Effects of various fixatives on the reactivity of plant cell tubulin and calmodulin in immunofluorescence microscopy

Summary We have examined the suitability of a variety of fixation regimes for immunofluorescence localization of tubulin and calmodulin in meristematic plant cells. Acrolein and most fixatives that contain glutaraldehyde (GA), while they have been employed by others in immunoenzyme, immunogold or immunofluorescence studies of plant endosperm, animal or plant tissue culture cells, cause unacceptably high background fluorescence of the dense cytoplasm of root meristem cells. Bifunctional imidoester and carbodiimide reagents do not give satisfactory results, either. Fixatives that have produced good results include paraformaldehyde (PFA) with the addition of picric acid or zinc salts or at high pH, a combination of PFA/GA/picric acid, and prefixation in PFA plus a monofunctional imidoester followed by PFA/GA. All of these cross-link the cytoplasm well enough so that cells can withstand isolation procedures, preserve antigenicity, allow antibody penetration and provide good contrast between specific and background fluorescence. The last two fixatives are of particular interest because of the potential they offer for immunoelectron microscopy of densely cytoplasmic, walled cells from tissues.     

Microinjection of fluorescent tubulin into dividing sea urchin cells

To follow the dynamics of microtubule (MT) assembly and disassembly during mitosis in living cells, tubulin has been covalently modified with the fluorochrome 5-(4,6-dichlorotriazin-2-yl)aminofluorescein and microinjected into fertilized eggs of the sea urchin Lytechinus variegatus. The changing distribution of the fluorescent protein probe is visualized in a fluorescence microscope coupled to an image intensification video system. Cells that have been injected with fluorescent tubulin show fluorescent linear polymers that assemble very rapidly and radiate from the spindle poles, coincident with the position of the astral fibers. No fluorescent polymer is apparent in other areas of the cytoplasm. When fluorescent tubulin is injected near the completion of anaphase, little incorporation of fluorescent tubulin into polymer is apparent, suggesting that new polymerization does not occur past a critical point in anaphase. These results demonstrate that MT polymerization is very rapid in vivo and that the assembly is both temporally and spatially regulated within the injected cells. Furthermore, the microinjected tubulin is stable within the sea urchin cytoplasm for at least 1 h since it can be reutilized in successive daughter cell spindles. Control experiments indicate that the observed fluorescence is dependent on MT assembly. The fluorescence is greatly diminished upon treatment of the cells with cold or colchicine agents known to cause the depolymerization of assembled MT. In addition, cells injected with fluorescent bovine serum albumin or assembly-incompetent fluorescent tubulin do not exhibit fluorescence localized in the spindle but rather appear diffusely fluorescent throughout the cytoplasm.     

Analysis of human megakaryocytic cells using dual-color immunofluorescence labeling

BACKGROUND: Megakaryocytes are classically identified by their cellular morphology and expression of platelet glycoproteins. METHODS: In this study, the expression of GPIIIa (CD61) on hemopoietic cells was analyzed by dual-fluorescence flow cytometry. RESULTS: All monocytic cells (CD14+) were shown to coexpress CD61. As the expression of platelet protein on these monocytic cells cannot be reduced by treating the cells with anticoagulant (ethlyenediaminetetraacetic acid [EDTA]), this observation is not simply due to platelet adhesion. When sorted CD61(lo)CD14+ cells were studied by light and electron microscopy, platelets or platelet fragments could not be detected on the cell surface. These cells were found to have typical monocytic morphology but no megakaryocytic characteristics. CONCLUSIONS: This finding demonstrates that without careful definition, the quantitation of megakaryocytic cells will be inappropriately high. A clear and unambiguous criteria for the identification of megakaryocytic cells is described based on the high expression of platelet glycoprotein (e.g., CD61(hi) or CD41(hi)) but not the monocyte marker (CD14(neg)).     

Gamma-tubulin colocalizes with microtubule arrays and tubulin paracrystals in dividing vegetative cells of higher plants

Summary The distribution of -tubulin throughout cell division is studied in several taxa of higher plants. -Tubulin is present along the whole length of microtubules (Mts) in every cell stage-specific Mt array such as the preprophase band, the preprophase-prophase perinuclear Mts, the kinetochore Mt bundles, the phragmoplast, and the telophase-interphase transition Mt arrays. -Tubulin follows with precision the Mt pattern, being absent from any other, Mt-free, cell site. In cells treated with anti-Mt drugs, -tubulin is present only on degrading or on reappearing Mt arrays, while it is totally absent from cells devoid of Mts. -Tubulin is also present in tubulin paracrystals, which are formed in colchicine-treated cells. These observations support the view that in higher plants -tubulin may not be a microtubule-organizing-center-specific protein, but it may play a certain structural and/or functional role being related to - and -tubulin.Abbreviations Mt microtubule - MTOC microtubule-organizing center - PPB preprophase band     

钙调素及钙调素相关蛋白在植物细胞中的研究进展

植物对一系列生物和非生物刺激所产生的反应都与细胞内Ca2+信号转导有关,而钙调素、钙调素相关蛋白则是Ca2+信号转导的下游靶蛋白。该文介绍了钙调素的结构及其在植物细胞中的分布,钙调素及钙调素相关蛋白在植物细胞中的表达等方面的最近研究进展。     

Demarcation of the cortical division zone in dividing plant cells

Somatic cytokinesis in higher plants involves, besides the actual construction of a new cell wall, also the determination of a division zone. Several proteins have been shown to play a part in the mechanism that somatic plant cells use to control the positioning of the new cell wall. Plant cells determine the division zone at an early stage of cell division and use a transient microtubular structure, the preprophase band (PPB), during this process. The PPB is formed at the division zone, leaving behind a mark that during cytokinesis is utilized by the phragmoplast to guide the expanding cell plate toward the correct cortical insertion site. This review discusses old and new observations with regard to mechanisms implicated in the orientation of cell division and determination of a cortical division zone.     

Localization of tubulin in the mitotic apparatus of mammalian cells by immunofluorescence and immunoelectron microscopy

Antitubulin antibody was used as an immunofluorescent and immunoelectron microscopic probe to localize tubulin in components of the mitotic apparatus of rat kangaroo (strain PtK₁) cells in vitro. In addition to the detection of tubulin in the spindle microtubules and centrioles, other structures were found to display specific staining including kinetochores, amorphous pericentriolar material and small virus-like particles associated with the centrioles. The kinetochores consisted of a densely stained outer layer about 400 Å thick which is separated from an inner layer of the same dimension by a lightly staining middle layer. Microtubules were primarily associated with the outermost plate of the kinetochore but tubulin was uniformly distributed in both outer and inner plates. Colcemid treatment prevented the assembly of spindle microtubules and resulted in specific alterations of the kinetochore but failed to diminish the staining of the kinetochores. These observations suggest that tubulin molecules may comprise an important structural component of the kinetochore.     

Exponential pattern of cell age distribution in dividing cells of plant meristems

In order to determine the pattern of cell age distribution in proliferating cells of Allium cepa roots we have measured by cytophotometry two cell size parameters, protein content and surface area projection, in cells that correspond to the entire proliferating population or only to the ana-telophase subpopulation. The size values of ana-telophase cells have been employed to construct theoretical size distributions for the entire proliferating cell population of the root meristem by assuming either a uniform or an exponential cell age distribution. Statistical comparison of theoretical distributions with the experimental one rules out a uniform cell age distribution and strongly favours an exponential age distribution similar to that found in bacteria.     

Probing microtubule organizing centres with MPM-2 in dividing cells of higher plants using immunofluorescence and immunogold techniques

Summary The localization in higher plant cells of phosphorylated proteins recognized by the monoclonal antibody MPM-2 was investigated, with particular attention to putative microtubule organizing centres (MTOCs). Immunofluorescence and immunogold electron microscopy showed that MPM-2 did not localize with most putative MTOCs in cells and protoplasts of the gymnospermPicea glauca and in cells of the angiospermVicia faba. The distribution of phosphoproteins detected by MPM-2 was similar during mitosis in both species. At late interphase and early prophase MPM-2 preferentially labelled nucleoli and the region around the condensing chromosomes but not the cytoplasm. General labelling of the cytoplasm followed dissolution of the nuclear envelope and by prometaphase centromeres stained strongly. At metaphase and very early anaphase kinetochores stained strongly by immunofluorescence but only weakly using immunogold; spindle microtubules (MTs) showed little staining. Kinetochore staining disappeared during anaphase and by telophase centromeres and loose regions of chromatin in reforming nuclei were labelled. Treatment with the anti-microtubular drug amiprophosmethyl (APM) showed that the phosphorylation/dephosphorylation cycle detected by MPM-2 proceeded independently of the mitotic spindle. Staining of centromeres/kinetochores with MPM-2 suggests that phosphorylation and dephosphorylation of this region of mitotic chromosomes may be involved in chromosome organization, chromatid separation and MT nucleation and/or attachment.Abbreviations APM amiprophos-methyl - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol-bis(-aminoethyl ether) - FITC fluorescein isothiocyanate - MT microtubule - MTOC microtubule organizing centre - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline - PBSB phosphate buffered saline with bovine serum albumin - PIPES piperazine-N,N-bis (2-ethanesulfonic acid) - PPB preprophase band - SPB spindle pole body - TRITC tetramethylrhodamine isothiocyanate     

Thermodynamics of calmodulin and tubulin binding to the vinca-alkaloid vinorelbine

Vinca-alkaloids, such as vinblastine, and some of their derivatives, for example vinorelbine, are widely used in clinical therapy of leukemia and several types of tumors. Their effects are associated with the disfunctioning of the mitotic spindle, which leads to mitosis blockage and a shutting down of the cell cycle. Their primary target is tubulin; however, recent research has shown that some of the vinca-alkaloids inhibit calmodulin binding to its targets. Vinca-alkaloids binding with other proteins could be responsible for their efficiency and neuroprotection. Here, we investigated the thermodynamics of vinorelbine interactions with calmodulin and tubulin. It was determined that, unlike the other vinca-alkaloids, both vinorelbine binding sites are located in the C-domain of calmodulin and they are characterized by association constants of 4.0 × 10⁵ and 5.4 × 10⁴ M⁻¹. At the same time, the thermodynamics of vinorelbine binding to tubulin are not much different from that of other vinca-alkaloids. These results will allow us to get a better insight on the reaction mechanisms of vinca-alkaloids on a secondary protein target.     

A unifying new model of cytokinesis for the dividing plant and animal cells

Cytokinesis ensures proper partitioning of the nucleocytoplasmic contents into two daughter cells. It has generally been thought that cytokinesis is accomplished differently in animals and plants because of the differences in the preparatory phases, into the centrosomal or acentrosomal nature of the process, the presence or absence of rigid cell walls, and on the basis of 'outside-in' or 'inside-out' mechanism. However, this long-standing paradigm needs further reevaluation based on new findings. Recent advances reveal that plant cells, similarly to animal cells, possess astral microtubules that regulate the cell division plane. Furthermore, endocytosis has been found to be important for cytokinesis in animal and plant cells: vesicles containing endocytosed cargo provide material for the cell plate formation in plants and for closure of the midbody channel in animals. Thus, although the preparatory phases of the cell division process differ between plant and animal cells, the later phases show similarities. We unify these findings in a model that suggests a conserved mode of cytokinesis.     

Tyrosine phosphorylation of plant tubulin

Phosphorylation of αβ-tubulins dimers by protein tyrosine kinases plays an important role in the regulation of cellular growth and differentiation in animal cells. In plants, however, the role of tubulin tyrosine phosphorylation is unknown and data on this tubulin modification are limited. In this study, we used an immunochemical approach to demonstrate that tubulin isolated by both immunoprecipitation and DEAE-chromatography is phosphorylated on tyrosine residues in cultured cells of Nicotiana tabacum. This opens up the possibility that tyrosine phosphorylation of tubulin could be involved in modulating the properties of plant microtubules.     

Colchicine-resistant assembly of tubulin in plant mitosis

Summary Tubulin distribution in c-mitoses (induced by 1 mM colchicine) has been studied by indirect immunofluorescence with monoclonal antibodies inAllium cepa L. meristems proliferating under steady state kinetics. Two hours after colchicine treatment was initiated tubulin is detected in approximately 25% of the cells as arrowheads on the kinetochores, as if these structures stabilize microtubules against disassembly. Total disassembly of microtubules occurs in 70% of the c-mitoses six hours after the initiation of the colchicine treatment, when restitution nuclei also start appearing. After 2 to 14 hours of colchicine treatment, tubulin is detected in about 30% of the c-mitoses, both in small kinetochores-like dots and in a strand which apparently connects sister kinetochores. Other larger microtubule-like structures, up to 20 m long, apparently unassociated with kinetochores, are assembled in the presence of cholchicine in c-mitoses after 10 hours. Such structures disappear when chromosomes decondense and the nuclear envelope reforms in the restitution nucleus; they do not seem to be related to interphase cortical microtubules which reappear in control telophase.     

Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate

Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state.     

Immunofluorescence microscopy of tubulin and microtubule arrays in plant cells. I. Preprophase band development and concomitant appearance of nuclear envelope-associated tubulin

The development of the preprophase band (PPB) of microtubules (MT) in meristematic plant cells was studied by using antibodies to pig brain tubulin and indirect immunofluorescence microscopy. The PPB is first visible as a wide band of MT that are arranged only slightly more densely than flanking MT of the cortical interphase array. MT progressively become more tightly packed together, and other cortical MT are no longer seen as the PPB matures. The surface of the nuclear envelope (NE) displays no tubulin fluorescence during interphase but begins to fluoresce in the early stages of PPB development, and its intensity progressively increases thereafter. The pattern at the NE is usually diffuse at first, suggesting the presence of nonpolymerized tubulin, but fibers along the NE can be resolved at later stages. MT, arranged either radially or as a meshwork, can occur between the nucleus and cell cortex, and sometimes appear to connect the PPB and NE directly. Isolated preprophase nuclei from cells ruptured during processing often retain the PPB in its normal orientation, indicating stable linkages between the nucleus and PPB. Fluorescent cross-bars perpendicular to the axis of the MT were resolved in some PPB, suggesting lateral linkages. This suggestion is reinforced by the presence of PPB that hold together as a ribbon of MT in certain preparations, allowing PPB to be isolated from the rest of the cytoplasm and the nucleus.