scholarly journals Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks.

1993 ◽  
Vol 123 (3) ◽  
pp. 691-705 ◽  
Author(s):  
T S Stappenbeck ◽  
E A Bornslaeger ◽  
C M Corcoran ◽  
H H Luu ◽  
M L Virata ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Cultured Cells ◽  
Sequence Similarity ◽  
Full Length ◽  
Carboxyl Terminus ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Carboxyl Terminal ◽  
Filament Networks ◽  
Vimentin Intermediate Filament

We previously demonstrated that truncated desmoplakin I (DP I) molecules containing the carboxyl terminus specifically coalign with and disrupt both keratin and vimentin intermediate filament (IF) networks when overexpressed in tissue culture cells (Stappenbeck, T. S., and K. J. Green. J. Cell Biol. 116:1197-1209). These experiments suggested that the DP carboxyl-terminal domain is involved either directly or indirectly in linking IF with the desmosome. Using a similar approach, we have now investigated the behavior of ectopically expressed full-length DP I in cultured cells. In addition, we have further dissected the functional sequences in the carboxyl terminus of DP I that facilitate the interaction with IF networks. Transient transfection of a clone encoding full-length DP I into COS-7 cells produced protein that appeared in some cells to associate with desmosomes and in others to coalign with and disrupt IF. Deletion of the carboxyl terminus from this clone resulted in protein that still appeared capable of associating with desmosomes but not interacting with IF networks. As the amino terminus appeared to be dispensable for IF interaction, we made finer deletions in the carboxyl terminus of DP based on blocks of sequence similarity with the related molecules bullous pemphigoid antigen and plectin. We found a sequence at the very carboxyl terminus of DP that was necessary for coalignment with and disruption of keratin IF but not vimentin IF. Furthermore, the coalignment of specific DP proteins along keratin IF but not vimentin IF was correlated with resistance to extraction by Triton. The striking uncoupling resulting from the deletion of specific DP sequences suggests that the carboxyl terminus of DP interacts differentially with keratin and vimentin IF networks.

Peripherin assembles into homopolymers in SW13 cells

1995 ◽  
Vol 108 (10) ◽  
pp. 3279-3284 ◽  
Author(s):  
C. Cui ◽  
P.J. Stambrook ◽  
L.M. Parysek
Keyword(s):  
Intermediate Filament ◽  
Network Formation ◽  
Full Length ◽  
Intermediate Filament Proteins ◽  
Filament Assembly ◽  
Mature Neurons ◽  
Carboxyl Terminal ◽  
Relationship Of ◽  
Filament Network ◽  
Carboxyl Tail

The properties of full-length and mutant peripherins were studied in intermediate filament-less SW13 cells to define regions of peripherin that are essential for initiation of filament assembly. A full-length rat peripherin gene transfected into SW13 cells resulted in filament formation, consistent with the close structural relationship of peripherin to other type III intermediate filament proteins that readily form homopolymers. Translation of full-length rat peripherin is initiated predominantly at the second of two inframe AUGs. Deletions within the amino terminus of wild-type peripherin abolished its ability to form filaments in SW13 cells. In contrast, deletion of the entire carboxyl-terminal tail of peripherin did not affect its ability to form filamentous arrays in transfected SW13 cells. These results indicate that, of the intermediate filament proteins that are expressed in mature neurons, only peripherin and alpha-internexin are capable of making homopolymer intermediate filaments. In addition, mutations of the carboxyl tail of peripherin generally do not interfere with filament network formation.

scholarly journals LOCALIZATION OF A PRIMATE-SPECIFIC ESTERASE USING IMMUNOFLUORESCENCE AND IMMUNOPEROXIDASE TECHNIQUES

1973 ◽  
Vol 21 (6) ◽  
pp. 559-567 ◽  
Author(s):  
GEORGIRENE D. VLADUTIU ◽  
PIERLUIGI E. BIGAZZI ◽  
NOEL R. ROSE
Keyword(s):  
Cultured Cells ◽  
Monkey Kidney ◽  
Close Apposition ◽  
Proximal Tubule Cells ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Golgi Region ◽  
Collecting Tubules ◽  
Convoluted Tubules ◽  
Bowman's Capsule

The techniques of mixed antibody immunoperoxidase and indirect immunofluorescence were used for the localization of an esterase isoenzyme in human and monkey tissue culture cells and in monkey kidney frozen sections. In cultured cells, the esterase was found to be cytoplasmic in close apposition to the nucleus with a distribution resembling that of the Golgi region. The esterase in monkey kidney was predominantly found in the cytoplasm of the proximal convoluted tubules. There was slight staining in the Bowman's capsule of the glomerulus and in the collecting tubules of the medulla. It is suggested that the kidney is actually producing the esterase in its proximal tubule cells.

scholarly journals Stiffening and inelastic fluidization in vimentin intermediate filament networks

Soft Matter ◽  
2019 ◽  
Vol 15 (36) ◽  
pp. 7127-7136 ◽  
Author(s):  
Anders Aufderhorst-Roberts ◽  
Gijsje H. Koenderink
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Shear Rheology ◽  
Cellular Processes ◽  
Filament Networks ◽  
Nonlinear Shear ◽  
Vimentin Intermediate Filament

Nonlinear shear rheology reveals that intermediate filaments balance two contradictory roles: mechanoprotection by stiffening and dynamic cellular processes through softening.

Nuclear import of cubitus interruptus is regulated by hedgehog via a mechanism distinct from Ci stabilization and Ci activation

Development ◽  
2000 ◽  
Vol 127 (14) ◽  
pp. 3131-3139 ◽  
Author(s):  
Q.T. Wang ◽  
R.A. Holmgren
Keyword(s):  
Nuclear Import ◽  
Full Length ◽  
Negative Regulators ◽  
Cell Fates ◽  
Cubitus Interruptus ◽  
Drosophila Wing ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
The Relationship

The Hedgehog (Hh) signal is transduced via Cubitus interruptus (Ci) to specify cell fates in the Drosophila wing. In the absence of Hh, the 155 kDa full-length form of Ci is cleaved into a 75 kDa repressor. Hh inhibits the proteolysis of full-length Ci and facilitates its conversion into an activator. Recently, it has been suggested that Hh promotes Ci nuclear import in tissue culture cells. We have studied the mechanism of Ci nuclear import in vivo and the relationship between nuclear import, stabilization and activation. We found that Ci rapidly translocates to the nucleus in cells close to the anteroposterior (AP) boundary and this rapid nuclear import requires Hh signaling. The nuclear import of Ci is regulated by Hh even under conditions in which Ci is fully stabilized. Furthermore, cells that exhibit Ci stabilization and rapid nuclear import do not necessarily exhibit maximal Ci activity. It has been previously shown that stabilization does not suffice for activation. Consistent with this finding, our results suggest that the mechanisms regulating nuclear import, stabilization and activation are distinct from each other. Finally, we show that cos2 and pka, two molecules that have been characterized primarily as negative regulators of Ci activity, also have positive roles in the activation of Ci in response to Hh.

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.

scholarly journals Antibody against the Carboxyl Terminus of Intimin α Reduces Enteropathogenic Escherichia coli Adherence to Tissue Culture Cells and Subsequent Induction of Actin Polymerization

2005 ◽  
Vol 73 (4) ◽  
pp. 2541-2546 ◽  
Author(s):  
Humberto M. Carvalho ◽  
Louise D. Teel ◽  
John F. Kokai-Kun ◽  
Alison D. O'Brien
Keyword(s):  
Escherichia Coli ◽  
Tissue Culture ◽  
Actin Polymerization ◽  
Carboxyl Terminus ◽  
Enteropathogenic Escherichia Coli ◽  
Lesion Formation ◽  
Culture Cells ◽  
Tissue Culture Cells

ABSTRACT The C-terminal third of intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of intimin α.

Desmin and Vimentin Intermediate Filament Networks: Their Viscoelastic Properties Investigated by Mechanical Rheometry

2009 ◽  
Vol 388 (1) ◽  
pp. 133-143 ◽  
Author(s):  
Michael Schopferer ◽  
Harald Bär ◽  
Bernhard Hochstein ◽  
Sarika Sharma ◽  
Norbert Mücke ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Viscoelastic Properties ◽  
Filament Networks ◽  
Vimentin Intermediate Filament

scholarly journals Motile Properties of Vimentin Intermediate Filament Networks in Living Cells

1998 ◽  
Vol 143 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Miri Yoon ◽  
Robert D. Moir ◽  
Veena Prahlad ◽  
Robert D. Goldman
Keyword(s):  
Intermediate Filament ◽  
Cell Shape ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
A Cell ◽  
Filament Networks ◽  
Green Fluorescent ◽  
Vimentin Intermediate Filament

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.

Nanomechanics of vimentin intermediate filament networks

Soft Matter ◽  
2010 ◽  
Vol 6 (9) ◽  
pp. 1910 ◽  
Author(s):  
Sarah Köster ◽  
Yi-Chia Lin ◽  
Harald Herrmann ◽  
David A. Weitz
Keyword(s):  
Intermediate Filament ◽  
Filament Networks ◽  
Vimentin Intermediate Filament
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