Shape transformation and cytoskeletal reorganization in activated non-mammalian thrombocytes

The nucleated thrombocytes of non-mammalian vertebrates are partially flattened, ovoid cells morphologically distinct from mammalian platelets, and the extent of their functional equivalence is unknown. To test whether they resemble platelets in having similar F-actin-based post-activation stages, rapid fixation/extraction/labeling methods were developed to reveal cytoskeletal organization in dogfish thrombocytes by confocal microscopy. Unactivated cells contained cortical F-actin plus denser F-actin co-localizing with outer marginal band (MB) microtubules. In the post-activation sequence, determined for the first time by continuous observation of individual thrombocytes following thrombin perfusion, cells rounded and blebbed, spread, and eventually flattened extensively. The MB twisted and then became disorganized, with microtubule bundles remaining centrally located and associated with nuclear clefts. In contrast, F-actin occupied blebs and outward-spreading cytoplasm, initially in spiky projections, then predominantly in stress fibers, and inhibitors of F-actin assembly or myosin ATPase blocked shape changes. Thus, the post-activation stages and cytoskeletal events observed in nucleated thrombocytes were found to parallel those of platelets.     

Molecular organization and in vivo function of the cytoskeleton of amphibian erythrocytes

One prominent cytoskeletal feature of non-mammalian vertebrate erythrocytes is the marginal band (MB), composed of microtubules. However, there have been several reports of MB-associated F-actin. We have further investigated the function of MB-associated F-actin, using newt erythrocytes having large, thick MBs. Confocal microscopy revealed a distinctive band of F-actin colocalizing point- by-point with MB microtubules. Furthermore, the F-actin band was present in isolated elliptical MBs, but absent in membrane skeletons lacking MBs. F-actin depolymerizing agents did not affect F-actin band integrity in isolated MBs, indicating its non-dynamic state. However, exposure to elastase resulted in F-actin removal and MB circularization. These results provide evidence of a strong association of F-actin with MB microtubules in mature ellipsoidal erythrocytes. To assess the true extent of mechanical stress on the cytoskeleton, erythrocytes were observed by video microscopy during flow in vivo. Moving with long axis parallel to flow direction, cells underwent reversible shape distortion as they collided vigorously with other erythrocytes and vessel walls. In addition, cells twisted into figure-8 shapes, a cytoskeletal property that may provide physiological advantages during flow. Our results, together with those of others, yield a consistent picture in which developing erythrocytes undergo transition from spheroids to immature discoids to mature ellipsoids. The causal step in discoid formation is biogenesis of circular MBs with sufficient flexural rigidity to determine cell shape. F-actin binding to MB microtubules then creates a composite system, enhancing flexural rigidity to produce and maintain ellipsoidal shape during the physical challenges of blood flow in vivo.     

Isolation and studies of the granules of the amebocytes of Limulus polyphemus,the horseshoe crab

Granules were isolated from the cytoplasm of the amebocytes of Limulus polyphemus, the horseshoe crab, by disruption of cells obtained from blood which had been drawn into 2 mM propranolol. The granules subsequently were purified by centrifugation through a sucrose gradient that contained heparin. Extracts of the granules were prepared by freezing and thawing the granule preparations in distilled water. Transmission and scanning electron microscopy of the granules revealed round or ovoid particles. However, only one type of granule appeared to be present. The ultraviolet spectrum of the extract of amebocyte granules demonstrated a peak at 277 nm at pH 7.4, and a shift into two peaks of 281 nm and 290 nm at alkaline pH. Analytical ultracentrifugation revealed a pattern similar to that observed with lysates prepared from intact amebocytes. Polyacrylamide gel electrophoresis, in the presence of urea at pH 4.5, demonstrated patterns similar to those observed with amebocyte lysate. Extracts of the granules were gelled by bacterial endotoxin. The blood of the horseshoe crab contains only one type of cell, the amebocyte. Previous studies have shown that the blood coagulation mechanism of Limulus is contained entirely within amebocytes. The current studies suggest that the granules, which pack the cytoplasm of these cells, contain all of the factors required for the coagulation of blood, including the clottable protein. The intracellularly localized coagulation system is released from amebocytes when their granules rupture during cell aggregation.     

Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.     

Association of centrioles with the marginal band of a molluscan erythrocyte

Continuous circumferential bundles of microtubules, or marginal bands (MBs), are best known as a prominent structural feature of all nonmammalian vertebrate erythrocytes and mammalian blood platelets. Since their discovery in the late 19th century, MBs have been thought to play a cellular morphogenetic role, but no cytological clues to the mechanism of MB biogenesis have been reported. In previous work we have established the presence of MBs in serveral invertebrate blood cell types, including amebocytes and coelomocytes of certain Arthropod species and erythrocytes of a Sipunculan. We report here the occurrence of MBs in erythrocytes of the ark Anadara transversa (Mollusca) and four closely related species. The MBs of these arks have a striking structural feature; each is physically associated with a pair of centrioles. The centrioles are identified as such on the basis of morphological criteria: size, cylindrical shape, right-angle orientation, pairing, and 9-triplet ultrastructure. This intimate association between centrioles and MBs suggests that centrioles may be MB-organizing centers and invites comparative investigation of their possible role in vertebrate erythrocyte and platelet morphogenesis.     

Endotoxin-induced degranulation of the Limulus amebocyte

Exocytosis and gelation of the granule contents of the amebocyte of Limulus polyphemus have been studied in a perfusion chamber observed with Nomarski differential interference contrast microscopy. Degranulation in response to bacterial endotoxin or the ionophore A23187 was significantly inhibited by the anion channel blocking agents suramin, SITS, DNDS and sodium isethionate. db-cAMP, PGI₂ and theophylline also succeeded in imparing degranulation of the amebocytes. All of the agents tested produced inhibition of degranulation which was readily reversed by washing the system free of the inhibitor and rechallenging the amebocytes with either endotoxin or the ionophore. After isolation in vitro, amebocytes underwent spontaneous degranulation in the absence of endotoxin at a rate 1–2 orders of magnitude slower than in the presence of endotoxin. Gelation of the clottable protein released from the amebocyte granules could occur in the absence of endotoxin. This is the first demonstration of gelation under endotoxin-free conditions.     

Colocalization of cortical microtubules and F-actin in<Emphasis Type="Italic">Dipodascus magnusii</Emphasis> using confocal laser scanning microscopy

Distribution of microtubules and F-actin in aerobically growing cells of Dipodascus magnusii, belonging to the class Saccharomycetes was analyzed using immunofluorescence microscopy and labeling with rhodamine-tagged phalloidin. A conspicuous system of permanent cytoplasmic microtubules was observed in association with multiple nuclei. In elongating cells, helices of cytoplasmic microtubules appeared at the cell cortex. In cells approaching cytokinesis transversely oriented microtubules were revealed at incipient division sites. Confocal laser scanning microscopy showed a continuity of these transverse microtubules with the remaining microtubule network. The actin system of D. magnusii consisted of patches and filaments. Patches were found to accumulate at the tips of growing cells. Bands of fine actin filaments were usually observed before F-actin rings were established. A close cortical association of microtubules with the F-actin ring was documented on individual optical sections of labeled cells. Cells with developing septa showed medial F-actin discs associated at both sides with microtubules. Colocalization of cytoplasmic microtubules with actin filaments at the cortex of dividing cells supports a role of both cytoskeletal components in controlling cell wall growth and septum formation in D. magnusii.     

Accumulation of Glucosylceramide in the Absence of the Beta-Glucosidase GBA2 Alters Cytoskeletal Dynamics

Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types.     

Culture of amebocytes in a nutrient mist bioreactor

Summary We report the first use of nutrient mist bioreactor (NMB) technology to culture animal cells. The nutrient mist approximated the amebocyte stem tissue’s natural environment, which is a thin layer of fluid in the gill leaflets of the horseshoe crabLimulus polyphemus. NMB culture was tried in an attempt to increase production of amebocytes, which are the source of theLimulus Amebocyte Lysate (LAL), the basis for a sensitive and commercially valuable endotoxin assay. Amebocyte growth in the nutrient mist bioreactor is comparable to growth in liquid medium. However, the current design of the bioreactor presents problems for primary cultures such as ours where a pyrogen-free environment is necessary and fungal decontamination is difficult.     

Posttranslationally modified tubulins and microtubule organization in hemocytes of the brine shrimp, Artemia franciscana

Crustaceans possess blood cells (hemocytes) that mediate organismal defense and are analogous to vertebrate leukocytes. In order to more fully characterize these types of cells, hemocytes of the branchiopod crustacean, Artemia franciscana, were analyzed. The data indicate that Artemia have one type of hemocyte, ranging in morphology from compact and spherical to flat and spreading when examined in vitro. Electron microscopy revealed many cytoplasmic granules in the hemocytes and only a limited number of other membrane-bound organelles. Centrioles and microtubules were also visible in thin sections of chemically fixed samples. The cytoplasm of spherical hemocytes was completely labeled by general antitubulin antibodies, but in flattened hemocytes packing of cytoskeletal elements was less tight and individual microtubules were observed. Probing of Western blots disclosed acetylated, tyrosinated, and detyrosinated tubulin isoforms in hemocyte homogenates, the first characterization of posttranslationally modified tubulins in this cell type. Acetylated tubulin was restricted to a subset of microtubules, whereas tyrosinated microtubules were displayed more abundantly. Staining obtained with antibody to detyrosinated tubulin was unusual because it was limited to the perinuclear region of hemocytes. Incubation of blood cells with a monoclonal antibody to gamma-tubulin yielded fluorescent dots sometimes in pairs, a pattern characteristic of centrosomes. The findings support the conclusion that Artemia hemocytes undergo rapid morphogenesis in vitro accompanied by extensive rearrangement of their microtubules, the latter probably indicative of cytoskeletal changes that occur during cell movement and phagocytosis. Additionally, the hemocytes contain posttranslationally modified alpha-tubulins and centrosome-associated gamma-tubulin, both with the potential to influence microtubule organization and function.     

Determination of bacterial number and biomass in the marine environment.

Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.     

Determination of bacterial number and biomass in the marine environment.

Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.     

Morphological alterations and cytoskeletal reorganization in opossum kidney (OK) cells during osmotic swelling and volume regulation

Cells from a variety of tissues regulate their volume when exposed to anisotonic conditions. After exposure of cells to hypotonic conditions, the rapid phase of cell swelling is followed by a slower phase of cell shrinkage towards the initial volume. The present study investigates morphological alterations of adherent and fully spread cells after exposure to hypotonic conditions and the reorganization of cytoskeletal components such as F-actin, actin-binding proteins, microtubules and intermediate-sized filaments. We used cells of a continuous epithelial cell line from the opossum kidney (OK cells), which were exposed to hypotonic conditions for a period of 60 min at 25° C. The osmolarity was reduced by 40% from 320 mosmol/l (isotonic conditions) to 192 mosmol/l (hypotonic conditions). The initial swelling after exposure of OK cells to hypotonic conditions caused enhanced ruffling membrane activity, formation of lamellipodia and an extended space between adjacent cells which was caused by a more rounded cell shape. Moreover, the height of cells located in the centre of cell clusters increased by 32±8% (mean value±SEM) as checked by morphometric analysis of the vertical distance between the apical and basolateral F-actin domain. Although the fluorescence intensity and organization of F-actin in a horizontal direction remained unaltered during cell swelling, we observed a loss of periodicity and irregular distribution of myosin aggregates and a partial rear-rangement of vimentin filaments in the form of short fragments. In all experiments the organization of microtubules was observed to be unaltered. The alterations described above were reversible during cell shrinkage towards the initial volume, i.e. at 60 min after exposure to hypotonic conditions cell morphology and cytoskeletal organization no longer differed from the corresponding controls which were kept under isotonic conditions for the whole experimental period. The results demonstrate that only certain intracellular cytoskeletal components are actively involved in cell swelling and regulatory volume decrease.     

The cytoskeletal system of nucleated erythrocytes. I. Composition and function of major elements

We have studied the dogfish erythrocyte cytoskeletal system, which consists of a marginal band of microtubules (MB) and trans-marginal band material (TBM). The TBM appeared in whole mounts as a rough irregular network and in thin sections as a surface-delimiting layer completely enclosing nucleus and MB. In cells incubated at 0 degrees C for 30 min or more, the MB disappeared but the TBM remained. MB reassembly occurred with rewarming, and was inhibited by colchicine. Flattened elliptical erythrocyte morphology was retained even when MBs were absent. Total solubilization of MB and TBM at low pH, or dissolution of whole anucleate cytoskeletons, yielded components comigrating with actin, spectrin, and tubulin standards during gel electrophoresis. Mass-isolated MBs, exhibiting ribbonlike construction apparently maintained by cross-bridges, contained four polypeptides in the tubulin region of the gel. Only these four bands were noticeably increased in the soluble phase obtained from cells with 0 degrees C- disassembled MBs. The best isolated MB preparations contained tubulin but no components comigrating with high molecular weight microtubule- associated proteins, spectrin, or actin. Actin and spectrin therefore appear to be major TBM constituents, with tubulin localized in the MB. The results are interpreted in terms of an actin- and spectrin- containing subsurface cytoskeletal layer (TBM), related to that of mammalian erythrocytes, which maintains cell shape in the absence of MBs. Observations on abnormal pointed erythrocytes containing similarly pointed MBs indicate further that the MB can deform the TBM from within so as to alter cell shape. MBs may function in this manner during normal cellular morphogenesis and during blood flow in vivo.     

Cytoskeletal interactions of synapsin I in non-neuronal cells

Synapsin I is a neuronal phosphoprotein involved in the localization and stabilization of synaptic vesicles. Recently, synapsin I has been detected in several non-neuronal cell lines, but its function in these cells is unclear. To determine the localization of synapsin I in non-neuronal cells, it was transiently expressed in HeLa and NIH/3T3 cells as an enhanced green fluorescent protein fusion protein. Synapsin I-enhanced green fluorescent protein colocalized with F-actin in both cell lines, particularly with microspikes and membrane ruffles. It did not colocalize with microtubules or vimentin and it did not cause major alterations in cytoskeletal organization. Synapsin Ia-enhanced green fluorescent protein colocalized with microtubule bundles in taxol-treated HeLa cells and with F-actin spots at the plasma membrane in cells treated with cytochalasin B. It did not noticeably affect F-actin reassembly following drug removal. Synapsin Ia-enhanced green fluorescent protein remained colocalized with F-actin in cells treated with nocodazole, and it did not affect reassembly of microtubules following drug removal. These results demonstrate that synapsin I interacts with F-actin in non-neuronal cells and suggest that synapsin I may have a role in regions where actin is highly dynamic.     

Distinct sites in E-cadherin regulate different steps in Drosophila tracheal tube fusion

We have investigated how E-cadherin controls the elaboration of adherens junction associated cytoskeletal structures crucial for assembling tubular networks. During Drosophila development, tracheal branches are joined at branch tips through lumens that traverse doughnut-shaped fusion cells. Fusion cells form E-cadherin contacts associated with a track that contains F-actin, microtubules, and Shot, a plakin that binds F-actin and microtubules. Live imaging reveals that fusion occurs as the fusion cell apical surfaces meet after invaginating along the track. Initial track assembly requires E-cadherin binding to beta-catenin. Surprisingly, E-cadherin also controls track maturation via a juxtamembrane site in the cytoplasmic domain. Fusion cells expressing an E-cadherin mutant in this site form incomplete tracks that contain F-actin and Shot, but lack microtubules. These results indicate that E-cadherin controls track initiation and maturation using distinct, evolutionarily conserved signals to F-actin and microtubules, and employs Shot to promote adherens junction-associated cytoskeletal assembly.     

Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria

We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > approximately 0.7 microm in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at approximately 1 microm/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.     

Limulus amebocyte clotting cascade: Roles of endotoxin and adenylate cyclase

Endotoxin, the lipopolysaccharide from the cell wall of Gram-negative bacteria, causes blood clotting in the horseshoe crab,Limulus polyphemus. Minute amounts of endotoxin stimulate the amebocytes in the blood to undergo exocytosis, which release the contents of their secretory granules to form a clot. An endotoxin-binding protein that possesses calmodulin-like activity has been isolated from the amebocyte plasma membrane. This endotoxin-binding protein can activate adenylate cyclase fromBordetella pertussis to the same extent as rat testes calmodulin. The effect of endotoxin and the endotoxin-binding protein on cyclic AMP synthesis inLimulus amebocytes was examined. Amebocytes exposed to endotoxin have increased levels of intracellular cyclic AMP. Amebocyte membranes contain an adenylate cyclase which is stimulated by NaF, guanosine (β,r-imido)triphosphate, and for skolin. This adenylate cyclase is also stimulated by the endotoxin-binding protein and calcium. Exposure of amebocytes to forskolin or dibutyryl cyclic AMP are stimulated to secrete clot components. Activation of adenylate cyclasein vivo by endotoxin via the endotoxin-binding protein may be one of the ways in which endotoxin stimulates secretion. It is suggested that endotoxin may have two actions in theLimulus system: (1) binding of endotoxin to the endotoxin-binding protein activates adenylate cyclase, promoting secretion by the amebocytes; and (2) endotoxin catalyzes a reaction on the secreted material to form a blood clot. This latter reaction is not elicited by forskolin or dibutyryl cyclic AMP.     

Microtubules and actin cytoskeleton in Cryptococcus neoformans compared with ascomycetous budding and fission yeasts

Actin cytoskeleton and microtubules were studied in a human fungal pathogen, the basidiomycetous yeast Cryptococcus neoformans (haploid phase of Filobasidiella neoformans), during its asexual reproduction by budding using fluorescence and electron microscopy. Staining with rhodamine-conjugated phalloidin revealed an F-actin cytoskeleton consisting of cortical patches, cables and cytokinetic ring. F-actin patches accumulated at the regions of cell wall growth, i. e. in sterigma, bud and septum. In mother cells evenly distributed F-actin patches were joined to F-actin cables, which were directed to the growing sterigma and bud. Some F-actin cables were associated with the cell nucleus. The F-actin cytokinetic ring was located in the bud neck, where the septum originated. Antitubulin TAT1 antibody revealed a microtubular cytoskeleton consisting of cytoplasmic and spindle microtubules. In interphase cells cytoplasmic microtubules pointed to the growing sterigma and bud. As the nucleus was translocated to the bud for mitosis, the cytoplasmic microtubules disassembled and were replaced by a short intranuclear spindle. Astral microtubules then emanated from the spindle poles. Elongation of the mitotic spindle from bud to mother cell preceded nuclear division, followed by cytokinesis (septum formation in the bud neck). Electron microscopy of ultrathin sections of chemically fixed and freeze-substituted cells revealed filamentous bundles directed to the cell cortex. The bundles corresponded in width to the actin microfilament cables. At the bud neck numerous ribosomes accumulated before septum synthesis. We conclude: (i) the topology of F-actin patches, cables and rings in C. neoformans resembles ascomycetous budding yeast Saccharomyces, while the arrangement of interphase and mitotic microtubules resembles ascomycetous fission yeast Schizosaccharomyces. The organization of the cytoskeleton of the mitotic nucleus, however, is characteristic of basidiomycetous yeasts. (ii) A specific feature of C. neoformans was the formation of a cylindrical sterigma, characterized by invasion of F-actin cables and microtubules, followed by accumulation of F-actin patches around its terminal region resulting in development of an isodiametrical bud.