Three-dimensional imaging of Toxoplasma gondii-host cell interactions within the parasitophorous vacuole.

The protozoan parasite Toxoplasma gondii is a representative of apicomplexan parasites that invades host cells through an unconventional motility mechanism. During host cell invasion it forms a specialized membrane-surrounded compartment that is called the parasitophorous vacuole. The interactions between the host cell and parasite membranes are complex and recent studies have revealed in more detail that both the host cell and the parasite membrane contribute to the formation of the parasitophorous vacuole. By using our a new specimen preparation technique that allows three-dimensional imaging of thick-sectioned internal cell structures with high-resolution, low-voltage field emission scanning electron microscopy, we were able to visualize continuous structural interactions of the host cell membrane with the parasite within the parasitophorous vacuole. Fibrous and tubular material extends from the host cell membrane and is connected to parasite membrane components. Shorter protrusions are also elaborated from the parasite. Several of these shorter fine protrusions connect to the fibrous material of the host cell membrane. The elaborate network may be used for modifications of the parasitophorous vacuole membrane that will allow utilization of nutrients from the host cell by the parasite while it is being protected from host cell attacks. The structural interactions between parasite and host cells undergo time-dependent changes, and a fission pore is the most prominent structure left connecting the parasite with the host cell. The fission pore is anchored in the host cell by thick structural components of unknown nature. The new information gained with this technique includes structural details of fibrous and tubular material that is continuous between the parasite and host cell and can be imaged in three dimensions. We present this technique as a tool to investigate more fully the complex structural interactions of the host cell and the parasite residing in the parasitophorous vacuole.     

Unconventional specimen preparation techniques using high resolution low voltage field emission scanning electron microscopy to study cell motility, host cell invasion, and internal cell structures in Toxoplasma gondii.

Apicomplexan parasites employ complex and unconventional mechanisms for cell locomotion, host cell invasion, and cell division that are only poorly understood. While immunofluorescence and conventional transmission electron microscopy have been used to answer questions about the localization of some cytoskeletal proteins and cell organelles, many questions remain unanswered, partly because new methods are needed to study the complex interactions of cytoskeletal proteins and organelles that play a role in cell locomotion, host cell invasion, and cell division. The choice of fixation and preparation methods has proven critical for the analysis of cytoskeletal proteins because of the rapid turnover of actin filaments and the dense spatial organization of the cytoskeleton and its association with the complex membrane system. Here we introduce new methods to study structural aspects of cytoskeletal motility, host cell invasion, and cell division of Toxoplasma gondii, a most suitable laboratory model that is representative of apicomplexan parasites. The novel approach in our experiments is the use of high resolution low voltage field emission scanning electron microscopy (LVFESEM) combined with two new specimen preparation techniques. The first method uses LVFESEM after membrane extraction and stabilization of the cytoskeleton. This method allows viewing of actin filaments which had not been possible with any other method available so far. The second approach of imaging the parasite's ultrastructure and interactions with host cells uses semithick sections (200 nm) that are resin de-embedded (Ris and Malecki, 1993) and imaged with LVFESEM. This method allows analysis of structural detail in the parasite before and after host cell invasion and interactions with the membrane of the parasitophorous vacuole as well as parasite cell division.     

Photodynamic therapy with pyropheophorbide-a methyl ester in human lung carcinoma cancer cell: efficacy,localization and apoptosis

Pyropheophorbide-a methyl ester (MPPa) is a semisynthetic photosensitizer derived from chlorophyll a. The absorption peak of MPPa in organic solvent and in cells was at 667 and 674 nm, respectively. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay showed that MPPa had no dark cytotoxicity. In vitro photodynamic activity was extensively evaluated using a human lung carcinoma cancer cell line (NCI-h446). MPPa exhibited no genotoxicity, as assayed by single-cell gel electrophoresis. Using confocal laser scanning microscopy and organelle-specific fluorescent probes, MPPa was found to localize in the intracellular membrane system, namely the endoplasmic reticulum, Golgi apparatus, lysosomes and mitochondria, in the NCI-h446 cells. Furthermore, nuclear staining and DNA gel electrophoresis revealed that DNA condensation and fragmentation occurred post-photodynamic therapy, indicating the cell death was in the apoptotic mode.     

SUBCELLULAR LOCALIZATION OF HEMATOPORPHYRIN DERIVATIVE IN BLADDER TUMOR CELLS IN CULTURE

Mitochondria have been implicated as a primary subcellular site of porphyrin localization and photodestruction. However, other organelles including the cell membrane, lysosomes and nucleus have been shown to be damaged by hematoporphyrin derivative (HpD) photosensitized destruction as well. In this study we attempted to follow the translocation of the fluorescent components of HpD in human bladder tumor cells (MGH-U1) in culture to determine whether specific subcellular localization occurs over time. Following a 30 min exposure to HpD the cellular fluorescence was examined immediately and 1, 2, 4, and 24 h after HpD removal using fluorescence microscopy and an interactive laser cytometer. The in vitro translocation of dye appeared to be fairly rapid with fluorescence present at the cell membrane and later (1-2 h) within a perinuclear area of the cytoplasm. To determine whether HpD had become concentrated into a specific subcellular organelle, these fluorescence distribution patterns were compared with fluorescent marker dyes specific for mitochondria, endoplasmic reticulum and other membranous organelles. The HpD fluorescence did not appear to be as discrete as the dyes specific for mitochondria or endoplasmic reticulum but appeared similar to the diffuse cytomembrane stain. Finally, the interaction between the fluorescent components of HpD and the cellular constituents was evaluated using a "fluorescence redistribution after photobleaching" technique. The results indicated that the mean lateral diffusion for HpD in MGH-U1 cells was 1.05 x 10(-8) cm2/s, a rate closer to that of lipid diffusion (10(-8)) than that of protein diffusion (10(-10)).(ABSTRACT TRUNCATED AT 250 WORDS)     

An aggregation-induced emission platform for efficient Golgi apparatus and endoplasmic reticulum specific imaging

As two important subcellular organelles in eukaryotic cells, the Golgi apparatus (GA) and endoplasmic reticulum (ER) have recently captivated much interest due to their considerable importance in many biofunctions and role as critical biomarkers for various diseases. The development of efficient GA- and ER-specific probes is of great significance, but remains an appealing yet significantly challenging task. Herein, we reported for the first time the construction of an aggregation-induced emission (AIE) platform for GA and ER fluorescent probes, termed as AIE-GA and AIE-ER, by facile synthesis and simple functionalization. Their excellent targeting specificity to GA or ER, remarkable photostability, high brightness, and low working concentration make AIE-GA and AIE-ER significantly impressive and superior to commercially available probes. Moreover, molecular docking calculations are performed to validate the targeting mechanism of the two AIE probes.

As two important subcellular organelles in eukaryotic cells, the Golgi apparatus (GA) and endoplasmic reticulum (ER) have recently captivated much interest due to their considerable importance in many biofunctions and role as critical biomarkers for various diseases.     

Multi-organelle-targeting pH-dependent NIR fluorescent probe for lysosomal viscosity

The normal operation of lysosome, mitochondria, Golgi apparatus and endoplasmic reticulum plays a significant role in maintaining cell homeostasis. Reflecting the state and function of lysosomes, viscosity is a pivotal parameter to assess the stability of microenvironment. Herein, based on TICT mechanism, a new NIR pH-dependent fluorescent probe DCIC with push-pull electronic moiety was synthesized to identify the lysosomes viscosity. In viscous media, DCIC was highly sensitive to viscosity, fluorescence intensity increased by 180 times as viscosity increased from 1.0 cp to 438.4 cp. In addition, DCIC have high localization ability for lysosome, mitochondria, Golgi apparatus, and endoplasmic reticulum and can monitor lysosomal viscosity fluctuations with laser confocal microscopy.     

Recent advances in mitochondria-and lysosomes-targeted small-molecule two-photon fluorescent probes

This review summarized the recent advances in small-molecule two-photon fl uorescent probes for monitoring a wide variety of biomolecules and changes inside micro-environment in mitochondria and lysosomes, or served as mitotracker and lysotracker with the assistance of two-photon microscopy.     

In vitro PHOTODYNAMIC EFFECTS OF LYSYL CHLORIN p6: CELL SURVIVAL, LOCALIZATION AND ULTRASTRUCTURAL CHANGES

The in vitro cell survival, localization and ultrastructural changes following irradiation were examined in 9L glioma cells sensitized with a new photosensitizer, lysyl chlorin p₆ (LCP). In clonogenic assays, LCP was 10–100-fold more phototoxic than photofrin II on a μg/mL basis. Lysyl chlorin p₆ uptake was blocked when cells were incubated at 2°C. In view of the chemical properties of LCP, this finding indicates that uptake probably occurred through the endocytic pathway. Fluorescence studies showed LCP localized in a region of the endocytic compartment similar in size, shape and distribution to that labeled by lucifer yellow CH (LY), as well as localizing diffusely throughout the perinuclear cytoplasm. Cells stained with both LY and LCP, however, had distinctly separate regions of staining. Lysyl chlorin p₆ localization differed from that of fluorescent probes labeling the mitochondria, Golgi apparatus and endoplasmic reticulum. Ultrastructural changes at both 2 and 30 min after laser irradiation were similar. Mitochondria were often condensed or swollen and also had constrictions and cytoplasmic invaginations. The Golgi apparatus, perinuclear space and rough endoplasmic reticulum (RER) were dilated. These data demonstrate that LCP localizes in a portion of the endosomal compartment, but that morphologic damage initially occurs in the mitochondria, Golgi apparatus and RER.     

一种基于萘酰亚胺的检测细胞内pH值的荧光探针及其生物成像应用

细胞内溶酶体的pH值对细胞自噬、吞噬、酶加工等各项生命活动有着重要影响. 细胞核是真核细胞中最大的细胞器, 控制着生物体内的遗传和代谢过程, 参与代谢过程的酶对pH值的变化很敏感. 因此, 研究细胞体内的pH值变化至关重要. 我们设计并以简单的两步反应合成了一种新型荧光探针 NpH-1. 该探针以萘酰亚胺作为荧光团, 以吗啉基团作为对pH值响应的位点, 通过光诱导电子转移(PET)机制调控荧光, 能够对pH值变化响应. 我们在缓冲范围为1.81到11.92的Britton-Robison缓冲液中测定了 NpH-1对pH值变化响应的光谱性质. 在pH值3.0道10.0的范围内, NpH-1能够对pH值的变化产生快速可逆的响应, 其p Ka值为5.41. 探针具有很高的光稳定性. NpH-1具有很低的细胞毒性, 能够用于活细胞成像. 我们用氯喹刺激HeLa细胞, 使细胞的pH值发生变化, 并用探针 NpH-1监测了这一过程中的pH值变化. 另外, 还对 NpH-1进行了溶酶体、线粒体、高尔基体、内质网和细胞核的共定位实验, 结果表明, 探针主要分布在溶酶体和细胞核中, 这意味着 NpH-1可以用于检测复杂细胞环境中的pH值变化.     

Coumarin‐based Fluorescent Probes for Selectively Targeting and Imaging the Endoplasmic Reticulum in Mammalian Cells

Developing improved fluorescent probes for imaging the endoplasmic reticulum (ER) is necessary for structure‐activity studies of this dynamic organelle. Two coumarin‐based compounds with sulfonamide side groups were synthesized and characterized as ER‐targeting probes. Their selectivity to target the ER in HeLa and GM07373 mammalian cells was shown with co‐localization experiments using commercially available probes that localize in the ER, mitochondria, or lysozymes. The hydrophobicity of the coumarin‐based probes was comparable to known probes that partition into the ER membrane. Their cytotoxicity in mammalian cells was low with IC50 values that range from 205 to 252 μm . The fluorescent quantum yields of the coumarin‐based probes when excited with 400 nm light were 0.60, and they have a much narrower emission spectrum (from 435 to 525 nm in methanol) than that of the only commercially available ER probe that is exited with 400 nm light (ER‐Tracker? Blue‐White DPX). Thus, the coumarin‐based probes are more useful for multicolor imaging with yellow and red emitting fluorophores. In addition to the above benefits, ER labeling was achieved with the coumarin‐based probes in both live cells and fixed cells, revealing their versatility for a wide range of cellular imaging applications.     

Tamoxifen subcellular localization; observation of cell-specific cytotoxicity enhancement by inhibition of mitochondrial ETC complexes I and III

Recently, a nongenomic cytotoxic component of the chemotherapeutic agent tamoxifen (TAM) has been identified that predominantly triggers mitochondrial events. The present study delineates the intracellular fate of TAM and studies its interaction with a spectrum of cell homeostasis modulators primarily relevant to mitochondria. The subcellular localization of TAM was assessed by confocal fluorescence microscopy. The effect of the modulators on TAM cytotoxicity was assessed by standard MTT assays. Our findings show that in estrogen receptor positive MCF7 breast adenocarcinoma cells and DU145 human prostate cancer cells, TAM largely accumulates in the mitochondria and endoplasmic reticulum, but not lysosomes. Our results further demonstrate that in MCF7, but not in DU145 cells, mitochondrial electron transport chain complex I and III inhibitors exacerbate TAM toxicity with an order of potency of myxothiazol ≥ stigmatellin > rotenone > antimycin A, suggesting a cell-specific cytotoxic interplay between mitochondrial complex I and III function and TAM action.     

A photoinduced electron transfer-based nanoprobe as a marker of acidic organelles in mammalian cells

Photoinduced electron transfer (PET)-based molecular probes have been successfully used for the intracellular imaging of the pH of acidic organelles. In this study, we describe the synthesis and characterization of a novel PET-based pH nanoprobe and its biological application for the signaling of acidic organelles in mammalian cells. A fluorescent ligand sensitive to pH via the PET mechanism that incorporates a thiolated moiety was synthesized and used to stabilize gold nanoparticles (2.4?±?0.6 nm), yielding a PET-based nanoprobe. The PET nanoprobe was unambiguously characterized by transmission electron microscopy, proton nuclear magnetic resonance, Fourier transform infrared, ultraviolet-visible absorption, and steady-state/time-resolved fluorescence spectroscopies which confirmed the functionalization of the gold nanoparticles with the PET-based ligand. Following a classic PET behavior, the fluorescence emission of the PET-based nanoprobe was quenched in alkaline conditions and enhanced in an acidic environment. The PET-based nanoprobe was used for the intracellular imaging of acidic environments within Chinese hamster ovary cells by confocal laser scanning microscopy. The internalization of the nanoparticles by the cells was confirmed by confocal fluorescence images and also by recording the fluorescence emission spectra of the intracellular PET-based nanoprobe from within the cells. Co-localization experiments using a marker of acidic organelles, LysoTracker Red DND-99, and a marker of autophagosomes, GFP-LC3, confirm that the PET-based nanoprobe acts as marker of acidic organelles and autophagosomes within mammalian cells.

Primary photodamage sites and mitochondrial events after Foscan photosensitization of MCF-7 human breast cancer cells

To determine the initial photodamage sites of Foscan-mediated photodynamic treatment, we evaluated the enzymatic activities in selected organelles immediately after light exposure of MCF-7 cells. The measurements indicated that the enzymes located in the Golgi apparatus (uridine 5'-diphosphate galactosyl transferase) and in the endoplasmic reticulum (ER) (nicotinamide adenine dinucleotide [reduced] [NADH] cytochrome c [cyt c] reductase) are inactivated by the treatment, whereas mitochondrial marker enzymes (cyt c oxidase and dehydrogenases) were unaffected. This indicates that the ER and the Golgi apparatus are the primary intracellular sites damaged by Foscan-mediated PDT in MCF-7 cells. We further investigated whether the specific mitochondria events could be associated with Foscan photoinduced cell death. The dose response profiles of mitochondrial depolarization and cytochrome c release immediately after Foscan-based PDT were very different from that of overall cell death. By 24 h post-PDT the fluence dependency was strikingly similar for both mitochondrial alterations and cell death. Therefore, although mitochondria are not directly affected by the treatment, they can be strongly implicated in Foscan-mediated MCF-7 cell death by late and indirect mechanism.     

Analysis of mitochondria isolated from single cells

Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users     

Three-dimensional ultrastructure of the ring stage of Plasmodium falciparum: evidence for export pathways.

The three-dimensional structure of the Plasmodium falciparum ring stage has been explored by reconstruction from serial sections and stereoscopic examination of tilted sections. The ring-like light microscopic appearance is related to the shape and contents of the biconcave discoidal parasite at this stage, its thick perimeter containing most of the ribosomes and its thin center containing smooth membrane organelles. The shapes of rings vary between flat and curved cuplike forms. The rough endoplasmic reticulum is a branched network continuous with the nuclear envelope. Evidence for a simple Golgi complex is seen in the presence on the outer nuclear envelope of a locus of coated vesicle budding associated with a single membranous cisterna or cluster of smooth vesicles. In middle and late stage rings this complex migrates along an extension of the nuclear envelope continuous with the rough endoplasmic reticulum. Evidence is also presented for a mechanism of exporting membrane from the parasite into the parasitophorous vacuole membrane and beyond into the red blood cell, by means of double-membraned vesicle-based exocytosis.     

Preparation of subcellular fractions from granulation tissue by density gradient centrifugation

The 7000 g pellet of homogenized mature sponge-induced granulation tissue was fractionated by centrifugation in a stepwise sucrose gradient in order to study the synthesis and secretion of collagen and other components of this tissue on the subcellular level. As indicated by chemical and enzymatic assays, by electron microscopy and by incorporation experiments, the collagen-synthethesizing rough endoplasmic reticulum fraction was isolated free from the secretory vesicles (smooth endoplasmic membranes and Golgi elements) and fibrous extracellular matrix. Collagen differed from other proteins in the distribution among the subcellular fractions. In pulse-chase experiment the translocation of 14C-labelled collagen was demonstrated from the rough endoplasmic reticulum through the secretory vesicles to extracellular fibrillar collagen. This fractionation method will be used to study the modulation of collagen synthesis and secretion in the reparative tissue.     

Subcellular distribution of rat pituitary homogenates by poly(ethylene glycol)-dextran countercurrent partitioning

Rat pituitary homogenates were subjected to two phase countercurrent partition in a poly(ethylene glycol)-dextran mixture using a simple apparatus with enhanced gravity to facilitate the phase separations. Assay of the fractions for organelle marker enzymes and prolactin after 17 transfers showed similar distributions for endoplasmic reticulum, lysosomes, prolactin granules and plasma membrane at the lowest dextran concentrations. Increasing the dextran concentrations had a differential effect on the various organelles. Excellent resolution of endoplasmic reticulum from the other organelles was obtained and marked organelle heterogeneity was demonstrated. Two-phase countercurrent partition thus offers an alternative approach to the subcellular fractionation of pituitary homogenates and should prove useful in separating endoplasmic reticulum from plasma membrane and other cell components.     

Fluxes in "free" and total zinc are essential for progression of intraerythrocytic stages of Plasmodium falciparum

Dynamic fluxes in the concentration of ions and small molecules are fundamental features of cell signaling, differentiation, and development. Similar roles for fluxes in transition metal concentrations are less well established. Here, we show that massive zinc fluxes are essential in the infection cycle of an intracellular eukaryotic parasite. Using single-cell quantitative imaging, we show that growth of the blood-stage Plasmodium falciparum parasite requires acquisition of 30 million zinc atoms per erythrocyte before host cell rupture, corresponding to a 400% increase in total zinc concentration. Zinc accumulates in a freely available form in parasitophorous compartments outside the food vacuole, including mitochondria. Restriction of zinc availability via small molecule treatment causes a drop in mitochondrial membrane potential and severely inhibits parasite growth. Thus, extraordinary zinc acquisition and trafficking are essential for parasite development.     

Long-term super-resolution imaging of mitochondrial dynamics

Monitoring dynamics of mitochondria has become an essential approach to explore the function of mitochondria in living cells with the emergence of super-resolution fluorescence microscopy. However, long-term super-resolution imaging of mitochondria is still challenging due to the lack of photostable fluorescent probes and stable mitochondria-specific markers which are not affected by the changes of mitochondrial membrane potential. Here, we introduce a method for long-term imaging mitochondrial dynamic through the SNAP-tag fluorogenic probe based on 4-azetidinyl-naphthalimide derivatives. Using structured illumination microscopy (SIM), we observed the fusion and fission of mitochondria over a course of 16 min at 109 nm resolution. Furthermore, the interactions as well as fusion between mitochondria and lysosomes were studied during mitophagy at the nanoscale. Convincingly, the combination of SNAP-tag fluorogenic probes and super-resolution fluorescence microscopy will offer a new way to monitor dynamic mitochondria in living cells.     

Long-term super-resolution imaging of mitochondrial dynamics

Monitoring dynamics of mitochondria has become an essential approach to explore the function of mitochondria in living cells with the emergence of super-resolution fluorescence microscopy. However, long-term super-resolution imaging of mitochondria is still challenging due to the lack of photostable fluorescent probes and stable mitochondria-specific markers which are not affected by the changes of mitochondrial membrane potential. Here, we introduce a method for long-term imaging mitochondrial dynamic through the SNAP-tag fluorogenic probe based on 4-azetidinyl-naphthalimide derivatives. Using structured illumination microscopy (SIM), we observed the fusion and fission of mitochondria over a course of 16 min at 109 nm resolution. Furthermore, the interactions as well as fusion between mitochondria and lysosomes were studied during mitophagy at the nanoscale. Convincingly, the combination of SNAP-tag fluorogenic probes and super-resolution fluorescence microscopy will offer a new way to monitor dynamic mitochondria in living cells.