Self-organization of a radial microtubule array by dynein-dependent nucleation of microtubules

Polarized radial arrays of cytoplasmic microtubules (MTs) with minus ends clustered at the cell center define the organization of the cytoplasm through interaction with microtubule motors bound to membrane organelles or chromosomes. It is generally assumed that the radial organization results from nucleation of MTs at the centrosome. However, radial MT array can also be attained through self-organization that requires the activity of a minus-end-directed MT motor, cytoplasmic dynein. In this study we examine the role of cytoplasmic dynein in the self-organization of a radial MT array in cytoplasmic fragments of fish melanophores lacking the centrosome. After activation of dynein motors bound to membrane-bound organelles, pigment granules, the fragments rapidly form polarized radial arrays of MTs and position pigment aggregates at their centers. We show that rearrangement of MTs in the cytoplasm is achieved through dynein-dependent MT nucleation. The radial pattern is generated by continuous disassembly and reassembly of MTs and concurrent minus-end-directed transport of pigment granules bearing the nucleation sites.     

Immunogold staining of microtubules in resting and activated platelets

A circumferential microtubule is known to support the discoid form of resting platelets, but its fate following exposure of the cells to aggregating agents is uncertain. The present study has employed an immunocytochemical approach to follow the fate of the circumferential microtubule in activated platelets. Monoclonal antibodies to tubulin and to vinculin and a polyclonal antibody to actin were incubated with isolated microtubule coils and stained with staphylococcal protein A coupled to immunogold in order to test their specificity. Thin sections of glycolmethacrylate embedded platelets before and after exposure to thrombin for 15, 30 and 60 s were stained with antibodies to tubulin and actin. Immunogold particles showed a high specificity for isolated MT coils stained for tubulin, modest intensity for actin, and none for vinculin. Gold particles were randomly distributed in thin sections of resting and activated platelets stained for actin. Immunogold was limited to the circumferential microtubule in resting platelets and constricted coils in thrombin-activated cells. The number of gold particles in areas of cytoplasm away from microtubules in platelets stained with antitubulin antibody increased slightly following thrombin activation, but the change was not significant. Results support the concept that microtubule coils supporting the discoid form of resting platelets do not dissolve following exposure of the cells to potent agonists.     

Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro.

High-molecular-weight components (HMW) specifically associated with microtubule protein purified from porcine brain tissue were separated from tubulin by DEAE-Sephadex ion exchange chromatography. Analysis by viscometry, sedimentation, and electron microscopy of the unfractionated microtubule protein, separated HMW and tubulin fractions, and reconstituted mixtures showed that HMW promoted formation of ring structures at 5 degrees and tubule polymerization at 37 degrees. The HMW reassociated with tubulin and was identified in thin sections as 18.9 x 5.6 nm projections attached to the microtubules with a longitudinal periodicity of 32.5 nm. These studies: (1) indicate that the HMW fraction stimulates microtubule assembly by facilitating the formation of ring structures which are apparently intermediates in polymerization, and (2) demonstrate that the HMW associates with microtubules as a structural component projecting from the surface of the microtubule wall.     

Op18 reveals the contribution of nonkinetochore microtubules to the dynamic organization of the vertebrate meiotic spindle

Accuracy in chromosome segregation depends on the assembly of a bipolar spindle. Unlike mitotic spindles, which have roughly equal amounts of kinetochore microtubules (kMTs) and nonkinetochore microtubules (non-kMTs), vertebrate meiotic spindles are predominantly comprised of non-kMTs, a large subset of which forms an antiparallel “barrel” array at the spindle equator. Though kMTs are needed to drive chromosome segregation, the contributions of non-kMTs are more mysterious. Here, we show that increasing the concentration of Op18/stathmin, a component of the chromosome-mediated microtubule formation pathway that directly controls microtubule dynamics, can be used to deplete non-kMTs in the vertebrate meiotic spindle assembled in Xenopus egg extracts. Under these conditions, kMTs and the spindle pole-associated non-kMT arrays persist in smaller spindles. In excess Op18, distances between sister kinetochores, an indicator of tension across centromeres, remain unchanged, even though kMTs flux poleward with a ≈30% slower velocity, and chromosomes oscillate more than in control metaphase spindles. Remarkably, kinesin-5, a conserved motor protein that can push microtubules apart and is required for the assembly and maintenance of bipolar meiotic spindles, is not needed to maintain spindle bipolarity in the presence of excess Op18. Our data suggest that non-kMTs in meiotic spindles contribute to normal kMT dynamics, stable chromosome positioning, and the establishment of proper spindle size. We propose that without non-kMTs, metaphase meiotic spindles are similar to mammalian mitotic spindles, which balance forces to maintain metaphase spindle organization in the absence of extensive antiparallel microtubule overlap at the spindle equator or a key mitotic kinesin.     

Visualization of biochemical networks in living cells

Functional annotation of novel genes can be achieved by detection of interactions of their encoded proteins with known proteins followed by assays to validate that the gene participates in a specific cellular function. We report an experimental strategy that allows for detection of protein interactions and functional assays with a single reporter system. Interactions among biochemical network component proteins are detected and probed with stimulators and inhibitors of the network. In addition, the cellular location of the interacting proteins is determined. We used this strategy to map a signal transduction network that controls initiation of translation in eukaryotes. We analyzed 35 different pairs of full-length proteins and identified 14 interactions, of which five have not been observed previously, suggesting that the organization of the pathway is more ramified and integrated than previously shown. Our results demonstrate the feasibility of using this strategy in efforts of genomewide functional annotation.     

Effects of a microtubule stabilizing agent on the response of platelets to vincristine

The discoid shape of blood platelets is supported by a circumferential bundle of microtubules. Removal of the microtubules by an antimitotic drug, vincristine, is associated with loss of lentiform appearance, formation of tubulin paracrystals, a depressed response to aggregating agents, and impaired secretory activity. Recent studies have suggested that the action of vincristine on platelet secretion and aggregation is directly related to its action on microtubules, while other work had indicated that the antimitotic drug prevents the release reaction by inhibiting prostaglandin synthesis. The present study has examined the influence of taxol, a microtubule stabilizing agent, on the response of platelets to vincristine. Taxol completely prevented vincristine- induced shape change, microtubule disassembly, and tubulin paracrystal formation, even at concentrations one-tenth that of the antimitotic drug. Pretreatment with vincristine to dissociate microtubules and convert tubulin to crystals before exposure to taxol did not affect altered shape or tubulin paracrystals, but did cause assembly of free pools of tubulin into tubular polymers. Studies of physiology confirmed that vincristine, in amounts that remove microtubules, depresses platelet aggregation and secretion, effects that could be overcome by increasing agonist concentration. Although completely preventing microtubule dissociation, taxol had no corrective influence on vincristine-induced inhibition of platelet function. Biochemical studies revealed that vincristine concentrations that disassembled microtubules and blocked secretion did not inhibit conversion of 14C- arachidonic acid to thromboxane B2. The findings suggest that vincristine inhibits platelet function through some mechanism other than disassembling microtubules, but the other mechanism does not involve inhibition of prostaglandin synthesis.     

Visualization of microtubules of cells in situ by indirect immunofluorescence.

Microtubule staining patterns can be visualized within cells in situ on the surface of fish scales from the squirrel fish, Holocentrus ascensionis, and the common goldfish, Carassius auratus, after incubation with antibodies to sea urchin tubulin and fluorescein-labeled goat antibodies to rabbit immunoglobulin G. Chromatophores in situ from both species reveal a radial microtubule framework that orients the alignment of pigment granules. Innervating fibers of erythrophores on the H. ascensionis scale can also be observed. In situ, pseudo-epithelial cells called scleroblasts show microtubule patterns with a remarkable degree of similarity within a selected region. Over 90% of the cells have a microtubule framework that is nearly superimposable from cell to adjacent cell. The microtubules in scleroblasts are few and form a simple radial framework with a localized microtubule organizing center (MTOC). Microtubules in scleroblasts in vitro emanate from localized MTOCs but are much less radially organized than in situ. Scleroblasts in situ on the scale of C. auratus show microtubules that curve abruptly into coalignment with phase striations on the fibrillary plate. The phase striations arise from the orthogonal plies of collagen in intimate association with the scleroblasts. The role of microtubules in scleroblasts may thus be to provide orientation for collagen fibrillogenesis, analogous to their role in orientation of cellulose fibers in plants. That cells in situ exhibit highly related and coordinated microtubule staining patterns reaffirms that the cytoskeleton plays an important role in the organization of differentiated tissues.     

Cryo-electron tomography reveals conserved features of doublet microtubules in flagella

The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin. The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the "beak" structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction.     

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Thrombocytosis and platelet hyperreactivity are known to be associated with malignancy; however, there have been no ultrastructure studies of platelets from patients with ovarian cancer. Here, we used electron cryotomography (cryo-ET) to examine frozen-hydrated platelets from patients with invasive ovarian cancer (n = 12) and control subjects either with benign adnexal mass (n = 5) or free from disease (n = 6). Qualitative inspections of the tomograms indicate significant morphological differences between the cancer and control platelets, including disruption of the microtubule marginal band. Quantitative analysis of subcellular features in 120 platelet electron tomograms from these two groups showed statistically significant differences in mitochondria, as well as microtubules. These structural variations in the platelets from the patients with cancer may be correlated with the altered platelet functions associated with malignancy. Cryo-ET of platelets shows potential as a noninvasive biomarker technology for ovarian cancer and other platelet-related diseases.Platelets are small anucleate multifunctional cells derived from megakaryocytes. Platelets circulate in the bloodstream and respond to vascular lesions (1). In their resting state, they adopt a discoidal shape and have an average lifespan of 5–7 d in humans (2). Platelets have increasingly been recognized as playing an important role in tumor growth and metastasis, in addition to their traditional roles in hemostasis (3–6). Thrombocytosis (platelet count >450,000/μL) is found in 31% of patients with ovarian cancer and is associated with a poor clinical prognosis (7, 8). In addition, platelets in patients with cancer are functionally altered, and often adopt a hyperreactive state (9). This platelet hyperreactivity helps explain the higher thrombosis risk in these patients (10). Furthermore, compelling preclinical and clinical data demonstrate that platelets actively promote tumor growth and metastasis through multiple pathways. Platelets form a physical shield to protect tumor cells from natural killer cell-mediated lysis; they facilitate the adhesion of tumor cells to the endothelium, allowing the critical extravasation step in the metastatic cascade to occur; and they release a plethora of bioactive molecules, including growth factors and cytokines stored in their secretory granules, that promote angiogenesis and tumor cell growth (11). It is shown in various experimental models that disruption of platelet–tumor interactions abolishes these effects (5, 9), improving clinical outcomes of patients.The relatively high incidence of thrombocytosis in these patient populations (12), which is related to cytokine signaling from both tumor and nontumor tissue (8), and the implication of platelets in cancer pathology (6, 8) suggested the possibility of ultrastructural perturbations in the platelets of patients with ovarian cancer. These perturbations could provide structural clues to improve our understanding of cancer-related hyperreactivity, thrombocytosis, or as-yet-unknown platelet defects, and could serve as potential biomarkers for screening for ovarian cancer. Prior attempts at examining platelet ultrastructure in diseases have been largely limited by methods that used plastic embedding and chemical fixation (13–15). The present investigation overcomes these technical limitations by using electron cryotomography (cryo-ET) to visualize human platelets in their naturally occurring pathophysiological states without chemical fixation or staining (16).     

Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling

B cell responses are initiated by the clustering of the B cell receptor (BCR) by the binding of multivalent antigens. Clustering leads to phosphorylation of tyrosines in the cytoplasmic domains of the BCR by the inner plasma membrane leaflet-associated Src-family kinase Lyn. At present, little is known about the earliest events after BCR clustering that precede the BCR's phosphorylation by Lyn. Here we use fluorescence resonance energy transfer (FRET) in living cells to detect the interaction of the BCR with a Lyn-based membrane-targeted reporter in the first several seconds after BCR clustering. The results showed that, within seconds of antigen binding, the BCR selectively and transiently associated with the Lyn construct and that this association preceded by several seconds the triggering of Ca2+ fluxes and could be prolonged by the engagement of the B cell coreceptor complex, CD19/CD21. Thus, FRET measurements in living B cells revealed highly dynamic and regulated antigen-induced changes in the plasma membrane, allowing association of the BCR with the earliest components of its signaling cascade.     

Formin proteins in megakaryocytes and platelets: regulation of actin and microtubule dynamics

The platelet and megakaryocyte cytoskeletons are essential for formation and function of these cells. A dynamic, properly organised tubulin and actin cytoskeleton is critical for the development of the megakaryocyte and the extension of proplatelets. Tubulin in particular plays a pivotal role in the extension of these proplatelets and the release of platelets from them. Tubulin is further required for the maintenance of platelet size, and actin is the driving force for shape change, spreading and platelet contraction during platelet activation. Whilst several key proteins which regulate these cytoskeletons have been described in detail, the formin family of proteins has received less attention. Formins are intriguing as, although they were initially believed to simply be a nucleator of actin polymerisation, increasing evidence shows they are important regulators of the crosstalk between the actin and microtubule cytoskeletons. In this review, we will introduce the formin proteins and consider the recent evidence that they play an important role in platelets and megakaryocytes in mediating both the actin and tubulin cytoskeletons.     

Feasibility of using marginal liver grafts in living donor liver transplantation

Liver transplantation(LT) is one of the most effective treatments for end-stage liver disease caused by related risk factors when liver resection is contraindicated. Additionally,despite the decrease in the prevalence of hepatitis B virus(HBV) over the past two decades,the absolute number of HBs Ag-positive people has increased,leading to an increase in HBV-related liver cirrhosis and hepatocellular carcinoma. Consequently,a large demand exists for LT. While the wait time for patients on the donor list is,to some degree,shorter due to the development of living donor liver transplantation(LDLT),there is still a shortage of liver grafts. Furthermore,recipients often suffer from emergent conditions,such as liver dysfunction or even hepatic encephalopathy,which can lead to a limited choice in grafts. To expand the pool of available liver grafts,one option is the use of organs that were previously considered "unusable" by many,which are often labeled "marginal" organs. Many previous studies have reported on the possibilities of using marginal grafts in orthotopic LT; however,there is still a lack of discussion on this topic,especially regarding the feasibility of using marginal grafts in LDLT. Therefore,the present review aimed to summarize the feasibility of using marginal liver grafts for LDLT and discuss the possibility of expanding the application of these grafts.     

Visualization of microtubules in interphase and mitotic plant cells of Haemanthus endosperm with the immuno-gold staining method.

A procedure is presented for the immunocytochemical visualization of microtubules in interphase and mitotic cells of Haemanthus endosperm. It includes preservation of microtubules (MTs) with glutaraldehyde and uses colloidal gold, coated with secondary antibodies, in a novel indirect-light microscopic technique: the immuno-gold staining method. This immunocytochemical stain allows us to follow the changes in distribution of MTs during mitosis with greater precision and specificity than allowed by other light microscopic techniques. Many aspects of MT arrangements, as reported from ultrastructural studies, are corroborated and extended. This demonstrates the reliability of the technique. In addition, a number of significant observations were made. These concern (i) the presence of a network of MTs in interphase cells, (ii) the transformation of this network into a spindle-like cage of MTs (the clear zone) surrounding the nucleus during prophase, (iii) the drastic rearrangement of MT distribution during prometaphase, (iv) new evidence for the formation of aster-like arrays of polar MTs during anaphase, and (v) the development of the phragmoplast.     

Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera.

A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the rat glucocorticoid receptor (GR). When GFP-GR is expressed in living mouse cells, it is competent for normal transactivation of the GR-responsive mouse mammary tumor virus promoter. The unliganded GFP-GR resides in the cytoplasm and translocates to the nucleus in a hormone-dependent manner with ligand specificity similar to that of the native GR receptor. Due to the resistance of the mutant GFP to photobleaching, the translocation process can be studied by time-lapse video microscopy. Confocal laser scanning microscopy showed nuclear accumulation in a discrete series of foci, excluding nucleoli. Complete receptor translocation is induced with RU486 (a ligand with little agonist activity), although concentration into nuclear foci is not observed. This reproducible pattern of transactivation-competent GR reveals a previously undescribed intranuclear architecture of GR target sites.     

Organization and energy-dependent growth of microtubules in cells.

The organization and growth of microtubules in cultured mouse macrophages and fibroblasts were examined by indirect immunofluorescence microscopy with antibodies to microtubule protein. In macrophages, microtubules converged at a samll region at the cytocenter. During depolymerization, and repolymerization, this region acted as a microtubule organizing center. Microtubule growth was energy-dependent, but unaffected by dibutyryl-adenosine 3':5'-cyclic monophosphate, cholera toxin, or dibutyryl-guanosine 3':5'-cyclic monophosphate. Fibroblasts, which did not show such a simple microtubule organization as macrophages, contained mainly one or two, but occasionally as many as four, organizing centers during repolymerization. These microtubule organizing centers often appeared as fluorescent rings with a dark center.     

活体肝移植中非标准供肝的可用性分析

目的 了解活体肝移植非标准供肝受体的并发症及生存率,分析活体肝移植中非标准供肝的可用性.方法 四川大学华西医院肝移植中心2001年1月-2007年12月完成成人间活体肝移植并资料齐全的患者92例.比较不同供体年龄、性别配型、供肝脂肪变程度、移植物质量与受体体质量比(GRWR)、肝中静脉重建情况,患者术后6个月的生存率及并发症发生率.根据不同资料分别采用单因素方差分析、X2检验或Cpx回归分析.结果 使用脂肪变性≥30%供肝的4例受体术后都出现了不明原因的肝功能衰竭,均在观察时间内死亡,而脂肪变性＜30%的受体中,术后6个月生存率为91.7%.虽然GRWR＜0.8%的受体生存率(86.9%)与GRWR≥0.8%的受体生存率(87.8%)比较,差异无统计学意义(X2=0.022,P＞0.05),但小供肝组流出道的重建情况与其生存率相关(X2=10.612,P＜0.01).供体年龄是否≥50岁和供、受体性别是否配型等对术后6个月并发症及生存率的影响差异均无统计学意义(P＞0.05).Cox回归分析结果显示,供肝脂肪变性≥30%对生存率有较大的影响.结论 供肝脂肪变性程度是影响受体术后并发症及生存率的重要因素.而以往认为的GRWR＜0.8%现已不是绝对的禁忌标准,但必须保证小供肝受体流出道通畅.供体年龄≥50岁、交叉性别供肝对受体术后并发症及生存率无影响.     

Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors

Many cellular cargoes move bidirectionally along microtubules, driven by teams of plus- and minus-end–directed motor proteins. To probe the forces exerted on cargoes during intracellular transport, we examined latex beads phagocytosed into living mammalian macrophages. These latex bead compartments (LBCs) are encased in membrane and transported along the cytoskeleton by a complement of endogenous kinesin-1, kinesin-2, and dynein motors. The size and refractive index of LBCs makes them well-suited for manipulation with an optical trap. We developed methods that provide in situ calibration of the optical trap in the complex cellular environment, taking into account any variations among cargoes and local viscoelastic properties of the cytoplasm. We found that centrally and peripherally directed forces exerted on LBCs are of similar magnitude, with maximum forces of ∼20 pN. During force events greater than 10 pN, we often observe 8-nm steps in both directions, indicating that the stepping of multiple motors is correlated. These observations suggest bidirectional transport of LBCs is driven by opposing teams of stably bound motors that operate near force balance.     

Visualization of AP-1 NF-kappaB ternary complexes in living cells by using a BiFC-based FRET

Protein-protein interactions are essential for maintaining cell structure and for executing almost all cellular processes. Determination of where and how each protein interacts with its partners provides significant insight into proteins' cellular roles. Although several assays, such as FRET and bimolecular fluorescence complementation (BiFC), have been developed and widely used for visualization and identification of protein interactions in living cells, there is no simple and convenient assay to visualize and identify multiple protein complexes in living cells. Because many signaling molecules often function as ternary complexes, availability of an assay for visualization and identification of ternary complexes will significantly expand the repertoire of protein interaction studies in living cells. By using the Fos-Jun-nuclear factor of activated T cells (NFAT) ternary complex as a model and the fluorescent proteins Cerulean and Venus, two mutant proteins of CFP and YFP with better folding and less environment sensitivity, as a donor and acceptor, respectively, we have combined a Venus-based BiFC system with Cerulean to develop a BiFC-based FRET (BiFC-FRET) assay for visualization of ternary complexes in living cells with a conventional three-filter FRET setup. We also have applied the BiFC-FRET to identify a ternary complex formed between Fos-Jun heterodimers and the NF-kappaB subunit, p65. This finding reveals a cross-talk between AP-1 and NF-kappaB. Thus, the BiFC-FRET represents a convenient assay for identification and visualization of ternary complexes in living cells.     

Taxol-induced anaphase reversal: evidence that elongating microtubules can exert a pushing force in living cells.

The effects of taxol on mitosis in Haemanthus endosperm were studied. Immuno-gold staining was used to visualize microtubules; observations on microtubule arrangements were correlated with studies in vivo. Mitosis is slowed down, but not arrested, by taxol over a wide range of concentrations. Taxol promotes the formation of abundant new microtubules and lateral association within and between microtubule arrays (spindle fibers). This leads to a pronounced reorganization of the spindle, especially at the polar regions. Chromosome arms may be pushed toward the equator in metaphase. Anaphase chromosomes, with their kinetochores still pointing to the poles, move backward before resuming their poleward migration. During anaphase, the interzone is depleted of microtubules and trailing chromosome arms are stretched and often torn apart by rapidly elongating polar microtubules. Fragments are transported away from the poles, apparently "riding" on the tips of microtubules. This provides evidence of "pushing" by elongating microtubules. The desynchronization of anaphase, often observed as one of the first effects of taxol, indicates that the anchorage of different kinetochore fibers varies. The data draw attention to modifications of spindle structure due to increased microtubule lateral associations and to the role of this process in spindle integrity and chromosome movement.