Microtubule dynamics.

Microtubules are fibrous elements in the cytoplasm of eukaryotic cells, where they perform a wide variety of functions. Microtubules are major organizers of the cell interior and are vitally involved in motility events such as chromosome migration during cell division. To fulfill their physiological function, microtubule arrays have to undergo dramatic changes in their spatial arrangement, and this depends to a large extent on the complex and special dynamic properties of the individual polymers. In this review we first describe the intrinsic dynamic properties of microtubules assembled in vitro from purified tubulin and examine the relationships between these properties and microtubule functions. Subsequent sections concern microtubule dynamics in vivo, their similarity and differences with microtubule dynamics in vitro, and the nature of the cellular regulators which act on microtubule assemblies in physiological conditions.

A reassessment of the factors affecting microtubule assembly and disassembly in vitro.

Current models of microtubule assembly from pure tubulin involve a nucleation phase followed by microtubule elongation at a constant polymer number. Both the rate of microtubule nucleation and elongation are thought to be tightly influenced by the free GTP-tubulin concentration, in a law of mass action-dependent manner. However, these basic hypotheses have remained largely untested due to a lack of data reporting actual measurements of the microtubule length and number concentration during microtubule assembly.Here, we performed simultaneous measurements of the polymeric tubulin concentration, of the free GTP-tubulin concentration, and of the microtubule length and number concentration in both polymerizing and depolymerizing conditions. In agreement with previous work we find that the microtubule nucleation rate is strongly dependent on the initial GTP-tubulin concentration. But we find that microtubule nucleation persists during microtubule elongation. At any given initial tubulin-GTP concentration, the microtubule nucleation rate remains constant during polymer assembly, despite the wide variation in free GTP-tubulin concentration. We also find a remarkable constancy of the rate of microtubule elongation during assembly. Apparently, the rate of microtubule elongation is intrinsic to the polymers, insensitive to large variations of the free GTP-tubulin concentration. Finally we observe that when, following assembly, microtubules depolymerize below the free GTP-tubulin critical concentration, the rate-limiting factor for disassembly is the frequency of microtubule catastrophe. At all time-points during disassembly, the microtubule catastrophe frequency is independent of the free GTP-tubulin concentration but, as the microtubule nucleation rate, is strongly dependent on the initial free GTP-tubulin concentration. We conclude that the dynamics of both microtubule assembly and disassembly depend largely on factors other than the free GTP-tubulin concentration. We propose that intrinsic structural factors and endogenous regulators, whose concentration varies with the initial conditions, are also major determinants of these dynamics.

Proliferation of LAMA-84 and LAMA-87 cell lines is modulated by autocrine loops involving M-CSF and TGF-beta.

The erythromegakaryocytic cell line (LAMA-84) and the erythroeosinophilic cell line (LAMA-87) were used to study receptor expression and receptor-mediated response to monocyte/macrophage colony-stimulating factor (M-CSF) and transforming growth factor beta (TGF-beta), two modulators of cell proliferation. As demonstrated by Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR), c-fms and M-CSF mRNA were expressed in both cell lines. M-CSF was detected in the supernatant of both cell lines and addition of a neutralizing anti-M-CSF antibody inhibited cell growth. The two LAMA cell lines were found to express TGF-beta1, -beta2, and -beta3 mRNAs and to secrete TGF-beta mostly in latent form. Addition of anti-TGF-beta antibodies to the culture medium increased their proliferation, whereas TGF-beta1 inhibited cell proliferation by downregulating the c-myc mRNA. These results show that the proliferation of both LAMA cell lines is positively and negatively regulated by autocrine mechanisms, implying the presence of M-CSF and TGF-beta, respectively. They suggest that similar autocrine loops could be involved in the growth regulation of leukemic cells in vivo.

Effects of retinoic acid on a new human erythromegakaryocytic cell line AP-217.

We have characterized a new human cell line AP-217, derived from the peripheral blood of a patient with chronic myeloid leukemia in blastic crisis. The analysis of cell surface antigens and ploidy showed that AP-217 was an erythro-megakaryocytic cell line. The effects of inducers of differentiation were studied and focused on retinoic acid (RA). Uninduced AP-217 cells produced a low level of hemoglobin (Hb) that showed a moderate but significant dose-dependent increase after 13 cis-RA induction (four times above the control at 10(-5) M). To outline this effect, AP-217 cells were cloned at limiting dilution. A subclone (clone 2) was isolated which expressed glycophorin A on 12% of cells, and showed a marked sensitivity to RA. After a 4 day induction with increasing concentrations of RA (1-10 x 10(-6) M) Hb production by clone 2 cells was enhanced 12 times over the control at the highest concentration (10(-5) M). No effect of RA on the Hb production of K-562 and HEL was observed. This increased Hb production occurred simultaneously with a growth inhibition in clonogenic cultures (20% reduction) associated with a drastic reduction of the colony size. Moreover, we demonstrated the expression of mRNA for the beta globin gene in clone 2 and AP-217-cells. This is the first report of a positive effect of RA on the erythroid differentiation of a human leukemic cell line.

Histone H1(0) expression is restricted to progenitor cells during human hematopoiesis.

The histone H1(0) accumulates in cells with little or no proliferative activity during the terminal phase of differentiation in adult tissues. The hematopoietic cell system is an interesting in vivo model to study the relationship between H1(0) and both the proliferative capacity and differentiation state of cells. Using immunofluorescence techniques, we have analyzed the distribution of histone H1(0) during human hematopoietic differentiation, in normal bone marrow cells and in cell lines representative of cells blocked at early stages of differentiation. In enriched bone marrow cell suspensions, H1(0) was not expressed in any cell population highly engaged into a differentiation pathway. However, more than 50% of cells from blastic population (CD34-positive cells) were expressing H1(0), whereas only 5% of CD34-negative cell population expressed H1(0). We show that H1(0) was also detected in almost all the cell lines studied. These results indicate that histone H1(0) is expressed in immature cells which, although committed, still retain several differentiation potentialities. For normal human hematopoiesis, cells expressing H1(0) belong to a population of cells that are largely quiescent, although having a high proliferative capacity. H1(0) is no longer present in terminally differentiating or differentiated cells with limited or no proliferative potential. Thus, we suggest that H1(0) accumulates in cells with little or no proliferative activity but which are able to resume cell proliferation if required. These results are in keeping with the hypothesis that H1(0) contributes to stabilize a chromatin structure in cells for which integrity and/or longevity are essential.

Desmoplakin expression and organization at human umbilical vein endothelial cell-to-cell junctions.

Desmoplakin is an intracellular component of desmosomes which plays a role in the anchorage of intermediate filaments to these structures. We report here that, despite the absence of desmosomes, cultured endothelial cells from human umbilical vein express desmoplakin I and II both at mRNA and protein level. Desmoplakin I/II are found only in the detergent insoluble fraction suggesting that most of the protein is linked to the cytoskeleton. Desmoplakin I/II could be detected by western blot only in long confluent cells even if desmoplakin mRNA levels are unchanged by cell confluency. This suggests that desmoplakin might be stabilized at protein level by its association with junctional components. Immunofluorescence confocal microscopy showed that desmoplakin codistributes with VE-cadherin and plakoglobin along the lateral cell membrane. In contrast, desmoplakin localization was distinct from that of PECAM, an endothelial specific junctional protein localized outside adherence junctions. Endothelial cells do not have keratins but they express vimentin. In confluent cells vimentin forms peripheral filaments which attach to the cell membrane in areas at desmoplakin localization. These data suggest that desmoplakin may participate in the molecular organization of interendothelial junctions by interacting with VE-cadherin and promoting vimentin anchorage. This new type of intercellular junction seems to correspond to the "complexus adhaerentes' described in vivo in lymphatic endothelium.

Selection and characterization of an erythroeosinophilic subclone (LAMA-87) and an eosinophilic subclone (LAMA-88) from the multipotential cell line LAMA-84.

The human leukemic cell line LAMA-84 was established and characterized as an erythromegakaryocytic cell line. In the present study we show that these cells can differentiate in estrone-treated athymic mice and give rise to an erythroeosinophilic cell line (LAMA-87). This new cell line expressed glycoporin A, alpha beta and gamma globin chain mRNA but also eosinophilic peroxidase. Hemin slightly increased the total hemoglobin production of the cells and phorbol diester (TPA), dimethyl sulfoxide (DMSO) and sodium butyrate (SB) increased the expression of megakaryocytic markers (gpIIb/IIIa complex). When inoculated into non-treated athymic mice, LAMA-87 cells can differentiate to give rise to eosinomonocytic cells (LAMA-88). This new cell line expresses eosinophilic peroxidase, Luxol fast blue stain and synthesizes lysozyme. Depending on the inducer used, LAMA-88 can differentiate along a monocytic lineage (TPA, DMSO, SB and vitamin D3). These three LAMA cell lines should be useful in further studies of the molecular regulation of the pluripotent cell commitment and may provide a model for the understanding of human hematopoiesis.

Serum-free medium allows the optimal growth of human megakaryocyte progenitors compared with human plasma supplemented cultures: role of TGF beta.

The growth of human megakaryocyte progenitors from human bone marrow (BM) cells was compared using a methylcellulose semisolid assay supplemented either by normal human plasma or by a serum-free medium. Far better growth of megakaryocyte colonies from CD34+ BM cells stimulated by interleukin 3 (IL-3) and interleukin 6 (IL-6) was observed in serum-free medium compared with human plasma supplemented cultures. These results were confirmed in liquid cultures using the same serum-free medium composition. The megakaryocytes were identified by using an immunocytochemical procedure after labeling with an anti-GPIIb-IIIa monoclonal antibody. High percentages (15 to 20%) of megakaryocytes were present in serum-free cultures stimulated by IL-3 alone or combined with IL-6. The absolute number of megakaryocytes in serum-free medium exceeds by 3.3 (IL-3 plus IL-6) to 4.4 (IL-3 alone) times the corresponding number of megakaryocytes observed in human plasma supplemented cultures. The optimal concentration of IL-3 alone was 5 ng/ml, and an optimal synergistic effect of IL-6 (5 ng/ml) was obtained when combined with a suboptimal dose of IL-3 (1 ng/ml). The poor growth of megakaryocyte colonies from CD34+ BM cells in human plasma suggested the presence of an inhibitory factor. When a neutralizing monoclonal antibody against transforming growth factor beta (TGF beta) is present in human plasma supplemented cultures of CD34+ BM cells, the number of megakaryocyte colonies is increased to the level observed in corresponding serum-free cultures. The high efficiency of this serum-free medium to promote the growth of human megakaryocytes will be useful to study the effects of regulators and platelet agonists acting on human megakaryocytes, without interference from factors in the serum.

Cryopreservation of human megakaryocytic progenitor cells (CFU-MK): influence of culture conditions.

The survival of human megakaryocytic progenitor cells (CFU-MK) after freezing using a two-step cooling technique was studied in a methylcellulose culture system, using different stimulating activities and their combinations. The best growth stimulating activity for CFU-MK was found in plasma from aplastic patients (PAP). PAP was roughly three times more potent than optimal doses of human recombinant interleukin 3 (IL3), itself two times more active than our batch of phytohemagglutinin stimulated leukocyte conditioned medium (PHA-LCM). The addition of erythropoietin (EPO) to PHA-LCM doubled the number of megakaryocytic colonies, but had no effect on the number of CFU-MK stimulated by IL3. Of the two batches of PAP used, one was optimal and the other was significantly improved by the addition of PHA-LCM. The percentage recovery of CFU-MK after freezing reached 70 to 80% in cultures stimulated by PHA-LCM and IL3 +/- EPO, and 58% in PAP-supplemented cultures. However, this last value was not significantly different from the previous ones. The percentage recovery of CFU-MK in each condition tested was similar to that obtained with the other myeloid progenitors, CFU-GM and BFU-E. However, the high CFU-MK stimulating activity of PAP is hindered by its high variability and shortage in supply. Thus it seems more appropriate to recommend the use of recombinant human IL3 because of its easily standardizable and consistent CFU-MK stimulating activity.

Toward an objective classification of cells in the immune system.

The relative abundance of individual proteins shared among clones of lymphocytes provides a meaningful basis for cellular classification. Twelve clones of T cells (obtained by limiting dilution) were analyzed by two-dimensional gel electrophoresis for polypeptide content and then evaluated by the computational technique known as principal component analysis. As a result, relatedness of the clones was established and expressed in terms of taxonomic distances. The data show that a comprehensive and objective classification of the cells involved in the immune system can be approached.

Immunophenotype of blast cells in chronic myeloid leukemia.

The immunophenotype of peripheral blood blast cells from 14 patients in the chronic phase of chronic myeloid leukemia (CML) was studied using a panel of monoclonal antibodies (McAb) directed against megakaryocytic, granulomonocytic, erythroid and lymphoid antigenic determinants. The blast cells were enriched by a simple bovine serum albumin (BSA) density-cut separation and cooled in liquid nitrogen. The study was done using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique on the thawed blast cells. A consistent pattern of reactivity with McAb was found in all patients, showing that blast cells were heterogeneous. A minor component of the blast cells react with platelet antibodies, most of them being labelled with anti-GPIIb-IIIa McAb. Anti-GPIb and Von Willebrand factor McAb detected 4 times fewer megakaryocytic blast cells, suggesting that these cells are located very early in the differentiation scheme. Two major blast cell compartments were labelled with early myelomonocytic (anti-CD13: MY7) and early erythroid (anti-CD36: FA6-152) McAb. The CD34 (My10) and DR antigens which are expressed by immature blast cells and myeloid progenitors of human bone marrow (BM) were present on more than 50% of the CML blast cells. Thus, the blast cells of chronic phase CML patients, showed the same cellular diversity as the increased progenitor cell compartment observed in this disease, and their differentiation stages seemed to be very closely related.

A metrological study of autoradiographs from two-dimensional gel electrophoresis.

Samples prepared from a single batch of labeled cells were subjected to two-dimensional gel electrophoresis and autoradiography. Three factors were varied: total quantity of protein, quantity of labeled protein, and exposure time. The mean background absorbance of the film remained identical (about 0.5 A) for all the treated series, whatever the exposure time and whether or not there were unlabeled proteins in the sample. Hence any spot with a peak A of the same order of magnitude can be seen. The standard deviation was about 0.05 A. Thus, the measurement precision is 2.5% of full scale for digitalization over 0 to 2 A. We derived experimental calibration curves, which are neither linear nor logarithmic because of the film response and which can be used on randomly chosen spots.

Molecular characterization of an abnormal fibrinogen by two-dimensional electrophoresis.

We examined normal and abnormal fibrinogen (fibrinogen "Grenoble") by two-dimensional gel electrophoresis to obtain data on possible defects at the molecular level. Fibrinogen Grenoble is characterized by an abnormal rate of fibrin monomer aggregation. The electrophoretic analysis revealed the presence of abnormal gamma chains. Two kinds of gamma chains can be detected in fibrinogen Grenoble: (a) normal gamma chains and (b) gamma chains Grenoble (gamma G) with a greater molecular mass but no modification in isoelectric point. The latter chain can be detected in whole plasma by two-dimensional gel electrophoresis. Metrological analysis was performed in an attempt to quantify observed differences between normal fibrinogen and fibrinogen Grenoble. On use of gels stained either with Coomassie Brilliant Blue or with silver, the partly qualified evaluation gives about 60% normal gamma chain and 40% gamma chain Grenoble.