scholarly journals Glucose induces the translocation of glycogen synthase to the cell cortex in rat hepatocytes

1997 ◽  
Vol 321 (1) ◽  
pp. 227-231 ◽  
Author(s):  
Josep M. FERNÁNDEZ-NOVELL ◽  
David BELLIDO ◽  
Senen VILARÓ ◽  
Joan J. GUINOVART
Keyword(s):  
Laser Scanning ◽  
Glycogen Synthase ◽  
Protein A ◽  
Rat Hepatocytes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Glycogen Deposition ◽  
In Cells

After incubation with glucose a dramatic change in the intracellular distribution of glycogen synthase was observed in rat hepatocytes. Confocal laser scanning microscopy showed that glycogen synthase existed diffusely in the cytosol of control cells, whereas in cells incubated with glucose it accumulated at the cell periphery. Colocalization analysis between glycogen synthase immunostaining and actin filaments showed that the change in glycogen synthase distribution induced by glucose correlated with a marked increase in the co-distribution of the two proteins, indicating that, in response to glucose, glycogen synthase moves to the actin-rich area close to the membrane. When glycogen synthase was immunostained with rabbit anti-(glycogen synthase) and Protein A–colloidal gold, few particles were observed close to the membrane in control cells. In contrast, in cells incubated with glucose most of the gold particles were found near the membrane, confirming that glycogen synthase had moved to the cell cortex. Furthermore, in agreement with the glycogen synthase distribution, glycogen deposition appeared to be more active at the periphery of the cell.

Probing Enhanced Cytochrome P450 2B1/2 Activity in Rat Hepatocyte Spheroids through Confocal Laser Scanning Microscopy

2001 ◽  
Vol 10 (3) ◽  
pp. 329-342 ◽  
Author(s):  
Emmanouhl S. Tzanakakis ◽  
Chang-Chun Hsiao ◽  
Taku Matsushita ◽  
Rory P. Remmel ◽  
Wei-Shou Hu
Keyword(s):  
Cytochrome P450 ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Constitutive Expression ◽  
Rat Hepatocytes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser

Cytochrome P450 (CYP450) enzymes are essential for xenobiotic metabolism. Although CYP450s are found in many tissues, CYP2B1/2 are primarily expressed in the rat liver. The constitutive expression in vivo of CYP2B1/2 is low but it is induced in the presence of various drugs such as phenobarbital (PB). In this study, CYP2B1/2 activity in cultured hepatocytes was assessed in situ with the introduction of a fluorogenic sub-strate, pentoxyresorufin. The product of 7-pentoxyresorufin-O-dealkylation (PROD), which is catalyzed specifically by CYP2B1/2, was detected using confocal laser scanning microscopy (CLSM). Primary hepatocytes cultured as monolayers on collagen-coated surfaces exhibited background PROD activity and minimal PB inducibility after 4 days in culture. In contrast, rat hepatocytes organized in compacted aggregates, or spheroids, exhibited higher levels of PROD activity and retained their ability for PB induction. The results from the CLSM analysis were verified by RT-PCR and Western immunoblotting analysis. Furthermore, CLSM in conjunction with image processing techniques and three-dimensional reconstruction revealed the localization of enhanced PROD activity in the center of spheroids. The results support the use of CLSM as a powerful tool for investigating CYP2B1/2 activity in cultured rat hepatocytes.

scholarly journals Proline-rich tyrosine kinase2 is involved in F-actin organization during in vitro maturation of rat oocyte

Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 859-867 ◽  
Author(s):  
Xiao-Qian Meng ◽  
Ke-Gang Zheng ◽  
Yong Yang ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Actin Filaments ◽  
Laser Scanning ◽  
Polar Body ◽  
Laser Scanning Microscopy ◽  
Meiotic Spindle ◽  
Confocal Laser ◽  
Cell Cortex ◽  
First Polar Body

Microfilaments (actin filaments) regulate various dynamic events during meiotic maturation. Relatively, little is known about the regulation of microfilament organization in mammalian oocytes. Proline-rich tyrosine kinase2 (Pyk2), a protein tyrosine kinase related to focal adhesion kinase (FAK) is essential in actin filaments organization. The present study was to examine the expression and localization of Pyk2, and in particular, its function during rat oocyte maturation. For the first time, by using Western blot and confocal laser scanning microscopy, we detected the expression of Pyk2 in rat oocytes and found that Pyk2 and Try402 phospho-Pyk2 were localized uniformly at the cell cortex and surrounded the germinal vesicle (GV) or the condensed chromosomes at the GV stage or after GV breakdown. At the metaphase and the beginning of anaphase, Pyk2 distributed asymmetrically both in the ooplasm and the cortex with a marked staining associated with the chromosomes and the region overlying the meiotic spindle. At telophase, Pyk2 was observed in the cleavage furrows in addition to its cortex and cytoplasm localization. The dynamics of Pyk2 were similar to that of F-actin, and this kinase was found to co-localize with microfilaments in several developmental stages during rat oocyte maturation. Microinjection of Pyk2 antibody demolished the microfilaments assembly and also inhibited the first polar body (PB1) emission. These findings suggest an important role of Pyk2 for rat oocyte maturation by regulating the organization of actin filaments.

scholarly journals Quantification of Protein A-Gold Staining for Peroxisomal Enzymes by Confocal Laser Scanning Microscopy

1999 ◽  
Vol 47 (10) ◽  
pp. 1343-1349 ◽  
Author(s):  
Naoko Ogiwara ◽  
Nobuteru Usuda ◽  
Masashi Yamada ◽  
Kohei Johkura ◽  
Kiyokazu Kametani ◽  
...  
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Protein A ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser

scholarly journals Stay in Touch—The Cortical ER of Moss Protonemata in Osmotic Stress Situations

Plants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 421 ◽  
Author(s):  
Dominik Harant ◽  
Ingeborg Lang
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Laser Scanning ◽  
Structural Changes ◽  
Physcomitrella Patens ◽  
Dynamic Network ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Natural State ◽  
Cell Cortex

Plasmolysis is usually introduced to cell biology students as a tool to illustrate the plasma membrane: hypertonic solutions cause the living protoplast to shrink by osmotic water loss; hence, it detaches from the surrounding cell wall. What happens, however, with the subcellular structures in the cell cortex during this process of turgor loss? Here, we investigated the cortical endoplasmic reticulum (ER) in moss protonema cells of Physcomitrella patens in a cell line carrying a transgenic ER marker (GFP-HDEL). The plasma membrane was labelled simultaneously with the fluorescent dye FM4-64 to achieve structural separation. By placing the protonemata in a hypertonic mannitol solution (0.8 M), we were able to follow the behaviour of the cortical ER and the protoplast during plasmolysis by confocal laser scanning microscopy (CLSM). The protoplast shape and structural changes of the ER were further examined after depolymerisation of actin microfilaments with latrunculin B (1 µM). In its natural state, the cortical ER is a dynamic network of fine tubes and cisternae underneath the plasma membrane. Under acute and long-term plasmolysis (up to 45 min), changes in the protoplast form and the cortical ER, as well as the formation of Hechtian strands and Hechtian reticula, were observed. The processing of the high-resolution z-scans allowed the creation of 3D models and gave detailed insight into the ER of living protonema cells before, during and after plasmolysis.

Actin microfilaments are associated with the migrating nucleus and the cell cortex in the green alga Micrasterias. Studies on living cells

1994 ◽  
Vol 107 (7) ◽  
pp. 1929-1934 ◽  
Author(s):  
U. Meindl ◽  
D. Zhang ◽  
P.K. Hepler
Keyword(s):  
Laser Scanning ◽  
Nuclear Migration ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cell Cortex ◽  
Actin Network ◽  
Cortical Actin ◽  
Electron Microscopic ◽  
Rhodamine Phalloidin ◽  
Growing Cell

Rhodamine-phalloidin or FITC-phalloidin has been injected in small amounts into living, developing cells of Micrasterias denticulata and the stained microfilaments visualized by confocal laser scanning microscopy. The results reveal that two different actin filament systems are present in a growing cell: a cortical actin network that covers the inner surface of the cell and is extended far into the tips of the lobes in both the growing and the nongrowing semicell; it is also associated with the surface of the chloroplast. The second actin system ensheathes the nucleus at the isthmus-facing side during nuclear migration. Its arrangement corresponds to that of the microtubule system that has been described in earlier electron microscopic investigations. The spatial correspondence between the distribution of actin filaments and microtubules suggests a cooperation between both cytoskeleton elements in generating the motive force for nuclear migration. The function of the cortical actin network is not yet clear. It may be involved in processes like transport and fusion of secretory vesicles and may also function in shaping and anchoring the chloroplast.

Internalized SP-A and lipid are differentially resecreted by type II pneumocytes

2000 ◽  
Vol 278 (3) ◽  
pp. L580-L590 ◽  
Author(s):  
Heide Wissel ◽  
Stefan Zastrow ◽  
Ekkehard Richter ◽  
Paul A. Stevens
Keyword(s):  
Laser Scanning ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Surfactant Protein ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Type Ii Cells ◽  
Type Ii ◽  
Type Ii Pneumocytes

Biochemical and morphological assays were developed to study surfactant protein A (SP-A) and lipid resecretion kinetics by isolated type II cells in vitro. After a 10-min uptake period with SP-A (3 μg/106 cells) in combination with liposomes (60 μg/106 cells), the cells were allowed to resecrete. After 5 min of resecretion, only 21.7 ± 4.6% of the internalized SP-A remained intracellularly compared with 54 ± 2.9% of the lipids. Extracellular SP-A present during the resecretion period partially inhibited resecretion (SP-A, 36% at 5 min; lipid, ∼16% at 5 min). Lipid resecretion was also dependent on the SP-A concentration present during the uptake period. Although, as shown by confocal laser scanning microscopy, after a 10-min uptake period at 37°C, most of the fluorescein isothiocyanate-labeled SP-A and rhodamine-phosphatidylethanolamine-labeled lipids colocalized within the cells, after an additional 10 min of resecretion, both the strength of the fluorescence signals and the extent of colocalization had markedly decreased. These data indicate that internalized lipid and SP-A can be resecreted rapidly by type II cells, likely via different pathways.

Surfactant protein A and lipid are internalized via the coated-pit pathway by type II pneumocytes

2001 ◽  
Vol 280 (1) ◽  
pp. L141-L151 ◽  
Author(s):  
Paul A. Stevens ◽  
Heide Wissel ◽  
Stefan Zastrow ◽  
Daniela Sieger ◽  
Klaus-Peter Zimmer
Keyword(s):  
Laser Scanning ◽  
Protein A ◽  
Early Time ◽  
Surfactant Protein ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lipid Uptake

Surfactant protein (SP) A and SP-A-mediated lipid uptake by isolated type II cells were investigated with biochemical and morphological methods. Inhibition of coated-pit function by potassium depletion severely reduced both SP-A and SP-A-mediated lipid internalization. Lipid uptake in the absence of SP-A was not affected. With confocal laser scanning microscopy and immunoelectron microscopy, SP-A and lipid predominantly (60%) colocalized in intracellular vesicles carrying early endosomal markers (EEA1) 5 min after endocytosis but were negative for the late endosomal or lysosomal marker LAMP-1. As estimated by subcellular fractionation, at this time point, 23% of the internalized SP-A and 45% of internalized lipid were localized within light (<0.38 M sucrose) fractions, which contain lamellar bodies and are positive for EEA1. The remaining label was predominantly found within EEA1-positive and plasma membrane-containing subfractions (≥1 M sucrose). We suggest that in isolated type II cells in vitro, SP-A and lipid are taken up together via the coated-pit pathway and that at early time points, both components reside in the same early endosomal compartment.

scholarly journals Aptamer-Aptamer Chimera for Targeted Delivery and ATP-Responsive Release of Doxorubicin into Cancer Cells

2021 ◽  
Vol 22 (23) ◽  
pp. 12940
Author(s):  
Ezaldeen Esawi ◽  
Walhan Alshaer ◽  
Ismail Sami Mahmoud ◽  
Dana A. Alqudah ◽  
Bilal Azab ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cancer Cell ◽  
Targeted Delivery ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Innovative Drug ◽  
Great Opportunity ◽  
Selective Delivery ◽  
In Cells

Aptamers offer a great opportunity to develop innovative drug delivery systems that can deliver cargos specifically into targeted cells. In this study, a chimera consisting of two aptamers was developed to deliver doxorubicin into cancer cells and release the drug in cytoplasm in response to adenosine-5′-triphosphate (ATP) binding. The chimera was composed of the AS1411 anti-nucleolin aptamer for cancer cell targeting and the ATP aptamer for loading and triggering the release of doxorubicin in cells. The chimera was first produced by hybridizing the ATP aptamer with its complementary DNA sequence, which is linked with the AS1411 aptamer via a poly-thymine linker. Doxorubicin was then loaded inside the hybridized DNA region of the chimera. Our results show that the AS1411–ATP aptamer chimera was able to release loaded doxorubicin in cells in response to ATP. In addition, selective uptake of the chimera into cancer cells was demonstrated using flow cytometry. Furthermore, confocal laser scanning microscopy showed the successful delivery of the doxorubicin loaded in chimeras to the nuclei of targeted cells. Moreover, the doxorubicin-loaded chimeras effectively inhibited the growth of cancer cell lines and reduced the cytotoxic effect on the normal cells. Overall, the results of this study show that the AS1411–ATP aptamer chimera could be used as an innovative approach for the selective delivery of doxorubicin to cancer cells, which may improve the therapeutic potency and decrease the off-target cytotoxicity of doxorubicin.

scholarly journals Branchial chloride cells in larvae and juveniles of freshwater tilapia Oreochromis mossambicus

1995 ◽  
Vol 198 (10) ◽  
pp. 2177-2184 ◽  
Author(s):  
J Li ◽  
J Eygensteyn ◽  
R Lock ◽  
P Verbost ◽  
A Heijden ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Specific Activity ◽  
Protein A ◽  
Oreochromis Mossambicus ◽  
Laser Scanning Microscopy ◽  
Chloride Cells ◽  
Confocal Laser ◽  
Larval Stages ◽  
Larvae And Juveniles ◽  
Secondary Lamellae

Branchial chloride cells in the developing larvae and juveniles of freshwater tilapia, Oreochromis mossambicus, were identified and the membrane Na+/K+-ATPase was localized in situ through binding of the fluorescent dye anthroylouabain. After co-labelling of the cells with the fluorescent probes DASPMI and Con-A-FITC, the mitochondria and apical crypt in the same chloride cells were visualized using confocal laser scanning microscopy. The high density of apical crypts indicated that many chloride cells were functional. The density of branchial chloride cells in larvae 10 days after hatching was approximately 6000 mm-2. An extremely high Na+/K+-ATPase specific activity of approximately 1500 &micro;mol Pi h-1 mg-1 was measured in the gills 10 days after hatching. With the development of secondary lamellae and hence an increase in the amount of branchial epithelial protein, a concomitant decrease in the specific activity of the enzyme in the gill tissues was observed. Total Na+/K+-ATPase activity increased markedly in the early life stages. Our data indicate that in larval stages of fish the gills form a functional ionoregulatory organ before they start functioning as a gas-exchange organ.

scholarly journals Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes.

1995 ◽  
Vol 131 (1) ◽  
pp. 137-150 ◽  
Author(s):  
R Mayer ◽  
J Kartenbeck ◽  
M Büchler ◽  
G Jedlitschky ◽  
I Leier ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Immunofluorescence Microscopy ◽  
Rat Hepatocytes ◽  
Laser Scanning Microscopy ◽  
Multi Drug Resistance ◽  
Confocal Laser ◽  
Double Immunofluorescence ◽  
Plasma Membrane Vesicles ◽  
Canalicular Membrane ◽  
Cdna Fragments

We have previously shown that the multi-drug resistance protein (MRP) mediates the ATP-dependent membrane transport of glutathione S-conjugates and additional amphiphilic organic anions. In the present study we demonstrate the expression of MRP in hepatocytes where it functions in hepatobiliary excretion. Analysis by reverse transcription-PCR of human and normal rat liver mRNA resulted in two expected cDNA fragments of MRP. Four different antibodies against MRP reacted on immunoblots with the glycoprotein of about 190 kD from human canalicular as well as basolateral hepatocyte membrane preparations. A polyclonal antibody directed against the carboxy-terminal sequence of MRP detected the rat homolog of MRP in liver. Double immunofluorescence microscopy and confocal laser scanning microscopy showed the presence of human MRP and rat Mrp in the canalicular as well as in the lateral membrane domains of hepatocytes. The transport function of the mrp gene-encoded conjugate export pump was assayed in plasma membrane vesicles with leukotriene C4 as a high-affinity glutathione S-conjugate substrate. The deficient ATP-dependent conjugate transport in canalicular membranes from TR- mutant rat hepatocytes was associated with a lack of amplification of one of the mrp cDNA fragments and with a selective loss of Mrp on immunoblots of canalicular membranes. Double immunofluorescence microscopy of livers from transport-deficient TR- mutant rats localized Mrp only to the lateral but not to the canalicular membrane. Our results indicate that the absence of Mrp or an isoform of Mrp from the canalicular membrane is the basis for the hereditary defect of the hepatobiliary excretion of anionic conjugates by the transport-deficient hepatocyte.

Sign in / Sign up

Export Citation Format

Share Document