Freeze-fracture of the normal and pathological megakaryocyte lineage in chronic megakaryocytic-granulocytic myelosis

Freeze-fracture and thin sections were performed on human bone marrow of chronic megakaryocytic-granulocytic myelosis (CMGM) to study the three-dimensional fine structure and maturation of normal and atypical megakaryocytes and thrombocytes. In the many normally maturing megakaryocytes the development of the demarcation membrane system (DMS) was best investigated by comparison of thin sections with freeze-fracture replicas. The DMS shows no connections with the Golgi apparatus or rough-surfaced endoplasmic reticulum, but originates from tubular infoldings of the plasma membrane. These infoldings are always in continuity with the extracellular space and form an intracellular membranous pool by branching and coalescing of flattened tubules from which finally the perforated cisternae of the DMS arise. Freeze-fracture of the normal thrombocytes confirms earlier findings. The abnormal giant platelets seen in CMGM display extensive areas of smooth membranes of a spongy structure consisting of dense tubules surrounded by the labyrinth of the surface-connected system. Their physiological significance in these atypical platelets remains unsolved.     

Thrombocytopoiesis--analysis by membrane tracer and freeze-fracture studies on fresh human and cultured mouse megakaryocytes

The origin of platelets (Pt) from megakaryocytes (MK) is beyond question, but the mechanism whereby Pts are released from the precursor cell is still debated. A widely-held theory claims that the MK plasma membrane invaginates to form demarcation membranes (DMS), which delineate Pt territories. Accordingly, Pts would be derived mostly from the periphery of the MK, and the MK and Pt plasma membranes would have to be virtually identical. Since, on morphologic grounds, this theory is untenable, several aspects of thrombocytopoiesis were reexamined with the help of membrane tracer and freeze-fracture analyses of freshly-collected human and cultured mouse MK. To our surprise, freeze-cleavage of the MK plasma membrane revealed that the vast majority of intramembranous particles (IMP) remained associated with the protoplasmic leaflet (P face), whereas the partition coefficient of IMPs of the platelet membrane was the reverse. This is the first time that any difference between MK and Pt membranes has been determined. Replicas of freeze-fractured MK that were in the process of thrombocytopoiesis revealed an additional novel phenomenon, i.e., numerous areas of membrane discontinuity that appeared to be related to Pt discharge. When such areas were small, the IMP were lined up along the margin of the crevice. At a later phase, a labyrinth of fenestrations was observed. Thin sections of MK at various stages of differentiation showed that Pt territories were fully demarcated before connections of the DMS with the surface could be found. Therefore, the Pt envelope is probably not derived from invaginations of the MK plasma membrane. When living, MK were incubated with cationic ferritin or peroxidase at 37 degrees C, the tracers entered into the DMS but did not delineate all membranes with which the DMS was in continuity, suggesting the existence of distinctive membrane domains. Interiorization of tracer was not energy-dependent, but arrested at low temperatures. At 4 degrees C the DMS remained empty, unless there was evidence that Pts had been released. In such instances, the tracers outlined infoldings of peripheral cytoplasm that was devoid of organelles. Thus, the majority of Pts seem to originate from the interior of the MK, and the surface membranes of the two cells differ in origin and structure. The observations do not only throw new light on the process of thrombocytopoiesis, but also strengthen the possibility that MKs and Pts may be subject to different stimuli.     

Wistar Furth Rat Megakaryocytes Lack Dense Compartments and Intercellular Plaques,Membranous Structures Rich in Cytoskeletal Proteins

Wistar Furth (WF) rats have an abnormal thrombopoietic phenotype with morphologically aberrant megakaryocytes, larger than normal mean platelet volume, and platelet alpha-granule protein deficiency. Here, ultrastructural comparisons of WF rat megakaryocytes to those of rats (Wistar) with normal platelet formation during stimulated megakaryocytopoiesis following 5-fluorouracil administration, have revealed a previously unrecognized membrane structure in normal rat megakaryocytes, and two additional abnormalities in WF megakaryocytes. The novel structures were zones of electron density on the cytoplasmic face of apposed plasma membranes of adjoining normal megakaryocytes. These modified focal adhesion-type contacts were distributed at intervals between adjacent megakaryocytes, and were spaced by deposits of extracellular material. These structures also were present between apposed plasma membranes of Wistar rat megakaryocytes in unperturbed marrows, but were absent between megakaryocytes of WF rats. The second WF rat megakaryocyte abnormality is the absence of cytoplasmic dense compartments, another specialized membranous structure that is continuous with the megakaryocyte demarcation membrane system. Both the intercellular plaques and dense compartments of Wistar rat megakaryocytes were found to be rich in cytoskeletal proteins including actin, α-actinin, talin, and vinculin as indicated by ultrastructural immunogold labeling. We hypothesize that an abnormality in cytoskeletal protein function may be responsible for the lack of these structures in the WF rat.     

Emperipolesis--a peculiar feature of megakaryocytes as evaluated in chronic myeloproliferative diseases by morphometry and ultrastructure

A combined morphometric and ultrastructural study was performed on so called emperipolesis or internal wandering of myeloid cells in the cytoplasm of large mature megakaryocytes. Measurements were made on material from a total of 115 patients comprising a normal control group and 5 groups with subtypes of chronic myeloproliferative diseases, including primary (essential, idiopathic) thrombocythemia (PTH). A significant increase in this peculiar phenomenon was noted in myeloproliferative disorders and especially in PTH where the frequency of emperipolesis showed a positive correlation with the number of anuclear cytoplasmic fragments sectioned, with the circular deviation of the shapes of megakaryocytes and with the extent to which the peripheral thrombocyte count was elevated. Electron microscopy in selected cases displayed the integrity of the plasma membranes of engulfed hematopoietic cells within the dilated cavities of the megakaryocytic demarcation membrane system (DMS) and no evidence of phagocytosis. Moreover there was a close relationship between engulfed myeloid cells and the presumptive sites of platelet shedding which had their openings from the cisternal lumina of the DMS. Our results demonstrate that emperipolesis of hematopoietic cells within megakaryocytes should not be regarded as a special nosological feature, but as an indication of enforced thrombocytogenetic activity which is expressed particularly in PTH.     

Disruption of microtubules in vivo by vincristine induces large membrane complexes and other cytoplasmic abnormalities in megakaryocytes and platelets of normal rats like those in human and Wistar Furth rat hereditary macrothrombocytopenias

Abnormal organization of platelet microtubules is associated with abnormal platelet formation in hereditary macrothrombocytopenias such as the gray platelet syndrome, May-Hegglin anomaly, and Epstein's syndrome, and that of the Wistar Furth rat, suggesting that aberrant microtubule organization may contribute to defective platelet formation in these clinical entities. Here, we examined the consequence of microtubule disruption on the organization of megakaryocyte cytoplasmic organelles using the microtubule depolymerizing agent, vincristine (VCR). Wistar rat bone marrow was fixed and processed for transmission electron microscopy after VCR administration alone, after 5-fluorouracil (5-FU) administration alone, or after 5-FU followed by intravenous injection of 0.1–1.0 mg/kg VCR for intervals of 30 min to 8 hr. 5-FU was given to increase megakaryocyte frequency to facilitate ultrastructural evaluations. VCR alone or in combination with 5-FU caused formation of large membrane complexes in the cytoplasm of Wistar rat megakaryocytes at all dosages studied, identical to those found in megakaryocytes of human hereditary macrothrombocytopenias and the Wistar Furth rat. The proportion of megakaryocytes with these large membrane complexes increased with time after 5-FU and VCR, and was maximal (～two-third of megakaryocytes) at VCR dosages of 0.75–1.0 mg/kg. The majorityof megakaryocytes displayed other abnormalities, including blebbing of plasma membranes, an increased number of dense compartments, dilated demarcation membrane (DMS) channels, which contained dense material immunocytochemically identified as secreted α-granule proteins, and an increased incidence of emperipolesis. Rats administered 5-FU alone did not demonstrate these abnormalities, with the exception of an increase in dense compartments. Platelets from rats treated with VCR aloene or 5-FU and VCR also showed abnormalities including membrane complexes, rounded shape, formation of tubulin paracrystals, development of membrane blebs, and the presence of proteinaceous material within the cisternae of the surface-connected canalicular system (SCCS). The membrane complexes in platelets of 5-FU-, VCR-treated Wistar rats as well as untreated Wistar Furth rats were composed of elements of both the SCCS and dense tubular system; membrane complexes in megakaryocytes of 5-FU-, VCR-treated rats were composed of both DMS and smooth endoplasmic reticulum. We conclude that intact microtubules play a major role in the organization of the megakaryocyte DMS and may contribute to the stability of megakaryocyte α-granules. © 1995 Wiley-Liss, Inc.     

Fusion-fission reorganization of membrane: a developing membrane model for thrombocytogenesis in megakaryocytes

Freeze-fracture and thin-section electron microscopy indicate that a sequence of fusion-fission leads to reorganization of membranes and the demarcation of platelets within the cytoplasm of megakaryocyte. Invagination of the megakaryocyte plasma membrane leads to the formation of tubular structures within the cytoplasm of megakaryocytes. Fusion of these tubular membranes in the plane of their long axes is followed by fission in the perpendicular plane. This results in the formation of two flat membranes, forming plasma membranes of two adjacent platelets. A similar fusion-fission reorganization of membranes could mediate a wide variety of other biologic phenomena. These observations also indicate that megakaryocytes are located in the subendothelial compartment of the marrow with their projections penetrating the endothelium and reaching the lumen. This direct contact with the circulation may serve as a means of receiving information as to the requirements of the body for platelet production.     

The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC

Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.     

Expression, activation, and subcellular localization of the Rap1 GTPase in cord blood-derived human megakaryocytes

The expression of the small GTPase Rap1 in human megakaryocytes (MKs) differentiated from cord blood (CB)-derived progenitors was investigated. High levels of Rap1 were detected in the majority of mature megakaryocytes independently of days of culture, while a very low percentage of immature megakaryocytes was found to express a small amount of the protein. Rap1 was predominantly detected on internal alpha-granule but not on the plasma membrane. By contrast, CD41 was clearly present on the peripheral plasma membrane, although it also displayed an intracellular localization similar to that of Rap1. Upon thrombin stimulation, both Rap1 and CD41 translocated to the periphery of the cell. At the opposite, RhoA GTPase and glycoprotein Ibalpha were predominantly located at the plasma membrane and did not undergo relocation upon thrombin stimulation. Thrombin induced a dose- and time-dependent activation of Rap1 in mature megakaryocytes. By using a confocal microscopy approach with a specific probe, active Rap1 was detected exclusively at the peripheral plasma membrane. These results demonstrate that expression of Rap1 occurs during maturation rather than differentiation of megakaryocytes from cord blood progenitor cells. Moreover, we demonstrate that thrombin-activated Rap1 is exclusively localized at the peripheral plasma membrane.     

Biochemical and morphological characterization of a plasma membrane-enriched fraction from bovine parathyroid cells

A fraction of enriched plasma membranes from bovine parathyroid cells has been prepared by differential centrifugation. Biochemical characterization shows that this fraction has a specific activity enrichment of 7.2-fold in ouabain-sensitive Na+-K+ ATPase, and 3.5-fold in 5'-nucleotidase. Less than 4% of the total mitochondria and lysosomes are present within the plasma membranes, while microsomal contamination accounts for 14% of total specific activity. Parathyroid hormone radioimmunoassay also reveals the presence of some secretory granules within the plasma membrane fraction. The characteristic morphological aspect of the unusual surface membrane is shown by freeze-fracture electron microscopy. In the enriched pellets, vesicles identified as having a plasma membrane origin have variable sizes, and 50% show an inside-out conformation. Even though the plasma membrane fraction described herein is not absolutely free from contamination by other subcellular components, this protocol represents the first attempt to purify surface membrane from parathyroid tissue and provide the starting material for understanding, at a molecular level, the properties of extracellular Ca2+ regulation and its coupling with secretion of parathyroid hormone.     

Rapid increase in plasma membrane chloride permeability during wound resealing in starfish oocytes

Plasma membrane wound repair is an important but poorly understood process. We used femtosecond pulses from a Ti-Sapphire laser to make multiphoton excitation-induced disruptions of the plasma membrane while monitoring the membrane potential and resistance. We observed two types of wounds that depolarized the plasma membrane. At threshold light levels, the membrane potential and resistance returned to prewound values within seconds; these wounds were not easily observed by light microscopy and resealed in the absence of extracellular Ca(2+). Higher light intensities create wounds that are easily visible by light microscopy and require extracellular Ca(2+) to reseal. Within a few seconds the membrane resistance is approximately 100-fold lower, while the membrane potential has depolarized from -80 to -30 mV and is now sensitive to the Cl(-) concentration but not to that of Na(+), K(+), or H(+). We suggest that the chloride sensitivity of the membrane potential, after wound resealing, is due to the fusion of chloride-permeable intracellular membranes with the plasma membrane.     

Polarized subcellular distribution of the 1-, 4- and 5-phosphatase activities that metabolize inositol 1,4,5-trisphosphate in intestinal epithelial cells.

In intestinal epithelial cells, Ins(1,4,5)P3 is metabolized both by an intracellular 5-phosphatase and by less specific extracellular phosphatases [Rubiera, Velasco, Michell, Lazo & Shears (1988) Biochem. J. 255, 131-137]. A total of 91% of intracellular Ins(1,4,5)P3 5-phosphatase was particulate, and was preferentially associated with plasma membranes rather than with other subcellular organelles. A soluble Ins(1,4,5)P3 3-kinase activity was also characterized, further supporting the idea that inositol phosphates are important in enterocyte function. We have studied the distribution of Ins(1,4,5)P3 phosphatase activities in basolateral and brush-border domains of the plasma membrane. Compared with homogenates, the extracellular phosphatases were 13-17-fold enriched in brush-border membranes, but only 2-fold enriched in basolateral membranes. The 1- and 4-phosphates of Ins(1,4,5)P3 were hydrolysed at equal rates by the extracellular phosphatases; these enzymes are proposed to have digestive functions. The intracellular particulate 5-phosphatase was 2-fold enriched in brush-border membranes and 13-fold enriched in basolateral membranes, at the same pole of the cell where Ins(1,4,5)P3 is believed to be generated. This is opposite to the polarized distribution of particulate 5-phosphatase in hepatocytes [Shears, Evans, Kirk & Michell (1988) Biochem. J. 256, 363-369]; these differences in subcellular distribution may be important in determining cell-specific metabolism of Ins(1,4,5)P3.     

Dense-cored membranous structures in smooth muscle cells of the vas deferens of the rat

Summary Smooth muscle cells from rat vas deferens were studied by electron microscopy. Vesicular and tubular membranous structures containing an electron-opaque material were found in the smooth muscle cells. Similar structures were also found in a subfraction (F3) of microsomes of vas deferens smooth muscle which was shown to be rich in both plasma membrane and putative endoplasmic reticulum markers. Treatment of the tissues with calcium-free Krebs solution containing EGTA prior to fixation eliminated almost completely the presence of these dense-cored membranous structures (DMS), whereas incubation of the subcellular membrane fraction with EGTA solution had no effect on the appearance of the DMS. Plasma membrane infoldings were found in the smooth muscle cells extending well into their interior. Horseradish peroxidase penetrates vesicles in a location similar to that of DMS in smooth muscle cells, suggesting that some of the DMS may be connected to the extracellular space. We conclude that the dense-core material within the DMS is calcium dependent. We also suggest that some of the DMS represent infoldings of the plasma membrane extending into the cell's interior.     

Biochemical characterization of plasma membranes and intracellular membranes isolated from human platelets using Percoll gradients

Two kinds of membranes (plasma membranes and intracellular membranes) have been separated from human platelets by fractionation on Percoll gradients (successively at pH 7.4 and pH 9.6). On alkaline Percoll gradient, plasma membranes floated at low density, as shown with specific markers such as [3H]concanavalin A and monoacylglycerol lipase, whereas intracellular membranes sedimented in the higher densities and displayed a 5.6-12.4-fold enrichment in NADH diaphorase, antimycin insensitive NADH-cytochrome-c oxidoreductase and Ca2+-ATPase. Another criterion allowing differentiation of two membrane populations of human platelets was their lipid composition, which showed a cholesterol/phospholipid molar ratio of 0.5 in plasma membranes against 0.2 in intracellular membranes. Phospholipid analysis of the two kinds of membranes displayed also quite different profiles, since phosphatidylcholine increased from 30-32% in the plasma membrane to 52-66% in the intracellular membranes. This was at the expense of sphingomyelin (20-23% in plasma membrane, against 6.8-7.7% in intracellular membranes) and of phosphatidylserine (12-13% in plasma membrane, against 2-6% in intracellular membranes). Other striking differences between plasma membranes and intracellular membranes were obtained by SDS-polyacrylamide gel electrophoresis, which revealed the absence of actin and myosin in the intracellular membrane, whereas both proteins were present in significant amounts in plasma membranes. Finally, intracellular membranes but not plasma membranes were able to incorporate calcium. These results suggest that intracellular membrane fractions are derived from the dense tubular system and plasma membranes should correspond to the whole surface membrane of human platelets.     

Effects of gangliosides on cell maturation of murine megakaryocytes in a liquid culture system

Formation of platelet-producing megakaryocytes, the cytoplasm of which showed the terminal stage of cell maturation, heavy granulation and platelet-fields delineated with demarcation membranes, was observed in a short-term culture system, using megakaryocyte-enriched bone marrow cell suspension. Approximately 6-8% of the megakaryocytes changed to the platelet-producing megakaryocytes during 12-hour incubation. In the presence of inhibitors of energy metabolism, formation of the platelet-producing megakaryocytes was inhibited, suggesting that the process is dependent on energy producing systems. Ganglioside GD1a increased both the number of total megakaryocytes and the ratio of the platelet-producing megakaryocytes to total megakaryocytes, while GM1 did not influence the number of total megakaryocytes, but increased the ratio. Gangliosides GM2, GM3 and GD1b showed little effect on either the number of total megakaryocytes or the ratio. The results suggest that ganglioside GD1a stimulates at least two steps of megakaryocyte maturation, the change of megakaryocytic progenitors to megakaryocytes and the subsequent maturation of megakaryocytes to the platelet-producing megakaryocytes, while GM1 stimulates only the latter step of the maturation.     

Sex and age alter plasma membranes of cultured fibroblasts

Human skin fibroblasts were taken from age-matched male and female subjects. The cells were then cultured under identical conditions and passage-number matched. Plasma membranes were isolated and membrane enzyme activities, lipid composition, and structure of isolated plasma membranes were measured in order to determine the presence of significant sex differences in human fibroblast membrane properties. The results indicated that plasma membranes from normal female subjects had a 1.6-fold and 3.6-fold higher cholesterol/phospholipid ratio and oleic acid (18:2) content than normal male subjects. The limiting anisotropy and the rotational relaxation time of fluorescence probe molecules such as trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene in the plasma membranes was not significantly different from fibroblasts of male versus female normal subjects. The total activity of plasma membrane (Na+, K+)-ATPase was significantly higher in female than male normal subjects. A potential 'membrane structural disorder', Huntington's disease, was confirmed in fibroblast membranes from male but not from female Huntington's disease subjects. The possibility that Huntington's disease was a 'premature membrane aging' phenomenon was considered. A comparison of plasma membrane enzymes, lipids, and structure from old and young Huntington's disease subjects did not show differences consistent with accelerated membrane aging as explaining the molecular basis for the disease. The age-dependent differences noted in aged Huntington's disease subjects: increased phosphatidylcholine/phosphatidylethanolamine ratio and sphingomyelin + lysophosphatidylcholine content of fibroblast plasma membranes were not significantly altered when compared to normal age-matched controls. However, (Na+, K+)-ATPase activity was significantly enhanced in fibroblast plasma membranes of older Huntington's disease subjects unlike those of control subjects. In conclusion, sex and age differences in membrane properties of cultured cells represent important potential variables in the elucidation of human genetic disorders that may be membrane-related.     

Effect of diameter on membrane capacity and conductance of sheep cardiac Purkinje fibers

Membrane electrical properties were measured in sheep cardiac Purkinje fibers, having diameters ranging from 50 to 300 mum. Both membrane capacitance and conductance per unit area of apparent fiber surface varied fourfold over this range. Membrane time constant, and capacitance per unit apparent surface area calculated from the foot of the action potential were independent of fiber diameter, having average values of 18.8 +/- 0.7 ms, and 3.4 +/- 0.25 muF/cm2, respectively (mean +/- SEM). The conduction velocity and time constant of the foot of the action potential also appeared independent of diameter, having values of 3.0 +/- 0.1 m/s and 0.10 +/- 0.007 ms. These findings are consistent with earlier suggestions that in addition to membrane on the surface of the fiber, there exists a large fraction of membrane in continuity with the extracellular space but not directly on the surface of the fiber. Combining the electrical and morphological information, it was possible to predict a passive length constant for the internal membranes of about 100 mum and a time constant for chaning these membranes in a passive 100-mum fiber of 1.7 ms.     

Synthesis of NAADP and cADPR in mitochondria.

Here we investigated whether cADPR and NAADP are synthesized in mitochondria. We found that ADPR-cyclase activity is present in mitochondria. In addition, we describe for the first time synthesis of NAADP in this intracellular organelle. ADPR-cyclase activities (V(MAX)) and NAADP synthesis in mitochondria were about 4-fold lower than that in plasma membranes. Otherwise, ADPR-cyclases in mitochondria and in plasma membranes have similar catalytic properties in terms of apparent K(m) for the substrate NGD and K(i) values for inhibition by dithiotreitol, beta-NAD, and nicotinamide. ADPR-cyclase in plasma membranes and to a lesser degree mitochondrial enzyme, was inhibited by Zn(2+) and Cu(2+); ADPR-cyclase from mitochondria was more stable upon thermal inactivation. CD38 antigen, determined by Western blot, was well-expressed in plasma membranes but was far less so (17-fold less) in mitochondria. The major difference between ADPR-cyclase activity in mitochondria and plasma membranes is that mitochondrial cyclase activity was increased by incubation with nonionic detergents. Conversely, the incubation with phosphatidylinositol-specific phosphodiesterase C (PI-PLC) released ADPR-cyclase activity from plasma membranes, but not from mitochondria. We conclude that ADPR-cyclase in mitochondria and in plasma membranes are both multifunctional enzymes with similar catalytic properties; however, the two ADPR-cyclases differ in the mode of anchoring to the membrane: by glycosylphosphoinositol anchor in plasma membranes and by hydrophobic interactions in mitochondria. In addition, synthesis of NAADP can also be found in intracellular organelles via mitochondria. We propose that independent mitochondrial cADPR and NAADP systems may have an intracrine signaling function that is not dependent on direct input by extracellular hormonal stimuli, but rather responds to changes of intermediary cellular metabolism.     

Macrophage adhesion on fibronectin evokes an increase in the elastic property of the cell membrane and cytoskeleton: an atomic force microscopy study

Interactions between cells and microenvironments are essential to cellular functions such as survival, exocytosis and differentiation. Cell adhesion to the extracellular matrix (ECM) evokes a variety of biophysical changes in cellular organization, including modification of the cytoskeleton and plasma membrane. In fact, the cytoskeleton and plasma membrane are structures that mediate adherent contacts with the ECM; therefore, they are closely correlated. Considering that the mechanical properties of the cell could be affected by cell adhesion-induced changes in the cytoskeleton, the purpose of this study was to investigate the influence of the ECM on the elastic properties of fixed macrophage cells using atomic force microscopy. The results showed that there was an increase (~50 %) in the Young’s modulus of macrophages adhered to an ECM-coated substrate as compared with an uncoated glass substrate. In addition, cytochalasin D-treated cells had a 1.8-fold reduction of the Young’s modulus of the cells, indicating the contribution of the actin cytoskeleton to the elastic properties of the cell. Our findings show that cell adhesion influences the mechanical properties of the plasma membrane, providing new information toward understanding the influence of the ECM on elastic alterations of macrophage cell membranes.     

Divergence of mechanisms regulating respiratory burst in blood and sputum eosinophils and neutrophils from atopic subjects

Eosinophil respiratory burst is an important event in asthma and related inflammatory disorders. However, little is known concerning activation of the respiratory burst NADPH oxidase in human eosinophils. Conversely, neutrophils are known to assemble NADPH oxidase in intracellular and plasma membranes. We hypothesized that eosinophils and neutrophils translocate NADPH oxidase to distinct intracellular locations, consistent with their respective functions in O(2)(-)-mediated cytotoxicity. PMA-induced O(2)(-) release assayed by cytochrome c was 3.4-fold higher in atopic human eosinophils than in neutrophils, although membrane-permeable dihydrorhodamine-123 showed similar amounts of release. Eosinophil O(2)(-) release was dependent on Rac, in that it was 54% inhibited by Clostridium difficile toxin B (400-800 ng/ml). In eosinophils stimulated with PMA, a pronounced shift of cytosolic Rac to p22(phox)-positive plasma membrane was observed by confocal microscopy, whereas neutrophils directed Rac2 mainly to intracellular sites coexpressing p22(phox). Similarly, ex vivo sputum eosinophils from asthmatic subjects exhibited predominantly plasma membrane-associated immunoreactivity for Rac, whereas sputum neutrophils exhibited cytoplasmic Rac2 staining. Thus, activated sputum eosinophils, rather than neutrophils, may contribute significantly to the pathogenesis of asthma by extracellular release of tissue-damaging O(2)(-). Our findings suggest that the differential modes of NADPH oxidase assembly in these cells may have important implications for oxidant-mediated tissue injury.     

Carbonic anhydrase II associated with plasma membrane in a human pancreatic duct cell line (CAPAN-1).

The subcellular distribution of carbonic anhydrase II, either throughout the cytosol or in the cytoplasm close to the apical plasma membrane or vesicular compartments, suggests that this enzyme may have different roles in the regulation of pH in intra- or extracellular compartments. To throw more light on the role of pancreatic carbonic anhydrase II, we examined its expression and subcellular distribution in Capan-1 cells. Immunocytochemical analysis by light, confocal, and electron microscopy, as well as immunoblotting of cell homogenates or purified plasma membranes, was performed. A carbonic anhydrase II of 29 kD associated by weak bonds to the inner leaflet of apical plasma membranes of polarized cells was detected. This enzyme was co-localized with markers of Golgi compartments. Moreover, the defect of its targeting to apical plasma membranes in cells treated with brefeldin A was indicative of its transport by the Golgi apparatus. We show here that a carbonic anhydrase II is associated with the inner leaflet of apical plasma membranes and with the cytosolic side of the endomembranes of human cancerous pancreatic duct cells (Capan-1). These observations point to a role for this enzyme in the regulation of intra- and extracellular pH.