Vacuolar Rupture Research Articles

Changes in Cytosolic ATP Levels and Intracellular Morphology during Bacteria-Induced Hypersensitive Cell Death as Revealed by Real-Time Fluorescence Microscopy Imaging

Hypersensitive cell death is known to involve dynamic remodeling of intracellular structures that uses energy released during ATP hydrolysis. However, the relationship between intracellular structural changes and ATP levels during hypersensitive cell death remains unclear. Here, to visualize ATP dynamics directly in real time in individual living plant cells, we applied a genetically encoded Förster resonance energy transfer (FRET)-based fluorescent ATP indicator, ATeam1.03-nD/nA, for plant cells. Intracellular ATP levels increased approximately 3 h after inoculation with the avirulent strain DC3000/avrRpm1 of Pseudomonas syringae pv. tomato (Pst), which was accompanied by the simultaneous disappearance of transvacuolar strands and appearance of bulb-like structures within the vacuolar lumen. Approximately 5 h after bacterial inoculation, the bulb-like structures disappeared and ATP levels drastically decreased. After another 2 h, the large central vacuole was disrupted. In contrast, no apparent changes in intracellular ATP levels were observed in the leaves inoculated with the virulent strain Pst DC3000. The Pst DC3000/avrRpm1-induced hypersensitive cell death was strongly suppressed by inhibiting ATP synthesis after oligomycin A application within 4 h after bacterial inoculation. When the inhibitor was applied 7 h after bacterial inoculation, cell death was unaffected. These observations show that changes in intracellular ATP levels correlate with intracellular morphological changes during hypersensitive cell death, and that ATP is required just before vacuolar rupture in response to bacterial infection.

The protein SdhA maintains the integrity of the Legionella -containing vacuole

Legionella pneumophila directs the formation of a specialized vacuole within host cells, dependent on protein substrates of the Icm/Dot translocation system. Survival of the host cell is essential for intracellular replication of L. pneumophila. Strains lacking the translocated substrate SdhA are defective for intracellular replication and activate host cell death pathways in primary macrophages. To understand how SdhA promotes evasion of death pathways, we performed a mutant hunt to identify bacterial suppressors of the ΔsdhA growth defect. We identified the secreted phospholipase PlaA as key to activation of death pathways by the ΔsdhA strain. Based on homology between PlaA and SseJ, a Salmonella protein associated with vacuole degradation, we determined the roles of SdhA and PlaA in controlling vacuole integrity. In the absence of sdhA, the Legionella-containing vacuole was unstable, resulting in access to the host cytosol. Both vacuole disruption and host cell death were largely dependent on PlaA. Consistent with these observations, the ΔsdhA strain colocalized with galectin-3, a marker of vacuole rupture, in a PlaA-dependent process. Access of ΔsdhA strains to the macrophage cytosol triggered multiple responses in the host cell, including degradation of bacteria, induction of the type I IFN response, and activation of inflammasomes. Therefore, we have demonstrated that the Legionella-containing vacuole is actively stabilized by the SdhA protein during intracellular replication. This vacuolar niche affords the bacterium protection from cytosolic host factors that degrade bacteria and initiate immune responses.

Non-apoptotic programmed cell death with paraptotic-like features in bleomycin-treated plant cells is suppressed by inhibition of ATM/ATR pathways or NtE2F overexpression

In plants, different forms of programmed cell death (PCD) have been identified, but they only partially correspond to those described for animals, which is most probably due to structural differences between animal and plant cells. Here, the results show that in tobacco BY-2 cells, bleomycin (BLM), an inducer of double-strand breaks (DSBs), triggers a novel type of non-apoptotic PCD with paraptotic-like features. Analysis of numerous PCD markers revealed an extensive vacuolization, vacuolar rupture, and chromatin condensation, but no apoptotic DNA fragmentation, fragmentation of the nuclei, or sensitivity to caspase inhibitors. BLM-induced PCD was cell cycle regulated, occurring predominantly upon G(2)/M cell cycle checkpoint activation. In addition, this paraptotic-like PCD was at least partially inhibited by caffeine, a known inhibitor of DNA damage sensor kinases ATM and ATR. Interestingly, overexpression of one NtE2F transcriptional factor, whose homologues play a dual role in animal apoptosis and DNA repair, reduced PCD induction and modulated G(2)/M checkpoint activation in BY-2 cells. These observations provide a solid ground for further investigations into the paraptotic-like PCD in plants, which might represent an ancestral non-apoptotic form of PCD conserved among animals, protists, and plants.

Changes in ultrastructure, protease and caspase-like activities during flower senescence in Lilium longiflorum

The last phase of flower development is senescence during which nutrients are recycled to developing tissues. The ultimate fate of petal cells is cell death. In this study we used the ethylene-insensitive Lilium longiflorum as a model system to characterize Lily flower senescence from the physiological, biochemical and ultrastructural point of view. Lily flower senescence is highly predictable: it starts three days after flower opening, before visible signs of wilting, and ends with the complete wilting of the corolla within 10 days. The earliest events in L. longiflorum senescence include a fall in fresh and dry weight, fragmentation of nuclear DNA and cellular disruption. Mesophyll cell degradation is associated with vacuole permeabilization and rupture. Protein degradation starts later, coincident with the first visible signs of tepal senescence. A fall in total protein is accompanied by a rise in total proteases, and also by a rise of three classes of caspase-like activity with activities against YVAD, DEVD and VEID. The timing of the appearance of these caspase-like activities argues against their involvement in the regulation of the early stages of senescence, but their possible role in the regulation of the final stages of senescence and cell death is discussed.

New Stages in the Program of Malaria Parasite Egress Imaged in Normal and Sickle Erythrocytes

The apicomplexan parasite Plasmodium falciparum causes malignant malaria. The mechanism of parasite egress from infected erythrocytes that disseminate parasites in the host at the end of each asexual cycle is unknown. Two new stages of the egress program are revealed: (1) swelling of the parasitophorous vacuole accompanied by shrinkage of the erythrocyte compartment, and (2) poration of the host cell membrane seconds before erythrocyte rupture because of egress. Egress was inhibited in dehydrated cells from patients with sickle cell disease in accord with experimental dehydration of normal cells, suggesting that vacuole swelling involves intake of water from the erythrocyte compartment. Erythrocyte membrane poration occurs in relaxed cells, thus excluding involvement of osmotic pressure in this process. Poration does not depend on cysteine protease activity, because protease inhibition blocks egress but not poration, and poration is required for the parasite cycle because the membrane sealant P1107 interferes with egress. We suggest the following egress program: parasites initiate water influx into the vacuole from the erythrocyte cytosol to expand the vacuole for parasite separation and vacuole rupture upon its critical swelling. Separated parasites leave the erythrocyte by breaching its membrane, weakened by putative digestion of erythrocyte cytoskeleton and membrane poration.

Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuolar rupture in real time by fluorescence microscopy

Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuolar rupture in real time by fluorescence microscopy

Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuole rupture in real time

Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen-induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram-negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin-3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high-content and high-throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.

PETAL SENESCENCE: NEW CONCEPTS FOR AGEING CELLS

Senescence in flower petals can be regarded as a form of programmed cell death (PCD), being a process where cells or tissues are broken down in an orderly and predictable manner, whereby nutrients are re-used by other cells, tissues or plant parts. The process of petal senescence shows many similarities to autophagic PCD in animal cells including a massive breakdown of protein, DNA and RNA, the formation of autophagic vacuoles for the breakdown of cytoplasm and organelles therein and, the eventual rupture of these vacuoles that kills the cell. Chromatin condensation and DNA and nuclear fragmentation (traditionally considered being apoptotic-like features) are observed in both autophagic animal cells and in senescing petal cells. We present a conceptional model underlying petal senescence that integrates elements that have been associated with both apoptotic and autophagic types of PCD

Liver Enzymes and Ultrastructure in Rabbit Haemorrhagic Disease (RHD)

Rabbit haemorrhagic disease (RHD) is caused by a calicivirus infection that kills most adult rabbits 24-72 h after viral inoculation. Two liver enzymes (AST, aspartate aminotransferase, and ALT, alanine aminotransferase) were monitored in blood samples of calicivirus-infected rabbits during the short course of RHD. Values of AST were used to differentiate three stages of hepatocellular degeneration in RHD: mild (up to 20-fold increase in AST), moderate (150-200-fold elevation of AST) and severe (more than 1000-fold elevation in AST). Liver samples of rabbits from these three biochemical stages of hepatocellular degeneration of RHD were studied by transmission electron microscopy to define the fine structure of the hepatocytes. In the mild hepatocellular degeneration there was proliferation (microvesiculation) of the smooth endoplasmic reticulum and swelling of mitochondria into spheroid bodies with loss of cristae. In moderate hepatocellular degeneration, vacuolization of cytoplasm and mitochondrial damage continued to be present, and there was also formation of autophagic vesicles. In the severe hepatocellular degeneration of RHD, the altered mitochondria also showed loss of density of their matrix; rupture of cytoplasmic vacuoles led to the formation of large vesicles. Marked depletion of liver glycogen was also found in this late stage of RHD. These data offer a correlation between biochemical and cytological features of the liver during the hepatocellular degeneration of RHD.

Epithelial transformations in the establishment of the blood–gas barrier in the developing chick embryo lung

The tall epithelium of the developing chick embryo lung is converted to a squamous one, which participates in formation of the thin blood-gas barrier. We show that this conversion occurred through processes resembling exocrine secretion. Initially, cells formed intraluminal protrusions (aposomes), and then transcellular double membranes were established. Gaps between the membranes opened, thus, severing the aposome from the cell. Alternatively, aposomes were squeezed out by adjacent cells or were spontaneously constricted and extruded. As a third mechanism, formation and fusion of severed vesicles or vacuoles below the aposome and their fusion with the apicolateral plasma membrane resulted in severing of the aposome. The atria started to form by progressive epithelial attenuation and subsequent invasion of the surrounding mesenchyme at regions delineated by subepithelial alpha-smooth muscle actin-positive cells. Further epithelial attenuation was achieved by vacuolation; rupture of such vacuoles with resultant numerous microfolds and microvilli, which were abscised to accomplish a smooth squamous epithelium just before hatching.

Selective Inhibition of a Two-step Egress of Malaria Parasites from the Host Erythrocyte

Escape from the host erythrocyte by the invasive stage of the malaria parasite Plasmodium falciparum is a fundamental step in the pathogenesis of malaria of which little is known. Upon merozoite invasion of the host cell, the parasite becomes enclosed within a parasitophorous vacuole, the compartment in which the parasite undergoes growth followed by asexual division to produce 16-32 daughter merozoites. These daughter cells are released upon parasitophorous vacuole and erythrocyte membrane rupture. To examine the process of merozoite release, we used P. falciparum lines expressing green fluorescent protein-chimeric proteins targeted to the compartments from which merozoites must exit: the parasitophorous vacuole and the host erythrocyte cytosol. This allowed visualization of merozoite release in live parasites. Herein we provide the first evidence in live, untreated cells that merozoite release involves a primary rupture of the parasitophorous vacuole membrane followed by a secondary rupture of the erythrocyte plasma membrane. We have confirmed, with the use of immunoelectron microscopy, that parasitophorous vacuole membrane rupture occurs before erythrocyte plasma membrane rupture in untransfected wild-type parasites. We have also demonstrated selective inhibition of each step in this two-step process of exit using different protease inhibitors, implicating the involvement of distinct proteases in each of these steps. This will facilitate the identification of the parasite and host molecules involved in merozoite release.

Fatty acids with certain structural characteristics are potent inhibitors of germination and inducers of cell death of powdery mildew spores

Fatty acids with certain structural characteristics inhibited the germination of Erysiphe polygoni spores. Inhibition of germination was associated with disruption of cellular organization, which included rupture of vacuoles, loss of structural order and condensation of cellular contents and finally considerable shrinkage of the spores. These events could be invoked at a low concentration, i.e. 20μM, of the fatty acids and were irreversible after 15min. Cellular disorganization was detected in a relatively short time, approx. 1h. In examining a group of fatty acids with different structural characteristics, it was found that the presence of a cis double bond was important for the inhibitory activities. Saturated and trans -monounsaturated fatty acids were ineffective. Also, at 20μM, polyunsaturated fatty acids had marginal activities and not all cis -monounsaturated fatty acids were inhibitory. It was noted that the fatty acids with the highest activities shared a common feature,. i.e. a double bond 6 or 7 carbons from either end of the carbon chain. The results suggest that the presence of a Δ6, Δ7, ω6, or ω7 double bond by the cis -monounsaturated fatty acids is an important characteristic for the potent inhibitory activity. In contrast, the presence of an acid group was apparently not a critical requirement for activity, because twocis -monounsaturated fatty alcohols tested also exhibited similar potent inhibitory and disruptive effects.Palmitoleic acid-induced spore shrinkage could be partially prevented by BaCl2 and fully prevented by spermine. These are compounds known to block certain K+ channels, suggesting the involvement of K+ channels in palmitoleic acid effects. Also, when the spores were stained with the K+ binding ratiometric dye, tetra (acetoxymethyl) ester benzofuran isophthalate (PBFI-AM), disappearance of fluorescence at 340nm was observed in spores treated with palmitoleic acid, indicating a loss of K+ in these spores.

Direct evidence of active and rapid nuclear degradation triggered by vacuole rupture during programmed cell death in Zinnia.

Differentiation into a tracheary element (TE) is a typical example of programmed cell death (PCD) in the developmental processes of vascular plants. In the PCD process the TE degrades its cellular contents and becomes a hollow corpse that serves as a water conduct. Using a zinnia (Zinnia elegans) cell culture we obtained serial observations of single living cells undergoing TE PCD by confocal laser scanning microscopy. Vital staining was performed and the relative fluorescence intensity was measured, revealing that the tonoplast of the swollen vacuole in TEs loses selective permeability of fluorescein just before its physical rupture. After the vacuole ruptured the nucleus was degraded rapidly within 10 to 20 min. No prominent chromatin condensation or nuclear fragmentation occurred in this process. Nucleoids in chloroplasts were also degraded in a similar time course to that of the nucleus. Degradations did not occur in non-TEs forced to rupture the vacuole by probenecid treatment. These results demonstrate that TE differentiation involves a unique type of PCD in which active and rapid nuclear degradation is triggered by vacuole rupture.

Neuropathologic findings of bromethalin toxicosis in the cat.

Ten random source male domestic shorthair cats, 2 to 6 years old and 3.0-4.4 kg body weight, were each given a single oral dose (1.5 mg/kg) of bromethalin (cat Nos. 1-5) or bait vehicle carrier (cat Nos. 6-10). Bromethalin-dosed cats developed a toxic syndrome characterized by ataxia, focal motor seizures, vocalization, decerebrate posture, decreased conscious proprioception, recumbency, depression, and semicoma. Bromethalin-dosed cats were euthanatized if seizure activity or hindlimb paralysis developed. Survival times were 48 hours (cat No. 1), 89 hours (cat No. 2), 90 hours (cat No. 3), and 97 hours (cat No. 4). Control cats (cat Nos. 6-10) and one bromethalin-dosed cat (cat No. 5) were euthanatized on day 20 after dosing. Spongy change (edema--characterized by the formation of vacuoles in extracellular spaces and myelin lamellae), hypertrophied fibrous astrocytes, and hypertrophied oligodendrocytes were observed in the white matter of the cerebrum, cerebellum, brain stem, spinal cord, and optic nerve of all bromethalin-dosed cats. Spongy change occasionally extended into contiguous cerebellar Purkinje cell layer and cerebral cortical gray matter. The severity of lesions varied among cats but was most pronounced in cat No. 5 (480 hours after dosing). A leukocytic inflammatory response, gitter cell (macrophage) response, or axonal degeneration was not observed in the vacuolated areas. Ultrastructural findings included separation of myelin lamellae at the interperiod lines with the formation of intramyelinic vacuoles (intramyelinic edema), rupture and coalescence of intramyelinic vacuoles into larger extracellular spaces (spongy change), and pronounced cytosolic edema of astrocytes and oligodendroglial cells.

Segmental Mediolytic Arteritis

We describe the histopathologic and ultrastructural changes in two cases of segmental mediolytic arteritis (SMA) and summarize the clinical and pathologic findings in previous reports. SMA is initiated by the transformation of the arterial smooth-muscle cytoplasmic contents into a maze of dilated vacuoles containing edema-like fluid. With vacuolar rupture, the smooth-muscle cells are disrupted and the mediolytic process completed. Mediolysis is accompanied by fibrin deposition and hemorrhages at the adventitio-medial junction and within the media. Inflammation is inconstant and limited to the periadventitial tissues. Transmural mediolysis leads to the formation of arterial wall gaps--defects in the vascular wall bridged by a serofibrinous layer. The serosal and intramural arteries and arterioles of the jejunum and the epicardial coronary arteries were the targets of SMA in this report. SMA occurs in two clinical settings: (a) in abdominal muscular arteries and arterioles of predominantly elderly patients presenting either with ischemic bowel disease or shock, and (b) in the coronary arteries of neonates in conditions associated with severe hypoxemia. We conclude that SMA is the result of an inappropriate vasospastic response expressed in a splanchnic vascular bed undergoing vasoconstriction as a response to shock or severe hypoxemia.

Chitin synthetase activity is bound to chitosomes anf to the plasma membrane in protoplasts of Saccharomyces cerevisiae

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacoular proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40–110 nm; bouyant density ( d) = 1.146 g/cm 3). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density ( d = 1.203 g/cm 3) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with 3H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100–250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.

Scanning electron microscopic investigations of the human umbilical artery intima

Umbilical cord arteries were investigated using a scanning electron microscope using different methods of preparation: Perfusion of one artery under pressure from a 100 cm water column caused artefacts and the preparatory work took at least 10 min after delivery. To shorten this time fully patent umbilical cords were double clamped and fixed immediately after birth. However, the removal of blood after fixation caused the endothelial layer to be lost. Therefore umbilical cords were double clamped, snap frozen and stored in liquid nitrogen until preparation. The endothelial lining of the fully patent umbilical artery at birth is composed of longitudinally arranged, spindle-shaped cells, connected by cellular junctions. The basement membrane contains numerous gaps. Because of these gaps postnatal vasoconstriction causes herniation of the subendothelial myofibroblasts forming subendothelial vacuoles. The vacuoles produce displacement of the endothelial cell cytoplasm towards the vascular lumina resulting in protuberances and blebs on the endothelial cells. Rupture of vacuoles leads to crater-like injuries. Beneath the basement membrane a thin layer of myofibroblasts is arranged longitudinally. Oblique or transversely ordered bundles of myofibroblasts are interposed at wide and irregular intervals. These transverse bundles are able to trigger localized contraction rings called "folds of Hoboken", the initial stage of postnatal arterial closure.

Characterization of chitosomes in Candida albicans protoplasts

A significant amount of highly zymogenic chitin synthetase of Candida albicans was found to be associated with cytoplasmic particles (chitosomes). These cellular particles were isolated from metabolically activated C. albicans protoplasts after disruption by a mild method that minimizes vacuole rupture and consequent contamination of cellular fractions by vacuolar proteinases. Contamination of chitosomes by soluble cytosolic enzymes, vacuolar proteinases, vacuolar membranes, or other endomembranous structures was minimal, as indicated by the virtual absence of the following marker enzymes; α-glucosidase, carboxypeptidase Y, α-mannosidase, mannosyltransferase, and NADPH-cytochrome c reductase. Secretory enzymes such as acid phosphatase and other membrane-bound enzymes such as X-prolyl-dipeptidyl-aminopeptidase and leucine aminopeptidase did not sediment with the chitosomal chitin synthetase. These results support the idea that, in C. albicans as well as in other fungi, chitosomes are well-defined secretory organelles containing zymogenic chitin synthetase and not artifactual vesicles derived from plasma membrane and other membranous systems during cell breakage.

The proper role of nerves in salivary secretion: a review.

Consideration of the history of this subject has created a perspective which helps one to appreciate how errors in thinking have occurred and why some have persisted, despite subsequent improvements in information. The most enduring misconception has been that the drying of the mouth under stress is due to sympathetic inhibitory fibers, a view that must be eradicated, for such fibers do not exist. The inhibition is due to central influences from higher centers acting on the salivary centers and thereby suppressing reflex activity. Wide variations exist in the neuro-effector arrangements and in the cellular responses in different glands from different species. Myoepithelial cells are usually contracted by both parasympathetic and sympathetic nerves. The blood vessels also receive a dual innervation, but parasympathetic impulses cause vasodilatation as part of secretion, whereas the sympathetic vasoconstrictor fibers are part of a more generalized vascular control system and not a direct part of the reflex secretory sympathetic pathway. Parasympathetic drive usually provides the main stimulus for fluid formation by parenchymal cells, whereas sympathetic nerves tend to increase the output of pre-formed components from certain cells. Absence of nerve impulses causes variable atrophic and other metabolic effects on the parenchymal cells. Evidence is beginning to accrue that certain nerve impulses may influence resynthetic activities. Vacuolation, often found experimentally after strong stimulation, also occurs to a variable extent in certain cells as a normal part of reflex secretion and may therefore have an effect on the components entering the saliva. If rupture of vacuoles occurs, then this may contribute to the salivary amylase that is present in the blood. Recent evidence points to the possibility that, even in a monomorphic gland, not all of the components necessarily enter nerve-induced saliva in "parallel" proportions. Consideration of current information has enabled some provisional generalizations about the roles of the secretory nerves to be suggested in the summary, but they must not be considered immutable.

THE ULTRASTRUCTURE OF LIPOSARCOMA

An ultrastructural study of 10 liposarcomas is reported. Four of the liposarcomas were wholly or predominantly of well-differentiated, lipoma-like or fibrosing type, 3 of myxoid type, 2 of round cell type and 1 pleomorphic type. The well-differentiated, lipoma-like liposarcomas showed cells with a few, large lipid droplets, few organelles and a peripherally located, fairly large nucleus, The well-differentiated liposarcomas of fibrosing type revealed mostly spindle-shaped, fibroblast-like cells, with abundant rough endoplasmic reticulum and inconspicuous lipid inclusions, surrounded by collagen. One well-differentiated liposarcoma contained an area which was similar to brown adipose tissue and hibernoma. The spindle and stellate shaped cells of the myxoid liposarcomas showed abundant rough endoplasmic reticulum and large smooth-membraned vacuoles filled with moderately dense amorphous material, which appeared to be extruded extracellularly by rupture of the vacuoles. Cytoplasmic lipid droplets were seen in most cells but were much less prominent than in the well-differentiated lipoma-like liposarcomas. Ultrastructurally there were many similarities between the myxoid and round cell liposarcoma, indicating a close relationship between the two types. The pleomorphic liposarcoma revealed cells with one or more large, irregular nuclei, numerous large vacuoles after dissolved lipids, abundant dilated cisternae of rough endoplasmic reticulum and rounded, electron-dense bodies corresponding to PAS-positive hyalin globules seen in the light microscope. The ultrastructural study suggests that the variegated cellular appearance of the different subtypes of liposarcoma reflects the wide cellular spectrum seen during the differentiation of adipose tissue and supports the view that all liposarcomas histogenetically represent a single entity.