Mitochondrial metabolism directs stemness and differentiation in P19 embryonal carcinoma stem cells.

The relationship between mitochondrial metabolism and cell viability and differentiation in stem cells (SCs) remains poorly understood. In the present study, we compared mitochondrial physiology and metabolism between P19SCs before/after differentiation and present a unique fingerprint of the association between mitochondrial activity, cell differentiation and stemness. In comparison with their differentiated counterparts, pluripotency of P19SCs was correlated with a strong glycolytic profile and decreased mitochondrial biogenesis and complexity: round, low-polarized and inactive mitochondria with a closed permeability transition pore. This decreased mitochondrial capacity increased their resistance against dichloroacetate. Thus, stimulation of mitochondrial function by growing P19SCs in glutamine/pyruvate-containing medium reduced their glycolytic phenotype, induced loss of pluripotent potential, compromised differentiation and became P19SCs sensitive to dichloroacetate. Because of the central role of this type of SCs in teratocarcinoma development, our findings highlight the importance of mitochondrial metabolism in stemness, proliferation, differentiation and chemoresistance. In addition, the present work suggests the regulation of mitochondrial metabolism as a tool for inducing cell differentiation in stem line therapies.

Adhesion, proliferation and differentiation of pluripotent stem cells on multi-walled carbon nanotubes.

This article studies the adhesion, growth and differentiation of stem cells on carbon nanotube matrices. Glass coverslips were coated with multi-walled carbon nanotube (MWNT) thin films using layer-by-layer self-assembling techniques. Pluripotent P19 mouse embryonal carcinoma stem cells were seeded onto uncoated or MWNT-coated glass coverslips and either maintained in an undifferentiated state or induced to differentiate by the addition of retinoic acid. The authors found that cell adhesion was increased on the MWNT-coated glass surfaces, and that the expression patterns of some differentiation markers were altered in cells grown on MWNTs. The results suggest that MWNTs will be useful in directing pluripotent stem cell differentiation for tissue engineering purposes.

Intermediate filament proteins in echinoderm coelomocytes.

The presence and organization of intermediate filament (IF) proteins in petaloid coelomocytes from two species of echinoderms, the sea urchin Strongylocentrotus droehachiensis and the sea cucumber Cucumaria frondosa, were studied. Two monoclonal antibodies (IFA and Ah6) and one polyclonal antibody (W3-1) that together recognize invertebrate as well as vertebrate IF proteins were used to probe coelomocytes by immunofluorescence and immunoblotting methods. All three antibodies cross-reacted with a single Mr 68,000 sea urchin lamin, as well as two putative lamin isoforms of approximately Mr 70,000 and 68,000 in sea cucumber coelomocytes. Both IFA and Ah6 labeled granular material in the cytoplasm of sea urchin coelomocytes; by contrast, IFA labeling revealed a striking network of reticular material irregularly arrayed within the central regions of the sea cucumber coelomocyte cytoplasm. In addition, foci of Ah6-positive material were present in coelomocyte nuclei from both species. Comparison of immunoblotting patterns among whole cell and isolated nuclear preparations suggest that the cytoplasmic IF-like material is composed of Mr 46,000 and 58,000 polypeptides, while Mr 215,000 and 185,000 proteins are candidates for the immunoreactive nuclear foci. Further study of the functions of these non-filamentous arrays of IF proteins may furnish valuable insights into the evolution of IF function within vertebrate cells, particularly with respect to certain cytoplasmic and nuclear regulatory functions with which IF proteins have been speculated to be involved.

Neuronal differentiation of NT2/D1 teratocarcinoma cells is accompanied by a loss of lamin A/C expression and an increase in lamin B1 expression.

Nuclear lamins are prominent elements of the nuclear matrix and are expressed in cell type-specific and differentiation state-specific patterns. A few observations have indicated that nervous tissue may display unusual patterns of lamin expression, in that some neurons appear to lack A-type lamins, which are generally prominently expressed in terminally differentiated, postmitotic cells. To investigate lamin expression patterns during the differentiation of a teratocarcinoma cell line into neurons, NT2/D1 cells were induced to differentiate with retinoic acid treatment. Lamin expression and organization during differentiation in vitro were examined by quantitative immunofluorescence and immunoblotting methods. Undifferentiated NT2/D1 cells were all strongly labeled with an anti-lamin B1 antibody, but displayed marked variation in A/C lamin immunoreactivity. After differentiation, neuronal nuclear envelopes were significantly more strongly labeled by anti-lamin B1 antibody than those of undifferentiated cells, but completely lacked A/C lamin immunoreactivity. In contrast, nonneuronal cells displayed a slight reduction in B1 lamin immunoreactivity, along with a distinct increase in A/C lamin levels. The loss of lamin A/C expression in NT2/D1 neurons is contrary to the pattern normally observed in most somatic cell types during early development and indicates that the nuclear matrix of some neurons, along with certain neuroendocrine and hematopoietic cells, is uniquely specialized in this regard.

Chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] disrupts microtubule organization, cell division, and early development of sea urchin embryos.

The herbicide CIPC [N-(3-chlorophenyl) carbamate] has been shown to disrupt microtubule organization in plants, apparently by interfering with the functioning of the microtubule organizing center. Very few studies have examined the effects of CIPC on animal cell microtubules and centrosomes, however, and the effects of this cytoskeletal disrupting agent on fertilization and early development have not been studied in detail. To address these questions, fertilized sea urchin eggs were cultured in the presence of CIPC until the prism stage, and perturbations in the cytoskeleton and development were examined. It was found that Lytechinus pictus embryos are sensitive to micromolar amounts of CIPC, and that a characteristic set of cytoskeletal and developmental deficits is produced as a result of exposure to this herbicide. Mitotic spindles were truncated and randomly oriented within zygotes and blastomeres, and cytokinesis was compromised, resulting in the production of blastomeres of various sizes and ploidy. Interestingly, in spite of these cytoskeletal and nuclear alterations, spindle poles at fourth cleavage retained their ability to interact with the plasma membrane in a manner similar to that normally characterizing the unequal division of macromeres and micromeres. CIPC treatment resulted in unequal cell divisions at atypical times, and skeletal spicule formation in these embryos was abnormal. These results indicate that CIPC may pose a significant health risk during mammalian embryogenesis; in addition, it may be a useful tool with which to study microtubule and centrosomal functioning during animal cell division-especially in those cell types that exhibit stereotypic patterns of cell division during early development.

Confocal microscopic observation of cytoskeletal reorganizations in cultured shark rectal gland cells following treatment with hypotonic shock and high external K+.

The dogfish shark (Squalus acanthias) rectal gland (SRG) cell has served as a model experimental system for investigating the relationship between the actin cytoskeleton and cell volume regulation. Previous reports employing conventional fluorescence microscopy of tissue slices have shown that cells exposed to high external K+ and hypotonically-induced cell swelling displayed a fading of F-actin staining intensity, particularly at the basolateral cell borders. However, spectroscopic measurement of the F-actin present in similarly treated rectal gland slices failed to demonstrate a net change in F-actin amount. In an effort to resolve the structural reorganizations of F-actin which may be occurring during high K+ and hypotonic shock treatments, we have used cultured SRG cells in conjunction with confocal microscopic immunocytochemical localization techniques to examine actin filament, microtubule, and cytokeratin filament dynamics under these two experimental conditions. The results reveal that F-actin in control cells exists in an array of parallel linear bundles (which do not appear to be stress fiber-like given their lack of staining for myosin II or alpha-actinin) that is reorganized to a punctate pattern in hypotonic shock and a dense meshwork in high K+. The linear bundle pattern of F-actin returns in cells undergoing regulatory volume decrease. Quantitative western blotting of F-actin in SRG cell detergent extracted cytoskeletons indicates no significant difference in the relative amounts of F-actin present in control, hypotonic shocked, or high K+ cells. Anti-tubulin and anti-cytokeratin labeling of the treated SRG cells suggest that these other major cytoskeletal elements are not significantly altered by the treatments. Taken together, our results reinforce the concept that there is an association between the structural organization of the actin cytoskeleton and cell volume regulation in the SRG epithelial cells.

Recruitment of maternal material during assembly of the zygote centrosome in fertilized sea urchin eggs.

Spindle poles of sea urchin embryos contain centrosomal material derived from maternal as well as paternal sources. To examine how maternal centrosomal material becomes recruited into spindle poles during the first cell cycle, fertilized sea urchin eggs were fixed and labeled with an anti-centrosomal antibody at sequential timepoints after insemination. Immunolabeling patterns demonstrate that the unfertilized egg contains small foci of immunoreactive material dispersed throughout the cytoplasm. Shortly after insemination, the diffuse foci coalesce to form a dense aggregate close to the sperm nucleus. Subsequently, centrosomal material spreads over the surface of the zygote nucleus and becomes partitioned into two masses during spindle pole formation. The involvement of the cytoskeleton in the translocation and targeting of maternal centrosomal material through the first cell cycle was examined by treating eggs with cytoskeletal disrupting agents, a general kinase inhibitor, and by re-inseminating fertilized eggs. These experiments indicate that the initially diffuse centrosomal material is transported centripetally to the sperm nucleus by the sperm aster and the centrosomal material is subsequently sequestered around the zygote nucleus by a microtubule-mediated mechanism. Remarkably, 6-dimethylaminopurine treatment shifted the targeting of maternal centrosomal material from the sperm nucleus to the female pronucleus; upon recovery, some of these zygotes formed spindle poles that flanked only the maternal chromosomes.

Conserved expression of a B-type nuclear lamin in different tissues of the sea urchin.

To study patterns of lamin expression in sea urchins, monoclonal and polyclonal antibodies were used to probe 2-D immunoblots of Strongylocentrotus purpuratus and Lytechinus pictus embryos and adult tissues. Three major charge isoforms of a single M(r) 70,000 lamin are common to different tissues of S. purpuratus, and are accompanied by two-to-four more variable minor charge variants. L. pictus expresses and M(r) 70,000 lamin that is distinct from that of S. purpuratus in pI, yet displays essentially the same pattern of major and minor charge isoforms. The results demonstrate that the single lamin gene product so far identified in sea urchin embryos appears to be constitutively expressed in most cells, and that three major charge isoforms are common to these different cell types.

Molecular characterization and expression patterns of a B-type nuclear lamin during sea urchin embryogenesis.

Developmentally regulated, tissue-specific patterns of nuclear lamin expression occur during vertebrate embryogenesis, but little is known regarding lamin ontogeny during the early development of other phyla. cDNA clones encoding a lamin from the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus have been identified, and the full coding region from the former has been sequenced. The predicted amino acid sequence indicates that this echinoderm lamin is more closely related to vertebrate B-type lamins than to dipteran fly and nematode lamins--the only other invertebrate lamins sequenced to date. Monoclonal and polyclonal antibodies to sea urchin lamin demonstrate that nuclei of unfertilized eggs and embryos exhibit relatively faint immunoreactivity until the differentiation of primary mesenchymal cells, the nuclear envelopes of which become strongly and selectively labeled by anti-lamin antibodies. Northern blots reveal stage-specific fluctuations in a single 4-kb lamin message during early development and, together with immunoblotting data, suggest that the increase in mesenchymal cell nuclear envelope immunoreactivity is due to a quantitative increase in a single type of lamin. These observations demonstrate that, similar to vertebrates, cell differentiation in invertebrates can be accompanied by a change in lamin expression patterns.

Hymenolepis diminuta: two morphologically distinct tegumental secretory mechanisms are present in the cestode.

The free-surface of the tapeworm's tegument was examined for morphological evidence of secretion after fixation by rapid freezing-freeze substitution and alternatively by immersion in low concentrations of glutaraldehyde maintained at room temperature. After low-aldehyde fixation, omega profiles were at the bases of tegumental microvilli, arguing for the participation of some of the ectocytoplasm's vesicles in secretion of their contents to the intestinal lumen. The almost instantaneous fixation provided by the rapid freezing-freeze substitution technique documents the constitutive production of 0.03 to 0.075-micron microvesicles from outpocketings from the plasma membrane of the tegumental brush border. Observed in secretory epithelial cells of other species, microvesicles are recognized as a secretory pathway for constituent molecules of surface membranes. We conclude that in addition to the primary route of merocrine exocytotic secretion provided by the fusion of the Golgi-derived, ectocytoplasmic vesicles at the bases of the brush border microvilli, tegumental microvesicles provide a second secretory pathway for endogenous macromolecules across the tegumental free surface.

Spindle pole centrosomes of sea urchin embryos are partially composed of material recruited from maternal stores.

The spindle poles of fertilized sea urchin eggs have commonly been modeled as being derived from the centrosomes of the fertilizing spermatozoon. Boveri's theory of fertilization, proposed at the turn of the century, states that the maternal centrosome is suppressed or inactivated during oogenesis and that the sperm centrosome is functionally dominant. In support of this proposal, more recent studies have shown that the sperm imports a determinant that is involved in centrosomal replication. Examination of sea urchin zygotes immunofluorescently labeled with a new anti-centrosomal antibody by quantitative confocal laser-scanning microscopy shows, however, that spindle pole centrosomes are not exclusively paternal structures, but additionally contain material derived from maternal pools. Furthermore, this maternal centrosomal material is divided among daughter blastomeres during cleavage. It therefore appears that although the sperm centrosome plays a dominant role in organizing the spindle poles, much of the centrosomal material within the spindle poles of the zygote is actually recruited from preexisting egg cytoplasmic stores. These data indicate that centrosomes of sea urchin embryos are biparentally derived, composite organelles.