scholarly journals Relative importance of βcyto- and γcyto-actin in primary mouse embryonic fibroblasts

2017 ◽  
Vol 28 (6) ◽  
pp. 771-782 ◽  
Author(s):  
Xiaobai Patrinostro ◽  
Allison R. O'Rourke ◽  
Christopher M. Chamberlain ◽  
Branden S. Moriarity ◽  
Benjamin J. Perrin ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Gene Knockout ◽  
T Antigen ◽  
Mouse Embryonic Fibroblasts ◽  
Double Knockout ◽  
Embryonic Fibroblasts ◽  
Primary Fibroblast ◽  
Primary Mouse ◽  
Primary Fibroblasts ◽  
Actin Isoform

The highly homologous β (βcyto) and γ (γcyto) cytoplasmic actins are hypothesized to carry out both redundant and unique essential functions, but studies using targeted gene knockout and siRNA-mediated transcript knockdown to examine βcyto- and γcyto-isoform–­specific functions in various cell types have yielded conflicting data. Here we quantitatively characterized actin transcript and protein levels, as well as cellular phenotypes, in both gene- and transcript-targeted primary mouse embryonic fibroblasts. We found that the smooth muscle αsm-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary βcyto- or β/γcyto-actin double-knockout fibroblasts. Gene targeting of βcyto-actin, but not γcyto-actin, led to greatly decreased cell proliferation, decreased levels of cellular ATP, and increased serum response factor signaling in primary fibroblasts, whereas immortalization induced by SV40 large T antigen supported fibroblast proliferation in the absence of βcyto-actin. Consistent with in vivo gene-targeting studies in mice, both gene- and transcript-targeting approaches demonstrate that the loss of βcyto-actin protein is more disruptive to primary fibroblast function than is the loss of γcyto-actin.

scholarly journals Gene Targeting of Cdc42 and Cdc42GAP Affirms the Critical Involvement of Cdc42 in Filopodia Induction, Directed Migration, and Proliferation in Primary Mouse Embryonic Fibroblasts

2006 ◽  
Vol 17 (11) ◽  
pp. 4675-4685 ◽  
Author(s):  
Linda Yang ◽  
Lei Wang ◽  
Yi Zheng
Keyword(s):  
Gene Targeting ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Cell Model ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Membrane Ruffling ◽  
Primary Mouse

Recent studies in Cdc42 knockout mouse embryonic stem (ES) cells and ES-derived fibroblastoid cell lines raise concern on a body of literature derived by dominant mutant expression approach in a variety of cell lines implicating mammalian Cdc42 as a key regulator of filopodia induction, directional migration and cell cycle progression. To resolve the physiological function of mammalian Cdc42, we have characterized the Cdc42−/− and Cdc42GAP−/− primary mouse embryonic fibroblasts (MEFs) produced by gene targeting as the Cdc42 loss- or gain-of-activity cell model. The Cdc42−/− cells were defective in filopodia formation stimulated by bradykinin and in dorsal membrane ruffling stimulated by PDGF, whereas the Cdc42GAP−/− cells displayed spontaneous filopodia. The Cdc42 loss- or gain-of-activity cells were defective in adhesion to fibronectin, wound-healing, polarity establishment, and migration toward a serum gradient. These defects were associated with deficiencies of PAK1, GSK3β, myosin light chain, and FAK phosphorylation. Furthermore, Cdc42−/− cells were defective in G1/S-phase transition and survival, correlating with deficient NF-κB transcription and defective JNK, p70 S6K, and ERK1/2 activation. These results demonstrate a different requirement of Cdc42 activity in primary MEFs from ES or ES-derived clonal fibroblastoid cells and suggest that Cdc42 plays cell-type–specific signaling roles.

scholarly journals S6K1 Is Indispensible for Stress-Induced Microtubule Acetylation and Autophagic Flux

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 929
Author(s):  
Aleksandra Hać ◽  
Karolina Pierzynowska ◽  
Anna Herman-Antosiewicz
Keyword(s):  
Degradation Process ◽  
Underlying Mechanism ◽  
Stress Conditions ◽  
Autophagic Flux ◽  
Ratiometric Fluorescence ◽  
Mouse Embryonic Fibroblasts ◽  
Double Knockout ◽  
Embryonic Fibroblasts ◽  
Microtubule Network ◽  
Tubulin Acetylation

Autophagy is a specific macromolecule and organelle degradation process. The target macromolecule or organelle is first enclosed in an autophagosome, and then delivered along acetylated microtubules to the lysosome. Autophagy is triggered by stress and largely contributes to cell survival. We have previously shown that S6K1 kinase is essential for autophagic flux under stress conditions. Here, we aimed to elucidate the underlying mechanism of S6K1 involvement in autophagy. We stimulated autophagy in S6K1/2 double-knockout mouse embryonic fibroblasts by exposing them to different stress conditions. Transient gene overexpression or silencing, immunoblotting, immunofluorescence, flow cytometry, and ratiometric fluorescence analyses revealed that the perturbation of autophagic flux in S6K1-deficient cells did not stem from impaired lysosomal function. Instead, the absence of S6K1 abolished stress-induced tubulin acetylation and disrupted the acetylated microtubule network, in turn impairing the autophagosome-lysosome fusion. S6K1 overexpression restored tubulin acetylation and autophagic flux in stressed S6K1/2-deficient cells. Similar effect of S6K1 status was observed in prostate cancer cells. Furthermore, overexpression of an acetylation-mimicking, but not acetylation-resistant, tubulin variant effectively restored autophagic flux in stressed S6K1/2-deficient cells. Collectively, S6K1 controls tubulin acetylation, hence contributing to the autophagic flux induced by different stress conditions and in different cells.

Effect of PRAK gene knockout on the proliferation of mouse embryonic fibroblasts

2009 ◽  
Vol 3 (4) ◽  
pp. 379-383 ◽  
Author(s):  
Xiaowei Gong ◽  
Aihua Liu ◽  
Xiaoyan Ming ◽  
Xu Wang ◽  
Daan Wang ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts

scholarly journals The piggyBac Transposon-Mediated Expression of SV40 T Antigen Efficiently Immortalizes Mouse Embryonic Fibroblasts (MEFs)

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e97316 ◽  
Author(s):  
Ning Wang ◽  
Wenwen Zhang ◽  
Jing Cui ◽  
Hongmei Zhang ◽  
Xiang Chen ◽  
...  
Keyword(s):  
T Antigen ◽  
Sv40 T Antigen ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Piggybac Transposon

scholarly journals Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts

2004 ◽  
Vol 91 (2) ◽  
pp. 423-428 ◽  
Author(s):  
Shayne A. Bellingham ◽  
Giuseppe D. Ciccotosto ◽  
B. Elise Needham ◽  
Lisa R. Fodero ◽  
Anthony R. White ◽  
...  
Keyword(s):  
Amyloid Precursor Protein ◽  
Cortical Neurons ◽  
Gene Knockout ◽  
Precursor Protein ◽  
Embryonic Fibroblasts ◽  
Primary Mouse ◽  
Cellular Copper

scholarly journals Alkbh2 protects against lethality and mutation in primary mouse embryonic fibroblasts

DNA Repair ◽  
2012 ◽  
Vol 11 (5) ◽  
pp. 502-510 ◽  
Author(s):  
Stephanie L. Nay ◽  
Dong-Hyun Lee ◽  
Steven E. Bates ◽  
Timothy R. O’Connor
Keyword(s):  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Primary Mouse

scholarly journals Parvovirus evades interferon-dependent viral control in primary mouse embryonic fibroblasts

Virology ◽  
2013 ◽  
Vol 442 (1) ◽  
pp. 20-27 ◽  
Author(s):  
Lisa M. Mattei ◽  
Susan F. Cotmore ◽  
Peter Tattersall ◽  
Akiko Iwasaki
Keyword(s):  
Mouse Embryonic Fibroblasts ◽  
Viral Control ◽  
Embryonic Fibroblasts ◽  
Primary Mouse

scholarly journals Nonviral Direct Conversion of Primary Mouse Embryonic Fibroblasts to Neuronal Cells

2012 ◽  
Vol 1 ◽  
pp. e32 ◽  
Author(s):  
Andrew F Adler ◽  
Christopher L Grigsby ◽  
Karina Kulangara ◽  
Hong Wang ◽  
Ryohei Yasuda ◽  
...  
Keyword(s):  
Neuronal Cells ◽  
Direct Conversion ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Primary Mouse

scholarly journals Functional specialization of calreticulin domains

2001 ◽  
Vol 154 (5) ◽  
pp. 961-972 ◽  
Author(s):  
Kimitoshi Nakamura ◽  
Anna Zuppini ◽  
Serge Arnaudeau ◽  
Jeffery Lynch ◽  
Irfan Ahsan ◽  
...  
Keyword(s):  
Storage Capacity ◽  
Gene Knockout ◽  
Cell Surface Receptor ◽  
Functional Specialization ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Inositol 1,4,5 Trisphosphate ◽  
Measurable Level ◽  
Surface Receptor ◽  
Lower Capacity

Calreticulin is a Ca2+-binding chaperone in the endoplasmic reticulum (ER), and calreticulin gene knockout is embryonic lethal. Here, we used calreticulin-deficient mouse embryonic fibroblasts to examine the function of calreticulin as a regulator of Ca2+ homeostasis. In cells without calreticulin, the ER has a lower capacity for Ca2+ storage, although the free ER luminal Ca2+ concentration is unchanged. Calreticulin-deficient cells show inhibited Ca2+ release in response to bradykinin, yet they release Ca2+ upon direct activation with the inositol 1,4,5-trisphosphate (InsP3). These cells fail to produce a measurable level of InsP3 upon stimulation with bradykinin, likely because the binding of bradykinin to its cell surface receptor is impaired. Bradykinin binding and bradykinin-induced Ca2+ release are both restored by expression of full-length calreticulin and the N + P domain of the protein. Expression of the P + C domain of calreticulin does not affect bradykinin-induced Ca2+ release but restores the ER Ca2+ storage capacity. Our results indicate that calreticulin may play a role in folding of the bradykinin receptor, which affects its ability to initiate InsP3-dependent Ca2+ release in calreticulin-deficient cells. We concluded that the C domain of calreticulin plays a role in Ca2+ storage and that the N domain may participate in its chaperone functions.

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