The Centrosome and Its Duplication Cycle

The centrosome was discovered in the late 19th century when mitosis was first described. Long recognized as a key organelle of the spindle pole, its core component, the centriole, was realized more than 50 or so years later also to comprise the basal body of the cilium. Here, we chart the more recent acquisition of a molecular understanding of centrosome structure and function. The strategies for gaining such knowledge were quickly developed in the yeasts to decipher the structure and function of their distinctive spindle pole bodies. Only within the past decade have studies with model eukaryotes and cultured cells brought a similar degree of sophistication to our understanding of the centrosome duplication cycle and the multiple roles of this organelle and its component parts in cell division and signaling. Now as we begin to understand these functions in the context of development, the way is being opened up for studies of the roles of centrosomes in human disease.     

Changes of Endocytotic Activities during the Cell Cycle of Dictyostelium Cells

Changes of endocytotic activities during the cell cycle of the cellular slime mould Dictyostelium discoideum Ax-2 were examined using the temperature-shift method for inducing synchronous growth. The activity of fluid-phase pinocytosis (FPP) was altered Ca²⁺-dependently and stimulated by EGTA. On the other hand, pinocytosis was greatly enhanced by addition of Bacteriological-peptone(BP) to the growth medium for Ax-2 cells, irrespective of the extracellular Ca²⁺-concentration. The maximal pinocytotic activity was attained in the presence of EGTA plus BP, the effects of the two substances being additive. The FPP activity was found to be high in cells in and just after the S phase, when the BP-induced fraction of pinocytosis was rather low. Thus the total activity for pinocytosis in the growth medium remained almost constant throughout the cell cycle, indicating that the rate of nutrient uptake through pinocytosis was not a limiting factor for cell cycle regulation. The change of phagocytotic activity during the cell cycle was somewhat similar to that of the FPP activity. Possible mechanisms of such cell-cycle related changes are discussed in relation to cytoskeletal proteins in the cell cortex. Some properties of BP-induced pinocytosis are also described.     

脑胶质瘤中心体的异常扩增及其与疾病分期的相关性

目的:探讨脑胶质瘤中心体扩增情况及其与疾病分期的相关性。方法:对40例胶质瘤标本和10例正常脑组织标本进行HE染色的检测;免疫组织化学检测Ki67和γ-微管蛋白的表达;使用免疫荧光染色技术检测中心体扩增情况。结果:不同标本中,HE染色呈现不同的细胞形态特征;Ki67在正常脑组织中没有表达,但在Ⅰ/Ⅱ、Ⅲ和Ⅳ级胶质瘤中的阳性表达率分别为65%、80%和100%,各组间差异具有统计学意义(P<0.01),说明Ki67的表达与胶质瘤级别相关。γ-微管蛋白在正常脑组织和Ⅰ/Ⅱ、Ⅲ、Ⅳ级胶质瘤中的表达率分别为30%、50%、80%和100%,各组间差异具有统计学意义(P<0.01),说明胶质瘤级别升高,中心体扩增率增加;免疫荧光检测显示,中心体扩增率和脑胶质瘤的级别呈正相关。结论:中心体扩增是脑胶质瘤的恶性程度的特征之一,并且与胶质瘤发病阶段有关,提示中心体扩增和脑胶质瘤的发展具有密切的相关性。     

Unusual polyamines in slime molds Physarum polycephalum and Dictyostelium discoideum

We analyzed the cellular contents of not only major polyamines but also minor polyamines in slime molds Physarum polycephalum and Dictyostelium discoideum. The presence of putrescine and spermidine in either plasmodia or myxamoebae of these molds as major polyamines was confirmed. In addition to these polyamines, appreciable amounts of 1,3-diaminopropane were detected in P. polycephalum and D. discoideum. Cadaverine and sym-homospermidine were detected in P. polycephalum even when the slime mold was cultured in a chemically defined growth medium. Spermine was not detected when these molds were grown in synthetic media. Other "unusual" polyamines such as norspermidine, norspermine, thermospermine, aminopropylcadaverine, and canavalmine were not detected in either mold.     

Dictyostelium discoideum CenB Is a Bona Fide Centrin Essential for Nuclear Architecture and Centrosome Stability

Centrins are a family of proteins within the calcium-binding EF-hand superfamily. In addition to their archetypical role at the microtubule organizing center (MTOC), centrins have acquired multiple functionalities throughout the course of evolution. For example, centrins have been linked to different nuclear activities, including mRNA export and DNA repair. Dictyostelium discoideum centrin B is a divergent member of the centrin family. At the amino acid level, DdCenB shows 51% identity with its closest relative and only paralog, DdCenA. Phylogenetic analysis revealed that DdCenB and DdCenA form a well-supported monophyletic and divergent group within the centrin family of proteins. Interestingly, fluorescently tagged versions of DdCenB were not found at the centrosome (in whole cells or in isolated centrosomes). Instead, DdCenB localized to the nuclei of interphase cells. This localization disappeared as the cells entered mitosis, although Dictyostelium cells undergo a closed mitosis in which the nuclear envelope (NE) does not break down. DdCenB knockout cells exhibited aberrant nuclear architecture, characterized by enlarged and deformed nuclei and loss of proper centrosome-nucleus anchoring (observed as NE protrusions). At the centrosome, loss of DdCenB resulted in defects in the organization and morphology of the MTOC and supernumerary centrosomes and centrosome-related bodies. The multiple defects that the loss of DdCenB generated at the centrosome can be explained by its atypical division cycle, transitioning into the NE as it divides at mitosis. On the basis of these findings, we propose that DdCenB is required at interphase to maintain proper nuclear architecture, and before delocalizing from the nucleus, DdCenB is part of the centrosome duplication machinery.Centrins (also known as caltractins) are small calcium-binding proteins of the EF-hand superfamily and are thought to have diversified by gene duplication (37). The first centrin was discovered in the unicellular green algae Tetraselmis striata more than 20 years ago (45). Since then, members of this family of proteins have been found in groups as diverse as yeasts, insects, plants, and humans, making these proteins essentially ubiquitous among eukaryotic cells (55). Furthermore, centrins have been included within the 347 “eukaryotic signature proteins” that are thought to be indispensable for the eukaryotic cell and share no similarities with prokaryotic proteins (21). Many lower eukaryotes have a single centrin gene (e.g., Saccharomyces cerevisiae and Chlamydomonas reinhardtii); however, up to three or four centrin paralogs have been found in higher eukaryotes (e.g., Xenopus laevis, Mus musculus, and Homo sapiens). Centrins have a high level of structural resemblance to calmodulin, exhibiting the characteristic two globular domains interconnected by a linker loop. Each globular domain in turn contains two helix-loop-helix motifs that, in calmodulins, bind calcium ions. However, in many centrins these motifs are slightly modified, and not all four of them have affinity for calcium in the normal range associated with signal transduction (33).Throughout the course of evolution, centrins have acquired multiple functionalities in addition to the archetypical role at the microtubule organizing center (MTOC). For example, the centrin of the flagellated green algae C. reinhardtii (CrCen) localizes to the basal bodies, to the fibers that interconnect the basal bodies and the nucleus, and to the axoneme. CrCen is required for normal basal body replication, segregation, and maturation (26). In addition, it plays an active role in the contraction of MTOC-related fibers (47, 57) and regulates the activity of the inner dynein arm in a calcium-regulated fashion (30).In the budding yeast Saccharomyces cerevisiae, centrin (ScCDC31) localizes primarily to a specialized region of the nuclear envelope (NE) called the half bridge (49), which is in close proximity to the MTOC (known as the spindle pole body [SPB]). Conditional mutants of ScCDC31 show cell cycle arrest and failure to duplicate the SPB (23, 49). CDC31 also binds the NEF2 complex and is required for efficient nucleotide excision repair. CDC31 mutants unable to bind to the complex showed an increased sensitivity to UV (1). In addition, CDC31 is involved in mRNA export through its interaction with SAC3 at the nuclear pore (11). Mammalian cells typically have four centrin paralogs; however, human cells express only three (HsCen1 to -3) and the fourth is a pseudogene (gene ID, 729338) (9, 13, 31, 36). All human centrins show partial localization at the centrioles, in a tissue-specific fashion (HsCen1) or ubiquitously (HsCen2 and -3) (29, 56). Knockdown of HsCen2 inhibits centriole duplication and induces cell division arrest in HeLa cells (46). Additionally, HsCen2 was shown to play a role similar to that of CDC31 in stimulating nucleotide excision repair by binding to xeroderma pigmentosum group C protein (38).The social amoeba Dictyostelium discoideum has emerged as a powerful model organism, in part because it is haploid, it is easy to propagate, and its genome has been recently completed (4, 8, 25). D. discoideum cells undergo a closed mitosis during which the NE remains intact. They also have multiple modes of cytokinesis (53), making them a very useful model for studying the cell division machinery. These cells lack basal bodies and have acentriolar centrosomes that are similar in their trilaminar core structure to yeast SPBs (17). However, D. discoideum interphase centrosomes are not embedded in the NE but are attached to it, and they are surrounded by a centrosomal corona analogous to the pericentriolar material of animal cell centrosomes (4). Centrosomal duplication in D. discoideum involves extensive structural changes and is synchronized with mitosis. It begins at early prophase, by increasing its size to about twice that of an interphase centrosome. At the prophase-prometaphase transition, the corona and the fibrous link to the nucleus are disassembled. This is followed by the insertion of the core into the NE. By metaphase, the two outer layers have come apart and migrated to opposite ends of the cell nucleus, where they organize the spindle. The anaphase-telophase transition marks the beginning of centrosomal maturation. The outer layers fold back into themselves, inducing the formation of a middle layer and a corona, and returning to the size of an interphase centrosome. Finally, the two maturing centrosomes transition out of the NE at the end of mitosis and reform the fibrous link that connects them to the NE (17, 52). It has recently been shown that the D. discoideum Sun1 protein is a key component of the fibrous link that bridges and anchors the centrosome to the cell nucleus (58). DdSun1 predominantly localizes to the nuclear membrane and links chromatin to other components of the fibrous link. Truncation or knockdown of DdSun1 promotes separation of the inner and outer NE membranes, inducing aberrant nuclear morphology and loss of the nucleus-centrosome connection (observed as protrusions of the outer NE membrane). Additionally, cells develop supernumerary centrosomes and aberrant spindles, leading to poor chromosome segregation. All this suggests that the centrosome-nucleus link is of extreme importance in maintaining the genetic stability of the cell.D. discoideum has two known centrin proteins, DdCenA (originally named DdCrp) and DdCenB. The initial characterization of DdCenA describes a very divergent centrin that localizes to the centrosomal corona and to the nucleus (5). The second centrin protein, known as DdCenB, was originally identified as a putative member of the centrin family based on sequence similarity by the Dictyostelium Genome Consortium and remained uncharacterized until now. In this work, we report the initial characterization of DdCenB, including molecular cloning, sequence analysis, cellular localization, and analysis of functional roles.     

Structural Changes of the Oxygen-evolving Complex in Photosystem II during the Catalytic Cycle

The oxygen-evolving complex (OEC) in the membrane-bound protein complex photosystem II (PSII) catalyzes the water oxidation reaction that takes place in oxygenic photosynthetic organisms. We investigated the structural changes of the Mn₄CaO₅ cluster in the OEC during the S state transitions using x-ray absorption spectroscopy (XAS). Overall structural changes of the Mn₄CaO₅ cluster, based on the manganese ligand and Mn-Mn distances obtained from this study, were incorporated into the geometry of the Mn₄CaO₅ cluster in the OEC obtained from a polarized XAS model and the 1.9-Å high resolution crystal structure. Additionally, we compared the S₁ state XAS of the dimeric and monomeric form of PSII from Thermosynechococcus elongatus and spinach PSII. Although the basic structures of the OEC are the same for T. elongatus PSII and spinach PSII, minor electronic structural differences that affect the manganese K-edge XAS between T. elongatus PSII and spinach PSII are found and may originate from differences in the second sphere ligand atom geometry.     

Dynamic Interplay of Spectrosome and Centrosome Organelles in Asymmetric Stem Cell Divisions

Stem cells have remarkable self-renewal ability and differentiation potency, which are critical for tissue repair and tissue homeostasis. Recently it has been found, in many systems (e.g. gut, neurons, and hematopoietic stem cells), that the self-renewal and differentiation balance is maintained when the stem cells divide asymmetrically. Drosophila male germline stem cells (GSCs), one of the best characterized model systems with well-defined stem cell niches, were reported to divide asymmetrically, where centrosome plays an important role. Utilizing time-lapse live cell imaging, customized tracking, and image processing programs, we found that most acentrosomal GSCs have the spectrosomes reposition from the basal end (wild type) to the apical end close to hub-GSC interface (acentrosomal GSCs). In addition, these apically positioned spectrosomes were mostly stationary while the basally positioned spectrosomes were mobile. For acentrosomal GSCs, their mitotic spindles were still highly oriented and divided asymmetrically with longer mitosis duration, resulting in asymmetric divisions. Moreover, when the spectrosome was knocked out, the centrosomes velocity decreased and centrosomes located closer to hub-GSC interface. We propose that in male GSCs, the spectrosome recruited to the apical end plays a complimentary role in ensuring proper spindle orientation when centrosome function is compromised.     

Single Centrosome Manipulation Reveals Its Electric Charge and Associated Dynamic Structure

The centrosome is the major microtubule-organizing center in animal cells and consists of a pair of centrioles surrounded by a pericentriolar material. We demonstrate laser manipulation of individual early Drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively charged organelle with a very low isoelectric region (3.1 ± 0.1). From this single-organelle electrophoresis, we infer an effective charge smaller than or on the order of 10³ electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. We show, however, that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find, on the one hand, that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter, and on the other hand, that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect that modulates its structural behavior via environmental pH. This methodology further proves useful for studying the action of different environmental conditions, such as the presence of Ca²⁺, over the thermally induced dynamic structure of the centrosome.     

Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae

The natural hosts of the bacterial pathogen Legionella pneumophila are amoebae and protozoa. In these hosts, as in human macrophages, the pathogen enters the cell through phagocytosis, then rapidly modifies the phagosome to create a compartment that supports its replication. We have examined L. pneumophila entry and behaviour during early stages of the infection of Dictyostelium discoideum amoebae. Bacteria were labelled with a red fluorescent marker, and selected proteins and organelles in the host were labelled with GFP, allowing the dynamics and interactions of L. pneumophila -containing phagosomes to be tracked in living cells. These studies demonstrated that entry of L. pneumophila is an actin-mediated process, that the actin-binding protein coronin surrounds the nascent phagosome but dissociates immediately after internalization, that ER membrane is not incorporated into a phagosome during uptake, that the newly internalized phagosome is rapidly transported about the cell on microtubules, that association of ER markers with the phagosome occurs in two steps that correlate with distinct changes in phagosome movement, and that the vacuolar H(+)-ATPase does not associate with mature replication vacuoles. These studies have clarified certain aspects of the infection process and provided new insights into the dynamic interactions between the pathogen and its host.     

Structural Changes in the Catalytic Cycle of the Na,K-ATPase Studied by Infrared Spectroscopy

Pig kidney Na⁺,K⁺-ATPase was studied by means of reaction-induced infrared difference spectroscopy. The reaction from E1Na₃⁺ to an E2P state was initiated by photolysis of P³-1-(2-nitrophenyl)ethyl ATP (NPE caged ATP) in samples that contained 3 mM free Mg²⁺ and 130 mM NaCl at pH 7.5. Release of ATP from caged ATP produced highly detailed infrared difference spectra indicating structural changes of the Na⁺,K⁺-ATPase. The observed transient state of the enzyme accumulated within seconds after ATP release and decayed on a timescale of minutes at 15°C. Several controls ensured that the observed difference signals were due to structural changes of the Na⁺,K⁺-ATPase. Samples that additionally contained 20 mM KCl showed similar spectra but less intense difference bands. The absorbance changes observed in the amide I region, reflecting conformational changes of the protein backbone, corresponded to only 0.3% of the maximum absorbance. Thus the net change of secondary structure was concluded to be very small, which is in line with movement of rigid protein segments during the catalytic cycle. Despite their small amplitude, the amide I signals unambiguously reveal the involvement of several secondary structure elements in the conformational change. Similarities and dissimilarities to corresponding spectra of the Ca²⁺-ATPase and H⁺,K⁺-ATPase are discussed, and suggest characteristic bands for the E1 and E2 conformations at 1641 and 1661 cm⁻¹, respectively, for αβ heterodimeric ATPases. The spectra further indicate the participation of protonated carboxyl groups or lipid carbonyl groups in the reaction from E1Na₃⁺ to an E2P state. A negative band at 1730 cm⁻¹ is in line with the presence of a protonated Asp or Glu residue that coordinates Na⁺ in E1Na₃⁺. Infrared signals were also detected in the absorption regions of ionized carboxyl groups.     

Structural Changes in the Vascular Cambium of Pinus strobus L. during an Annual Cycle

The changes in the ultrastructure of cambial cells of easternwhite pine (Pinus strobus L.) during an annual cycle are observedand recorded as are relationships of cambial cells during dormancyand at resumption of cambial activity. Cambial activity wasresumed late in March or early in April, when a few cells dividedpericlinally. Cambial activity reached a maximum during thelatter part of May with 15 to 20 undifferentiated cells present.In July it declined markedly, and the number of undifferentiatedcells equalled that of the dormant period. The xylem and phloemtissue cells produced late in the annual cycle overwinteredat varying developmental stages. In October cambial cells structurallyresembled dormant cells. The number of dormant cells in easternwhite pine cambium varied from 6 to 10. Active cells were characterizedby a large central vacuole, by an abundance of all cell organelles,and by thin cell walls. Dormant cells were characterized bynumerous small vacuoles, by structurally and quantitativelymodified cell organelles, and by relatively thick cell walls.     

Correlation between plaque size and spore size in Dictyostelium discoideum

Haploid spores usually lead to the production of small plaques when plated under standard conditions; diploid spores usually lead to the formation of large plaques.     

The migration stage of Dictyostelium: Behavior without muscles or nerves

Abstract Glycoproteins are providing to be quite common in prokaryotes. Those is S-layers are the best understood in terms of structure. Numerous eubacteria produce non-S-layer glycoproteins about which relatively little is known. The glycans on such protein and the nature and sites of their linkages to protein are novel in those glycoproteins which have been examined in any detail. The possible functions of the glycans are mostly not understood. Eubacterial non-S-layer glycoproteins and the glycosylation systems producing them observe more attention.     

Structural requirements of dictyopyrones isolated from Dictyostelium spp. in the regulation of Dictyostelium development and in anti-leukemic activity

Cellular slime molds are fascinating to the field of developmental biology, and have long been used as excellent model organisms for the study of various aspects of multicellular development. We have recently isolated alpha-pyronoids, named dictyopyrones A-D (1-4), from various species of Dictyostelium cellular slime molds, and it was shown that compound 3 may regulate Dictyostelium development. In this study, we synthesized dictyopyrones A-D (1-4) and their analogues, investigated the physiological role of the molecules in cell growth and morphogenesis in D. discoideum, and further verified their effects on human leukemia K562 cells. Nitrogen-containing compounds 22 and 37 strongly inhibited cell growth in K562 leukemia cells, indicating that these compounds may be utilized as novel lead compounds for anti-leukemic agents.     

Centrosome duplication: of rules and licenses

Most microtubule arrays in animal cells, including the bipolar spindle required for cell division, are organized by centrosomes. Thus, strict control of centrosome numbers is crucial for accurate chromosome segregation. Each centrosome comprises two centrioles, which need to be duplicated exactly once in every cell cycle. Recent work has begun to illuminate the mechanisms that regulate centriole duplication. First, genetic and structural studies concur to delineate a centriole assembly pathway in Caenorhabditis elegans. Second, the protease Separase, previously known to trigger sister chromatid separation, has been implicated in a licensing mechanism that restricts centrosome duplication to a single occurrence per cell cycle. Finally, Plk4 (also called Sak), a member of the Polo kinase family, has been identified as a novel positive regulator of centriole formation.     

Circadian Changes in Anxiolysis-Related Behavior of Syrian Hamsters. Correlation with Hypothalamic GABA Release

The diurnal variations in anxiolysis and exploratory behavior were examined in a plus-maze paradigm in Syrian hamsters exposed to 14 h light: 10 h dark photoperiods or to constant darkness for 3 days. The percent of time spent in open arms and the percent of entries to open arms (both indexes of anxiety-related behavior) as well as the number of crosses to both arms (an index of locomotor behavior) showed significant daily variations under the two lighting conditions, maxima being found at night (2400-0400 h). Flumazenil (5 mg/kg) injected at the middle of the light (at 1600 h) or dark period (at 0400 h) decreased by 39-54 % anxiolysis-related behavior without affecting locomotor activity significantly. 3 H-?-aminobutyric acid (GABA) release from preoptic-medial basal hypothalamic explants obtained from hamsters exposed to 14 h light: 10 h dark photoperiods attained maximal values at 2400-0400 h. The results further supported the existence of circadian changes in anxiolysis-related behavior in Syrian hamsters that correlated with an increased hypothalamic GABA release.