Search

Megakaryocytes (Mks) are unique cells in the human body in that they carry a single and polyploid nucleus. It is therefore of interest to understand their nuclear ultrastructure. PML oncogenic domains (PODs) were described in several types of eukaryotic cells using human autoantibodies which recognize nuclear antigens with a specific speckled pattern (dots) in indirect immunofluorescence (IF). Two main antigens, PML and Sp 100, usually colocalize and concentrate in these nuclear subdomains. We investigated the presence of PODs using IF and immunoelectron microscopy (IEM) in cells from megakaryocytic lineage: the HEL cell line and human cultured Mks. Antibodies against PML, Sp100, and anti-nuclear dots were used in single and double labeling. PODs were identified in HEL cells and in human Mks, and their ultrastructure was characterized. We then used IF to quantify PODs within Mks and showed that their number increased proportionally to nuclear lobularity. In summary, we report the identification of PODs in human Mks at an ultrastructural level and an increase in PODs number in parallel with Mk ploidy. We show that endomitosis not only leads to DNA increase but also to the multiplication of at least one of the associated nuclear structures.

The effect of calmodulin antagonists on endocytosis, transcytosis, recycling, and transport to the Golgi apparatus from both the apical and the basolateral plasma membrane of polarized Madin-Darby canine kidney cells has been investigated by using the plant toxin ricin as a membrane marker. The calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) stimulated apical endocytosis of ricin, whereas basolateral endocytosis was unaffected. A stimulation of the apical uptake of the fluid-phase marker horseradish peroxidase by calmodulin antagonists was also found both by biochemical and by ultrastructural studies. Furthermore, W-7 reduced the recycling of ricin to the apical plasma membrane, whereas the recycling to the basolateral plasma membrane was not changed. Transport of ricin to the Golgi apparatus was also selectively affected by the calmodulin antagonist W-7. After basolateral endocytosis of ricin, transport to the Golgi apparatus was reduced, whereas after apical endocytosis the fraction of endocytosed ricin transport to the Golgi apparatus was increased. Transcytosis of ricin from the basolateral to the apical pole was increased in the presence of calmodulin antagonists, whereas these compounds did not have any significant effect on the apical to basolateral transcytosis. Thus, the results obtained indicate that calmodulin is involved in regulation of apical endocytosis and recycling as well as in transcytosis of ricin from the basolateral plasma membrane. Furthermore, the data suggest that calmodulin plays a role in regulation of ricin transport to the Golgi apparatus.

Nerve cells detect and respond to multiple extrinsic guidance cues during development and regeneration using a motile growth cone. Navigational decisions may be required of the growth cone when different guidance cues are encountered simultaneously. We have tested the relative potencies of two opposing cues by presenting Xenopus spinal cord nerve cells growing on a micropatterned laminin culture substratum with an orthogonal DC electric field. Substrata composed of repeating 25-micron laminin tracks and spaces failed to influence the position of neuritogenesis from nerve cell soma. Once established, however, growth cone movement was constrained by laminin tracks such that neurites of 65% of cells were aligned after 5 h in vitro. Two hours after the application of a 100-140 mV/mm DC field the majority of cells remained aligned with the laminin tracks. Around 70% of Xenopus neurites normally orient cathodally on homogenous laminin substrata; therefore the galvanotropic response was impeded by prior exposure to a patterned laminin substrate. However, a proportion of aligned neurites did orient cathodally and evidence of a response to both directional cues was even found within the same cell. Video-enhanced contrast, differential interference contrast (VEC-DIC) microscopy was used to examine the detailed behavior of growth cones on micropatterned laminin substrata. The present study has demonstrated that growth cones can detect and integrate at least two morphogenetic guidance cues simultaneously. The strength of the galvanotropic response in Xenopus growth cones, however, was often insufficient to override established adhesive guidance in this model system.

Models for cocultures of parenchymal (PC) and nonparenchymal cells (NPC) of the liver relied on mixing the cells in a two-dimensional configuration or on establishing spheroidal aggregates. In vivo hepatic nonparenchymal cells, such as endothelial cells and Kupffer cells, are separated from parenchymal cells by extracellular matrix (ECM). Due to their location outside of the space of Disse they can form a barrier toward the sinusoid. Hepatocytes are attached to ECM of the space of Disse via two opposing sinusoidal surfaces. No three-dimensional coculture model reflecting this specific microenvironment of the liver cell plates in vivo has been available to date. We designed a three-dimensional model by positioning NPC on top of PC enclosed as a monolayer within a collagen sandwich. A gas-permeable membrane support can be used to allow the supply of oxygen to the resulting cell plate also from underneath the cell layers. Morphological analysis was performed by inverse and cross-sectional studies by light microscopy, scanning, and transmission electron microscopy of the coculture model. Cuboidal hepatocytes formed confluent layers below the NPC layer. They regularly expressed bile canaliculi at intercellular contact zones. Both sinusoidal surfaces expressed microprojections. Characteristic NPC including endothelial cells, Kupffer cells, and Ito cells completely covered the second matrix layer within a week. Kupffer cells were located on top of endothelial cells. Ito cells were intermingled and could be identified by their intracytoplasmic lipid droplets. LPS stimulation of cocultures resulted in a depression of albumin secretion. Phase I and phase II metabolites of the cytochrome P-450 1A1 substrate ethoxyresorufin were generated independently from the presence of cocultured NPC. This study describes the development of a novel three-dimensional coculture model, which intends to mimic more closely the microenvironment of the hepatic sinusoid by respecting the specific plate structure of the liver parenchyma. The model could serve as a complex tool to study potential collaborations between PC and NPC of the liver.

The synaptonemal complex (SC) is a meiosis-specific, tripartite structure essential for synapsis of homologous chromosomes; it contains a central element positioned between two lateral elements and transversal filaments connecting the lateral elements. In mammals, a major constituent of the transversal filament is known: the SCP1 protein. It contains a long central coiled-coil motif and the molecules are probably organized as dimers, each forming a coiled-coil fiber. We have now developed a new sensitive procedure for immunoelectron microscopy of synaptonemal complex proteins and determined the exact localization of the two nonhelical ends of the SCP1 protein within the mouse synaptonemal complex. We found that the N-terminal end of the SCP1 protein is located within the central element of the synaptonemal complex, whereas the C-terminal end is close to or within the lateral element of the synaptonemal complex. This result supports the notion that SCP1 is an extended filamentous protein and that the two molecules of the putative SCP1 dimer are likely to have the same polarity. The observation that the N-termini are confined to the central element indicated that SCP1 dimers, anchored in opposite lateral elements, could establish contact with each other in the central element via their N-termini. To test this possibility we used the yeast two-hybrid system and found that the N-terminal end of the SCP1 protein indeed strongly interacted with itself, but not with other protein domains tested. We therefore suggest that a transversal filament consists of one or more pairs of SCP1 dimers, each pair being organized in a head-to-head arrangement with the C-termini anchored in the lateral elements and the two N-termini being joined in the central element.

Fibrillar collagens represent the most abundant extracellular matrix components surrounding fibroblasts. Although there is a large heterogeneity in the collagen composition and in the physiological functions of different tissues, interactions between cells and native collagens monomers are mediated by only two integrins, the alpha1beta1 and alpha2beta1 integrins. In tissue, fibroblasts are exposed to collagen polymers, supramolecular assemblies which might play a role on the availability of the cell-binding sites at the surface of the fibrils. We have addressed this issue by investigating the patterns of adhesion structures in normal human skin fibroblasts exposed to collagen monomers or polymers. Our results showed that cell morphology, cell adhesion pattern, actin organization, and distribution of integrin subunits, talin, vinculin, and phosphotyrosine-containing proteins are dependent on the supramolecular organization of the collagens. In particular, compared to monomers, collagen polymers induced a looser organization of the actin network and a linear clustering of integrins, talin, vinculin, and phosphotyrosine-containing proteins. These results emphasize the role of the physical state of collagen on cellular interactions and underline the role of the extracellular matrix in the phenotypic modulation of fibroblasts. Furthermore, our studies suggest the existence of a local heterogeneity in the biological activity of collagen fibrils.

Human fibroblasts cultured in an anchored collagen matrix reorganize the matrix and develop stress. Upon experimental release of the matrix, cells contract the matrix, a phenomenon that has been studied as an in vitro model of wound contraction. We have found that treatment of fibroblasts in stressed collagen matrices with vanadate and platelet-derived growth factor (PDGF) in serum-containing medium inhibits the ability of cells to contract the matrix. Vanadate/PDGF/serum stimulation did not block contraction immediately, but rather resulted in generation of an inhibitory signal that developed over a period of 80 min. The signal was highly specific since other factors such as lysophosphatidic acid and epidermal growth factor were unable to replace PDGF or serum. The presence of vanadate also blocked dephosphorylation of p-Tyr-PDGF receptors after PDGF/serum stimulation and caused accumulation of tyrosine-phosphorylated proteins in the cells. In parallel experiments, fibroblasts in monolayer culture were found to undergo reorganization of their actin cytoskeleton when treated with vanadate in the presence of PDGF and serum-containing medium. Our results suggest that a p-Tyr signaling pathway is important in the regulation of wound contraction.

Through immunohistochemical studies we have identified the cell-surface proteoglycan, NG2, on blood vessels throughout the rat embryo. The particular cell type expressing this chondroitin sulfate proteoglycan, however, is dependent upon tissue location. Microvessels within the rat CNS express NG2 on endothelial cells, while in blood vessels outside the CNS, NG2 is found on smooth muscle cells. To analyze what role NG2 might play in these blood vessels, an enzymatic dissociation protocol was used to establish primary cultures of vascular smooth muscle cells from Postnatal Day 3 rat aorta. In this study we demonstrate the involvement of NG2 in the mitogenic and chemoattractant responses of smooth muscle cells to PDGF. In assays measuring either DNA synthesis or cell migration, treatment of smooth muscle cells with anti-NG2 immunoglobulins decreased their responses to PDGF-AA but had no effect upon their ability to react to PDGF-BB. These results support a role for NG2 in potentiating signaling through the alpha PDGF receptor in vascular smooth muscle cells. The presence of the proteoglycan on a large subpopulation of these cells could provide an enhanced response to the growth factor in times of active normal growth or in pathological conditions, such as arterial injury or atherosclerosis.

Astrocytes in aging periventricular brain regions and in cysteamine-treated neonatal brain cell cultures contain cytoplasmic inclusions that exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic peroxidase activity. In order to delineate the cellular constituents participating in the biogenesis of these astrocytic inclusions, colocalization of FITC-immunolabeled organelles to the red autofluorescent granules was analyzed using a laser scanning confocal imaging system. Areas of true colocalization exhibited yellow fluorescence which persisted in Z-axis image reconstructions. We observed no colocalization of catalase- and PMP70-positive peroxisomes or the Golgi apparatus to the autofluorescent inclusions. The rough endoplasmic reticulum exhibited infrequent dot-like regions of colocalization consistent with previous electron microscopic observations. A minority of early endosomes colocalized to the autofluorescent inclusions in perinuclear cytoplasm. There was extensive colocalization of lgp120-labeled lysosomes to larger autofluorescent granules in close proximity to the nucleus whereas smaller autofluorescent granules often remained unlabeled. A macroautophagic process involving lysosomes and to lesser extent, early endosomes and the rough endoplasmic reticulum, may participate in the biogenesis of autofluorescent astrocytic inclusions in cysteamine-treated glial cultures and in the aging periventricular brain.

Benzylphenoxyethanamine derivatives are known to display antiproliferative activities on tumor cell lines consistently with their binding affinity to the microsomal antiestrogen binding site. In the present study we show that pyrrolidinobenzylphenoxyethanamine, a new efficient compound of this series, exhibits reversible effects on exponentially growing adult bovine aortic endothelial cells inducing (1) lamellated cytoplasmic inclusions, (2) cell proliferation inhibition, (3) dose-dependent transition delay of cells in the G0-G1 phase of the cell cycle. Complete reversal of these effects is achieved only by withdrawing the drug from the medium. The ultrastructural cellular modifications disappeared, and flow cytometry and thymidine incorporation analysis showed the effect on degree of synchronization of this one-step methodology.