Organelles In Intact Cells Research Articles

Intranuclear inclusions in hepatocellular carcinoma contain autophagy-associated proteins and correlate with prolonged survival.

For decades, intranuclear inclusions in many normal and neoplastic cells have been considered to be mere invaginations of cytoplasm into the nucleus without any notable function or influence on disease. We investigated such inclusions in 75 specimens of hepatocellular carcinoma (HCC). In this context we demonstrate that these inclusions are true inclusions, completely closed and delimited by the nuclear membrane, containing degenerate cell organelles and lysosomal proteins. Moreover, their occurrence was positively associated with patient survival but not with tumour grade or stage. In a standardised area a mean of 124 inclusions per specimen was present in the tumorous liver tissue in contrast to 5 inclusions in the non‐tumorous adjacent section and 89% of all scrutinised HCC showed at least one membrane‐bound nuclear inclusion. Ultrastructural characterisation by transmission electron microscopy revealed degenerative materials such as residues of lysosomes, endoplasmic reticulum and Golgi apparatus within the inclusions. Due to the fact that the content of the inclusions appears to be more condensed than cytoplasm and contains fewer intact cell organelles, we assume that they are not mere invaginations of cytoplasm. Three dimensional (3D) reconstruction of isolated and immunofluorescence stained nuclei showed that the inclusions are completely located within the nucleus without any connection to the cytoplasm. The limiting membrane of the inclusions contained lamin B suggesting nuclear membrane origin. The content of the inclusions stained for the autophagy‐associated proteins p62, ubiquitin, LC3B, cathepsin B and cathepsin D. Triple immunofluorescence staining followed by 3D reconstruction revealed co‐localisation of p62, ubiquitin and LC3B in the same inclusion. Our observations uncover that these inclusions are real inclusions completely surrounded by the nucleus. We propose that the presence of autophagy‐associated proteins and proteases within the inclusions contribute to beneficial survival.

High-Throughput Screening Using Photoluminescence Probe to Measure Intracellular Calcium Levels.

Aequorin, a 22kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10μM, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.

Interaction of Mitochondria with the Endoplasmic Reticulum and Plasma Membrane in Calcium Homeostasis, Lipid Trafficking and Mitochondrial Structure.

Studying organelles in isolation has been proven to be indispensable for deciphering the underlying mechanisms of molecular cell biology. However, observing organelles in intact cells with the use of microscopic techniques reveals a new set of different junctions and contact sites between them that contribute to the control and regulation of various cellular processes, such as calcium and lipid exchange or structural reorganization of the mitochondrial network. In recent years, many studies focused their attention on the structure and function of contacts between mitochondria and other organelles. From these studies, findings emerged showing that these contacts are involved in various processes, such as lipid synthesis and trafficking, modulation of mitochondrial morphology, endoplasmic reticulum (ER) stress, apoptosis, autophagy, inflammation and Ca handling. In this review, we focused on the physical interactions of mitochondria with the endoplasmic reticulum and plasma membrane and summarized present knowledge regarding the role of mitochondria-associated membranes in calcium homeostasis and lipid metabolism.

Bin/Amphiphysin/Rvs (BAR) Family Members Bend Membranes in Cells

We provide direct evidence that Bin/Amphiphysin/Rvs (BAR) family members bend the steady state membrane architecture of organelles in intact cells. In response to inducible BAR molecular actuators, organelles exhibit distinct changes to the orientation and degree of their membrane curvature. This rapidly inducible system may offer a mechanism by which to better understand the structure-function relationship of intracellular organelles.

Bin/Amphiphysin/Rvs (BAR) family members bend membranes in cells.

We provide direct evidence that Bin/Amphiphysin/Rvs (BAR) family members bend the steady state membrane architecture of organelles in intact cells. In response to inducible BAR molecular actuators, organelles exhibit distinct changes to the orientation and degree of their membrane curvature. This rapidly inducible system may offer a mechanism by which to better understand the structure-function relationship of intracellular organelles.

The Use of Aequorin and Its Variants for Ca2+ Measurements

Ca(2+)-sensitive photoproteins are ideal agents for measuring the Ca(2+) concentration ([Ca(2+)]) in intracellular organelles because they can be modified to include specific targeting sequences. Aequorin was the first Ca(2+)-sensitive photoprotein probe used to measure the [Ca(2+)] inside specific intracellular organelles in intact cells. Aequorin is a 22-kDa protein produced by the jellyfish Aequorea victoria. On the binding of Ca(2+) to three high-affinity sites in aequorin, an irreversible reaction occurs in which the prosthetic group is released and a photon is emitted. Aequorin has become widely used for intracellular Ca(2+) measurements because it offers many advantages: For example, it can be targeted with precision, functions over a wide range of [Ca(2+)], and shows low buffering capacity. In this article we describe the main characteristics of the aequorin probe and review the reasons why it is widely used to measure intracellular [Ca(2+)].

Quantitative Analysis of Three-Dimensional Fluorescence Localization Microscopy Data

Identifying the three-dimensional molecular organization of subcellular organelles in intact cells has been challenging to date. Here we present an analysis approach for three-dimensional localization microscopy that can not only identify subcellular objects below the diffraction limit but also quantify their shape and volume. This approach is particularly useful to map the topography of the plasma membrane and measure protein distribution within an undulating membrane.

Orphan Nuclear Receptor TR3 (Nur77) Binds and Converts the Phenotype of Bcl-B, an Anti-Apoptotic Bcl-2-Family Protein Predominantly Expressed in Human Plasma Cells.

TR3 (Nur77) is an orphan member of the steroid/retinoid family of Nuclear Receptors (NRs) that translocates from nucleus to cytosol, where it binds Bcl-2 and converts its phenotype from anti-apoptotic to pro-apoptotic (Li, et al. Science 289: 1159, 2000; Lin, et al CELL 116: 527, 2004). We surveyed TR3 (Nur77) for interactions with all six human anti-apoptotic Bcl-2-family proteins: Bcl-2, Bcl-XL, Mcl-1, Bcl-W, Bfl-1, and Bcl-B. We observed that Bcl-2, Bfl-1, and Bcl-B interact with TR3 as determined by co-immunoprecipitation assays using lysates from transfected cells and by GST pull down assays using GST-Bcl-2-family fusion proteins. In contrast, Bcl-XL, Bcl-W, and Mcl-1 displayed little or no binding to TR3 (Nur77).Co-localization experiments using fluorescent protein tagging and confocal microscopy corroborated these findings, showing co-localization of a fragment of TR3 (Nur77) that accumulates in cytosol (rather than nucleus) [TR3 lacking the DNA-binding domain [DDBD]) with Bcl-2, Bfl-1, and Bcl-B on mitochondria and other intracellular organelles in intact cells, but not co-localization with Bcl-XL, Mcl-1 or Bcl-W. Co-expression of Bcl-2, Bfl-1, or Bcl-B with TR3DDBD by transfection resulted in robust apoptosis induction, while co-expression of Bcl-XL, Bcl-W, or Mcl-1 with TR3DDBD did not. In contrast to results obtained in TR3DDBD co-expression studies, expressing any of the anti-apoptotic Bcl-2-family proteins (Bcl-2, Bfl-1, Bcl-B, Bcl-XL, Mcl-1, Bcl-W) individually resulted in suppression of apoptosis induced by Staurosporine, illustrating the role of TR3 in converting the phenotypes of Bcl-2, Bfl-1 and Bcl-B from protector to killer.Because binding of TR3 (Nur77) to Bcl-B appeared to be strongest among the Bcl-2-family proteins, we focused on this Bcl-B for additional studies. (Ke, N. et al. J. Biol Chem 276: 12481, 2001. Using monospecific antibodies, the endogenous Bcl-B protein was localized in human tissues, revealing predominant expression in plasma cells. Several myeloma cell lines also expressed Bcl-B protein, as determined by immunoblotting. Stimulating myeloma cell line RPMI8226 with ionomycin and phorbol ester TPA (agents that induce TR3 expression and accumulation in cytosol) resulted in association of endogenous TR3 with endogenous Bcl-B, as determined by co-immunoprecipitation experiments. Reducing endogenous Bcl-B levels using small interfering RNA (siRNA) reduced apoptosis induced by transfected TR3DDBD as well as apoptosis induced by a synthetic peptide that mimics TR3.We conclude that cytosolic TR3 (Nur77) interacts selectively with certain anti-apoptotic members of the Bcl-2-family (Bcl-2, Bfl-1, Bcl-B), converting them from protectors to killers. The ability of TR3 (Nur77) to convert Bcl-B suggests a possible novel strategy for triggering apoptosis of Bcl-B-expressing cells, which may be of utility for eradicating long-lived autoantibody-producing plasma cells or for killing malignant myeloma cells. (Supported by NIH GM60554 and SASS Foundation).

Hepatocyte apoptosis: Is transforming growth factor-B1 the kiss of death?

In previous studies hepatocytes undergoing cell death by apoptosis but not normal hepatocytes in rat liver showed immunostaining for transforming growth factor β1 (TGF-β1). Staining was much stronger with antibodies recognizing the pro-region of TGF-β1 than the mature peptide itself. Therefore we investigated the ability of both forms of TGF-β1 to induce apoptosis in primary cultures of rat hepatocytes. Mature TGF-β1 induced rounding up of the cells and fragmentation into multiple vesicles. As revealed by the DNA-specific stain H33258, the chromatin of these cells condensed and segregated into masses at the nuclear membrane; this was obviously followed by fragmentation of the nucleus. Ultrastructurally the cytoplasm was well preserved, as demonstrated by the presence of intact cell organelles. These features strongly suggest the occurrence of apoptosis. Quantification of nuclei with condensed chromatin revealed that mature TGF-β1 was 30-fold more effective than the TGF-β1 latency-associated protein complex. Finally, we administered TGF-β1 in vivousing an experimental model in which regression of rat liver was initiated by a short preceding treatment with the hepatomitogen cyproterone acetate. Two doses of TGF-β1, each 1 nM/kg, augmented the incidence of apoptotic hepatocytes 5-fold. Equimolar doses of TGF-β1 latency-associated protein complex were ineffective. These studies suggest that TGF-β1 is involved in the initiation of apoptosis in the liver and that the mature form of TGF-β1 is the active principle.

Induction of apoptosis in cultured hepatocytes and in the regressing liver by transforming growth factor-β1 occurs without activation of an endonuclease

In previous studies in vivo apoptotic liver cells were found to be positive for transforming growth factor-β1 (TGF-β1). In hepatocyte cultures TGF-β1 induced rounding up and fragmentation of the cells into multiple vesicles. As revealed by the DNA specific stain H33258 the chromatin of these cells condensed and segregated into masses at the nuclear membrane, followed by nuclear fragmentation. Ultrastructurally the cytoplasm was well preserved as demonstrated by the presence of intact cell organelles. These features strongly suggest the occurrence of apoptosis. Furthermore we administered TGF-β1 in vivo using an experimental model in which regression of the liver was initiated by a short preceding treatment with the hepatomitogen cyproterone acetate (CPA). Two doses of 1 nM TGF- β1/ kg each augmented the incidence of apoptotic hepatocytes 5-fold. These studies strongly suggest that TGF-β1 is involved in the initiation of apoptosis in the liver In TGF-β1 treated hepatocytes both from the liver and monolayer culture no DNA fragmentation into oligosomes could be detected. Comparison of nuclei in which endonuclease was activated by Ca 2+ with apoptotic nuclei revealed no obvious similarities, as demonstrated by FACS analysis, H33258 staining and electron microscopy. Thus, apoptosis induced by a growth inhibitor obviously occurs without activation of an endonuclease.

Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor beta 1.

In previous studies hepatocytes undergoing cell death by apoptosis but not normal hepatocytes in rat liver showed immunostaining for transforming growth factor beta 1 (TGF-beta 1). Staining was much stronger with antibodies recognizing the pro-region of TGF-beta 1 than the mature peptide itself. Therefore we investigated the ability of both forms of TGF-beta 1 to induce apoptosis in primary cultures of rat hepatocytes. Mature TGF-beta 1 induced rounding up of the cells and fragmentation into multiple vesicles. As revealed by the DNA-specific stain H33258, the chromatin of these cells condensed and segregated into masses at the nuclear membrane; this was obviously followed by fragmentation of the nucleus. Ultrastructurally the cytoplasm was well preserved, as demonstrated by the presence of intact cell organelles. These features strongly suggest the occurrence of apoptosis. Quantification of nuclei with condensed chromatin revealed that mature TGF-beta 1 was 30-fold more effective than the TGF-beta 1 latency-associated protein complex. Finally, we administered TGF-beta 1 in vivo using an experimental model in which regression of rat liver was initiated by a short preceding treatment with the hepatomitogen cyproterone acetate. Two doses of TGF-beta 1, each 1 nM/kg, augmented the incidence of apoptotic hepatocytes 5-fold. Equimolar doses of TGF-beta 1 latency-associated protein complex were ineffective. These studies suggest that TGF-beta 1 is involved in the initiation of apoptosis in the liver and that the mature form of TGF-beta 1 is the active principle.

Ultrastructure of the guinea pig pancreas in acute hypercalcemia.

The effect of acute hypercalcemia on pancreatic ultrastructure and the ultrastructural localization of calcium during hypercalcemia were studied in the guinea pig pancreas. After 3 h of i.v. calcium infusion (0.6 mmol/kg/h), hypertrophy and distention of the Golgi apparatus and an increased number of condensing vacuoles were seen. At 6 h, vacuolar fusion and displacement of zymogen granules occurred. At 9 h, irregular distribution of zymogen granules, indentation of the nucleus with chromatin clumping, and inclusion of intact cell organelles were present. Disruption of the plasma membrane and release of cell organelles into the interstitial space were seen. Control animals receiving saline solution (0.9% NaCl) revealed normal pancreatic ultrastructure. The serum ionized calcium values were 0.65 +/- 0.36 mM in controls and 0.71 +/- 0.14, 0.79 +/- 0.21, and 1.22 +/- 0.50 mM at 3, 6, and 9 h of calcium infusion, respectively. The ultrastructural localization of calcium was performed with the pyroantimonate staining technique after 3 h of calcium and saline infusion. Large calcium deposits were found in calcium-treated animals along the plasma membrane and in the Golgi region. The findings indicate that calcium exerts a strong stimulatory effect that eventually leads to the degeneration of the pancreatic acinar cell.

Visualization of acidic organelles in intact cells by electron microscopy.

We report the synthesis of a probe that permits the visualization by electron microscopy of acidic organelles in intact cells. This probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), is a basic congener of dinitrophenol that readily diffuses into intact cells. Its primary and tertiary amino groups (apparent pKa, 10.6) allow it to be concentrated in acidic organelles and to be retained there after fixation with aldehydes. The dinitroarene moiety of DAMP can then be localized with mouse monoclonal antibodies directed against dinitrophenol. The antibodies, in turn, can be visualized by light or electron microscopy by reaction with rabbit anti-mouse antibodies coupled to rhodamine or horseradish peroxidase, respectively. We have used these methods to show that DAMP concentrates in a variety of membrane-bound structures in cultured fibroblasts, including classic multivesicular bodies (resembling lysosomes), intermediate-sized vesicles with multiple shapes (resembling endosomes), and an abundant population of very small spherical vesicles. A small fraction of coated vesicles is labeled with DAMP. Labeling with DAMP does not occur when the pH gradient of fibroblasts is disrupted by the ionophore monensin or the weak base chloroquine. DAMP should be a useful probe for exploring the assembly, distribution, and function of acidic organelles by electron microscopy.

Low Temperature-Induced Alterations in the Chloroplast and Microsomal Membranes of Dunaliella salina

The metabolic regulation of membrane lipid composition has been examined using the cell wall-less, unicellular green alga Dunaliella salina (UTEX 1644) as a model system. Low temperature stress was employed to initiate and study the regulatory response.When cultures growing logarithmically at 30 degrees C were chilled to 12 degrees C, cell division ceased for approximately 100 hours, and then the cells resumed logarithmic growth at a slower rate. The phospholipid, glycolipid and protein content, on a per cell basis, was, in each case, approximately 20% higher in cells grown at 12 degrees C. The volume of the 12 degrees C-acclimated cells was 2.8 times that of 30 degrees C-grown cells. The quantity of chloroplast membrane, as determined by morphometric analysis, was 20% greater, whereas the content of microsomal membrane material was more elevated, being approximately 2.8 times that of 30 degrees C-grown cells.Lipid compositional analyses were carried out on purified chloroplasts and microsomes isolated from Dunaliella grown at 30 and 12 degrees C and also from cells 12 and 60 hours following a shift from 30 to 12 degrees C. In both chloroplast and microsomal phospholipids fatty acid unsaturation increased during acclimation to low temperature. Generally, microsomal phospholipids responded more quickly and to a greater extent than did chloroplast phospholipids. Despite these alterations, little change in the relative proportions of phospholipid classes was observed in either cell fraction.In sharp contrast to the pattern of phospholipid change, chloroplast glycolipids responded to low temperature by significantly increasing the proportion of one specific class, digalactosyl diglycerides, relative to monogalactosyl diglycerides, while showing minimal change in fatty acid distribution within any given glycolipid class.The ease and rapidity with which Dunaliella cells can be manipulated with respect to environmental stress and isolation of intact cell organelles makes it particularly well suited for research on intermembrane lipid dynamics within the plant cell.

Nucleic Acids From Subcellular Fractions of N-Nitrosodiethylamine-Induced Hepatoma in Mice

During eight successive isologous passages of hepatoma induced in male C3HA mice by N-nitrosodiethylamine, no common features of tumor progression were observed, although both the mitotic pattern and ploidy differed from generation to generation. These additional cytologic criteria allowed the biochemical examination of material least changed due to tumor progression. Tumor nDNA's were characterized by greater actinomycin D (AD)- and acridine orange (AO)-binding abilities than were normal nDNA's; this could have resulted from a higher proportion of double-stranded regions in tumor DNA. Isolated tumor deoxyribonucleoprotein had both lower template activity in an RNA polymerase system and fewer AD- and AO-binding sites, when compared with the activity and sites from normal mouse liver. RNA-DNA hybridization data with the above-mentioned findings showed that in hepatoma, part of the nuclear genome was repressed. Also, RNA "new classes" appeared and a certain proportion of nuclear genes controlling mitochondrial protein biosynthesis were derepressed in tumor mitochondria. The hybridization of mitochondrial RNA (mtRNA) and DNA revealed new classes of pulse-labeled RNA's in in vitro-incubated liver mitochondria that were absent from intact cell organelles; the hybridization properties of in vivo- and in vitro-formed hepatoma mtRNA's were similar. Competition and hybridization experiments demonstrated that in tumor mitochondria in vivo, some new classes of RNA existed. Hepatoma mitochondrial mRNA had a higher metabolic stability than did normal mRNA.