Coherent Optical Signatures of Electron Microbunching in Laser-Driven Plasma Accelerators.

We report observations of coherent optical transition radiation interferometry (COTRI) patterns generated by microbunched ∼200-MeV electrons as they emerge from a laser-driven plasma accelerator. The divergence of the microbunched portion of electrons, deduced by comparison to a COTRI model, is ∼9× smaller than the ∼3 mrad ensemble beam divergence, while the radius of the microbunched beam, obtained from COTR images on the same shot, is <3 µm. The combined results show that the microbunched distribution has estimated transverse normalized emittance ∼0.4 mm mrad.

Developing personal sense of risk: a grounded theory in men with hypertension.

BACKGROUND: For older men with hypertension living in rural areas, non-adherence to treatment is a common phenomenon. The experience on risk perception of individuals with their condition is recognized as playing a critical role in promoting treatment adherence. However, the experience on risk perception in a cohort of older men with hypertension seems unclear. AIM: To develop a theory concerning risk perception experience in a cohort of older men with hypertension from a rural area of Thailand. METHODS: A grounded theory approach was used. Semi-structured, face-to-face interviews with 29 hypertensive older men were conducted in Thailand using purposive and theoretical sampling methods. The grounded theory analytical method that included initial and focused coding, and constant comparison was used to analyse the data. FINDINGS: 'Developing a personal sense of risk' emerged as a core category, which incorporated the related four subprocesses: comparing healthcare provider information with stories of people with hypertension, comparing one's own situation with stories of people with hypertension, changing personal sense of risk and changing risk-related behaviour. Older men selected to focus on one particular outcome, using the selected outcome to monitor their risk. CONCLUSION: This investigation provides a theory for healthcare providers to understand older men's perceptions of personal risk for complications of hypertension. A personal sense of risk influences risk-related behaviour change. IMPLICATIONS FOR NURSING AND HEALTH POLICY: The findings can be used in assessing a personal sense of risk and promoting treatment adherence in older men with hypertension. Effective storytelling intervention, a standard tool for assessment personal sense of risk in older men with hypertension, should be developed. Hypertension care policy needs to be developed for individualized approaches.

Cultural perceptions and clinical experiences of nursing students in Eastern Turkey.

AIM: This study explored Turkish nursing students' perceptions of providing care to patients culturally different from themselves. BACKGROUND: Increasing migration will increase the need for nurses to provide care across cultural groups. METHODS: Twenty one students in the second year of a 4-year nursing programme participated in 3 focus groups. Data were analysed using directed content analysis. Research questions were based on Campinha-Bacote's model. RESULTS: Three themes were identified: perceived cultural barriers, perceived cultural facilitators and identifying culturally sensitive actions. Generally, students were able to define culture but were unable to say how culture would affect nursing practice. DISCUSSION: Students were unprepared to practice in a multicultural setting. Cultural awareness is insufficient for determining how to respond to cultural differences. LIMITATIONS: The study is limited by its restriction to a single school of nursing and a single curriculum. CONCLUSIONS: The multiple, ongoing political, religious and ethnic conflicts will require nurses to provide care to patients from other cultural groups, in some instances to people identified as adversaries to the group the nurse may represent. Understanding cultural differences is insufficient to do this effectively. IMPLICATIONS FOR NURSING EDUCATION: Learning culturally competent care requires opportunities to provide, be guided through and reflect on care to individuals from different cultural groups. IMPLICATIONS FOR ORGANIZATIONAL AND PUBLIC POLICIES: Standards for culturally competent care should be adopted by all care delivery settings. Public and organizational policies openly declaring healthcare settings as cultural safe zones, and explicit organizational commitment to culturally safe care, would set clear expectations for providers and help ensure a positive patient experience.

Planning and decision making about the future care of older group home residents and transition to residential aged care.

BACKGROUND: Planning for future care after the death of parental caregivers and adapting disability support systems to achieve the best possible quality of life for people with intellectual disability as they age have been important issues for more than two decades. This study examined perceptions held by family members, group home staff and organisational managers about the future of older residents and the decisions made that a move to residential aged care was necessary. METHODS: Grounded Dimensional Analysis was used to guide data collection and analysis by an interdisciplinary research team. Three sets of interviews over a period of 18 months were conducted with a family member, house supervisor and the programme manager for each of seventeen older group home residents in Victoria. For the eight people for whom it was decided a move was necessary and the six who eventually moved focussed questions were asked about the decision-making process. RESULTS: While plans for lifelong accommodation in a group home proved unfounded, key person succession plans were effective. However, decisions to move to a residential aged care facility where necessary were made in haste and seen as a fait accompli by involved family members. CONCLUSIONS: Although family members take seriously their mandate to oversee well-being of their older relative, they have little knowledge about their rights or avenues to safeguard untimely or inappropriate decisions being made by professionals.

A morphological study of plasma and phagosome membranes during endocytosis in Acanthamoeba.

Particle ingestion by Acanthamoeba is rapid. Within 40 s bound particles can be surrounded by pseudopods, brought into the cytoplasm, and released as phagosomes into the cytoplasmic stream. In electron micrographs the phagosome appears as a flasklike invagination of the surface. Separation from the surface occurs by fragmentation of the attenuated "neck+ of the invagination. The separated phagosome membrane has a three- to fourfold greater density of intramembrane particles than the plasma membrane from which it derives. This change is evident within 15 min of ingestion and is detectable while the membrane is still tightly apposed to the particle. There is no direct evidence for the mechanism of this increase; no increase in particle density was seen in the membrane at an early stage in the forming phagosomes still connected to the surface. These morphological observations are consistent with chemical analyses, to be reported in a separate communication, that show that the phagosome membrane has a higher protein to phospholipid ratio and a higher glycosphingolipid content than the plasma membrane. Enlarged phagosomes (presumptive phagolysosomes) show multiple small vesiculations of characteristic morphology. The small vesicles are postulated to be the major route of membrane return to the cell surface.

Pinocytosis in Acanthamoeba castellanii. Kinetics and morphology.

The uptake of radioactively labeled albumin, inulin, leucine, and glucose by Acanthamoeba castellanii (Neff strain) was measured. The uptake is linear with time and appears to be continuous under the conditions of these experiments. Uptake is abolished at 0 degrees C. No evidence for saturation of the uptake mechanism was obtained with either albumin or leucine. Each of the four tracer molecules enters the ameba at a similar rate when the uptake is calculated as volume of fluid ingested per unit time. The data suggest that each of these molecules enters the cell by pinocytosis. The highest rate of uptake was obtained with cells in their usual culture medium containing proteose peptone, glucose, and salts but pinocytosis also continued at a reduced rate in a simple salt solution. The calculated volume of fluid taken in during pinocytosis in culture medium was about 2 microl/hr per 10(6) cells. The route of uptake was examined in the electron microscope using horseradish peroxidase (HRP) as a tracer. HRP activity was found exclusively within membrane profiles within the cytoplasm, confirming the pinocytotic mode of uptake. An estimate of the rate of surface membrane turnover due to pinocytosis was made using the biochemical and morphological data obtained. This estimate suggests that the plasma membrane turnover of one cell is on the order of several times an hour.

The fine structure of Acanthamoeba castellanii (Neff strain). II. Encystment.

Encysting cells of Acanthamoeba castellanii, Neff strain, have been examined with the electron microscope. The wall structure and cytoplasmic changes during encystment are described. The cyst wall is composed of two major layers: a laminar, fibrous exocyst with a variable amount of matrix material, and an endocyst of fine fibrils in a granular matrix. The two layers are normally separated by a space except where they form opercula in the center of ostioles (exits for excysting amebae). An additional amorphous layer is probably present between the wall and the protoplast in the mature cyst. Early in encystment the Golgi complex is enlarged and contains a densely staining material that appears to contribute to wall formation. Vacuoles containing cytoplasmic debris (autolysosomes) are present in encysting cells and the contents of some of the vacuoles are deposited in the developing cyst wall. Lamellate bodies develop in the mitochondria and appear in the cytoplasm. Several changes are associated with the mitochondrial intracristate granule. The nucleus releases small buds into the cytoplasm, and the nucleolus decreases to less than half its original volume. The cytoplasm increases in electron density and its volume is reduced by about 80%. The water expulsion vesicle is the only cellular compartment without dense content in the mature cyst. The volume fractions of lipid droplets, Golgi complex, mitochondria, digestive vacuoles, and autolysosomes have been determined at different stages of encystment by stereological analysis of electron micrographs. By chemical analyses, dry weight, protein, phospholipid, and glycogen are lower and neutral lipid is higher in the mature cyst than in the trophozoite.

The fine structure of Acanthamoeba castellanii. I. The trophozoite.

The fine structure of the trophozoite of Acanthamoeba castellanii (Neff strain) has been studied. Locomotor pseudopods, spikelike "acanthopodia," and microprojections from the cell surface are all formed by hyaline cytoplasm, which excludes formed elements of the cell and contains a fine fibrillar material. Golgi complex, smooth and rough forms of endoplasmic reticulum, digestive vacuoles, mitochondria, and the water-expulsion vesicle (contractile vacuole) are described. A canicular system opening into the water-expulsion vesicle contains tubules about 600 A in diameter that are lined with a filamentous material. The tubules are continuous with unlined vesicles or ampullae of larger diameter. Centrioles were not observed, but cytoplasmic microtubules radiate from a dense material similar to centriolar satellites and are frequently centered in the Golgi complex. Cytoplasmic reserve materials include both lipid and glycogen, each of which amounts to about 10% of the dry weight.

Acanthamoeba discriminates internally between digestible and indigestible particles.

The capacity of Acanthamoeba to distinguish nutritive yeast particles from non-nutritive plastic beads during phagocytosis was investigated. When cells were allowed to phagocytose yeast to capacity, endocytosis stopped and subsequent presentation of particles (either yeast or beads) did not result in further uptake. By contrast, when cells were allowed to phagocytose plastic beads to capacity and a second dose of particles was presented (either yeast or beads), the cells exocytosed the internal particles and took up new ones. Yeast rendered indigestible by extensive chemical cross-linking were taken up at rates similar to those of untreated yeast, but, like beads, they were exocytosed when a second dose of particles was presented. The results show that an internal distinction is made between vacuoles containing yeast and vacuoles containing plastic beads, and they are consistent with the hypothesis that the presence within the vacuoles of material capable of being digested prevents exocytosis.

Hydrolase secretion is a consequence of membrane recycling.

Acanthamoeba releases lysosomal hydrolases continuously into the culture medium. This release is specific for lysosomal hydrolases, but not other cellular proteins, and is energy dependent. The secreted hydrolases can be separated into two groups on the basis of their secretion kinetics: one is secreted at approximately 15% of the cellular activity per hour and the other at approximately 5%. Intracellularly the lysosomal hydrolases are restricted almost exclusively to secondary lysosomes where the hydrolases demonstrate a differential pH-dependent binding to membrane. Hydrolase secretion is not the result of secondary lysosomes' fusing with the plasma membrane since soluble and particulate lysosomal contents are not released at the same rate. Together the data suggest that the secreted hydrolases are trapped in shuttle vesicles that cycle membrane from secondary lysosomes to the cell surface. The inner membrane and content of these vesicles undergo a marked pH shift when, following fragmentation from lysosomes, these vesicles fuse with plasma membrane. This rapid pH shift and the differential pH-dependent membrane binding of hydrolases appear to account for the heterogeneous hydrolase secretion kinetics.

Distribution of chitin in the yeast cell wall. An ultrastructural and chemical study.

The distribution of chitin in Saccharomyces cervisiae primary septa and cell walls was studied with three methods: electron microscopy of colloidal gold particles coated either with wheat germ agglutinin or with one of two different chitinases, fluorescence microscopy with fluorescein isothiocyanate derivatives of the same markers, and enzymatic treatments of [14C]glucosamine-labeled cells. The septa were uniformly and heavily labeled with the gold-attached markers, an indication that chitin was evenly distributed throughout. To study the localization of chitin in lateral walls, alkali-extracted cell ghosts were used. Observations by electron and fluorescence microscopy suggest that lectin-binding material is uniformly distributed over the whole cell ghost wall. This material also appears to be chitin, on the basis of the analysis of the products obtained after treatment of 14C-labeled cell ghosts with lytic enzymes. The chitin of lateral walls can be specifically removed by treatment with beta-(1 leads to 6)-glucanase containing a slight amount of chitinase. During this incubation approximately 7% of the total radioactivity is solubilized, about the same amount liberated when lateral walls of cell ghosts are completely digested with snail glucanase yield primary septa. It is concluded that the remaining chitin, i.e., greater than 90% of the total, is in the septa. The facilitation of chitin removal from the cell wall by beta-(1 leads to 6)-glucanase indicates a strong association between chitin and beta-(1 leads to 6)-glucan. Covalent linkages between the two polysaccharides were not detected but cannot be excluded.

Plasma membrane association of Acanthamoeba myosin I.

Myosin I accounted for approximately 2% of the protein of highly purified plasma membranes, which represents about a tenfold enrichment over its concentration in the total cell homogenate. This localization is consistent with immunofluorescence analysis of cells that shows myosin I at or near the plasma membrane as well as diffusely distributed in the cytoplasm with no apparent association with cytoplasmic organelles or vesicles identifiable at the level of light microscopy. Myosin II was not detected in the purified plasma membrane fraction. Although actin was present in about a tenfold molar excess relative to myosin I, several lines of evidence suggest that the principal linkage of myosin I with the plasma membrane is not through F-actin: (a) KI extracted much more actin than myosin I from the plasma membrane fraction; (b) higher ionic strength was required to solubilize the membrane-bound myosin I than to dissociate a complex of purified myosin I and F-actin; and (c) added purified myosin I bound to KI-extracted plasma membranes in a saturable manner with maximum binding four- to fivefold greater than the actin content and with much greater affinity than for pure F-actin (apparent KD of 30-50 nM vs. 10-40 microM in 0.1 M KCl plus 2 mM MgATP). Thus, neither the MgATP-sensitive actin-binding site in the NH2-terminal end of the myosin I heavy chain nor the MgATP-insensitive actin-binding site in the COOH-terminal end of the heavy chain appeared to be the principal mechanism of binding of myosin I to plasma membranes through F-actin. Furthermore, the MgATP-sensitive actin-binding site of membrane-bound myosin I was still available to bind added F-actin. However, the MgATP-insensitive actin-binding site appeared to be unable to bind added F-actin, suggesting that the membrane-binding site is near enough to this site to block sterically its interaction with actin.

Morphometric analysis of volumes and surface areas in membrane compartments during endocytosis in Acanthamoeba.

Stereologic analysis was made of cell surface membrane (PM) and two interrelated cytoplasmic membrane systems, the vacuole membranes (VM) and small vesicle membranes (SVM). Volumes and surface areas of the three membrane compartments were measured during steady-state pinocytosis, when membrane recycling is rapid, and during phagocytosis, when a shift to a lower rate of membrane uptake by endocytosis occurs (B. Bowers, 1977, Exp. Cell Res. 110:409). Total membrane area in the three compartments was 3.2 micrometers 2/micrometers 3 of protoplasmic volume and was constant throughout the experiments. In pinocytosing cells, 32% of the membrane was in the PM, 25% in the vM, and 43% in the SVM. The vacuole compartment occupies approximately 20% of the total cell volume, and the small vesicle, approximately 3%. As the endocytic uptake of membrane from the surface decreased, there was an increase in PM area and a marked decrease in SVM area. The VM area remained constant even though "empty" vacuoles were almost completely replaced by newly formed phagosomes within 45 min. This demonstrates directly a rapid flux of membrane though this compartment. A model, taking into consideration these and other data on Acanthamoeba, is proposed to account for the observed membrane shifts. The data suggest that the vacuolar (digestive) system of Acanthamoeba is central to cellular control of endocytosis and membrane recycling.

Visualization of melanosome dynamics within wild-type and dilute melanocytes suggests a paradigm for myosin V function In vivo.

Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va-) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid ( approximately 1.5 microm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va-dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This "capture" model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an approximately 120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, approximately 0.14 microm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do not drive obvious spreading of melanosomes over 90 min. We conclude that long-range, bidirectional, microtubule-dependent melanosome movements, coupled with actomyosin Va-dependent capture of melanosomes in the periphery, is the predominant mechanism responsible for the centrifugal transport and peripheral accumulation of melanosomes in mouse melanocytes. This mechanism represents an alternative to straightforward transport models when interpreting other myosin V mutant phenotypes.

Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae.

Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.

Complete nucleotide sequence and deduced polypeptide sequence of a nonmuscle myosin heavy chain gene from Acanthamoeba: evidence of a hinge in the rodlike tail.

We have completely sequenced a gene encoding the heavy chain of myosin II, a nonmuscle myosin from the soil ameba Acanthamoeba castellanii. The gene spans 6 kb, is split by three small introns, and encodes a 1,509-residue heavy chain polypeptide. The positions of the three introns are largely conserved relative to characterized vertebrate and invertebrate muscle myosin genes. The deduced myosin II globular head amino acid sequence shows a high degree of similarity with the globular head sequences of the rat embryonic skeletal muscle and nematode unc 54 muscle myosins. By contrast, there is no unique way to align the deduced myosin II rod amino acid sequence with the rod sequence of these muscle myosins. Nevertheless, the periodicities of hydrophobic and charged residues in the myosin II rod sequence, which dictate the coiled-coil structure of the rod and its associations within the myosin filament, are very similar to those of the muscle myosins. We conclude that this ameba nonmuscle myosin shares with the muscle myosins of vertebrates and invertebrates an ancestral heavy chain gene. The low level of direct sequence similarity between the rod sequences of myosin II and muscle myosins probably reflects a general tolerance for residue changes in the rod domain (as long as the periodicities of hydrophobic and charged residues are largely maintained), the relative evolutionary "ages" of these myosins, and specific differences between the filament properties of myosin II and muscle myosins. Finally, sequence analysis and electron microscopy reveal the presence within the myosin II rodlike tail of a well-defined hinge region where sharp bending can occur. We speculate that this hinge may play a key role in mediating the effect of heavy chain phosphorylation on enzymatic activity.

The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle.

The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.

Chitin synthetase distribution on the yeast plasma membrane.

Purified, intact yeast plasma membranes were allowed to synthesize chitin, and the nascent chains of polysaccharide were observed either by the fluorescence produced with a brightener or by autoradiography. By both methods, it was concluded that the newly formed chitin emerged at many sites on each membrane. Thus, the synthetase that catalyzes chitin formation has a similar distribution. Since chitin synthetase is found mainly in a zymogen form, these results confirm the hypothesis that initiation of the chitinous primary septum of Saccharomyces occurs by localized activation of the uniformly distributed zymogen.

A survey of people with intellectual disabilities living in residential aged care facilities in Victoria.

BACKGROUND: Australia's national ageing policy recognises that people ageing with intellectual disability (ID) require particular attention, yet there is no policy framework concerning this population. This study describes the distribution and characteristics of people with ID in residential aged care in Victoria, provides insights into the pathways they take into aged care, and gives some indications of how facilities adapt to their needs. METHOD: A postal survey was sent to 826 residential aged care facilities in Victoria, seeking information from directors about their residents with ID. Facilities that responded were fairly representative of all facilities in Victoria. FINDINGS: Residents with ID were younger, had entered at an earlier age and remained longer than other residents. Their reported dependency profile was similar to the general aged care population, although the incidence of dementia was lower. Primary areas of concern identified by providers were: inability to fit into the resident community, lack of participation in activities and lack of meaningful relationships. CONCLUSION: This study provides a first glimpse into how older people with ID find their way into aged care and how others view their experiences once there. It suggests that further investigation is required into the accuracy of assessment undertaken prior to entry to more clearly understand whether residents with ID are inappropriately placed in residential aged as a result of a shortage of disability accommodation and inadequate resources to support aging in place for those in such accommodation.

Localization of lipophosphonoglycan in membranes of Acanthamoeba by using specific antibodies.

Antibodies against two electrophoretically distinct forms of lipophosphonoglycan (LPG) were produced in rabbits. Antibody specificity was demonstrated by the coupled antibody 125I-protein A assay (Adair et al., J. Cell Biol. 79:281-285, 1978). Indirect immunofluorescent labeling of intact Acanthamoeba showed that antibodies to both LPG components had the same uniform distribution on the cell surface. Both antibodies also bound to the cytoplasmic surface of isolated phagosomes. The location of LPG in other membranes of the amoeba was demonstrated on sections by the unlabeled antibody method. Although LPG was absent from the nuclear membrane, virtually all of the internal vacuole membranes were labeled, including the contractile vacuole. Antibodies directed against LPG were utilized to label lipophosphonoglycan in the plasma membrane of living amoebae. Labeled membrane was internalized and then localized by immunofluorescence in cytoplasmic vacuoles within 10 min of incubation. Although these results are evidence for exchange between plasma and cytoplasmic vacuolar membranes, the contractile vacuole remained unlabeled and can be considered, therefore, a separate membrane compartment. Concanavalin A also was bound and internalized by the amoeba, but electron microscopy showed that this label caused pronounced membrane perturbation, limiting its usefulness as a membrane marker in this system.