Spherical Organelles Research Articles

An acridone based fluorescent dye for lipid droplet tracking and cancer diagnosis

Lipid droplets (LDs) are spherical organelles that localize in the cytosol of eukaryotic cells. Different proteins are embedded on the surface of LDs, so LDs play a vital role in the physiological activities of cells. The dysregulation of LDs is associated with various human diseases, such as diabetes and obesity. Therefore, it is essential to develop a fluorescent dye that labels LDs to detect and monitor illnesses. In this study, we developed the compound BDAA12C for staining LDs in cells. BDAA12C exhibits excellent LD specificity and low toxicity, enabling us to successfully stain and observe the fusion of LDs in A549 cancer cells. Furthermore, we also successfully distinguished A549 cancer cells and MRC-5 normal cells in a co-culture experiment and in normal and tumour tissues. Interestingly, we found different localizations of BDAA12C in well-fed and starved A549 cancer cells and consequently illustrated the transfer of fatty acids (FAs) from LDs to mitochondria to supply energy for β-oxidation upon starvation. Therefore, BDAA12C is a promising LD-targeted probe for cancer diagnosis and tracking lipid trafficking within cells.

Insights into the evolution of photoreceptor oil droplets in frogs and toads.

Photoreceptor oil droplets (ODs) are spherical organelles placed most commonly within the inner segment of the cone photoreceptors. Comprising neutral lipids, ODs can be either non-pigmented or pigmented and have been considered optically functional in various studies. Among living amphibians, ODs were only reported to occur in frogs and toads (Anura), while they are absent in salamanders and caecilians. Nonetheless, the limited understanding of their taxonomic distribution in anurans impedes a comprehensive assessment of their evolution and relationship with visual ecology. We studied the retinae of 134 anuran species, extending the knowledge of the distribution of ODs to 46 of the 58 currently recognized families, and providing a new perspective on this group that complements the available information from other vertebrates. The occurrence of ODs in anurans shows a strong phylogenetic signal, and our findings revealed that ODs evolved at least six times during the evolutionary history of the group, independently from other vertebrates. Although no evident correlation was found between OD occurrence, adult habits and diel activity, it is inferred that each independent origin involves distinct scenarios in the evolution of ODs concerning photic habits. Furthermore, our results revealed significant differences in the size of the ODs between nocturnal and arrhythmic anurans relative to the length of the cones' outer segment.

Optical diffraction tomography for assessing single cell models in angular light scattering.

Angularly resolved light scattering (ALS) has become a useful tool for assessing the size and refractive index of biological scatterers at cellular and organelle length scales. Sizing organelle populations with ALS relies on Mie scattering theory models, which require significant assumptions about the object, including spherical scatterers and a homogeneous medium. These assumptions may incur greater error at the single cell level, where there are fewer scatterers to be averaged over. We investigate the validity of these assumptions using 3D refractive index (RI) tomograms measured via optical diffraction tomography (ODT). We compute the angular scattering on digitally manipulated tomograms with increasingly strong model assumptions, including RI-matched immersion media, homogeneous cytosol, and spherical organelles. We also compare the tomogram-computed angular scattering to experimental measurements of angular scattering from the same cells to ensure that the ODT-based approach accurately models angular scattering. We show that enforced RI-matching with the immersion medium and a homogeneous cytosol significantly affects the angular scattering intensity shape, suggesting that these assumptions can reduce the accuracy of size distribution estimates.

Possible means of swimming of red algae spores

This article proposes a model for swimming of red algae spores. The model considers a released spore in unbound water as a spherical particle enclosing a liquid incompressible cytosol, in which oscillates a solid spherical organelle. An analysis of the solutions of the Navier-Stokes equations for the cytosol flow caused by the organelle motion within the cell is presented in the limit of small Reynolds number. It is shown that in the case when the cytosol has Newtonian or Maxwell properties, the spore may swim only when the forward and backward trajectories of the organelle are different. In the case of the shear thinning cytosol properties the spore may swim also when the organelle trajectories are the same, but the velocities of forward and backward movements of the organelle should differ. Such a cell may swim in a straight line. The swimming of the model spores completely satisfies experimental data.

In situ polymerization of polypyrrole in oil body for efficient solar-driven freshwater collection

Solar-driven interfacial water evaporation can be exploited for freshwater generation with clean and pollution-free characteristics. The transport of water molecules from bulk water to the surface of the absorber is a key factor for efficient water evaporation. Herein, an oil body (OB), a spherical organelle extracted from safflower seeds, was used as supporting materials due to its low density and hydrophilic surface. After in situ polymerization of polypyrrole (PPy) in the interior of OB, an OB-PPy absorber with excellent photothermal performance and water transporting ability was obtained. When adding OB-PPy into sewage or seawater, it shows higher water evaporation rate and freshwater collection capabilities than PPy alone. The highest evaporation rate (2.11 kg m-2 h−1) and evaporation efficiency (92.02 %) can be obtained in OB-PPy containing rainwater. The freshwater collection rate can reach 1.38 kg m-2 h−1 when using a homemade freshwater collection device. Moreover, OB-PPy exhibits long term stability and reusability. Interestingly, we have found that OB-PPy lyophilized powder could absorb atmospheric water and produce freshwater from humid air. Therefore, OB-PPy holds great potential for solving the problem of freshwater resource shortage.

STED Nanoscopy Imaging of Cellular Lipid Droplets Employing a Superior Organic Fluorescent Probe

Lipid droplets (LDs) are spherical organelles that participate in numerous biological processes. In order to visualize LDs on the nanoscale, nanoscopy fluorescence imaging is considered as the most attractive technique but is substantially limited by the characteristics of fluorescent probes. Thus, the development of a superior fluorescent probe that is capable of nanoscopy fluorescence imaging has attracted enormous attention. Herein, a benzodithiophene-tetraoxide-based molecule Lipi-BDTO has been developed that can easily undergo the stimulated emission depletion (STED) process and displays high photostability. These two characteristics of fluorescent probes finely satisfy the requirements of STED nanoscopy imaging. Indeed, applying the probe for STED imaging achieves a high resolution of 65 nm, belonging to one of the leading results of LDs fluorescence imaging. Furthermore, the high photostability of this fluorescent probe enables it to monitor the dynamics of LDs by time-lapse STED imaging as well as to visualize the three-dimensional (3D) spatial distribution of LDs by 3D STED imaging. Notably, the resolution of the 3D STED image represents one of the best LDs fluorescence imaging results so far. Besides STED nanoscopy imaging, the superior utility of this fluorescent probe has been also demonstrated in two-color 3D confocal imaging and four-color confocal imaging.

Interaction of bacterial extracellular microvesicles with eukaryotic cells.

Bacterial extracellular microvesicles (BMV) are formed by nonpathogenic, pathogenic and opportunistic bacteria. BMV are spherical bilayer-membrane organelles containing different cargoes: lipopolysaccharides, pathogen associated molecular patterns (PUMP), DNA, RNA, signal molecules, proteins, antibiotic resistance factors, virulence factors, toxins providing various immune response options and conducive to the survival and pathogen dissemination in the human body. BMVs secretion play an important role in the ability of microorganisms to cause various diseases. BMV are involved in biofilms formation, help bacteria to obtain nutrition in a nutrient-poor conditions, to evade the host's immune response, provide communication and surviving in a stressful environment during infection inside the host. The heterogeneity of the biogenesis mechanisms causes differences in the BMV and their characteristics including virulence rate. BMVs host cells entering is mediated by several mechanisms and helps to activate innate and adaptive immune reactions. This review focuses on interaction study of BMV with various eukaryotic cells types including neutrophils, dendritic cells, macrophages, epithelial, endothelial cells. This interaction depends on bacteria species, type of target cell and number of vesicles and can lead to different responses: non-immunogenic, pro-inflammatory, cytotoxic. Subcellular and molecular mechanisms related to the involvement of extracellular microvesicles in host's immune response modulation are presented. Stimulation of immune response is provided by increased secretion of proinflammatory cytokines and chemokines. In some cases BMV use mechanisms to evade immune surveillance: anti-inflammatory cytokines secretion, alterations of phagocytosis and chemotaxis of macrophages, increasing the proteolytic cleavage of CD14 on the macrophage surface, alterations of antigen-presenting function of dendritic cells, T-cell proliferation suppression, reducing the pro-inflammatory cytokines secretion, evasion of host-immune cells direct interactions, destruction of neutrophilic traps. These features allow bacterial cells to survive in the human body, increase their invasive potential, and reduce the excessive inflammatory reactions leading to death of the pathogen itself and life-threatening damage of tissues and organs of the host. Further studies of these mechanisms will improve existing therapeutic approaches to the infectious diseases treatment.

Abstract LB-368: Determining and characterizing differential biodistribution of tumor derived exosomes by non-invasive nuclear imaging

Abstract Introduction: New theory of “seeds (cancer cells) and soil (distant tissue)” transpired as scientists acquainted exosomes as seeds that cruise to distant organs and release growth factors (fertilizers) to acclimate the “foreign soil” and prepare the metastatic niche. Exosomes are nano-sized (30-150nm) spherical organelles, derived from the endosomal system for intercellular communications. Tumor derived exosomes (TDEs) play an imperative role in tumor growth, stromal reorganization, cancer stemness, neovascularization, and metastatic niche formation by providing proteins, lipids, mRNA, miRNA etc. to tumor microenvironment. Despite intense research in the field, only few studies have investigated exosome biodistribution and thus very little known about their in vivo trafficking, that is also impeding their biomedical application as diagnostic and prognostic marker and as a potential therapeutic delivery vehicle. Also, it would be of interest to investigate the biodistribution of exosomes isolated from tumor cells after different treatment. Application of nuclear imaging techniques, such as single-photon emission computed tomography (SPECT) using radio isotopically labeled exosomes have indisputable advantages over fluorescent imaging regarding tissue penetration and quantitative measurement of the biodistribution. Methods: To determine in vivo trafficking of TDEs and whether exosomes from treated cells are distributed differently, we isolated exosomes from hypoxic (1% O2) 4T1 breast cancer cells without any treatment (control), and treated with GW2580 or HET0016 drug. GW2580, a selective small molecule kinase inhibitor of colony stimulating factor 1-receptor (CSF-1R) and HET0016 that is a highly selective inhibitor of CYP4A in arachidonic acid pathway, have been shown to decrease tumor growth and metastasis. We labeled these three groups of exosomes with radio I-131 and injected them in 4T1 tumor bearing and non-tumor bearing Balb/c mice. Then, we followed up the radio I-131labeled exosome distribution for 3 consecutive days with SPECT. Results: More exosomes were localized in the lungs in tumor bearing mice compared to non-tumor bearing mice. Interestingly, control exosomes were more localized in the metastatic site that is in the lungs than the primary breast tumor. We observed TDEs from untreated cells were localized more in metastatic site than that exosomes from the GW2580 and HET0016 treated cells. Conclusion: This study demonstrates that treatment of tumor cells with drugs that inhibit tumor growth and metastasis potentially yield exosomes with less protumorigenic and less metastatic characteristics and become less homing to metastatic site. TDEs are more efficiently visualized and quantified in vivo by radio-isotope labelling of exosomes. The knowledge attained from this study will open up new horizon in exosome research and biological nanotechnology for diagnosis and prognosis and targeted treatment. Citation Format: Mohammad Harun Rashid, Thaiz Borin, Kartik Angara, Roxan Ara, Achyut Bhagelu, Ali Arbab. Determining and characterizing differential biodistribution of tumor derived exosomes by non-invasive nuclear imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-368.

Extracellular Vesicles as a Platform for Glioma Therapeutic Development.

Normal and pathologic cells secrete extracellular vesicles (EV), which are defined as 30-2,000 nm spherical organelles. It has been found that EV mediate various biological functions including cellular remodeling and export of biomolecules, extracellular communication, immune stimulation and suppression, and modulation of the cellular microenvironment. EV secreted by human glioma cells contain a wealth of tumor-specific proteins and nucleic acids that can be isolated from patients with these neoplasms. Thus, EV contribute to the development of biomarkers, and additionally have certain therapeutic potential for possible use in neuro-oncology and neurosurgery.

Structure and properties of oil bodies in diatoms.

Diatoms accumulate triacylglycerols in spherical organelles called oil bodies when exposed to nutrient deprivation conditions. Oil body biology in diatoms has attracted significant attention due to the complexity of the intracellular organelles and the unique combination of genes generated by the evolutionary history of secondary endosymbiosis. The demand for biofuel production has further increased the interest in and importance of a better understanding of oil body biology in diatoms, because it could provide targets for genetic engineering to further enhance their promising lipid accumulation. This review describes recent progress in studies of the structure and properties of diatom oil bodies. Firstly, the general features of diatom oil bodies are described, in particular, their number, size and morphology, as well as the quantity and quality of lipids they contain. Subsequently, the diatom oil body-associated proteins, which were recently discovered through oil body proteomics, are introduced. Then, the metabolic pathways responsible for the biogenesis and degradation of diatom oil bodies are summarized. During biogenesis and degradation, oil bodies interact with other organelles, including chloroplasts, the endoplasmic reticulum and mitochondria, suggesting their dynamic nature in response to environmental changes. Finally, the functions of oil bodies in diatoms are discussed.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

Optical influence of oil droplets on cone photoreceptor sensitivity.

ABSTRACTOil droplets are spherical organelles found in the cone photoreceptors of vertebrates. They are generally assumed to focus incident light into the outer segment, and thereby improve light catch because of the droplets' spherical lens-like shape. However, using full-wave optical simulations of physiologically realistic cone photoreceptors from birds, frogs and turtles, we find that pigmented oil droplets actually drastically reduce the transmission of light into the outer segment integrated across the full visible wavelength range of each species. Only transparent oil droplets improve light catch into the outer segments, and any enhancement is critically dependent on the refractive index, diameter of the oil droplet, and diameter and length of the outer segment. Furthermore, oil droplets are not the only optical elements found in cone inner segments. The ellipsoid, a dense aggregation of mitochondria situated immediately prior to the oil droplet, mitigates the loss of light at the oil droplet surface. We describe a framework for integrating these optical phenomena into simple models of receptor sensitivity, and the relevance of these observations to evolutionary appearance and loss of oil droplets is discussed.

Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles.

Centrosomes are highly dynamic, spherical organelles without a membrane. Their physical nature and their assembly are not understood. Using the concept of phase separation, we propose a theoretical description of centrosomes as liquid droplets. In our model, centrosome material occurs in a form soluble in the cytosol and a form that tends to undergo phase separation from the cytosol. We show that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This nonequilibrium feature also allows for multiple stable centrosomes, a situation that is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the Caenorhabditis elegans embryo, and it also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, the model can describe unequal centrosome sizes observed in cells with perturbed centrioles. We also propose an interpretation of the molecular details of the involved proteins in the case of C. elegans. Our example suggests a general picture of the organization of membraneless organelles.

Artificially-induced organelles are optimal targets for optical trapping experiments in living cells.

Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads.

Composition and properties of the surface of oil bodies recovered from Echium plantagineum

Neutral-lipids within oilseeds are most commonly stored in oil bodies, small spherical organelles with oleosin proteins inserted through a phospholipid monolayer. Oil bodies extracted from Echium plantagineum are highly enriched in polyunsaturated fatty acids and are stable to coalescence and oxidation. This stability has been attributed to the strong association between the phospholipid monolayer and oleosin proteins. To better understand this association the phospholipid fatty acyl groups of E. Plantagineum oil bodies were determined for the first time; a large proportion (≈70%) of saturated fatty acids were present, and this may aid in oleosin anchorage and thus contributes to oil body stability. The effect of oil body washing on surface charge was also observed (using turbidity, zeta and streaming potentials), and dependent on the washing protocol, E. Plantagineum oil bodies had an isoelectric point of pH 4–5. This is significantly different to pI values for oil bodies from a range of other seeds reported in the literature using isoelectric focusing; a possible explanation for this discrepancy is discussed.

Use of LysoTracker dyes: A flow cytometric study of autophagy

The flow cytometric use of LysoTracker dyes was employed to investigate the autophagic process and to compare this with the upregulation of autophagy marker, the microtubule-associated protein LC3B. Although the mechanism of action of LysoTracker dyes is not fully understood, they have been used in microscopy to image acidic spherical organelles, and their use in flow cytometry has not been thoroughly investigated in the study of autophagy. This investigation uses numerous autophagy-inducing agents including chloroquine (CQ), rapamycin, low serum (<1%) RPMI, and nutrient starvation to induce autophagy in Jurkat T-cell leukemia and K562 erythromyeloid cell lines. LC3B showed an increase with CQ treatment although this was different to LysoTracker signals in terms of dose and time. Rapamycin, low serum (<1%) RPMI, and nutrient starvation induction of autophagy also induced an increase in LysoTracker and LC3B signals. CQ also induced apoptosis in cell lines, which was blocked by pan-caspase inhibitor z-VAD resulting in a reduction in cells undergoing apoptosis and a subsequent upregulation of autophagic markers LC3B and lysosomal dye signals. Given that LC3B and LysoTracker are measuring different biological events in the autophagic process, they surprisingly both upregulated during autophagic process. This study, however, shows that although LysoTracker dyes do not specifically label lysosomes or autophagosomes within the cell, they allow the simultaneous measurement of an autophagy-related process and other live-cell functions, which are not possible with the standard LC3B antibody-labeling technique. This method has the advantage of other live-cell LCB-GFP-tagged experiments in that be used to analyze patient cells as well as easier to use and significantly less costly.

Mitochondria: isolation, structure and function

Mitochondria are complex organelles constantly undergoing processes of fusion and fission, processes that not only modulate their morphology, but also their function. Yet the assessment of mitochondrial function in skeletal muscle often involves mechanical isolation of the mitochondria, a process which disrupts their normally heterogeneous branching structure and yields relatively homogeneous spherical organelles. Alternatively, methods have been used where the sarcolemma is permeabilized and mitochondrial morphology is preserved, but both methods face the downside that they remove potential influences of the intracellular milieu on mitochondrial function. Importantly, recent evidence shows that the fragmented mitochondrial morphology resulting from routine mitochondrial isolation procedures used with skeletal muscle alters key indices of function in a manner qualitatively similar to mitochondria undergoing fission in vivo. Although these results warrant caution when interpreting data obtained with mitochondria isolated from skeletal muscle, they also suggest that isolated mitochondrial preparations might present a useful way of interrogating the stress resistance of mitochondria. More importantly, these new findings underscore the empirical value of studying mitochondrial function in minimally disruptive experimental preparations. In this review, we briefly discuss several considerations and hypotheses emerging from this work.

Statistics of Active Transport in Xenopus Melanophores Cells

The transport of cell cargo, such as organelles and protein complexes in the cytoplasm, is determined by cooperative action of molecular motors stepping along polar cytoskeletal elements. Analysis of transport of individual organelles generated useful information about the properties of the motor proteins and underlying cytoskeletal elements. In this work, for the first time (to our knowledge), we study collective movement of multiple organelles using Xenopus melanophores, pigment cells that translocate several thousand of pigment granules (melanosomes), spherical organelles of a diameter of ∼1 μm. These cells disperse melanosomes in the cytoplasm in response to high cytoplasmic cAMP, while at low cAMP melanosomes cluster at the cell center. Obtained results suggest spatial and temporal organization, characterized by strong correlations between movement of neighboring organelles, with correlation length of ∼4 μm and pair lifetime ∼5 s. Furthermore, velocity statistics revealed strongly non-Gaussian velocity distribution with high velocity tails demonstrating exponential behavior suggestive of strong velocity correlations. Depolymerization of vimentin intermediate filaments using a dominant-negative vimentin mutant or actin with cytochalasin B reduced correlation of behavior of individual particles. Based on our analysis, we concluded that steric repulsion is dominant, but both intermediate filaments and actin microfilaments are involved in dynamic cross-linking organelles in the cytoplasm.

A series of naphthalimide derivatives as intra and extracellular pH sensors

A series of new naphthalimide derivatives were synthesized and studied. Three of the materials ( SM1, SM2, and SM3) possess methacrylate(s) moieties as pH sensor monomers, enabling these compounds to be polymerized with other monomers for thin film preparation for extracellular pH sensing. Herein, poly(2-hydroxyethyl methacrylate)- co-poly(acrylamide) (PHEMA- co-PAM) was chosen as the polymer matrix. Structure influences on pH responses and p K a values were studied. The film P3 composed of the sensing moiety SM3 has a p K a close to the usual biological environmental pH of ∼7. It was used as an extracellular pH sensor to monitor pH change during the metabolism of prokaryotic Escherichia coli ( E. coil). On the other hand, the three sensor monomers are new intracellular biomarkers to sense lysosomes of eukaryotic cells since (1) their p K a values are in a range of 5.9–6.8; (2) their emission intensities at acidic conditions (such as at pH 5) are much stronger than those at a neutral condition of pH 7; (3) lysosomes range in size from 0.1 to 1.2 μm in diameter with pH ranging from 4.5 to 5.0, which is much more acidic than the pH value of the cytoplasm (usually with a pH value of ∼7.2); and (4) the acidity of lysosomes enables a protonation of the amino groups of the pH probes making the sensors emit brightly in acidic organelles by inhibiting the photo-induced electron transfer from the amino groups to the fluorophores. Lysosome sensing was demonstrated using live human brain glioblastoma U87MG cell line, human cervical cancer HeLa cell line, and human esophagus premalignant CP-A and CP-D cell lines by observations of small acidic spherical organelles (lysosomes) and significant colocalizations (82–95%) of the sensors with a commercially available lysosome-selective staining probe LysoTracker Red ® under confocal fluorescence microscopy.

Identification and localization of a caleosin in olive (Olea europaea L.) pollen during in vitro germination

In plant organs and tissues, the neutral storage lipids are confined to discrete spherical organelles called oil bodies. Oil bodies from plant seeds contain 0.6–3% proteins, including oleosins, steroleosins, and caleosins. In this study, a caleosin isoform of ∼30 kDa was identified in the olive pollen grain. The protein was mainly located at the boundaries of the oil bodies in the cytoplasm of the pollen grain and the pollen tube. In addition, caleosins were also visualized in the cytoplasm at the subapical zone, as well as in the tonoplast of vacuoles present in the pollen tube cytoplasm. The cellular behaviour of lipid bodies in the olive pollen was also monitored during in vitro germination. The number of oil bodies decreased 20-fold in the pollen grain during germination, whereas the opposite tendency occurred in the pollen tube, suggesting that oil bodies moved from one to the other. The data suggest that this pollen caleosin might have a role in the mobilization of oil bodies as well as in the reorganization of membrane compartments during pollen in vitro germination.

Structure and distribution of organelles and cellular location of calcium transporters in Neurospora crassa.

We wanted to examine the cellular locations of four Neurospora crassa proteins that transport calcium. However, the structure and distribution of organelles in live hyphae of N. crassa have not been comprehensively described. Therefore, we made recombinant genes that generate translational fusions of putative organellar marker proteins with green or red fluorescent protein. We observed putative endoplasmic reticulum proteins, encoded by grp-78 and dpm, in the nuclear envelope and associated membranes. Proteins of the vacuolar membrane, encoded by vam-3 and vma-1, were in an interconnected network of small tubules and vesicles near the hyphal tip, while in more distal regions they were in large and small spherical vacuoles. Mitochondria, visualized with tagged ARG-4, were abundant in all regions of the hyphae. Similarly, we tagged the four N. crassa proteins that transport calcium with green or red fluorescent protein to examine their cellular locations. NCA-1 protein, a homolog of the SERCA-type Ca(2+)-ATPase of animal cells, colocalized with the endoplasmic reticulum markers. The NCA-2 and NCA-3 proteins are homologs of Ca(2+)-ATPases in the vacuolar membrane in yeast or in the plasma membrane in animal cells. They colocalized with markers in the vacuolar membrane, and they also occurred in the plasma membrane in regions of the hyphae more than 1 mm from the tip. The cax gene encodes a Ca(2+)/H(+) exchange protein found in vacuoles. As expected, the CAX protein localized to the vacuolar compartment. We observed, approximately 50 to 100 mum from the tip, a few spherical organelles that had high amounts of tagged CAX protein and tagged subunits of the vacuolar ATPase (VMA-1 and VMA-5). We suggest that this organelle, not described previously in N. crassa, may have a role in sequestering calcium.