Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

Summary Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division. divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all.     

Suppression of Rac1 activity at the apical membrane of MDCK cells is essential for cyst structure maintenance

Using MDCK cells that constitutively express a Förster resonance energy transfer biosensor, we found that Rac1 activity is homogenous at the entire plasma membrane in early stages of cystogenesis, whereas in later stages Rac1 activity is higher at the lateral membrane than at the apical plasma membrane. If Rac1 is activated at the apical membrane in later stages, however, the monolayer cells move into the luminal space. In these cells, tight junctions are disrupted, accompanied by mislocalization of polarization markers and disorientation of cell division. These observations indicate that Rac1 suppression at the apical membrane is essential for the maintenance of cyst structure.     

Cyclin D2 in the basal process of neural progenitors is linked to non-equivalent cell fates

Asymmetric cell division plays an indispensable role during corticogenesis for producing new neurons while maintaining a self-renewing pool of apical progenitors. The cellular and molecular determinants favouring asymmetric division are not completely understood. Here, we identify a novel mechanism for generating cellular asymmetry through the active transportation and local translation of Cyclin D2 mRNA in the basal process. This process is regulated by a unique cis-regulatory sequence found in the 3' untranslated region (3'UTR) of the mRNA. Unequal inheritance of Cyclin D2 protein to the basally positioned daughter cell with the basal process confers renewal of the apical progenitor after asymmetric division. Conversely, depletion of Cyclin D2 in the apically positioned daughter cell results in terminal neuronal differentiation. We demonstrate that Cyclin D2 is also expressed in the developing human cortex within similar domains, thus indicating that its role as a fate determinant is ancient and conserved.     

Exposure to the electrofusion process can increase the immunogenicity of human cells

The cellular products obtained following electrofusion (EF) of dendritic cells (DC) and tumour cells have shown promise as cancer vaccines. The immunogenicity of these preparations has been attributed to the presence of small numbers of DC-tumour hybrids and the contribution of the non-hybrid tumour cells present has received little attention. In this report, we investigated the effect of the EF process on the immunogenicity of allogeneic human cells, in particular the colorectal cell line, SW620. EF conditions were optimised to yield the maximum number of DC-SW620 hybrids co-expressing tumour associated antigen (TAA) and DC associated antigens. Exposure of SW620 to EF induced significant increases (P<0.05) in apoptosis and necrosis. Pre-exposure of SW620 to the EF buffer alone [0.3 M glucose, 0.1 mM Ca(CH₃COO)₂ and 0.5 mM Mg(CH₃COO)₂] resulted in significant increases in TAA uptake by DC during co-culture (P<0.05). DC phenotype was, however, not altered by exposure to EF treated tumour cells. In co-cultures of PBMC responders with SW620, the levels of IFN release and cytotoxic activity were significantly increased (P<0.05) by pre-exposure of the SW620 to EF. Pre-exposure of allogeneic non-T cells, the colorectal cell line Lovo and a breast cancer cell line (MCF7) to EF also significantly (P<0.05) increased the levels of IFN release by responding PBMC. These results demonstrate that the EF process itself can increase the immunogenicity of at least some human cell types independently of hybrid formation. These findings suggest that EF protocols should be evaluated with regard to the possibility that DC-tumour hybrids may not contribute all, or even most, of the immunostimulatory capacity present in preparations of EF treated cells.BH and GR contributed equally as senior authors     

Vrp1p functions in both actomyosin ring-dependent and Hof1p-dependent pathways of cytokinesis

Vrp1p/verprolin/End5p is a Saccharomyces cerevisiae proline-rich protein, structurally and functionally related to human Wiskott–Aldrich syndrome protein-interacting protein. Vrp1p is required for viability at 37°C, but not 24°C. Here, we show that loss of Vrp1p ( vrp1Δ ) leads to a 3–4-fold delay in cytokinesis, wide bud necks, abnormal actomyosin rings, and aberrant septa even at 24°C. Like other mutations affecting the actomyosin ring, vrp1Δ is synthetic lethal with deletion of HOF1 (or CYK2 ), which encodes a protein related to mammalian proline serine threonine phosphatase-interacting protein and Schizosaccharomyces pombe Cdc15p required for an actomyosin ring-independent pathway of cytokinesis in S. cerevisiae . At 37°C, vrp1Δ cells rapidly cease dividing and exhibit a novel terminal phenotype: a single large bud, two well-separated nuclei, and an interphase microtubule array. The arrested cells have a persistent ring containing both actin and myosin at the bud neck. Many also exhibit some polarisation of cortical actin patches to the bud neck. Vrp1p binds an SH3-domain-containing fragment of Hof1p in vitro . Vrp1p is required in vivo for Hof1p relocalisation to a single ring at the bud neck prior to cytokinesis at 37°C, but not at 24°C. Vrp1p thus acts in both actomyosin ring formation and function, as well as in Hof1p localisation during cytokinesis.     

Grandparental stem cells in leech segmentation: Differences in CDC42 expression are correlated with an alternating pattern of blast cell fates

Embryonic segmentation in clitellate annelids (oligochaetes and leeches) is a cell lineage-driven process. Embryos of these worms generate a posterior growth zone consisting of 5 bilateral pairs of identified segmentation stem cells (teloblasts), each of which produces a column of segmental founder cells (blast cells). Each blast cell generates a lineage-specific clone via a stereotyped sequence of cell divisions, which are typically unequal both in terms of the relative size of the sister cells and in the progeny to which they give rise. In two of the five teloblast lineages, including the ventralmost, primary neurogenic (N) lineage, the blast cells adopt two different fates, designated nf and ns, in exact alternation within the blast cell column; this is termed a grandparental stem cell lineage. To lay groundwork for investigating unequal divisions in the leech Helobdella, we have surveyed the Helobdella robusta genome for genes encoding orthologs of the Rho family GTPases, including the rho, rac and cdc42 sub-families, which are known to be involved in multiple processes involving cell polarization in other systems. We find that, in contrast to most other known systems the Helobdella genome contains two cdc42 orthologs, one of which is expressed at higher levels in the ns blast cells than in nf blast cells. We also demonstrate that the asymmetric divisions of the primary nf and ns blast cells are regulated by the polarized distribution of the activated form of the Cdc42 protein, rather than by the overall level of expression. Our results provide the first molecular insights into the mechanisms of the grandparental stem cell lineages, a novel, yet evolutionarily ancient stem cell division pattern. Our results also provide an example in which asymmetries in the distribution of Cdc42 activity, rather than in the overall levels of Cdc42 protein, are important regulating unequal divisions in animal cells.     

Involvement of Rho GTPases and their effectors in the secretory process of PC12 cells

Intrathymic maturation of thymocytes is essential for the proper formation of T-cell repertoire. This process involves two major biochemical pathways, one initiated by the recognition of MHC/peptide by the T-cell receptor and the other mediated by glucocorticoids. These hormones seem to affect thymocyte maturation by increasing the threshold of TCR-mediated positive and negative selection, and by inducing apoptosis of nonselected thymocytes. We have previously reported that an SV40-immortalized murine thymic epithelial cell line, namely 2BH4, was able to protect thymocytes from dexamethasone-induced apoptosis. Here we show that this protection is independent of cell-to-cell contact and does not seem to involve a Bcl-2-mediated resistance, since incubation of thymocytes with 2BH4 cells or its supernatant does not interfere with the levels of this antiapoptotic molecule. The protection conferred by 2BH4 cells, or by a primary culture of thymic stromal cells, is specific for the CD4(+)CD8(-) and CD4(-)CD8(+) single-positive thymocytes, whereas the broad-spectrum caspase inhibitor z-VAD-fmk blocks apoptosis induced by dexamethasone in all thymocyte subpopulations. Our results suggest that positively selected single-positive thymocytes are still susceptible to glucocorticoid-induced apoptosis but are protected from it through the action of a heat-stable protein(s) released by thymic stromal cells.     

p85 binds to G-actin in a Ca(2+)/calmodulin-dependent manner, thus regulating the initiation of cytokinesis in tetrahymena

Tetrahymena p85 is localized to the presumptive division plane before the formation of contractile ring microfilaments. p85 binds to calmodulin in a Ca(2+)-dependent manner and both proteins colocalize to the division furrow. Inhibition of the binding of p85 and Ca(2+)/calmodulin prevents both the localization of p85 and calmodulin to the division plane and the formation of the contractile ring, suggesting that the interaction of p85 and Ca(2+)/calmodulin is important in the formation of the contractile ring. We investigated the mechanisms of the formation of contractile ring, and the relationship among p85, CaM, and actin using co-sedimentation assay: p85 binds to G-actin in a Ca(2+)/calmodulin-dependent manner, but does not bind to F-actin. Therefore, we propose that a Ca(2+)/calmodulin signal and its target protein p85 are cooperatively involved in the recruitment of G-actin to the division plane and the formation of the contractile ring.     

Secreted MUC1 mucins lacking their cytoplasmic part and carrying sialyl-Lewis a and x epitopes from a tumor cell line and sera of colon carcinoma patients can inhibit HL-60 leukocyte adhesion to E-selectin-expressing endothelial cells

A secreted MUC1 mucin from the spent medium of the colon carcinoma cell line COLO 205 carrying sialyl-Lewis a and x epitopes (H-CanAg) was purified by trichloroacetic acid precipitation and Superose 6 gel filtration. The purified H-CanAg inhibited adhesion of the leukocyte cell line HL-60 to E-selectin transfected COS-1 cells or interleukin-1β (IL-1β)-activated human umbilical vein endothelial cells. Sera from two patients with advanced colon carcinoma containing high concentrations of sialyl-Lewis a and x activity inhibited HL-60 cell adhesion to E-selectin-expressing COS-1 cells and IL-1β-activated endothelial cells. After affinity column absorption of the sialyl-Lewis a activity, the sera also lost most of their sialyl-Lewis x activity and at the same time their adhesion inhibitory effect. A large part of the sialyl-Lewis a/x activity in the two patients was found in fractions containing mucins having a MUC1 apoprotein, as shown by its size, and reactivity with the two anti-MUC1 apoprotein monoclonal antibodies, Ma552 and HMFG-2. The cell-adhesion inhibitory effect of the purified sialyl-Lewis a-carrying MUC1 mucin fraction from the sera of the two patients was stronger than that of smaller sized sialyl-Lewis a-carrying mucin-type glycoproteins also found in the patient sera. The MUC1 mucin fraction secreted by the COLO 205 cells and from the two sera were all shown to lack their C-terminal portion, in contrast to the MUC1 mucin from cells. It is hypothesized that sialyl-Lewis a- and/or x-containing mucins, especially MUC1, secreted by tumors can interact with E-selectin on endothelial cells and thus inhibit leukocyte adhesion. © 1996 Wiley-Liss, Inc.