Analysis of Lysophagic Flux in Cultured Cells using Lyso-Keima v1

2021 ◽  
Author(s):  
Vinay V. Eapen ◽  
Sharan Swarup ◽  
Melisa Hoyer ◽  
Harper not provided JW
Keyword(s):  
Tissue Culture ◽  
Cultured Cells ◽  
Confocal Imaging ◽  
Signaling Proteins ◽  
Membrane Rupture ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Luminal Side ◽  
Galectin 3 ◽  
Autophagy Pathway

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe a facile method for monitoring lysophagy using the acid sensitive fluorophore mKeima, affixed onto Galectin-3, which allows for the monitoring of lysophagic flux by Flow cytometry, Western blotting or Confocal imaging. This method, which we have termed Lyso-Keima, serves as a facile and quantitative assay for monitoring lysophagy in tissue culture cells.

Analysis of Lysophagic Flux in Cultured Cells using Lyso-Keima v2

2021 ◽  
Author(s):  
Vinay V. Eapen ◽  
Sharan Swarup ◽  
Melisa Hoyer ◽  
Harper JW
Keyword(s):  
Tissue Culture ◽  
Cultured Cells ◽  
Confocal Imaging ◽  
Signaling Proteins ◽  
Membrane Rupture ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Luminal Side ◽  
Galectin 3 ◽  
Autophagy Pathway

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe a facile method for monitoring lysophagy using the acid sensitive fluorophore mKeima, affixed onto Galectin-3, which allows for the monitoring of lysophagic flux by Flow cytometry, Western blotting or Confocal imaging. This method, which we have termed Lyso-Keima, serves as a facile and quantitative assay for monitoring lysophagy in tissue culture cells.

Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe v1

2021 ◽  
Author(s):  
Vinay V. Eapen ◽  
Sharan Swarup ◽  
Melissa Hoyer ◽  
Harper not provided JW
Keyword(s):  
Confocal Imaging ◽  
Signaling Proteins ◽  
Membrane Rupture ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Luminal Side ◽  
Selective Elimination ◽  
Galectin 3 ◽  
Autophagy Pathway ◽  
Lysosomal Proteins

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe our method for monitoring galectin-3 puncta clearance as a proxy for turnover of damaged lysosomes via immunofluorescence and confocal imaging.

Immunofluorescence of Galectin-3 Puncta after lysosomal damage with LLoMe v2

2021 ◽  
Author(s):  
Vinay V. Eapen ◽  
Sharan Swarup ◽  
Melissa Hoyer ◽  
Harper not provided JW
Keyword(s):  
Confocal Imaging ◽  
Signaling Proteins ◽  
Membrane Rupture ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Luminal Side ◽  
Selective Elimination ◽  
Galectin 3 ◽  
Autophagy Pathway ◽  
Lysosomal Proteins

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe our method for monitoring galectin-3 puncta clearance as a proxy for turnover of damaged lysosomes via immunofluorescence and confocal imaging.

Immunofluorescence of autophagic cargo receptors and p-TBK1 at LAMP1 lysosomes during lysophagy v1

2021 ◽  
Author(s):  
Vinay V. Eapen ◽  
Sharan Swarup ◽  
Melissa Hoyer ◽  
Harper not provided not provided JW
Keyword(s):  
Confocal Imaging ◽  
Signaling Proteins ◽  
Membrane Rupture ◽  
Culture Cells ◽  
Luminal Side ◽  
Selective Elimination ◽  
Galectin 3 ◽  
Autophagy Pathway ◽  
Protein Recruitment ◽  
Lysosomal Proteins

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe a method for monitoring protein recruitment to damaged lysosomes via immunofluorescence and confocal imaging.

Analysis of Lysophagic Flux in Cultured Induced Neurons using RFP-GFP-galectin3 v1

2021 ◽  
Author(s):  
Vinay Eapen ◽  
Harper not provided not provided JW ◽  
Melissa Hoyer ◽  
sharan_swarup not provided
Keyword(s):  
Confocal Imaging ◽  
Signaling Proteins ◽  
Membrane Rupture ◽  
Culture Cells ◽  
Luminal Side ◽  
Selective Elimination ◽  
Galectin 3 ◽  
Autophagy Pathway ◽  
Induced Neurons ◽  
Lysosomal Proteins

Lysophagy-the selective elimination of damaged lysosomes by the autophagy pathway-is a critical housekeeping mechanism in cells. This pathway surveils lysosomes and selectively demarcates terminally damaged lysosomes for elimination. Among the most upstream signaling proteins in this pathway are the glycan binding proteins-Galectins-which recognize N and O linked glycan chains on the luminal side of transmembrane lysosomal proteins. These glycosyl modifications are only accessible to galectin proteins upon extensive lysosomal membrane rupture and serve as a sensitive measure of lysosomal damage and eventual clearance by selective autophagy. Indeed, prior work has shown that immunofluorescence of Galectin-3 serves as a convenient proxy for lysophagic flux in tissue culture cells (Aits et al., 2015; Maejima et al., 2013). Here we describe our method for monitoring GFP positive RFP-GFP-galectin-3 GFP positive puncta clearance as a proxy for turnover of damaged lysosomes via immunofluorescence and confocal imaging.

scholarly journals Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells.

1984 ◽  
Vol 98 (6) ◽  
pp. 2126-2132 ◽  
Author(s):  
M C Beckerle
Keyword(s):  
Tissue Culture ◽  
Cultured Cells ◽  
Cytoplasmic Matrix ◽  
High Voltage Electron Microscopy ◽  
Polystyrene Beads ◽  
Voltage Electron ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
A Cell ◽  
Path Lengths

Microinjected 0.26-micron fluorescent, carboxylated microspheres were found to display classical saltatory motion in tissue culture cells. The movement of a given particle was characterized by a discontinuous velocity distribution and was unaffected by the activity of adjacent particles. The microspheres were translocated at velocities of up to 4.7 micron/s and sometimes exhibited path lengths greater than 20 micron for a single saltation . The number of beads injected into a cell could range from a few to over 500 with no effect on the cell's ability to transport them. Neither covalent cross-linking nor preincubation of the polystyrene beads with various proteins inhibited the saltatory motion of the injected particles. The motion of the injected beads in cultured cells was reversibly inhibited by the microtubule poison nocodazole, under conditions in which actin-rich, nitrobenzoxadiazol - phallacidin -staining structures remain intact. Whole-cell high voltage electron microscopy of microinjected cells that were known to be moving the fluorescent microspheres revealed that the beads were embedded in the cytoplasmic matrix and did not appear to be membrane bound. The enhanced detectability of the fluorescent particles over endogenous organelles and the ability to modify the surfaces of the beads before injection may enable more detailed studies on the mechanism of saltatory particle motion.

Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

1969 ◽  
Vol 27 ◽  
pp. 376-377
Author(s):  
A. M. Watrach
Keyword(s):  
Tissue Culture ◽  
Small Proportion ◽  
Chicken Embryo ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Morphologic Characteristics ◽  
Infectious Laryngotracheitis ◽  
Laryngotracheitis Virus ◽  
In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

Sulfhydryl bond formation is a prerequisite for proper cycling of centrosomes and chromosomes in mammalian tissue culture cells

1993 ◽  
Vol 51 ◽  
pp. 342-343
Author(s):  
Heide Schatten ◽  
Neidhard Paweletz ◽  
Ron Balczon
Keyword(s):  
Tissue Culture ◽  
Cell Cycle Progression ◽  
Group Formation ◽  
Sulfhydryl Group ◽  
Mammalian Tissue ◽  
Mitotic Chromosomes ◽  
Microtubule Network ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Transmission Electron

To study the role of sulfhydryl group formation during cell cycle progression, mammalian tissue culture cells (PTK2) were exposed to 100¼M 2-mercaptoethanol for 2 to 6 h during their exponential phase of growth. The effects of 2-mercaptoethanol on centrosomes, chromosomes, microtubules, membranes and intermediate filaments were analyzed by transmission electron microscopy (TEM) and by immunofluorescence microscopy (IFM) methods using a human autoimmune antibody directed against centrosomes (SPJ), and a mouse monoclonal antibody directed against tubulin (E7). Chromosomes were affected most by this treatment: premature chromosome condensation was detected in interphase nuclei, and the structure in mitotic chromosomes was altered compared to control cells. This would support previous findings in dividing sea urchin cells in which chromosomes are arrested at metaphase while the centrosome splitting cycle continues. It might also support findings that certairt-sulfhydryl-blocking agents block cyclin destruction. The organization of the microtubule network was scattered probably due to a looser organization of centrosomal material at the interphase centers and at the mitotic poles.

UDP-D-glucose 4-epimerase activity of the tissue culture of white poplars (Populus alba L. var. pyramidalis)

1982 ◽  
Vol 47 (5) ◽  
pp. 1530-1536 ◽  
Author(s):  
Ladislav Bilisics ◽  
Štefan Karácsonyi ◽  
Marta Kubačková
Keyword(s):  
Tissue Culture ◽  
Cell Walls ◽  
Enzyme Preparation ◽  
Uridine Diphosphate ◽  
Populus Alba ◽  
Activity Change ◽  
White Poplar ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Galactose Content

The presence of UDP-D-glucose 4-epimerase (EC 5.1.3.2) in the culture tissue of white poplar was evidenced. As found, the partially purified enzyme preparation contained UDP-D-glucose glucosyltransferase, UDP-D-galactose galactosyltransferase and non-specific enzymes able to cleave the uridine-diphosphate saccharides into the appropriate hexose monophosphates. The activity change of UDP-D-glucose 4-epimerase in tissue culture cells during the growth was in accord with changes in D-galactose content in cell walls and indicated the possibility to regulate the formation of polysaccharides containing D-galactose at the level of production of UDP-D-galactose in cells.

Sign in / Sign up

Export Citation Format

Share Document