Apoptotic and Nonapoptotic Caspase Functions in Animal Development

A developing animal is exposed to both intrinsic and extrinsic stresses. One stress response is caspase activation. Caspase activation not only controls apoptosis but also proliferation, differentiation, cell shape, and cell migration. Caspase activation drives development by executing cell death or nonapoptotic functions in a cell-autonomous manner, and by secreting signaling molecules or generating mechanical forces, in a noncell autonomous manner.Programmed cell death or apoptosis occurs widely during development. During C. elegans development, 131 cells die by caspase CED-3-dependent apoptosis; however, ced-3 mutants do not show significant developmental defects (Ellis and Horvitz 1986). In contrast, studies on caspase mutants in mouse and Drosophila have revealed caspases’ roles in development. During development, cells are exposed to extrinsic and intrinsic stresses, and caspases are activated as one of multiple stress responses that ensure developmental robustness (Fig. 1). Caspases actively regulate animal development through both apoptosis and nonapoptotic functions that involve cell–cell communication in developing cell communities (Miura 2011). This chapter focuses on the in vivo roles of caspases in development and regeneration.Open in a separate windowFigure 1.Caspase activation during development. An embryo undergoes intrinsic and extrinsic stress, which activates caspases to execute both apoptotic and nonapoptotic functions, including cell differentiation and dendrite pruning. Apoptotic cells affect the shape and behavior of their neighboring cells. Caspase-activated cells are shown in dark gray.     

Polarity in Stem Cell Division: Asymmetric Stem Cell Division in Tissue Homeostasis

Many adult stem cells divide asymmetrically to balance self-renewal and differentiation, thereby maintaining tissue homeostasis. Asymmetric stem cell divisions depend on asymmetric cell architecture (i.e., cell polarity) within the cell and/or the cellular environment. In particular, as residents of the tissues they sustain, stem cells are inevitably placed in the context of the tissue architecture. Indeed, many stem cells are polarized within their microenvironment, or the stem cell niche, and their asymmetric division relies on their relationship with the microenvironment. Here, we review asymmetric stem cell divisions in the context of the stem cell niche with a focus on Drosophila germ line stem cells, where the nature of niche-dependent asymmetric stem cell division is well characterized.Asymmetric cell division allows stem cells to self-renew and produce another cell that undergoes differentiation, thus providing a simple method for tissue homeostasis. Stem cell self-renewal refers to the daughter(s) of stem cell division maintaining all stem cell characteristics, including proliferation capacity, maintenance of the undifferentiated state, and the capability to produce daughter cells that undergo differentiation. A failure to maintain the correct stem cell number has been speculated to lead to tumorigenesis/tissue hyperplasia via stem cell hyperproliferation or tissue degeneration/aging via a reduction in stem cell number or activity (Morrison and Kimble 2006; Rando 2006). This necessity changes during development. The stem cell pool requires expansion earlier in development, whereas maintenance is needed later to sustain tissue homeostasis.There are two major mechanisms to sustain a fixed number of adult stem cells: stem cell niche and asymmetric stem cell division, which are not mutually exclusive. Stem cell niche is a microenvironment in which stem cells reside, and provides essential signals required for stem cell identity (Fig. 1A). Physical limitation of niche “space” can therefore define stem cell number within a tissue. Within such a niche, many stem cells divide asymmetrically, giving rise to one stem cell and one differentiating cell, by placing one daughter inside and another outside of the niche, respectively (Fig. 1A). Nevertheless, some stem cells divide asymmetrically, apparently without the niche. For example, in Drosophila neuroblasts, cell-intrinsic fate determinants are polarized within a dividing cell, and subsequent partitioning of such fate determinants into daughter cells in an asymmetric manner results in asymmetric stem cell division (Fig. 1B) (see Fig. 3A and Prehoda 2009).Open in a separate windowFigure 1.Mechanisms of asymmetric stem cell division. (A) Asymmetric stem cell division by extrinsic fate determinants (i.e., the stem cell niche). The two daughters of stem cell division will be placed in distinct cellular environments either inside or outside the stem cell niche, leading to asymmetric fate choice. (B) Asymmetric stem cell division by intrinsic fate determinants. Fate determinants are polarized in the dividing stem cells, which are subsequently partitioned into two daughter cells unequally, thus making the division asymmetrical. Self-renewing (red line) and/or differentiation promoting (green line) factors may be involved.In this review, we focus primarily on asymmetric stem cell divisions in the Drosophila germ line as the most intensively studied examples of niche-dependent asymmetric stem cell division. We also discuss some examples of stem cell division outside Drosophila, where stem cells are known to divide asymmetrically or in a niche-dependent manner.     

Wnt Signaling in Bone Development and Disease: Making Stronger Bone with Wnts

The skeleton as an organ is widely distributed throughout the entire vertebrate body. Wnt signaling has emerged to play major roles in almost all aspects of skeletal development and homeostasis. Because abnormal Wnt signaling causes various human skeletal diseases, Wnt signaling has become a focal point of intensive studies in skeletal development and disease. As a result, promising effective therapeutic agents for bone diseases are being developed by targeting the Wnt signaling pathway. Understanding the functional mechanisms of Wnt signaling in skeletal biology and diseases highlights how basic and clinical studies can stimulate each other to push a quick and productive advancement of the entire field. Here we review the current understanding of Wnt signaling in critical aspects of skeletal biology such as bone development, remodeling, mechanotransduction, and fracture healing. We took special efforts to place fundamentally important discoveries in the context of human skeletal diseases.The skeleton has many important functions related to human health. Aside from the classical functions of the skeleton in structural support and movement, the bone matrix forms a major reservoir of calcium and other inorganic ions, and bone cells are active regulators of calcium homeostasis. Recent data suggest that bone cells can secrete hormones (e.g., FGF23 and osteocalcin) and likely play a physiologically significant role in regulating phosphate and energy homeostasis. It has emerged that Wnt signaling plays a major role controlling multiple aspects of skeletal development and maintenance. Thus, understanding how the Wnt pathway controls skeletal growth and homeostasis has broad implications for human health and disease.Cartilage and bone define the skeleton and are produced by chondrocytes and osteoblasts, respectively. During embryonic development, bones are formed by two distinct processes: intramembranous and endochondral ossification (Fig. 1A). A number of cranial bones and the lateral portion of the clavicles are formed by intramembranous ossification. In this process, mesenchymal progenitor cells condense and differentiate directly into bone-forming osteoblasts. The majority of bones in our body are formed by endochondral ossification, during which mesenchymal progenitor cells condense and differentiate first into cartilage-forming chondrocytes to generate an avascular template of the future bone. Chondrocytes in these templates undergo a program of proliferation and progressive cellular maturation. Eventually, they exit the cell cycle and become pre-hypertrophic, then terminally differentiating into hypertrophic chondrocytes, which are eliminated ultimately by apoptosis. Hypertrophic chondrocytes produce a matrix that is calcified and functions as a scaffold for new bone formation. Concomitant with chondrocyte hypertrophy, osteoclasts, osteoblasts, and blood vessels migrate in from perichondral regions and remodel this template into bone.Open in a separate windowFigure 1.Mechanisms of skeleton formation. (A) Bones can form by either intramembranous or endochondral ossification. Both processes are initiated by the condensation of mesenchymal cells. During intramembranous ossification, mesenchymal cells differentiate directly into osteoblasts and deposit bone. During endochondral ossification, mesenchymal cells differentiate into chondrocytes and first make a cartilage intermediate. Chondrocytes in the center of the bone initiate a growth plate, stop proliferating, and undergo hypertrophy. Hypertrophic chondrocytes mineralize their matrix and undergo apoptosis, attracting blood vessels and osteoblasts that remodel the intermediate into bone. (B) The first histologic sign of synovial joint formation is the gathering and flattening of cells, forming the interzone. Cavitation occurs within the presumptive joint separating the two cartilaginous structures. Remodeling and maturation proceed to give rise to the mature synovial joint. Wnt signaling plays a significant role in controlling almost all aspects of skeleton formation. Osteoblasts (purple); chondrocytes (blue); osteochondroprogenitor cells (brown).The developing skeletal elements are often segmented to form joints, which are required to support mobility. Synovial joints, which allow movement via smooth articulation between bones, form when chondrogenic cells in a newly formed cartilage undergo a program of dedifferentiation and flattening to form an interzone (Fig. 1B). Cavitation occurs within the flattened cells, allowing physical separation of the skeletal elements, and the formation of the synovial cavity. Cells and tissues in and around the interzone are remodeled at the same time to form the articular cartilage and other joint structures. Failure to form or maintain joints leads to joint fusion or osteoarthritis, a major skeletal disease.Following its formation, bone remains a regenerative tissue and is maintained during postnatal life by continuous remodeling. This highly active, homeostatic process is required for its functions and is controlled by three cell types: osteoblasts on the bone surface that deposit new bone matrix; osteocytes embedded in bone that are terminally differentiated from osteoblasts and function as mechanical and metabolic sensors; and the matrix-resorbing osteoclasts (Fig. 2). Osteoblasts are derived from mesenchymal stem cells (MSCs), whereas osteoclasts differentiate from hematopoietic progenitors. Decreased bone mass may be due to reduced osteoblast function or elevated osteoclast activity, and, conversely, increased bone mass may result from increased osteoblast function or decreased osteoclast activity. The precise balance of formation and resorption is critical for maintaining normal bone mass, and alterations in this balance lead to common bone diseases such as osteoporosis and osteopetrosis.Open in a separate windowFigure 2.Anatomy of bone. Cortical and trabecular bone represent the two major forms of bone. Osteoblasts (dark purple) are present on the surface and form new bone. Osteocytes (brown) are terminally differentiated osteoblasts that have become embedded in bone and communicate information to one another and to cells on the surface to regulate bone homeostasis. Osteoclasts (blue) are of hematopoietic origin and catabolize bone. A major function of Wnt/β-catenin signaling in osteoblasts is to suppress RANKL and to promote OPG production, thereby inhibiting osteoclast formation.There are two major bone types, cortical and trabecular, which show different anatomical properties (Fig. 2). Cortical (or compact) bone is the solid, densely packed bone that forms the outer layer of most bones and gives strength and rigidity. Trabecular (or cancellous) bone is present mostly in the marrow cavities of long bones and is the dominant bone type in vertebral bodies. Trabecular bone forms a porous, cobweb-like network of trabeculae whose large surface area is thought to facilitate the metabolic activity of bones mediated by osteoblasts and osteoclasts. Trabeculae are sites of active remodeling and will often orient in the direction of mechanical loading, dissipating the energy of loading and adding to bone strength. It is trabecular bone, rather than cortical bone, which is most severely affected in osteoporosis.The Wnt/β-catenin pathway plays a major role in controlling skeletal development and homeostasis, which are the focus of this work. We focus not only on differentiation of skeletal cells and formation of skeletal tissues, but also on the role of the Wnt/β-catenin signaling pathway on bone homeostasis, mechanotransduction, and wound healing, paying particular attention to human and mouse studies.     

Schwann Cells: Development and Role in Nerve Repair

Schwann cells develop from the neural crest in a well-defined sequence of events. This involves the formation of the Schwann cell precursor and immature Schwann cells, followed by the generation of the myelin and nonmyelin (Remak) cells of mature nerves. This review describes the signals that control the embryonic phase of this process and the organogenesis of peripheral nerves. We also discuss the phenotypic plasticity retained by mature Schwann cells, and explain why this unusual feature is central to the striking regenerative potential of the peripheral nervous system (PNS).The myelin and nonmyelin (Remak) Schwann cells of adult nerves originate from the neural crest in well-defined developmental steps (Fig. 1). This review focuses on embryonic development (for additional information on myelination, see Salzer 2015). We also discuss how the ability to change between differentiation states, a characteristic attribute of developing cells, is retained by mature Schwann cells, and explain how the ability of Schwann cells to change phenotype in response to injury allows the peripheral nervous system (PNS) to regenerate after damage.Open in a separate windowFigure 1.Main transitions in the Schwann cell precursor (SCP) lineage. The diagram shows both developmental and injury-induced transitions. Black uninterrupted arrows, normal development; red arrows, the Schwann cell injury response; stippled arrows, postrepair reformation of myelin and Remak cells. Embryonic dates (E) refer to mouse development. (Modified from Jessen and Mirsky 2012; reprinted, with permission and with contribution from Y. Poitelon and L. Feltri.)     

Chromosome Dynamics during Mitosis

The primary goal of mitosis is to partition duplicated chromosomes into daughter cells. Eukaryotic chromosomes are equipped with two distinct classes of intrinsic machineries, cohesin and condensins, that ensure their faithful segregation during mitosis. Cohesin holds sister chromatids together immediately after their synthesis during S phase until the establishment of bipolar attachments to the mitotic spindle in metaphase. Condensins, on the other hand, attempt to “resolve” sister chromatids by counteracting cohesin. The products of the balancing acts of cohesin and condensins are metaphase chromosomes, in which two rod-shaped chromatids are connected primarily at the centromere. In anaphase, this connection is released by the action of separase that proteolytically cleaves the remaining population of cohesin. Recent studies uncover how this series of events might be mechanistically coupled with each other and intricately regulated by a number of regulatory factors.In eukaryotic cells, genomic DNA is packaged into chromatin and stored in the cell nucleus, in which essential chromosomal processes, including DNA replication and gene expression, take place (Fig. 1, interphase). At the onset of mitosis, the nuclear envelope breaks down and chromatin is progressively converted into a discrete set of rod-shaped structures known as metaphase chromosomes (Fig. 1, metaphase). In each chromosome, a pair of sister kinetochores assembles at its centromeric region, and their bioriented attachment to the mitotic spindle acts as a prerequisite for equal segregation of sister chromatids. The linkage between sister chromatids is dissolved at the onset of anaphase, allowing them to be pulled apart to opposite poles of the cell (Fig. 1, anaphase). At the end of mitosis, the nuclear envelope reassembles around two sets of segregated chromatids, leading to the production of genetically identical daughter cells (Fig. 1, telophase).Open in a separate windowFigure 1.Overview of chromosome dynamics during mitosis. In addition to the crucial role of kinetochore–spindle interactions, an intricate balance between cohesive and resolving forces acting on sister chromatid arms (top left, inset) underlies the process of chromosome segregation. See the text for major events in chromosome segregation.Although the centromere–kinetochore region plays a crucial role in the segregation process, sister chromatid arms also undergo dynamic structural changes to facilitate their own separation. Conceptually, such structural changes are an outcome of two balancing forces, namely, cohesive and resolving forces (Fig. 1, top left, inset). The cohesive force holds a pair of duplicated arms until proper timing of separation, otherwise daughter cells would receive too many or too few copies of chromosomes. The resolving force, on the other hand, counteracts the cohesive force, reorganizing each chromosome into a pair of rod-shaped chromatids. From this standpoint, the pathway of chromosome segregation is regarded as a dynamic process, in which the initially robust cohesive force is gradually weakened and eventually dominated by the resolving force. Almost two decades ago, genetic and biochemical studies for the behavior of mitotic chromosomes converged productively, culminating in the discovery of cohesin (Guacci et al. 1997; Michaelis et al. 1997; Losada et al. 1998) and condensin (Hirano et al. 1997; Sutani et al. 1999), which are responsible for the cohesive and resolving forces, respectively. The subsequent characterizations of these two protein complexes have not only transformed our molecular understanding of chromosome dynamics during mitosis and meiosis, but also provided far-reaching implications in genome stability, as well as unexpected links to human diseases. In this article, I summarize recent progress in our understanding of mitotic chromosome dynamics with a major focus on the regulatory networks surrounding cohesin and condensin. I also discuss emerging topics and attempt to clarify outstanding questions in the field.     

The Signaling Mechanisms Underlying Cell Polarity and Chemotaxis

Chemotaxis—the directed movement of cells in a gradient of chemoattractant—is essential for neutrophils to crawl to sites of inflammation and infection and for Dictyostelium discoideum (D. discoideum) to aggregate during morphogenesis. Chemoattractant-induced activation of spatially localized cellular signals causes cells to polarize and move toward the highest concentration of the chemoattractant. Extensive studies have been devoted to achieving a better understanding of the mechanism(s) used by a neutrophil to choose its direction of polarity and to crawl effectively in response to chemoattractant gradients. Recent technological advances are beginning to reveal many fascinating details of the intracellular signaling components that spatially direct the cytoskeleton of neutrophils and D. discoideum and the complementary mechanisms that make the cell''s front distinct from its back.Chemotaxis—the directed movement of cells in a gradient of chemoattractant—allows leukocytes to seek out sites of inflammation and infection, amoebas of Dictyostelium discoideum (D. discoideum) to aggregate, neurons to send projections to specific regions of the brain to find their synaptic partners, yeast cells to mate, and fibroblasts to move into the wound space (Fig. 1). In each case, chemoattractant-induced activation of spatially localized cellular signals causes cells to polarize and move toward the highest concentration of the chemoattractant. During chemotaxis, filamentous actin (F-actin) is polymerized asymmetrically at the upgradient edge of the cell (leading edge), providing the necessary force to thrust projections of the plasma membrane in the proper direction (see Mullins 2009). Neutrophilic leukocytes (neutrophils), for instance, can polarize and move up very shallow gradients, with a chemoattractant concentration ∼2% higher at the front than the back (Fig. 2) (Devreotes and Zigmond 1988). To restrict actin polymerization to the leading edge in such a shallow gradient, neutrophils must create a much steeper internal gradient of regulatory signals. In addition, distinctive actin–myosin contractile complexes are also formed at the sides and back of the cells (Fig. 2). The ability to create such distinctive segregation of actin assemblies enables neutrophils to move nearly 50 times more quickly than fibroblasts. The polarization response is self-organizing, which occurs even when the attractant concentration is uniform and apparently stimulating all portions of the plasma membrane at the same intensity; in the absence of a gradient, the direction of polarity is random, but all cells can be induced to polarize (Fig. 2). Thus, neutrophil polarization to chemoattractant stimulation represents a striking example of symmetry breaking from an unpolarized state to a polarized one.Open in a separate windowFigure 1.Examples of chemotaxis. (A) A human neutrophil chasing a Staphylococcus aureus microorganism on a blood film among red blood cells, notable for their dark color and principally spherical shape (imaged by David Rogers, courtesy of Thomas P. Stossel). Bar, 10 µm. Chemotaxis is also necessary for (B) D. discoideum to form multicellular aggregates during development (courtesy of M.J. Grimson and R.L. Blanton, Texas Tech University), and (C) for axons to find their way in the developing nervous system. Photo provided by Kathryn Tosney, University of Miami.Open in a separate windowFigure 2.(A–D) Polarization of a neutrophil in response to gradient of chemoattractant. Nomarski images of unpolarized neutrophil responding to a micropipette containing the chemoattractant fMLP (white circle) at (A) 5 s, (B) 30 s, (C) 81 s, and (D) 129 s of stimulation. Bar = 5 µm. (Figure is taken from Weiner et al. 1999, with permission.) Human neutrophils stimulated with fMLP show highly polarized morphology and asymmetric cytoskeletal assemblies. (E–G) Human neutrophils were stimulated by a uniform concentration of fMLP (100 nM) and fixed 2 min after stimulations. Fixed cells were stained for F-actin with rhodamine-phalloidin (E, red) and an antibody raised against activated myosin II (phosphorylated specifically at Ser19, p[19]-MLC) (F, green). These fluorescent images are merged with Nomarski image in (G). Bars, 10 µm.To enter an infected tissue, neutrophils require chemoattractants produced by host cells and microorganisms to migrate to the sites and infection and inflammation. Neutrophil chemotaxis also contributes to many inflammatory and autoimmune diseases, including rheumatoid arthritis, ischemia-reperfusion syndrome, acute respiratory distress, and systemic inflammatory response syndromes. Although the critical physiological functions of neutrophils have made their chemoattractants and chemoattractant receptors targets of intense investigation, understanding of the neutrophil polarity and directional migration has until recently lagged behind that of other cells. Over the past decade, experimentation with knockout mice and human neutrophil cell lines has begun to shed light on the complex intracellular signals responsible for neutrophil polarity. In this article, I summarize recent advances in the study of chemotactic signals in neutrophils, with some of the discussion also devoted to a related model—chemotaxis of D. discoideum. These soil amoebas grow as single cells, but on starvation chemotax into multicellular aggregates in response to secreted chemoattractants such as adenosine 3′,5′-monophosphate (cAMP).     

Toll-Like Receptor Signaling

Toll-like receptors sense pathogen-associated molecular patterns (e.g., lipopolysaccharides) and trigger gene-expression changes that ultimately eradicate the invading microbes.Toll-like receptors (TLRs) are protective immune sentries that sense pathogen-associated molecular patterns (PAMPs) such as unmethylated double-stranded DNA (CpG), single-stranded RNA (ssRNA), lipoproteins, lipopolysaccharide (LPS), and flagellin. In innate immune myeloid cells, TLRs induce the secretion of inflammatory cytokines (Newton and Dixit 2012), thereby engaging lymphocytes to mount an adaptive, antigen-specific immune response (see Fig. 1) that ultimately eradicates the invading microbes (Kawai and Akira 2010).Open in a separate windowFigure 1.TLR signaling (simplified view).Identification of TLR innate immune function began with the discovery that Drosophila mutants in the Toll gene are highly susceptible to fungal infection (Lemaitre et al. 1996). This was soon followed by identification of a human Toll homolog, now known as TLR4 (Medzhitov et al. 1997). To date, 10 TLR family members have been identified in humans, and at least 13 are present in mice. All TLRs consist of an amino-terminal domain, characterized by multiple leucine-rich repeats, and a carboxy-terminal TIR domain that interacts with TIR-containing adaptors. Nucleic acid–sensing TLRs (TLR3, TLR7, TLR8, and TLR9) are localized within endosomal compartments, whereas the other TLRs reside at the plasma membrane (Blasius and Beutler 2010; McGettrick and O’Neill 2010). Trafficking of most TLRs from the endoplasmic reticulum (ER) to either the plasma membrane or endolysosomes is orchestrated by ER-resident proteins such as UNC93B (for TLR3, TLR7, TLR8, and TLR9) and PRAT4A (for TLR1, TLR2, TLR4, TLR7, and TLR9) (Blasius and Beutler 2010). Once in the endolysosomes, TLR3, TLR7, and TLR9 are subject to stepwise proteolytic cleavage, which is required for ligand binding and signaling (Barton and Kagan 2009). For some TLRs, ligand binding is facilitated by coreceptors, including CD14 and MD2.Following ligand engagement, the cytoplasmic TIR domains of the TLRs recruit the signaling adaptors MyD88, TIRAP, TRAM, and/or TRIF (see Fig. 2). Depending on the nature of the adaptor that is used, various kinases (IRAK4, IRAK1, IRAK2, TBK1, and IKKε) and ubiquitin ligases (TRAF6 and pellino 1) are recruited and activated, culminating in the engagement of the NF-κB, type I interferon, p38 MAP kinase (MAPK), and JNK MAPK pathways (Kawai and Akira 2010; Morrison 2012). TRAF6 is modified by K63-linked autoubiquitylation, which enables the recruitment of IκB kinase (IKK) through a ubiquitin-binding domain of the IKKγ (also known as NEMO) subunit. In addition, a ubiquitin-binding domain of TAB2 recognizes ubiquitylated TRAF6, causing activation of the associated TAK1 kinase, which then phosphorylates the IKKβ subunit. Pellino 1 can modify IRAK1 with K63-linked ubiquitin, allowing IRAK1 to recruit IKK directly. TLR4 signaling via the TRIF adaptor protein leads to K63-linked polyubiquitylation of TRAF3, thereby promoting the type I interferon response via interferon regulatory factor (IRFs) (Hacker et al. 2011). Alternatively, TLR4 signaling via MyD88 leads to the activation of TRAF6, which modifies cIAP1 or cIAP2 with K63-linked polyubiquitin (Hacker et al. 2011). The cIAPs are thereby activated to modify TRAF3 with K48-linked polyubiquitin, causing its proteasomal degradation. This allows a TRAF6–TAK1 complex to activate the p38 MAPK pathway and promote inflammatory cytokine production (Hacker et al. 2011). TLR signaling is turned off by various negative regulators: IRAK-M and MyD88 short (MyD88s), which antagonize IRAK1 activation; FADD, which antagonizes MyD88 or IRAKs; SHP1 and SHP2, which dephosphorylate IRAK1 and TBK1, respectively; and A20, which deubiquitylates TRAF6 and IKK (Flannery and Bowie 2010; Kawai and Akira 2010).Open in a separate windowFigure 2.TLR signaling. (Adapted with kind permission of Cell Signaling Technology [http://www.cellsignal.com].)Deregulation of the TLR signaling cascade causes several human diseases. Patients with inherited deficiencies of MyD88, IRAK4, UNC93B1, or TLR3 are susceptible to recurrent bacterial or viral infections (Casanova et al. 2011). Chronic TLR7 and/or TLR9 activation in autoreactive B cells, in contrast, underlies systemic autoimmune diseases (Green and Marshak-Rothstein 2011). Furthermore, oncogenic activating mutations of MyD88 occur frequently in the activated B-cell-like subtype of diffuse large B-cell lymphoma and in other B-cell malignancies (Ngo et al. 2011). Inhibitors of various TLRs or their associated kinases are currently being developed for autoimmune or inflammatory diseases and also hold promise for the treatment of B-cell malignancies with oncogenic MyD88 mutations. Many TLR7 and TLR9 agonists are currently in clinical trials as adjuvants to boost host antitumor responses in cancer patients (Hennessy et al. 2010).     

Break-Induced DNA Replication

Recombination-dependent DNA replication, often called break-induced replication (BIR), was initially invoked to explain recombination events in bacteriophage but it has recently been recognized as a fundamentally important mechanism to repair double-strand chromosome breaks in eukaryotes. This mechanism appears to be critically important in the restarting of stalled and broken replication forks and in maintaining the integrity of eroded telomeres. Although BIR helps preserve genome integrity during replication, it also promotes genome instability by the production of loss of heterozygosity and the formation of nonreciprocal translocations, as well as in the generation of complex chromosomal rearrangements.The break-copy mode of recombination (as opposed to break-join), was initially proposed by Meselson and Weigle (1961). Break-copy recombination, now more commonly known as recombination-dependent DNA replication or break-induced replication (BIR), is believed to account for restarting replication at broken replication forks and may also play a central role in the maintenance of telomeres in the absence of telomerase. BIR has been studied in various model systems and has been invoked to explain chromosome rearrangements in humans. This review focuses primarily on mechanistic studies in Escherichia coli and its bacteriophages, T4 and λ, in the budding yeasts Saccharomyces cerevisiae and Kluyveromyces lactis and on apparently similar, but less well-documented, mechanisms in mammalian cells.Homology-dependent repair of DNA double-strand breaks (DSBs) occur by three major repair pathways (Pâques and Haber 1999) (Fig. 1). When both ends of the DNA share substantial homology with a donor template (a sister chromatid, a homologous chromosome, or an ectopically located segment), repair occurs almost exclusively by gene conversion (GC). If the DSB is flanked by direct repeats, then a second repair process, single-strand annealing (SSA), can occur as 5′ to 3′ resection of the DSB ends exposes complementary sequences that can anneal to each other and repair the break by the formation of a deletion. However, when only one DSB end shares homology with a donor sequence, repair occurs by BIR. There are two BIR pathways, one dependent on Rad51 recombinase and the other independent of Rad51.Open in a separate windowFigure 1.Three major repair pathways of homology-dependent recombination. Noncrossover (NCO) and crossover (CO) events are indicated. Black triangles represent resolution of Holliday junctions (HJs). Dashed lines represent new DNA synthesis. GC, gene conversion; SSA, single-strand annealing; BIR, break-induced replication.     

Physical Model of Cellular Symmetry Breaking

Cells can polarize in response to external signals, such as chemical gradients, cell–cell contacts, and electromagnetic fields. However, cells can also polarize in the absence of an external cue. For example, a motile cell, which initially has a more or less round shape, can lose its symmetry spontaneously even in a homogeneous environment and start moving in random directions. One of the principal determinants of cell polarity is the cortical actin network that underlies the plasma membrane. Tension in this network generated by myosin motors can be relaxed by rupture of the shell, leading to polarization. In this article, we discuss how simplified model systems can help us to understand the physics that underlie the mechanics of symmetry breaking.Symmetry breaking in physics is an old well-known concept. It is based on energy considerations: A symmetrical system can lose its symmetry if an asymmetrical state has a lower energy. The initial symmetrical state can be either unstable or metastable. In the latter case, there is an energy barrier to be overcome before symmetry breaking occurs. An external trigger can drive the system from its symmetrical to its asymmetrical state, but simple noise can also do so if its amplitude is sufficiently high. A simple example is a clown balancing on a ball: When the clown is standing on top of the ball, the system has a cylindrical symmetry (Fig. 1A). However, this state is unstable: The slightest perturbation will cause the clown to fall down in some direction, breaking the cylindrical symmetry (Fig. 1B). Imagine now that the ball is slightly flat on its base, giving more stability to the clown. Such a state is metastable: The clown can make small excursions safely (Fig. 1C,D), but if he moves too much (i.e., generates too much “noise”), he will fall down in this case also (Fig. 1E,F).Open in a separate windowFigure 1.Illustration of symmetry breaking with a clown standing on a balloon. In (A), the clown is in unstable equilibrium and the situation is symmetrical. However, any movement will make him fall down and the system (clown + balloon) then loses its symmetry. (B) If the balloon is slightly flat on its base (C–F), then the system is metastable, i.e., a slight perturbation of the clown will not break the symmetry (C, D), whereas a larger perturbation will destabilize the clown (F).Symmetry breaking is ubiquitous in physics, and can lead to phase transitions or pattern formation. It is also an important theme in cell biology, in which polarization is crucial for proper functioning of the cell. Cell polarization typically occurs in response to certain external or internal triggers. A well-known example is chemotaxis, in which a chemical gradient leads to polarization and directed movement of bacterium cells. Polarization also occurs during cytokinesis, in which intracellular stimuli triggered by the mitotic spindle determine the position of the cleavage furrow (Burgess and Chang 2005). Interestingly, cells conserve the ability to polarize even in the absence of an asymmetric signal (Devreotes and Zigmond 1988). For example, chemotactic cells that are presented a uniform concentration of chemoattractant polarize and move in random directions. Another example is blebbing, the spontaneous appearance of bare membrane bulges in some cells.Symmetry breaking in biological systems is a complex phenomenon, because biological systems are always out of equilibrium. Hence, symmetry breaking is not just a transition to a state of lower potential energy. Instead, active, dynamic processes must be considered that feed energy into the system. A biochemical explanation for symmetry breaking was given by Alan Turing. In a seminal paper in 1952 (Turing 1952), he showed that patterns can be generated by simple chemical reactions if the reactants have different diffusion rates. To make this clear, he considered the hypothetical situation in which the morphology of a cell (or cell clump) is determined by two chemical substances (called morphogens). These morphogens also control their own production rate: One enhances morphogen production (the activator) and the other inhibits morphogen production (the inhibitor). It was shown that a spatially homogeneous distribution of morphogens is unstable if the activator diffuses more slowly than the inhibitor. In this case, small stochastic concentration fluctuations are amplified, leading to a chemical instability (“a Turing instability”) and the formation of concentration gradients (or patterns). Reaction–diffusion models of the Turing type have been widely explored to explain polarization and biological development (Gierer and Meinhardt 1972; Sohrmann and Peter 2003; Wedlich-Soldner and Li 2003).Although reaction–diffusion models have proven to be very successful, there is increasing evidence that cell polarization is not only a matter of biochemistry; mechanical aspects play an important role too. Recent work suggests that spontaneous polarization can also be driven by a mechanical instability of the actomyosin cortex of cells. In the remainder of this review, we focus on such mechanical instabilities.     

Holliday Junction Resolvases

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

The Spatiotemporal Organization of ErbB Receptors: Insights from Microscopy

Signal transduction is regulated by protein–protein interactions. In the case of the ErbB family of receptor tyrosine kinases (RTKs), the precise nature of these interactions remains a topic of debate. In this review, we describe state-of-the-art imaging techniques that are providing new details into receptor dynamics, clustering, and interactions. We present the general principles of these techniques, their limitations, and the unique observations they provide about ErbB spatiotemporal organization.Signal transduction is associated with dramatic spatial and temporal changes in membrane protein distribution. Although the biochemical events downstream of membrane receptor activation are often well characterized, the initiating events within the plasma membrane remain unclear. Many cell surface receptors have been shown to redistribute into clusters in response to ligand binding (Metzger 1992). Therefore, correlating membrane receptor activation with dynamics and aggregation state is essential to understanding cell signaling.The role of receptor aggregation is of particular interest in the case of receptor tyrosine kinases (RTKs). It is generally accepted that ligand binding to the extracellular domain of RTKs induces dimerization, whether ligand- or receptor-mediated (Lemmon and Schlessinger 2010). However, there is evidence that some RTKs exist as oligomers in the absence of ligand, whereas others require higher-order oligomerization for activation (Lemmon and Schlessinger 2010). Understanding the fundamental interactions that regulate RTK signaling still remains an important focus in the field.Over the past decade, imaging technologies and biological tools have developed to a point such that questions about protein dynamics, clustering, and interactions can now be addressed in living cells (Fig. 1). These techniques reveal information about protein behavior on a spatial and temporal scale that is not provided by traditional biochemical assays. In this review, we will discuss the application of these advanced imaging technologies to the study of the ErbB family of RTKs.Open in a separate windowFigure 1.Summary of imaging techniques for quantifying receptor clustering, dynamics, and interactions.     

Cell–Matrix Interactions in Mammary Gland Development and Breast Cancer

The mammary gland is an organ that at once gives life to the young, but at the same time poses one of the greatest threats to the mother. Understanding how the tissue develops and functions is of pressing importance in determining how its control mechanisms break down in breast cancer. Here we argue that the interactions between mammary epithelial cells and their extracellular matrix (ECM) are crucial in the development and function of the tissue. Current strategies for treating breast cancer take advantage of our knowledge of the endocrine regulation of breast development, and the emerging role of stromal–epithelial interactions (Fig. 1). Focusing, in addition, on the microenvironmental influences that arise from cell–matrix interactions will open new opportunities for therapeutic intervention. We suggest that ultimately a three-pronged approach targeting endocrine, growth factor, and cell-matrix interactions will provide the best chance of curing the disease.Cellular interactions with the ECM are one of the defining features of metazoans (Huxley-Jones et al. 2007). Matrix proteins are among the most abundant in the body, and are integral components of cell regulation and developmental programs operating in all tissues. They provide structure and support to tissues, and they interact with cells through diverse receptors to guide development, patterning, and cell fate decisions (Streuli 2009). Together with cytokines and growth factors, and cell–cell interactions, the ECM determines whether cells survive, proliferate, differentiate, or migrate, and it influences cell shape and polarity (Streuli and Akhtar 2009). Cell–ECM interactions also are central in the assembly of the matrix itself, and in determining ECM organization and rigidity (Kadler et al. 2008; Kass et al. 2007). The cell–matrix interface is therefore pivotal in controlling both cell function and tissue structure, which together build organs into operational structures. Thus, elucidating precisely how the matrix directs cell phenotype is crucial for understanding mechanisms of development and disease.Mammary gland tissue contains epithelium and stroma (​(Fig.Fig. 2). Mammary epithelial cells (MEC) form collecting ducts and, in pregnancy and lactation, milk-secreting alveoli (or lobules). The mammary epithelium is bilayered, with the inner luminal cells facing a central apical cavity and surrounded by the outer basal, myoepithelial cells. It also harbors stem and progenitor cells, which are the source of both luminal and myoepithelial cells (Visvader 2009). The epithelium is ensheathed by one of the main types of ECM, basement membrane (BM), which separates epithelium from stroma, and profoundly influences the development and biology of the gland (Streuli 2003). The stroma includes fibrous connective tissue ECM proteins, and a wide variety of cell types, including inter- and intralobular fibroblasts, adipocytes, endothelial cells, and innate immune cells (both macrophages and mast cells). The stroma is the support network for the epithelium, providing both nutrients and blood supply, and immune defenses, as well as physical structure to the gland. Importantly, each of the different stromal cell types secrete instructive signals that are crucial for various aspects of the development and function of the epithelium (Sternlicht 2006).Open in a separate windowFigure 1.Mammary gland development. Whole mounts of (A) virgin and (B) mid-pregnant mouse mammary gland. The thin, branched epithelial ducts that are characteristic of nonpregnant gland undergo dramatic alterations in pregnancy, when new types of epithelial structures, the milk-producing alveoli, emerge. The huge amount of proliferation that accompanies this change occurs in a discrete and controlled fashion. The formation of ducts and alveoli is under three types of environmental control. The first is long-range endocrine hormones, which includes estrogen, progesterone, glucocorticoids, and prolactin. The second is locally acting growth factors, which arise from stromal–epithelial conversation, and includes amphiregulin, FGF, HGF, and IGF. Finally, microenvironmental adhesive signals from adjacent cells (e.g., via cadherins) and from the ECM (e.g., integrin) have an equally central role in all aspects of mammary development and function. Importantly, the proliferation that occurs in breast cancer is not well controlled, indicating not only defects in growth signaling, but also in cellular organization. Chronologically, breast cancer drugs were initially developed against endocrine regulators, e.g., estrogen, and more recently against the stromal/epithelial regulators, e.g., receptor tyrosine kinases. A complete control of the disease will only happen when therapies targeting the microenvironmental adhesion breast regulators, e.g., cell–matrix interactions, are formulated, and used in combination.Open in a separate windowFigure 2.Ducts and alveoli in early pregnancy. Transverse section of ducts surrounded by a thick layer of collagenous (stromal) connective tissue containing fibroblasts and the fat pad. Also visible are small alveoli, which fill the fat pad by the time the gland lactates, but note that they are not surrounded collagen. A capillary is evident, and macrophages and mast cells are also present, though they require specific staining to visualize. A basement membrane is present directly at the basal surface of both ductal and alveolar epithelium (see Fig. 3).BMs surround three cell types in the mammary gland: the epithelium, the endothelium of the vasculature, and adipocytes (Fig. 3). These ECMs are thin, ∼100-nm thick sheets of glycoproteins and proteoglycans, which are constructed around an assembled polymer of laminins and a cross-linked network of collagen IV fibrils (Yurchenco and Patton 2009). Laminins form αβγ trimers, and in the breast at least four distinct isoforms are present: laminin-111, -322, and -511 and -521 (previously known as LM-1, 5, 10, and 11) (Aumailley et al. 2005; Prince et al. 2002). Similarly, BM proteoglycans are diverse and show complexity in their GAG chain modifications that vary with development of the mammary gland, though the major species is perlecan (Delehedde et al. 2001). BM proteins interact with MEC via integrins and transmembrane proteoglycans dystroglycan and syndecan, which all couple to the cytoskeleton and assemble signaling platforms to control cell fate (Barresi and Campbell 2006; Morgan et al. 2007). The best-studied MEC BM receptors are integrins, which are αβ heterodimers: they include receptors for collagen (α1β1 and α2β1), LM-111, -511, -521 (α3β1, α6β1, and α6β4), LM-322 (α3β1 and α6β4), and in some MECs fibronectin and vitronectin (α5β1 and β3 integrins) (Naylor and Streuli 2006). BM proteoglycans have a further signaling role via their capacity to bind growth factors and cytokines: They act both as a reservoir and a delivery vehicle to GF receptors, thereby controlling the passage of GFs across the BM (Iozzo 2005). Because of these diverse roles, the BM is a dominant regulator of the mammary epithelial phenotype.Open in a separate windowFigure 3.Alveolar and ductal architecture of breast epithelia shown through fluorescence and histological images. (A) An alveolus from a lactating mammary gland, showing luminal epithelial cells with cell–cell adhesion junctions (green, E-cadherin) and cell–matrix interactions (red, laminin-111). The central lumen is where milk collects. (B) The duct of a nonpregnant gland is stained with an antibody to laminin (brown) and counterstained with hematoxylin. Note that the laminin-containing basement membrane surrounds the ductal epithelial cells, and outside this lie collagenous connective tissue and adipocytes. Figure B courtesy of Dr. Rama Khokha.Apart from the endothelium and adipocytes, which contact BMs, the mammary stromal cells are mostly solitary and embedded within a fibrous ECM. Stromal matrix components include collagens type I and III, proteoglycans and hyaluronic acid, fibronectin and tenascins, and the composition varies with development and pregnancy (Schedin et al. 2004). Not a great deal is known about the specific interactions between breast stromal cells and their ECM, or how the matrix composition and density determines stromal cell function. However, it is becoming evident that the stromal matrix exerts a powerful influence on malignant breast epithelial cells, which invade the stroma and are further transformed by exposure to this distinct microenvironment (Kumar and Weaver 2009; Streuli 2006).In this article we focus on cell–matrix interactions within mammary epithelium, and reveal known and possible mechanisms for its control on ductal development, alveolar function, and cancer progression.