Pseudovacuoles--immobilized by high-pressure freezing--are associated with blebbing in walker carcinosarcoma cells.

By applying high pressure freezing and freeze-substitution, we observed large inclusions of homogeneous appearance in the front of locomoting Walker carcinosarcoma cells that have not been described earlier. Live cell imaging revealed that these inclusions were poor in lipids and nucleic acids but had a high lysine (and hence protein) content. Usually one such structure 2-5 mum in size was present at the front of motile Walker cells, predominantly in the immediate vicinity of newly forming blebs. By correlating the lysine-rich areas in fixed and embedded cells with electron microscopic pictures, inclusions could be assigned to confined, faintly stained cytoplasmic areas that lacked a surrounding membrane; they were therefore called pseudovacuoles. After high-pressure freezing and freeze substitution, pseudovacuoles appeared to be filled with 20 nm large electron-transparent patches surrounded by 12 and 15 nm large particles. The heat shock protein Hsp90 was identified by peptide sequencing as a major fluorescent band on SDS-PAGE of lysine-labelled Walker cell extracts. By immunofluorescence, Hsp90 was found to be enriched in pseudovacuoles. Colocalization of the lysine with a potassium-specific dye in living cells revealed that pseudovacuoles act as K+ stores in the vicinity of forming blebs. We propose that pseudovacuoles might support blebbing by locally regulating the intracellular hydrostatic pressure.

Deficiency in parvalbumin, but not in calbindin D-28k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells.

The Ca(2+)-binding proteins parvalbumin (PV) and calbindin D-28k (CB) are key players in the intracellular Ca(2+)-buffering in specific cells including neurons and have profound effects on spatiotemporal aspects of Ca(2+) transients. The previously observed increase in mitochondrial volume density in fast-twitch muscle of PV-/- mice is viewed as a specific compensation mechanism to maintain Ca(2+) homeostasis. Since cerebellar Purkinje cells (PC) are characterized by high expression levels of the Ca(2+) buffers PV and CB, the question was raised, whether homeostatic mechanisms are induced in PC lacking these buffers. Mitochondrial volume density, i.e. relative mitochondrial mass was increased by 40% in the soma of PV-/- PC. Upregulation of mitochondrial volume density was not homogenous throughout the soma, but was selectively restricted to a peripheral region of 1.5 microm width underneath the plasma membrane. Accompanied was a decreased surface of subplasmalemmal smooth endoplasmic reticulum (sPL-sER) in a shell of 0.5 microm thickness underneath the plasma membrane. These alterations were specific for the absence of the "slow-onset" buffer PV, since in CB-/- mice neither changes in peripheral mitochondria nor in sPL-sER were observed. This implicates that the morphological alterations are aimed to specifically substitute the function of the slow buffer PV. We propose a novel concept that homeostatic mechanisms of components involved in Ca(2+) homeostasis do not always occur at the level of similar or closely related molecules. Rather the cell attempts to restore spatiotemporal aspects of Ca(2+) signals prevailing in the undisturbed (wildtype) situation by subtly fine tuning existing components involved in the regulation of Ca(2+) fluxes.

Cytochemical localization of hyaluronan in rat and human skin mast cell granules.

Rat and human skin were processed either by osmium tetroxide/microwave fixation followed by embedding in epoxy resin or by glutaraldehyde/microwave fixation and low-temperature embedding in Lowicryl K4M. Hyaluronan-binding proteins and link proteins (LP) were isolated from bovine nasal cartilage, coupled to 15-20-nm gold particles and employed as markers in a one-step post-embedding procedure for identifying hyaluronan (hyaluronic acid) at the ultrastructural level. Mast cell granules of both species were labeled. The specificity of the hyaluronan-binding probes was demonstrated by treatment of sections with testicular hyaluronidase, Streptomyces hyaluronidase, and chondroitinase ABC, and pre-incubation of probes with hyaluronan oligosaccharides. The results suggest that mast cell granules are a rich source of hyaluronan; this finding may account for the striking concurrence of hyaluronan accumulation with a mastocytotic condition in many tissues undergoing pathologic changes.

Embryonic central nervous system angiogenesis does not involve blood-borne endothelial progenitors.

We asked, whether, in the blood of avian embryos, endothelial precursor cells circulate that actually contribute to the growing vascular system in and around the central nervous system (CNS). We compared the morphology and distribution of QH1-positive cells after transplantation of quail paraxial mesoderm, after blood transfusion, in quail-chick parabiosis, or after quail bone-marrow transplantation. After head mesoderm transplantation from quail to chick, we observed sprouting endothelial cells (ECs), capillary tube formation, and chimeric endothelial lining of large arteries in the host brain. These QH1-positive quail cells showed EC morphologies that demonstrated three different aspects of CNS angiogenesis: invasion by means of filopodia, clonal proliferation and tube formation, and integration into preexisting EC layers. After blood transfusion or in chick-quail parabiosis, blood-borne QH1+ cells were found in the lumen of but not integrated into the wall of the host vascular system. Neither were QH1+ cells observed in the capillary walls of parabiotic chick chorioallantoic membranes. In both cases, the quail cells showed typical macrophage morphology. In chicks that had received quail bone marrow transplants onto their chorioallantoic membranes, QH1+ cells with macrophage, but not EC shape were occasionally seen near the inoculation site. We conclude that (1) blood-borne cells do not become ECs or directly contribute to angiogenesis inside, or in vascular plexuses around the CNS during embryonic development; (2) blood-borne cells do not contribute to the intraneural macrophage population of the embryonic CNS.

Ultrastructural localization of hyaluronan in myelin sheaths of the rat central and rat and human peripheral nervous systems using hyaluronan-binding protein-gold and link protein-gold.

Neural tissue of central (rat spinal cord) and peripheral origin (rat sciatic nerve, nerve fascicles of rat skin and iris and of human conjunctiva) was processed by osmium tetroxide/microwave fixation and embedded in epoxy resin. Hyaluronan-binding proteins and link proteins coupled to 15-20-nm gold particles were used as markers in a one-step post-embedding procedure for identifying hyaluronan (hyaluronic acid) at the ultrastructural level. All myelin sheaths in both rat and human material were found to be intensely labelled. The specificity of the hyaluronan-binding probes was demonstrated by the total loss of labelling following treatment of sections with hyaluronidase or by preincubating either the probes with hyaluronan oligosaccharides or the sections with unlabelled hyaluronan-binding protein. The identified hyaluronan appears to be located extracellularly, but is precise role here remains to be elucidated.

Association of hyaluronan with rat vascular endothelial and smooth muscle cells.

We investigated the distribution of hyaluronan (hyaluronic acid) in rat vascular tissue fixed by an osmium tetroxide (or glutaraldehyde) microwave technique and embedded in epoxy resin (or Lowicryl K4M), using hyaluronan binding proteins coupled to 15-20-nm gold particles as ultrastructural markers in a one-step post-embedding procedure. The intra- and extracellular aspects of vascular endothelial and smooth muscle cell plasma membranes revealed distinct labeling, with a high affinity for caveolae being manifested in both cell types and in all kinds of vessels. Hyaluronan was also localized intercellularly in areas characterized by extensive endothelial cell interdigitation. Intracellularly, moderate staining of nuclear heterochromatin was observed.

Thymic lymphopoiesis: protected from, or influenced by, external stimulation?

The mechanisms regulating thymic lymphopoiesis are still a matter of debate. Intracortical proliferation and differentiation of thymocytes are thought to be controlled by locally produced humoral factors and close contact with epithelial, possibly also phagocytic, cells, and restricted by products of the major histocompatibility complex. The observation of a translocation of intraabdominally introduced PVP-coated silica particles (Percoll) via parathymic lymph vessels and through the thymic capsule into the cortical parenchyma demonstrates that the thymic cortex is accessible to materials carried with the transcapsular flux of interstitial fluid, and that this barrier is less effective than the blood-thymus barrier. The proliferative activity of cortical thymocytes following an intraabdominal injection of particulate tetanus toxoid was compared in sites adjacent to, and distant from, parathymic lymph nodes. Absolute numbers of DNA-synthesizing thymocytes were found to be much higher in cortical areas close to the lymph nodes, where lymphatic vessels are most numerous, than on the opposite sides of the thymic lobes. Taken together, these findings indicate that--in addition to intrinsic control mechanisms--cortical thymocyte production may be influenced by peripheral stimulation to some extent, and that materials from sites which are drained by parathymic lymph nodes may be important in this respect.

Hypothalamic accessory nuclei and their relation to the angiotensinergic and vasopressinergic systems.

The existence and colocalization of angiotensin II- and vasopressin-like immunoreactivity in individual magnocellular cell groups of the hypothalamus has been demonstrated by using immunocytochemical methods. These neurosecretory magnocellular groups consist of the paraventricular nucleus and the supraoptic nucleus, as well as different accessory cell groups. The fibers from the neurons of the accessory nuclei project directly to adjacent blood vessels and do not comigrate with the hypothalamo-neurohypophysial fiber pathway. On the basis of these findings it can be concluded that in the hypothalamus two different angiotensinergic and vasopressinergic neurosecretory systems exist: (1) an intrinsic hypothalamic and (2) a hypothalamo-neurohypophysial system. The distribution of the accessory cell groups in the hypothalamus is shown in a 3D reconstruction which includes the connection of these magnocellular nuclei with the vascular system in this area.

Zone-specific cell biosynthetic activity in mature bovine articular cartilage: a new method using confocal microscopic stereology and quantitative autoradiography.

A new methodology was developed to measure spatial variations in chondrocyte/matrix structural parameters and chondrocyte biosynthetic activity in articular cartilage. This technique is based on the use of a laser scanning confocal microscope that can "optically" section chemically fixed, unembedded tissue. The confocal images are used for morphometric measurement of stereologic parameters such as cell density (cells/mm3), cell volume fraction (%), surface density (l/cm), mean cell volume (micron3), and mean cell surface area (micron2). Adjacent pieces of tissue are simultaneously processed for conventional liquid emulsion autoradiography, and a semiautomated grain counting program is used to measure the silver grain density at regions corresponding to the same sites used for structural measurements. An estimate of chondrocyte biosynthetic activity in terms of grains per cell is obtained by dividing the value for grain density by that for cell density. In this paper, the newly developed methodology was applied to characterize the zone-specific behavior of adult articular cartilage in the free-swelling state. Cylinders of young adult bovine articular cartilage were labelled with either [3H]proline or [35S]sulfate, and chondrocyte biosynthesis and structural parameters were measured from the articular surface to the tidemark. The results showed that chondrocytes of the radial zone occupied twice the volume and surface area of the chondrocytes of the superficial zone but were 10 times more synthetically active. This efficient and unbiased technique may prove useful in studying the correlation between mechanically induced changes in cell form and biosynthetic activity within inhomogeneous tissue as well as metabolic changes in cartilage due to ageing and disease.

Chondrocyte biosynthesis correlates with local tissue strain in statically compressed adult articular cartilage.

In this study, we investigated the depth-dependent metabolic and structural responses of adult articular cartilage to large-strain, static, unconfined compression. Changes in cell biosynthetic activity and several morphometry-based structural parameters (cell density, cell volume fraction, cell surface-area density, mean cell surface area, and mean cell volume) were measured at eight sites representing different depth-zones between the articular surface and the cartilage/bone border. In addition, local axial strain in the superficial, transitional, upper radial, and lower radial zones was estimated on the basis of the change in cell density values. Static compression of articular cartilage revealed a highly heterogeneous deformation profile through the depth of the sample as well as zone-specific changes in biosynthetic activity, as reflected by incorporation of [3H]proline. The axial strains in the top layers were greater than the applied surface-to-surface strain, whereas axial strains adjacent to the cartilage/bone border were significantly less than the applied strain. Zonal changes in cell density and axial strain that occurred during static compression correlated well with alterations in metabolic activity. These coordinated changes between cell biosynthesis and cartilage structure suggest that zone-specific variations in mechanical stimuli could be responsible for spatially varied patterns of cartilage metabolic activity under load.

Morphology of the stromal surface and endothelium using two different microkeratomes.

PURPOSE: To compare stromal surface and endothelial morphology after keratectomies and after laser in situ keratomileusis (LASIK), using two different microkeratomes. METHODS: Keratectomies (160-microm and 400-microm) were performed on 82 enucleated porcine eyes using the Chiron Automated Corneal Shaper (52 eyes) and the Microtech Turbokeratome (30 eyes). LASIK procedures of -9.00 D, -27.00 D, and -36.00 D were performed with a Schwind excimer laser. The corneas were immediately fixed in glutaraldehyde or stained with alizarin red and trypan. Scanning electron microscopy was then performed. RESULTS: All keratectomies performed with the Chiron microkeratome displayed a relatively smooth surface. The quality of the keratectomies with the manually advanced Microtech microkeratome was variable, with a high incidence (4 of 9) of incomplete cuts and irregular surfaces. In the eyes in which the stromal laser ablation was performed, a thin layer of condensed stroma (pseudomembrane formation) was seen. Vital staining did not indicate endothelial damage. CONCLUSIONS: The surface morphology was unacceptable for one of the microkeratomes tested. Keratectomies of 160 to 400 microm and LASIK up to -36.00 D did not acutely alter endothelial morphology in porcine eyes.

Transplantation of cultured adult human or porcine corneal endothelial cells onto human recipients in vitro. Part II: Evaluation in the scanning electron microscope.

PURPOSE: To evaluate the morphology of endothelial monolayers, which have been regrafted onto the denuded Descemet's membrane, with scanning electron microscopy (SEM). METHODS: Material derived from each of the experimental groups described in part I of this investigation was evaluated in the current study. Recipient corneas, denuded of their native endothelium by mechanical, chemical, or physical debridement, were examined to assess the effectiveness of each technique in killing and removing cells. Porcine or human donor corneal endothelial cells maintained in monolayer culture for up to 10 passages then were seeded onto the denuded Descemet's membranes of recipients in the absence or presence of fibroblast growth factor (FGF). The monolayers thereby established were examined in the SEM, and the morphologic status of individual cells compared with that manifested in normal human donor corneas maintained for 4 weeks in organ culture (reference control). Isolated and cultured human keratocytes regrafted onto the denuded Descemet's membranes of recipient corneas served as nonendothelial control specimens. Tissue was processed for examination in the SEM according to standard techniques. RESULTS: Each of the three methods used to strip recipient corneas of their native endothelium was effective and elicited no gross structural damage to Descemet's membrane. Some small focal defects within this latter layer were, however, observed, these being encountered at higher frequency after mechanical debridement than after chemical or physical stripping. Porcine or human endothelial cells seeded onto the denuded Descemet's membranes of recipient corneas formed stable monolayers. The morphologic status of regrafted cells corresponded to that manifested in monolayer cultures before seeding, porcine ones always being more differentiated than their human counterparts. Poorly differentiated human endothelial cells had a slender, elongated, fibroblast-like appearance, whereas more highly differentiated ones manifested broad, flat, polygonal profiles. Monolayers covered the entire corneal surface and impinged to a variable degree onto the trabecular meshwork, at which juncture cells always assumed a less well-differentiated morphology. FGF consistently effected an increase in differentiation status, and as this became augmented, the capacity of monolayers to violate the corneal-trabecular meshwork border was correspondingly repressed. Seeded keratocytes formed dense, multilayered sheaths, resembling retrocorneal membranes, across the entire corneal surface, trabecular meshwork, and iris root. The surface characteristics of the constituent cells were quite distinct from those manifested by endothelial cells, even the least well-differentiated ones. CONCLUSION: Regrafting of human corneal endothelial cells onto the denuded Descemet's membranes of recipients resulted in the formation of stable monolayers. Because the morphologic status of seeded cells closely mimicked that manifested in monolayer cultures before transplantation, it may be anticipated that efforts to refine and optimize culturing conditions would yield improvements in this parameter after regrafting. If these expectations can be realized, then the possibility of successfully establishing a "new" and functional endothelium on recipient corneas destined for clinical grafting may well be brought to fruition in the not-too-distant future.

Assessment of corneal wound repair in vitro.

The time course for the closure of linear wounds produced in cultured cell monolayers following either mechanical debridement or excimer laser photoablation were compared. Wounds were produced in the central zone of a confluent layer of bovine corneal epithelial cells, keratocytes, or endothelial cells, and the residual area of the wound measured at regular intervals. Differences in the rate of wound closure between cell types were demonstrated. For epithelial cells the rate of resurfacing after mechanical or excimer laser damage was similar; for keratocytes there was a lag period before cell slide began after laser injury, but the subsequent rate of resurfacing was then similar to mechanical wounds; only for endothelial layers was a significantly reduced rate of closure observed after photoablation as compared to mechanical wounding. In this model of wound healing it appears that it is the mechanism of removal of the extracellular matrix formed by endothelial cells that produces the major difference in the rate of wound closure between mechanical injury or photoablation.

Protrusive activity, cytoplasmic compartmentalization, and restriction rings in locomoting blebbing Walker carcinosarcoma cells are related to detachment of cortical actin from the plasma membrane.

The dynamic events at the front of locomoting blebbing Walker carcinosarcoma cells [Keller and Bebie, Cell Motil. Cytoskeleton 33:241-251, 1996] are interpreted on the basis of an analysis of the actin cytoskeleton and its relationship to the plasma membrane in fixed cells using a novel double-staining procedure. The data show that blebs are formed where cortical actin is locally depolymerized and/or by detachment of the plasma membrane from more or less intact cortical actin layers. Dissociation between the cortical actin layer and the plasma membrane, which is stimulated by microtubule disassembly, is achieved by forward movement of the plasma membrane, rather than by retraction of the actin layer. Therefore, the detached actin layers form a boundary between the newly forming protrusions and the rest of the cell. They can be associated with "constriction rings," which we have termed "restriction rings." Detached actin layers can impede entry of organelles and the nucleus into the protrusions and thereby compartmentalize the cytoplasm. Later, detached cortical actin layers depolymerize, allowing for relaxation of the restriction rings and for forward movement of cytoplasmic organelles and the nucleus. Actin may repolymerize along the detached plasma membrane allowing for a new cycle to occur. Estimates indicate that the actin polymerization/depolymerization cycles may be largely confined to the front of blebbing cells. The findings suggest that the dynamic events at the front of blebbing metazoan cells are similar to those previously found in Amoeba proteus [Grebecki, Protoplasma, 154:98-111, 1990] but different from those found in lamellipodia.

Actin accumulation in pseudopods or in the tail of polarized walker carcinosarcoma cells quantitatively correlates with local folding of the cell surface membrane.

We determined the actin distribution and the relationship between actin and the cell surface membrane in polarized Walker carcinosarcoma cells showing lamellipodia or blebs at the front in order to get a better insight into actin's role in shape changes and cell locomotion. Using two different techniques, we found that actin is mainly present as a submembraneous layer. The actin concentration detectable in the cytoplasm was about 16X lower. F-actin staining was increased mainly at the contracted tail and to a lesser extent in lamellipodia. However, there is also accumulation of the cell surface membrane at these sites. The quantitative analysis of electron micrographs showed that the apparent accumulation of F-actin at the tail and in the leading lamellipodia was, on the average, fully explained by increased membrane folding. The cell membrane as well as the cortical actin may fold and unfold during shape changes and polarized cells have reserves of plasma membrane as well as of cortical actin at the tail. In addition, the cells may show spots where the surface membrane was dissociated from the cortical actin layer. Polarized cells showed no increase in actin within the blebs or at the basis of lamellipodia. In this respect, the distribution of polymerized actin was different from other currently studied locomoting metazoan cells. So far, the data are difficult to reconcile with models, postulating that polymerized actin within the protrusions is the direct force driving the membrane forward.

Ultrastructural distribution of hyaluronan in rat cornea.

The ultrastructural distribution of hyaluronan (hyaluronic acid) in rat cornea was investigated using tissue processed by osmium tetroxide/microwave fixation and epoxy resin embedding or by glutaraldehyde/microwave fixation and lowicryl K4M embedding. Hyaluronan-binding proteins (HABP) and link proteins (LP) coupled to 15-20 nm gold particles were used as novel ultrastructural markers in a one-step post-embedding procedure. These probes labelled the intra- and extracellular aspects of corneal epithelial and endothelial plasma membranes, as well as those of stromal keratocytes. In the corneal epithelium, wing and superficial cell plasma membranes were intensely stained, whereas basal cells exhibited a weak reaction on their stromal-facing surfaces and an intermediate one on their lateroapical aspects. In the corneal endothelium labelling was associated primarily with the apical and lateral plasma membranes. The labelling intensity associated with keratocyte plasma membranes was weak.

Growth characteristics of central and peripheral bovine corneal epithelial cells in vitro.

Primary cultures of pure bovine corneal epithelial sheets, isolated from either central or peripheral areas of the cornea and grown on an extracellular matrix, exhibited major differences in relation to their respective growth characteristics and morphology. After 15 days in culture, cells of peripheral origin covered a 40% greater area and were 2.75 times more numerous than those of central origin. Most peripheral cells were small with a polygonal morphology, whereas central cells varied considerably in both size and shape, although areas consisting of large cells were regularly observed. Differences in the rates of proliferation between central and peripheral corneal epithelial cells were maintained throughout the first and second subcultures. However, the growth rates were considerably lower in second passage cultures of both central and peripheral cells when compared with those of first passage. The growth characteristics of primary cultures of pure epithelial sheets were confirmed by further studies on corneal buttons in culture. Our results in vitro strongly support the concept of a slow but steady physiological movement of increasingly differentiated cells from the periphery of the cornea towards the centre, resulting in a constant renewal of epithelial cells in vivo.

Ultrastructural association of hyaluronan with rat unmyelinated nerve fibres.

A potential association between hyaluronan and unmyelinated nerve fibres in the PNS (rat skin and iris) and CNS (rat retinal inner plexiform and nerve fibre layers) was investigated at the ultrastructural level using two different hyaluronan-binding probes (link protein-gold and aggrecan-gold). Neuronal and glial cell plasma membranes, as well as the periaxonal space in between, were specifically labelled, suggesting that hyaluronan is secreted by these cells and utilized locally. Intracelluarly, weak labelling of mitochondrial profiles was observed.

Effects of the myosin inhibitor 2,3-butanedione monoxime (BDM) on cell shape, locomotion and fluid pinocytosis in human polymorphonuclear leucocytes.

We investigated the role of myosin in polymorphonuclear leucocyte (PMN) shape changes, locomotion, and fluid pinocytosis using the myosin inhibitor 2,3 butanedione monoxime (BDM). Treatment of resting spherical PMNs with BDM produced spheroid cells showing small continuous shape changes (IC(50)=15.5 m m BDM) and occasionally small blebs. Cell polarity, as induced by the chemotactic peptide fNLPNTL or by colchicine, and locomotion were completely suppressed (IC(50)=8.4 to 10 m m). Suppression of fNLPNTL- or colchicine-induced cell polarity produced spheroid cells, suppression of PMA-induced shape changes and fluid pinocytosis produced non-motile spherical cells (IC(50)=25 to 30 m m BDM). BDM suppressed formation of lamellipodia but not formation of blebs. Suppression of microvilli by BDM as observed in resting spherical cells was partially antagonized by PMA. The results suggest that myosin is involved in stabilizing the shape of resting spherical cells, including microvilli, and that myosin is required for cell polarity, locomotion, fluid pinocytosis and for formation of lamellipodia, but not for formation of blebs.

Relationship between astrocytic processes and "perineuronal nets" in rat neocortex.

"Perineuronal nets" (PNs) ensheath a subtype of inhibitory neurons in the mammalian neocortex. In the light of the proposal that PNs consist of glial processes, we have analyzed the relationship between intracellularly injected glial cells and PNs in the rat neocortex. Glial cells were injected iontophoretically with Lucifer Yellow in lightly fixed tissue slices and PNs were visualized with the lectin from Vicia villosa. Using confocal laser scanning microscopy, glial processes and PNs were identified as distinct structures. Lectin labeling was consistently associated with the extracellular space interposed between LY-labeling was consistently associated with the extracellular space interposed between LY-labeled astrocyte processes and neurons. Of the different types of glial cells injected, only the densely-ramifying protoplasmic astrocytes extended processes which could be traced to contact PNs. These protoplasmic astrocytes also sent out processes to adjacent neurons not ensheathed by PNs, and to capillaries. The present data strongly suggests that PNs do not consist of glial processes but rather support the idea that PNs represent specialized extracellular material interposed between the surface of some inhibitory interneurons and astrocytic processes.