three broader evolutionary mechanisms are occurring. Sexual conflict (hatched lines) occurs through the simultaneous action of male–male competition and female choice, or male–male competition and natural selection. SAC, sexually antagonistic coevolution. See text for explanation.     

Helicase Loading at Chromosomal Origins of Replication

Loading of the replicative DNA helicase at origins of replication is of central importance in DNA replication. As the first of the replication fork proteins assemble at chromosomal origins of replication, the loaded helicase is required for the recruitment of the rest of the replication machinery. In this work, we review the current knowledge of helicase loading at Escherichia coli and eukaryotic origins of replication. In each case, this process requires both an origin recognition protein as well as one or more additional proteins. Comparison of these events shows intriguing similarities that suggest a similar underlying mechanism, as well as critical differences that likely reflect the distinct processes that regulate helicase loading in bacterial and eukaryotic cells.Replicative DNA helicases are ring-shaped molecules with a central cavity through which DNA passes as they unwind DNA. Their loading at replication origins is a critical and highly regulated event in chromosomal replication. The DNA helicase is the first of the replication fork proteins recruited to and loaded onto origins of replication, and the loaded helicase is required for the recruitment of the rest of the replication machinery (Remus and Diffley 2009; Kaguni 2011). Indeed, the replicative DNA helicase links the replication machinery to the parental DNA (O’Donnell 2006). In Escherichia coli cells, the DnaB replicative helicase binds to primase (DnaG) and the sliding clamp loader, which in turn binds the DNA polymerases. Although the polymerases are also linked to the template DNA by sliding clamps, when these interactions are broken, the polymerases’ association with the sliding clamp loader and the helicase keeps them at the site of replication. The interactions that tether the DNA polymerases to the eukaryotic replication fork are less clear but very likely involve direct and indirect interactions with the Mcm2–7 replicative helicase (Calzada et al. 2005).Helicase loading is carefully regulated to control the location and frequency of replication initiation. In eukaryotic cells, helicase loading is tightly restricted to the G₁ phase of the cell cycle. This constraint is a key part of the mechanisms that ensure that no origin can initiate more than once per cell cycle (Siddiqui et al. 2013; Zielke et al. 2013). In addition, the sites of eukaryotic replicative helicase loading define the potential sites of replication initiation in the cell (but not all loaded helicases are used during a given S phase [Rhind and Gilbert 2012]). Although the central regulated event in bacterial chromosome duplication is the recruitment of the ATP-bound initiator protein DnaA (Skarstad and Katayama 2013), the loading of the replicative helicase represents a key committed step during initiation.Here we will discuss the mechanism of helicase loading in bacteria and eukaryotic cells. Much of the discussion will focus on studies in the bacterium E. coli, the yeast Saccharomyces cerevisiae, and the frog Xenopus laevis, in which the events of helicase loading are best understood. Comparison of these mechanisms shows important similarities and differences between the domains of life. In both bacteria and eukaryotic cells, multiple AAA⁺ proteins use ATP binding and hydrolysis to direct helicase loading and both helicases are initially loaded in an inactive form. On the other hand, the eukaryotic helicase is loaded around double-stranded DNA (dsDNA) and as a double hexamer, whereas the bacterial helicase is loaded around single-stranded DNA (ssDNA) as a single hexamer. These distinctions are very likely due to the very different regulatory mechanisms of DNA replication in bacteria and eukaryotic cells.Before describing the process of helicase loading, it is relevant to know that the essential function of replicative helicases is to unwind the parental duplex DNA using the energy provided by the hydrolysis of a nucleoside triphosphate (Patel et al. 2011). Replicative DNA polymerases then copy each parental DNA strand to duplicate the genome. That replicative DNA helicases are hexameric (and sometimes heptameric) structures that have a positively charged, central channel provides a framework for how these molecular machines work. Several models that describe the mechanism of unwinding have been considered. One is the steric or strand-exclusion model, in which the helicase excludes one strand of DNA while the other passes through the central cavity as the enzyme moves. Because these enzymes can bind and translocate on duplex DNA without unwinding (Kaplan et al. 2003), two additional models have been proposed. In the ploughshare model, duplex DNA enters the central cavity and exits as unwound DNA by virtue of a domain/protein that acts as a ploughshare or pin, which disrupts the hydrogen bonds of DNA as it is pumped through the enzyme (reviewed in Takahashi et al. 2005). A second model, the DNA-pumping model, was proposed on the basis of the double-hexameric forms of SV40, Mcm2–7, and other replicative helicases (Mastrangelo et al. 1989; Remus et al. 2009). This model proposes that the two helicases pump duplex DNA toward one another, resulting in torsional strain that forces the two strands apart, at which point they exit the central channel as two ssDNA loops. Current evidence supports the steric exclusion model (Jezewska et al. 1998b; Kaplan 2000; Galletto et al. 2004; Fu et al. 2011). Of interest, the E. coli enzyme moves in the 5′-to-3′ direction relative to the engaged ssDNA that passes through the central cavity, whereas archaeal and eukaryotic enzymes move in the 3′-to-5′ direction.

Table 1.

Replicative DNA helicases of free-living organisms are hexameric

How Do Astrocytes Participate in Neural Plasticity?

Work over the past 20 years has implicated electrically nonexcitable astrocytes in complex neural functions. Despite controversies, it is increasingly clear that many, if not all, neural processes involve astrocytes. This review critically examines past work to identify the commonalities among the many published studies of neuroglia signaling. Although several studies have shown that astrocytes can impact short-term and long-term synaptic plasticity, further work is required to determine the requirement for astrocytic Ca²⁺ and other second messengers in these processes. One of the roadblocks to the field advancing at a rapid pace has been technical. We predict that the novel experimental tools that have emerged in recent years will accelerate the field and likely disclose an entirely novel path of neuroglia signaling within the near future.The year 2014 represents the 20th anniversary of a pair of papers published by the writers that provided the first indication that astrocytes actively signal to neurons, and that these glial cells have the potential to be participants in the control of neural circuit function and behavior (Nedergaard 1994; Parpura et al. 1994). Although we have taken independent paths, we come together on this anniversary to discuss what we have learned since 1994, where we see the field progressing, and, finally, to discuss some key steps that we believe should be taken in the next decade to begin to further clarify the diverse roles that astrocytes play in brain function in health and disease.Without knowledge of one another’s work, we published a pair of papers that provided the first demonstration that physiological changes in astrocytes influence neurons: stimulated Ca²⁺ changes in astrocytes led to delayed Ca²⁺ responses in neurons (Nedergaard 1994; Parpura et al. 1994). To set the backdrop to these studies, this was a period of explosive growth in Ca²⁺ imaging that resulted from the availability of fluorescent Ca²⁺ indicators and sensitive cameras to permit low-light-level imaging of intracellular biochemistry. As a consequence, there had been several recently published studies revealing that astrocytes show Ca²⁺ elevations in response to mechanical contact or even to the addition of neurotransmitters, such as glutamate (Cornell-Bell et al. 1990). However, the functional downstream consequences were unknown. In our studies, which used a combination of different stimuli—mechanical, optical, as well as a chemical transmitter bradykinin—we were able to show a robust impact of the astrocytic Ca²⁺ signal on the adjacent neurons (Nedergaard 1994; Parpura et al. 1994). Despite differences in mechanistic conclusions, these papers stimulated revised thinking about roles of astrocytes in the brain—perhaps these glial cells were actively signaling, albeit on a slower time scale, to modulate neurons, circuits, and, ultimately, behavior.The subsequent two decades have been spent examining the signaling of astrocytes to neurons in more intact systems. As a consequence of this work, it is clear that astrocytes play critical roles in actively modulating brain function, although we are still putting the pieces of the puzzle in place to understand where, when, and how this process occurs naturally in vivo and when dysfunction can lead to disorders of the brain.The field has gone through an explosive growth that has consisted of several phases. Initially, there was the dish phase in which the potential of the astrocyte was revealed in cell culture. These studies were essential as they captured our imagination and stimulated new thinking; however, they were limited by the fact that the properties of astrocytes can be different in vitro and in vivo. Next, we had the in situ phase, in which we asked whether similar processes could be detected in situ in acutely isolated brain slices. Then we began asking about roles in vivo through Ca²⁺ imaging together with two-photon microscopy as well as with molecular genetic alterations to permit the inhibition and stimulation of astrocytes. Each of these phases has offered unique insights, challenges, and opportunities for the field.Through all of these phases, an emergent picture is developing in which it is without doubt that astrocytes play important roles in vivo but that the mechanisms are so diverse and complex that an understanding is still to emerge.Some of the major challenges that we have faced and continue to face include: What are the endogenous signals of these glial cells? How can we stimulate astrocytes in a physiologically relevant manner? How can we inhibit astrocytes to determine when they are needed for brain function? And last, but by no means least, how diverse are astrocytes?We are still at the early days of understanding astrocytes, and patience regarding functional interpretation is required. We make this statement because there have been apparently contradictory conclusions drawn from different studies. The individual observations are important as they help provide a fuller picture of the biology of these complex cells. However, the jury still needs further evidence before definitive conclusions can be drawn. For example, a plethora of studies have shown that Ca²⁺ signals stimulate gliotransmission and the consequent modulation of neurons and synapses (see Agulhon et al. 2010), the field was rocked. We do not question the data of the study; instead, we believe this is an important piece of information that ultimately needs to be put in context. In contrast, additional studies have shown that IP₃ receptors are important for other aspects of astrocyte-induced synaptic modulation. A more recent study has shown that, in addition to Ca²⁺ release from internal stores, the influx of Ca²⁺ through transient receptor potential (TRP) channels is important for gliotransmission (Shigetomi et al. 2013). Clearly, such influx sites were unlikely to have been affected by the IP₃R₂ knockout and were shown to regulate d-serine release from the astrocyte. Another piece of the puzzle is added. Undoubtedly, there will be further twists and turns, but that is the joy of discovery, and it should be embraced.

Table 1.

Effect of Ca²⁺ signaling on excitatory or inhibitory potentials, slow inward current, synaptic failure, or neural bistability

Variation in Adult Plant Phenotypes and Partitioning among Seed and Stem-Borne Roots across Brachypodium distachyon Accessions to Exploit in Breeding Cereals for Well-Watered and Drought Environments

Seedling roots enable plant establishment. Their small phenotypes are measured routinely. Adult root systems are relevant to yield and efficiency, but phenotyping is challenging. Root length exceeds the volume of most pots. Field studies measure partial adult root systems through coring or use seedling roots as adult surrogates. Here, we phenotyped 79 diverse lines of the small grass model Brachypodium distachyon to adults in 50-cm-long tubes of soil with irrigation; a subset of 16 lines was droughted. Variation was large (total biomass, ×8; total root length [TRL], ×10; and root mass ratio, ×6), repeatable, and attributable to genetic factors (heritabilities ranged from approximately 50% for root growth to 82% for partitioning phenotypes). Lines were dissected into seed-borne tissues (stem and primary seminal axile roots) and stem-borne tissues (tillers and coleoptile and leaf node axile roots) plus branch roots. All lines developed one seminal root that varied, with branch roots, from 31% to 90% of TRL in the well-watered condition. With drought, 100% of TRL was seminal, regardless of line because nodal roots were almost always inhibited in drying topsoil. Irrigation stimulated nodal roots depending on genotype. Shoot size and tillers correlated positively with roots with irrigation, but partitioning depended on genotype and was plastic with drought. Adult root systems of B. distachyon have genetic variation to exploit to increase cereal yields through genes associated with partitioning among roots and their responsiveness to irrigation. Whole-plant phenotypes could enhance gain for droughted environments because root and shoot traits are coselected.Adult plant root systems are relevant to the size and efficiency of seed yield. They supply water and nutrients for the plant to acquire biomass, which is positively correlated to the harvest index (allocation to seed grain), and the stages of flowering and grain development. Modeling in wheat (Triticum aestivum) suggested that an extra 10 mm of water absorbed by such adult root systems during grain filling resulted in an increase of approximately 500 kg grain ha⁻¹ (Manschadi et al., 2006). This was 25% above the average annual yield of wheat in rain-fed environments of Australia. This number was remarkably close to experimental data obtained in the field in Australia (Kirkegaard et al., 2007). Together, these modeling and field experiments have shown that adult root systems are critical for water absorption and grain yield in cereals, such as wheat, emphasizing the importance of characterizing adult root systems to identify phenotypes for productivity improvements.Most root phenotypes, however, have been described for seedling roots. Seedling roots are essential for plant establishment, and hence, the plant’s potential to set seed. For technical reasons, seedlings are more often screened than adult plants because of the ease of handling smaller plants and the high throughput. Seedling-stage phenotyping may also improve overall reproducibility of results because often, growth media are soil free. Seedling soil-free root phenotyping conditions are well suited to dissecting fine and sensitive mechanisms, such as lateral root initiation (Casimiro et al., 2003; Péret et al., 2009a, 2009b). A number of genes underlying root processes have been identified or characterized using seedlings, notably with the dicotyledonous models Arabidopsis (Arabidopsis thaliana; Mouchel et al., 2004; Fitz Gerald et al., 2006; Yokawa et al., 2013) and Medicago truncatula (Laffont et al., 2010) and the cereals maize (Zea mays; Hochholdinger et al., 2001) and rice (Oryza sativa; Inukai et al., 2005; Kitomi et al., 2008).Extrapolation from seedling to adult root systems presents major questions (Hochholdinger and Zimmermann, 2008; Chochois et al., 2012; Rich and Watt, 2013). Are phenotypes in seedling roots present in adult roots given developmental events associated with aging? Is expression of phenotypes correlated in seedling and adult roots if time compounds effects of growth rates and growth conditions on roots? Watt et al. (2013) showed in wheat seedlings that root traits in the laboratory and field correlated positively but that neither correlated with adult root traits in the field. Factors between seedling and adult roots seemed to be differences in developmental stage and the time that growing roots experience the environment.Seedling and adult root differences may be larger in grasses than dicotyledons. Grass root systems have two developmental components: seed-borne (seminal) roots, of which a number emerge at germination and continue to grow and branch throughout the plant life, and stem-borne (nodal or adventitious) roots, which emerge from around the three-leaf stage and continue to emerge, grow, and branch throughout the plant life. Phenotypes and traits of adult root systems of grasses, which include the major cereal crops wheat, rice, and maize, are difficult to predict in seedling screens and ideally identified from adult root systems first (Gamuyao et al., 2012).Phenotyping of adult roots is possible in the field using trenches (Maeght et al., 2013) or coring (Wasson et al., 2014). A portion of the root system is captured with these methods. Alternatively, entire adult root systems can be contained within pots dug into the ground before sowing. These need to be large; field wheat roots, for example, can reach depths greater than 1.5 m depending on genotype and environment. This method prevents root-root interactions that occur under normal field sowing of a plant canopy and is also a compromise.A solution to the problem of phenotyping adult cereal root systems is a model for monocotyledon grasses: Brachypodium distachyon. B. distachyon is a small-stature grass with a small genome that is fully sequenced (Vogel et al., 2010). It has molecular tools equivalent to those available in Arabidopsis (Draper et al., 2001; Brkljacic et al., 2011; Mur et al., 2011). The root system of B. distachyon reference line Bd21 is more similar to wheat than other model and crop grasses (Watt et al., 2009). It has a seed-borne primary seminal root (PSR) that emerges from the embryo at seed germination and multiple stem-borne coleoptile node axile roots (CNRs) and leaf node axile roots (LNRs), also known as crown roots or adventitious roots, that emerge at about three leaves through to grain development. Branch roots emerge from all root types. There are no known anatomical differences between root types of wheat and B. distachyon (Watt et al., 2009). In a recent study, we report postflowering root growth in B. distachyon line Bd21-3, showing that this model can be used to answer questions relevant to the adult root systems of grasses (Chochois et al., 2012).In this study, we used B. distachyon to identify adult plant phenotypes related to the partitioning among seed-borne and stem-borne shoots and roots for the genetic improvement of well-watered and droughted cereals (Fig. 1; Krassovsky, 1926; Navara et al., 1994), nitrogen, phosphorus (Tennant, 1976; Brady et al., 1995), oxygen (Wiengweera and Greenway, 2004), soil hardness (Acuna et al., 2007), and microorganisms (Sivasithamparam et al., 1978). Of note is the study by Krassovsky (1926), which was the first, to our knowledge, to show differences in function related to water. Krassovsky (1926) showed that seminal roots of wheat absorbed almost 2 times the water as nodal roots per unit dry weight but that nodal roots absorbed a more diluted nutrient solution than seminal roots. Krassovsky (1926) also showed by removing seminal or nodal roots as they emerged that “seminal roots serve the main stem, while nodal roots serve the tillers” (Krassovsky, 1926). Volkmar (1997) showed, more recently, in wheat that nodal and seminal roots may sense and respond to drought differently. In millet (Pennisetum glaucum) and sorghum (Sorghum bicolor), Rostamza et al. (2013) found that millet was able to grow nodal roots in a dryer soil than sorghum, possibly because of shoot and root vigor.Open in a separate windowFigure 1.B. distachyon plant scanned at the fourth leaf stage, with the root and shoot phenotypes studied indicated. Supplemental Table S1.