pERp1 is significantly up-regulated during plasma cell differentiation and contributes to the oxidative folding of immunoglobulin

Plasma cells can synthesize and secrete thousands of Ig molecules per second, which are folded and assembled in the endoplasmic reticulum (ER) and are likely to place unusually high demands on the resident chaperones and folding enzymes. We have discovered a new resident ER protein (pERp1) that is a component of the BiP chaperone complex. PERp1 is substantially up-regulated during B to plasma cell differentiation and can be induced in B cell lines by some UPR activators, arguing that it represents a potentially new class of conditional UPR targets. In LPS-stimulated murine splenocytes, pERp1 interacted covalently via a disulfide bond with IgM monomers and noncovalently with other Ig assembly intermediates. Knockdown and overexpression experiments revealed that pERp1 promoted correct oxidative folding of Ig heavy chains and prevented off-pathway assembly intermediates. Although pERp1 has no homology with known chaperones or folding enzymes, it possesses a thioredoxin-like active site motif (CXXC), which is the signature of oxidoreductases. Mutation of this sequence did not affect its in vivo activity, suggesting that pERp1 is either a unique type of oxidoreductase or a previously unidentified class of molecular chaperone that is dedicated to enhancing the oxidative folding of Ig precursors.     

Z-ring force and cell shape during division in rod-like bacteria

The life cycle of bacterial cells consists of repeated elongation, septum formation, and division. Before septum formation, a division ring called the Z-ring, which is made of a filamentous tubulin analog, FtsZ, is seen at the mid cell. Together with several other proteins, FtsZ is essential for cell division. Visualization of strains with GFP-labeled FtsZ shows that the Z-ring contracts before septum formation and pinches the cell into two equal halves. Thus, the Z-ring has been postulated to act as a force generator, although the magnitude of the contraction force is unknown. In this article, we develop a mathematical model to describe the process of growth and Z-ring contraction in rod-like bacteria. The elasticity and growth of the cell wall is incorporated in the model to predict the contraction speed, the cell shape, and the contraction force. With reasonable parameters, the model shows that a small force from the Z-ring (8 pN in Escherichia coli) is sufficient to accomplish division.     

Asymmetric cancer cell division regulated by AKT

Human tumors often contain slowly proliferating cancer cells that resist treatment, but we do not know precisely how these cells arise. We show that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating "G0-like" progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell division results from asymmetric suppression of AKT/PKB kinase signaling in one daughter cell during telophase of mitosis. Moreover, inhibition of AKT signaling with small-molecule drugs can induce asymmetric cancer cell division and the production of slow proliferators. Cancer cells therefore appear to continuously flux between symmetric and asymmetric division depending on the precise state of their AKT signaling network. This model may have significant implications for understanding how tumors grow, evade treatment, and recur.     

Oral feeding of isolated lectins from red kidney bean stimulates rat small intestinal mucosal DNA synthesis and crypt cell division

A lectin preparation containing enterokinase inhibitor purified or partially purified from red kidney bean (RKB) when fed to weanling rats was shown to cause small intestinal hyperplasia. To see if this hyperplastic effect on the rat small intestine was due to the mitogenic properties of the isolated lectin, male weanling rats were fed a chow containing 0.1% of the isolated lectin for six days. Age-matched control rats were fed regular chow. Both control and lectin-fed rats were sacrificed at one, two, three, four, and six days after the start of lectin feeding. The proximal small intestinal mucosa of rats fed lectin showed gradual increases in protein and DNA contents throughout the experimental period. Morphological studies showed marked increases in crypt depth from days I through 6 in these rats with essentially no change in mucosal thickness or villous height. DNA synthetic activity peaked at day 2, but was higher than control throughout the experimental period. Labeling index was 0.36±0.03 in duodenum of controls as compared to 0.45±0.02 in duodenum of weanling rats fed lectin for two days. These results demonstrate that RKB lectin stimulates overall DNA synthetic activity and increases crypt cell proliferation on the small intestine of weanling rats. The observed mucosal hyperplasia is probably due to increases in crypt cell population as shown by the increase in crypt depth.This study was supported by a grant from the U.S. Agency for International Development.     

Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes.     

Physical mechanisms of ESCRT-III–driven cell division

Living systems propagate by undergoing rounds of cell growth and division. Cell division is at heart a physical process that requires mechanical forces, usually exerted by assemblies of cytoskeletal polymers. Here we developed a physical model for the ESCRT-III–mediated division of archaeal cells, which despite their structural simplicity share machinery and evolutionary origins with eukaryotes. By comparing the dynamics of simulations with data collected from live cell imaging experiments, we propose that this branch of life uses a previously unidentified division mechanism. Active changes in the curvature of elastic cytoskeletal filaments can lead to filament perversions and supercoiling, to drive ring constriction and deform the overlying membrane. Abscission is then completed following filament disassembly. The model was also used to explore how different adenosine triphosphate (ATP)-driven processes that govern the way the structure of the filament is changed likely impact the robustness and symmetry of the resulting division. Comparisons between midcell constriction dynamics in simulations and experiments reveal a good agreement with the process when changes in curvature are implemented at random positions along the filament, supporting this as a possible mechanism of ESCRT-III–dependent division in this system. Beyond archaea, this study pinpoints a general mechanism of cytokinesis based on dynamic coupling between a coiling filament and the membrane.

Cell division is one of the most fundamental requirements for the existence of life on Earth. During division the material from a single cell is divided into two separate daughter cells. This is an inherently physical process. Living cells have evolved multiple ways to apply mechanical forces for this purpose. In general, division is thought to be achieved by proteins that assemble into long polymeric filaments at the cytoplasmic side of the cell membrane. These filaments then undergo a series of energy-driven changes in their form and organization to deform the associated membrane and/or guide cell wall assembly. However, the physical mechanisms by which this type of nonequilibrium protein self-assembly produces the mechanical work needed to reshape and cut soft surfaces remain underexplored.Although the mechanisms of division differ across the tree of life, recent data support the idea that eukaryotic cells likely arose from the symbiosis of an archaeal cell and an alphaproteobacterial cell, where the archaeal host gave rise to the eukaryotic cell body and the associated proteobacteria went on to become mitochondria (1–3). Because of this, many physical processes that control eukaryotic cell division are likely to have originated in archaea. In particular, ESCRT-III filaments, which drive cell division in a subgroup of archaea called TACK (thaumarchaeota, aigarchaeota, crenarchaeota and korachaeota) archaea, also catalyze the final step of cell division in many eukaryotes (4, 5).Here we develop a physical model to study the dynamics of archaeal cell division by ESCRT-III filaments. In archaea, ESCRT-III proteins polymerize into at least two distinct filamentous rings that likely form a copolymer that is adsorbed on the cytoplasmic side of the cell membrane. The first filamenteous ring (called CdvB) serves as a template for the assembly of a contractile ring (made of CdvB1 and CdvB2 proteins) (Fig. 1A). Recently it has been shown that the contractile CdvB1/2 ring is free to exert forces to reshape the membrane only once the template CdvB ring has been removed (6). As cytokinesis proceeds, the contractile CdvB1/2 ESCRT-III filament is then disassembled.Open in a separate windowFig. 1.Computational model. (A) Division of an archaeon S. acidocaldarius. The cell membrane is indicated with a dashed line, the template filament (CdvB) is fluorescently labeled in magenta, while the constricting ESCRT-III filament (CdvB1) is labeled in green. (B) The initial ESCRT-III filament state (CdvB + CdvB1/2) is modeled as a single helical filament with a target radius equal to the cell radius Rcell. The filament is attached to the inside of the fluid vesicle that represents the archaeal cell. To constrict, upon CdvB degradation, the ESCRT-III filament (CdvB1/2) reduces its target radius to Rtarget, which results in a new target state of a tighter helix. The filament model itself consists of triplet subunits that are connected to each other via nine bonds whose lengths control the filament curvature (Inset and SI Appendix). (C) An example of a cell division simulation. The target radius of the filament is instantaneously decreased to 5% of the original cell radius. The filament is then disassembled from both ends at a rate 102×vdis=6.7/τ (τ is the molecular dynamics unit of time). The filament first forms a superhelix that consists of multiple short helices of alternating chiralities (shown in the box). As the superhelix contracts and disassembles, it pulls the membrane into a tight neck, which spontaneously breaks (Movie S1).Here we investigate different energy-driven protocols that can lead to the contraction and disassembly of an ESCRT-III ring in contact with a deformable cell. We quantify the rates, reliability, and symmetry of the resulting cell division processes. We then compare the dynamics of cell division predicted in simulations with those observed via live imaging of the archeon Sulfolobus acidocaldarius, the closest archaeal relative to eukaryotic cells that can be easily cultured in a laboratory. This comparison identifies a single regime of filament remodeling that matches the experimental data remarkably well.Our simulations identify a physical mechanism for reshaping and splitting cells in which division is driven by the supercoiling of the filament. This differs from models of division described previously but, given the generality of our modeling approach, suggests a possible role for this process in cytoskeletal-induced membrane deformation events across biological systems. In this way, our analysis should help inform efforts to design synthetic nanomachinery that can drive the division of synthetic cells (7–9).     

Microenvironmental modulation of asymmetric cell division in human lung cancer cells

Normal tissue homeostasis is maintained through asymmetric cell divisions that produce daughter cells with differing self-renewal and differentiation potentials. Certain tumor cell subfractions can self-renew and repopulate the heterogeneous tumor bulk, suggestive of asymmetric cell division, but an equally plausible explanation is that daughter cells of a symmetric division subsequently adopt differing cell fates. Cosegregation of template DNA during mitosis is one mechanism by which cellular components are segregated asymmetrically during cell division in fibroblast, muscle, mammary, intestinal, and neural cells. Asymmetric cell division of template DNA in tumor cells has remained elusive, however. Through pulse-chase experiments with halogenated thymidine analogs, we determined that a small population of cells within human lung cancer cell lines and primary tumor cell cultures asymmetrically divided their template DNA, which could be visualized in single cells and in real time. Template DNA cosegregation was enhanced by cell–cell contact. Its frequency was density-dependent and modulated by environmental changes, including serum deprivation and hypoxia. In addition, we found that isolated CD133⁺ lung cancer cells were capable of tumor cell repopulation. Strikingly, during cell division, CD133 cosegregated with the template DNA, whereas the differentiation markers prosurfactant protein-C and pan-cytokeratins were passed to the opposing daughter cell, demonstrating that segregation of template DNA correlates with lung cancer cell fate. Our results demonstrate that human lung tumor cell fate decisions may be regulated during the cell division process. The characterization and modulation of asymmetric cell division in lung cancer can provide insight into tumor initiation, growth, and maintenance.     

Actin filament debranching regulates cell polarity during cell migration and asymmetric cell division

The formation of the branched actin networks is essential for cell polarity, but it remains unclear how the debranching activity of actin filaments contributes to this process. Here, we showed that an evolutionarily conserved coronin family protein, the Caenorhabditis elegans POD-1, debranched the Arp2/3-nucleated actin filaments in vitro. By fluorescence live imaging analysis of the endogenous POD-1 protein, we found that POD-1 colocalized with Arp2/3 at the leading edge of the migrating C. elegans neuroblasts. Conditional mutations of POD-1 in neuroblasts caused aberrant actin assembly, disrupted cell polarity, and impaired cell migration. In C. elegans one-cell−stage embryos, POD-1 and Arp2/3, moved together during cell polarity establishment, and inhibition of POD-1 blocked Arp2/3 motility and affected the polarized cortical flow, leading to symmetric segregation of cell fate determinants. Together, these results indicate that F-actin debranching organizes actin network and cell polarity in migrating neuroblasts and asymmetrically dividing embryos.

Cell polarity is a fundamental feature of virtually all eukaryotic cells and plays crucial roles in a wide range of cellular processes, including cell motility, asymmetric cell division, and cell signaling (1). The establishment of cell polarity involves the asymmetric assembly of distinct cellular components to perform specialized functions. The actin-related protein (Arp) 2/3 complex-dependent branched actin networks and the pushing force they produce provide the principal means for cells to remodel the plasma membrane during cellular polarization (2). For example, in the leading edge of a migrating cell, the local Arp2/3-nucleated actin polymerization powers asymmetric projections of the plasma membrane (3). During asymmetric cell division of the Caenorhabditis elegans zygote, an actomyosin flow is central to the transport of the polarity PAR proteins into defined subcellular domains (4).Actin filaments'' continuous assembly must be balanced by actin depolymerization to ensure a constant supply of actin monomers for new growth. The Arp2/3 complex potency in actin nucleation empowers this complex as an essential regulator to organize the actin cytoskeleton. While Arp2/3 by itself is biochemically inactive, interactions with nucleation-promoting factors (NPFs) such as the Wiskott Aldrich syndrome protein (WASP)/WASP family verproline-homologous (WASP/WAVE) family proteins shift the Arp2/3 complex from its open, inactive conformation to a closed, active conformation (5, 6). The conformationally activated Arp2/3 complex then binds to the side of preexisting actin filaments to nucleate a branch from the mother filament (7–12). Conversely, nucleation by Arp2/3 can be inhibited by several binding partners, including glia maturation factor (GMF), Gadkin, Arpin, and Coronin, whose activities replenish available pools of actin monomers and Arp2/3 complexes for sustained actin assembly (13–18).The coronin family proteins are conserved actin regulators (19). The phylogenetic analysis grouped coronin genes into three types (19, 20). The best-characterized coronin is the Type I coronin (e.g., Coronin 1B) that binds to actin filaments through the β-propeller structure and to the Arp2/3 complex via its N terminus. These interactions block the docking of Arp2/3 onto actin filaments or facilitate debranching the existing actin network (20). Coronin 1B simultaneously interacts with the Slingshot phosphatase to dephosphorylate and activate ADF/Cofilin proteins that sever actin filaments, thereby promoting the actin network disassembly (13). Despite significant progress on Type I coronin, the activity and function of other coronins remain unclear. In particular, Type III coronins, known as POD-1 in C. elegans and Drosophila or Coronin7 in Dictyostelium and humans, contain two tandem coronin repeats, making them distinct from other coronins (19–21). POD-1 was biochemically isolated from C. elegans oocytes (22), and its mutations disrupted the polarity and architecture in early C. elegans embryos and impaired midlife touch sensitivity of the nematode (21, 23). However, it remains unclear how the Type III coronin functions. The Drosophila homolog of POD-1 is required for correct axon guidance, and the purified Dpod-1 cross-links the actin and microtubule cytoskeletons (24), whereas the mammalian Coronin7 was implicated in the Golgi morphology and function (25, 26), demonstrating the functional divergence of this family of coronin. Here, we show that the C. elegans POD-1 debranches Arp2/3-nucleated actin filaments in vitro and that POD-1 regulates cell polarity by remodeling the actin cytoskeleton during cell migration and asymmetric cell division.     

Age-dependent regulation of mammalian DNA synthesis and cell division in vivo by glucocorticoids

The impaired ability to initiate DNA synthesis and cell division in vivo in submandibular glands of aging rats may be the consequence of both decreased sensitivity to isoproterenol treatment and increased susceptibility to regulation by endogenous glucocorticoids.     

Cis-hydroxyproline-induced inhibition of pancreatic cancer cell growth is mediated by endoplasmic reticulum stress

AIM: To investigate the biological effects of cishydroxyproline (CHP) on the rat pancreatic carcinoma cell line DSL6A, and to examine the underlying molecular mechanisms. METHODS: The effect of CHP on DSL6A cell proliferation was assessed by using BrdU incorporation. The expression of focal adhesion kinase (FAK) was characterized by Western blotting and immunofluorescence. Induction of endoplasmic reticulum (ER) stress was investigated by using RT-PCR and Western blotting for the glucose-related protein-78 (GRP78) and growth arrest and DNA inducible gene (GADD153). Cell viability was determined through measuring the metabolic activity based on the reduction potential of DSL6A cells. Apoptosis was analyzed by detection of caspase-3 activation and cleavage of poly(ADP-ribose) polymerase (PARP) as well as DNA laddering. RESULTS: In addition to inhibition of proliferation, incubation with CHP induced proteolytic cleavage of FAK and a delocalisation of the enzyme from focal adhesions, followed by a loss of cell adherence. Simultaneously, we could show an increased expression of GRP78 and GADD153, indicating a CHP-mediated activation of the ER stress cascade in the DSL6A cell line. Prolonged incubation of DSL6A cells with CHP finally resulted in apoptotic cell death. Beside L-proline, the inhibition of intracellular proteolysis by addition of a broad spectrum protease inhibitor could abolish the effects of CHP on cellular functions and the molecular processes. In contrast, impeding the activity of apoptosis-executing caspases had no influence on CHP-mediated cell damage. CONCLUSION: Our data suggest that the initiation of ER stress machinery by CHP leads to an activation of intracellular proteolytic processes, including caspaseindependent FAK degradation, resulting in damaging pancreatic carcinoma cells.     

Force generation by a dynamic Z-ring in Escherichia coli cell division

FtsZ, a bacterial homologue of tubulin, plays a central role in bacterial cell division. It is the first of many proteins recruited to the division site to form the Z-ring, a dynamic structure that recycles on the time scale of seconds and is required for division to proceed. FtsZ has been recently shown to form rings inside tubular liposomes and to constrict the liposome membrane without the presence of other proteins, particularly molecular motors that appear to be absent from the bacterial proteome. Here, we propose a mathematical model for the dynamic turnover of the Z-ring and for its ability to generate a constriction force. Force generation is assumed to derive from GTP hydrolysis, which is known to induce curvature in FtsZ filaments. We find that this transition to a curved state is capable of generating a sufficient force to drive cell-wall invagination in vivo and can also explain the constriction seen in the in vitro liposome experiments. Our observations resolve the question of how FtsZ might accomplish cell division despite the highly dynamic nature of the Z-ring and the lack of molecular motors.     

Melatonin alleviates cadmium-induced cellular stress and germ cell apoptosis in testes

Increasing evidence demonstrates that melatonin has an anti-apoptotic effect in somatic cells. However, whether melatonin can protect against germ cell apoptosis remains obscure. Cadmium (Cd) is a testicular toxicant and induces germ cell apoptosis. In this study, we investigated the effects of melatonin on Cd-evoked germ cell apoptosis in testes. Male ICR mice were intraperitoneally (i.p.) injected with melatonin (5 mg/kg) every 8 hr, beginning at 8 hr before CdCl(2) (2.0 mg/kg, i.p.). As expected, acute Cd exposure resulted in germ cell apoptosis in testes, as determined by terminal dUTP nick-end labeling (TUNEL) staining. Melatonin significantly alleviated Cd-induced testicular germ cell apoptosis. An additional experiment showed that spliced form of XBP-1, the target of the IRE-1 pathway, was significantly increased in testes of mice injected with CdCl(2). GRP78, an endoplasmic reticulum (ER) chaperone, and CHOP, a downstream target of the PERK pathway, were upregulated in testes of Cd-treated mice. In addition, acute Cd exposure significantly increased testicular eIF2α and JNK phosphorylation, indicating that the unfolded protein response (UPR) pathway was activated by CdCl(2). Interestingly, melatonin almost completely inhibited Cd-induced ER stress and the UPR in testes. In addition, melatonin obviously attenuated Cd-induced heme oxygenase (HO)-1 expression and protein nitration in testes. Taken together, these results suggest that melatonin alleviates Cd-induced cellular stress and germ cell apoptosis in testes. Melatonin may be useful as pharmacological agents to protect against Cd-induced testicular toxicity.     

Analysis of cell division patterns in the Arabidopsis shoot apical meristem

The stereotypic pattern of cell shapes in the Arabidopsis shoot apical meristem (SAM) suggests that strict rules govern the placement of new walls during cell division. When a cell in the SAM divides, a new wall is built that connects existing walls and divides the cytoplasm of the daughter cells. Because features that are determined by the placement of new walls such as cell size, shape, and number of neighbors are highly regular, rules must exist for maintaining such order. Here we present a quantitative model of these rules that incorporates different observed features of cell division. Each feature is incorporated into a “potential function” that contributes a single term to a total analog of potential energy. New cell walls are predicted to occur at locations where the potential function is minimized. Quantitative terms that represent the well-known historical rules of plant cell division, such as those given by Hofmeister, Errera, and Sachs are developed and evaluated against observed cell divisions in the epidermal layer (L1) of Arabidopsis thaliana SAM. The method is general enough to allow additional terms for nongeometric properties such as internal concentration gradients and mechanical tensile forces.The Arabidopsis shoot apical meristem (SAM) is a structure at the tip of the shoot that is responsible for generating almost all of the above-ground tissue of the plant (1). Its epidermal and subepidermal cells are organized into layers with very few cells moving between layers (2, 3). When these cells expand they do so laterally, pushing other cells toward the periphery of the meristem. Division in these cells is anticlinal such that each layer remains one cell thick. The underlying mechanism determining the location of new cell walls is unknown but the qualitative properties of meristematic cell division are well documented (4–8). Perhaps the best known summary is Errera’s rule, derived following observations of soap bubble formation. In the modern interpretation, the plane of division corresponds to the shortest path that will halve the mother cell. Errera, in fact, wrote that the wall would be a surface “mit constanter mittlerer Krümmung (= Minimalfläche) [with constant mean curvature (= minimal area)]” (4). Because this does not specify a location for the new cell wall, more recent authors have added to this that the mother cell divides evenly (9, 10). With this modification, Errera’s rule is easily quantifiable.A second observation is Hofmeister’s rule: New cell walls usually form in a plane normal to the principal axis of cell elongation (5). This rule is more difficult to quantify, because the principal axis of cell elongation is often confused with the direction of growth. Cells are asymmetrical and hence a principal direction of cell elongation can easily be calculated (e.g., the principal axis of inertia or principal component of a segmentation). The assumption is often made that because the cell is more elongated in one direction that the primary growth of the cell has been along that direction, but this is not necessarily the case, because the elongation may be derived from a prior cell division. For example, if a symmetrical square divides into two rectangular cells, this does not mean that the two daughter cells have grown primarily along their longer axis. Quantification of cell growth direction is much more difficult: It requires the observation of matching points over time and varies with the internal and external tensile forces on the cell. It is not clear whether the instantaneous direction of cell growth or the longer-term average (e.g., as measured over a significant fraction of a cell generation) is more directly relevant to forming the division plane. Under compression, single cells tend to divide in a plane perpendicular to the principal axis of the stress tensor (11), which could indicate a mechanical basis for cell wall placement.Other observations are that new cell walls form in a plane perpendicular to existing cell walls (6), that cell walls tend to avoid four-way junctions (7), and that cell division planes tend to be staggered, like bricks in a wall (8). Because chemical signals can be induced by physical interactions such as mechanical stress and strain it is conceivable that these geometric indicators are merely emergent properties of the underlying physicochemical interaction processes that drives cell division. Although most of the geometric observations tend to be true most of the time, none of them is true all of the time, and it is not possible for all of them to be true at once. For example, the actual growth direction is rarely in alignment with the principal geometric axis of the cell, and hence the division cannot simultaneously satisfy shortest length and perpendicularity requirements. Such conflicting results can in principle be resolved by minimizing a sum of potential functions (12), and insight can often be gained into the underlying mechanisms by examining the results of the optimization. Additionally, recent work by Besson and Dumais (9) suggests that cell division in plants is inherently random. The new wall tends to find a global minimum length, but in situations where there are multiple similar local minima the global minimum is not necessarily chosen.Previously we looked at cell divisions in the shoot meristem using 2D maximum intensity projections (13). Some of the results from that work may have been biased owing to the inconsistent perspective on cells in the peripheral zone compared with the center created by projecting a 3D object into 2D space. Because the meristem is dome-shaped, when projecting the meristem from the top the cells in the center are viewed perpendicularly, whereas the cells toward the edges are viewed at an angle. This nonperpendicular viewing angle distorts the lengths of the cell walls and the angles at which the walls join each other. To rectify that problem the geometry of the cells must be examined in 3D. Here we expand on earlier work with more comprehensive 3D image processing techniques to analyze the division patterns in the local tangent plane. By using the image processing software MorphoGraphX (14) we were able to reconstruct the cell boundaries in the first layer of a growing SAM.Having a 3D model of the structure of the epidermal (L1) layer over time allowed us to generate a model composed of a set of functions, each incorporating a different feature from the observed cell divisions. The functions each contribute a single term to a greater potential function and new walls are predicted to form where the combined potential is reduced. This model also brought to light some of the shortcomings of previously proposed plant cell division rules. Additionally, these data allowed us to make the observations reported below of the dynamics of cell expansion and division in different regions of the SAM.     

Self-assembly and sorting of acentrosomal microtubules by TACC3 facilitate kinetochore capture during the mitotic spindle assembly

Kinetochore capture by dynamic kinetochore microtubule fibers (K fibers) is essential for proper chromosome alignment and accurate distribution of the replicated genome during cell division. Although this capture process has been extensively studied, the mechanisms underlying the initiation of this process and the proper formation of the K fibers remain largely unknown. Here we show that transforming acidic coiled-coil–containing protein 3 (TACC3) is essential for kinetochore capture and proper K-fiber formation in HeLa cells. To observe the assembly of acentrosomal microtubules more clearly, the cells were released from higher concentrations of nocodazole into zero or lower concentrations. We find that small acentrosomal TACC3–microtubule aster formation near the kinetochores and binding of the asters with the kinetochores are the initial steps of the kinetochore capture by the acentrosomal microtubules, and that the sorting of kinetochore-captured acentrosomal microtubules with centrosomal microtubules leads to the capture of kinetochore by centrosomal microtubules from both spindle poles. We demonstrate that the sorting of the TACC3-associated microtubules with the centrosomal microtubules is a crucial process for spindle assembly and chromosome movement. These findings, which are also supported in the unperturbed mitosis without nocodazole, reveal a critical TACC3-dependent acentrosomal microtubule nucleation and sorting process to regulate kinetochore–microtubule connections and provide deep insight into the mechanisms of mitotic spindle assembly and chromosome alignment.To ensure proper segregation of the chromosomes into its two daughter cells during proliferation, the chromosomes of a mother cell must be captured by its assembling mitotic spindle through attachment of the chromosome kinetochores and the dynamic spindle microtubules (1). A “search-and-capture” model was proposed long ago, in which the dynamic spindle microtubules nucleated from the centrosomes search for and capture the chromosome kinetochores (2). Previous studies showed that the kinetochores are initially captured by the spindle-pole–nucleated microtubules with their lateral side (3, 4). Once captured, the kinetochores with their chromosomes are transported along the microtubules toward a spindle pole, and the microtubules shrink at their plus ends until the establishment of the end-on attachment (4, 5). However, this model is insufficient to explain the initial connection of the kinetochore and the spindle microtubules in the centrosome-independent spindle assembly process. Recent studies in Xenopus extracts indicated that microtubules are nucleated near the chromosomes and self-organize into a spindle (6). A new model for acentrosomal spindle assembly has been raised in mouse oocytes, in which self-organized microtubule organizing centers (MTOCs) replace the centrosome function (7). The somatic cells may also use the centrosome-independent pathway for their spindle assembly (8–10). In Drosophila cells, the centrosome-independent assembled kinetochore fibers can be captured by centrosomal microtubules (11–13).Previous studies have shown that transforming acidic coiled-coil–containing protein 3 (TACC3) is essential for the mitotic spindle assembly and chromosome alignment, but the mechanism remains largely unknown (14–18). Here we reveal that TACC3-dependent small microtubule aster formation and sorting near the kinetochores contribute to correct microtubule–kinetochore connections.     

Duplication and segregation of the actin (MreB) cytoskeleton during the prokaryotic cell cycle

The bacterial actin homolog MreB exists as a single-copy helical cytoskeletal structure that extends between the two poles of rod-shaped bacteria. In this study, we show that equipartition of the MreB cytoskeleton into daughter cells is accomplished by division and segregation of the helical MreB array into two equivalent structures located in opposite halves of the predivisional cell. This process ensures that each daughter cell inherits one copy of the MreB cytoskeleton. The process is triggered by the membrane association of the FtsZ cell division protein. The cytoskeletal division and segregation events occur before and independently of cytokinesis and involve specialized MreB structures that appear to be intermediates in this process.     

食管鳞癌组织中线粒体和细胞核DNA的提取和鉴定

目的:研究在同一组织中同时提取线粒体DNA(mtDNA)和核DNA(nDNA)的方法的有效性.方法:同一组织中既含有mtDNA又含有nDNA,利用试剂盒同时提取出mtDNA和nDNA,用琼脂糖凝胶电泳,聚合酶链反应(PCR)的方法,对所提取的mtDNA和nDNA进检测.结果:用同一组织从线粒体中得到mtDNA,从细胞核中得到nDNA.与用传统方法分别提取mtDNA和nDNA效果一样,并且两者相关性强,相比较更有说服力.结论:同一组织中能同时提取mtDNA和nDNA,既节省组织又节省时间和费用,为DNA的研究提供了较好的实验方法.