scholarly journals MRTF transcription and Ezrin-dependent plasma membrane blebbing are required for entotic invasion

2017 ◽  
Vol 216 (10) ◽  
pp. 3087-3095 ◽  
Author(s):  
Laura Soto Hinojosa ◽  
Manuel Holst ◽  
Christian Baarlink ◽  
Robert Grosse
Keyword(s):  
Plasma Membrane ◽  
Cell Invasion ◽  
Serum Response Factor ◽  
Critical Role ◽  
Actin Dynamics ◽  
Actin Cortex ◽  
Tightly Coupled ◽  
Related Transcription Factor ◽  
Nuclear Shuttling ◽  
The Impact

Entosis is a nonapoptotic form of cell death initiated by actomyosin-dependent homotypic cell-in-cell invasion that can be observed in malignant exudates during tumor progression. We previously demonstrated formin-mediated actin dynamics at the rear of the invading cell as well as nonapoptotic plasma membrane (PM) blebbing in this cellular motile process. Although the contractile actin cortex involved in bleb-driven motility is well characterized, a role for transcriptional regulation in this process has not been studied. Here, we explore the impact of the actin-controlled MRTF–SRF (myocardin-related transcription factor–serum response factor) pathway for sustained PM blebbing and entotic invasion. We find that cortical blebbing is tightly coupled to MRTF nuclear shuttling to promote the SRF transcriptional activity required for entosis. Furthermore, PM blebbing triggered SRF-mediated up-regulation of the metastasis-associated ERM protein Ezrin. Notably, Ezrin is sufficient and important to sustain bleb dynamics for cell-in-cell invasion when SRF is suppressed. Our results highlight the critical role of the actin-regulated MRTF transcriptional pathway for bleb-associated invasive motility, such as during entosis.

scholarly journals MMP3 activity rather than cortical stiffness determines NHE1-dependent invasiveness of melanoma cells

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Dennis Keurhorst ◽  
Ivan Liashkovich ◽  
Fabian Frontzek ◽  
Svenja Nitzlaff ◽  
Verena Hofschröer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Invasion ◽  
Time Lapse ◽  
Actin Dynamics ◽  
Cell Stiffness ◽  
Migration And Invasion ◽  
Boyden Chamber ◽  
Cortical Actin ◽  
Structural Element ◽  
Mmp Inhibitor

Abstract Background Both cell adhesion and matrix metalloproteinase (MMP) activity depend on pH at the cell surface. By regulating extracellular juxtamembrane pH, the Na+/H+ exchanger NHE1 plays a significant part in human melanoma (MV3) cell migration and invasion. Because NHE1, besides its pH-regulatory transport function, also serves as a structural element tying the cortical actin cytoskeleton to the plasma membrane, we investigated whether NHE1 affects cortical stiffness of MV3 cells, and how this makes an impact on their invasiveness. Methods NHE1 overexpressing MV3 cells were compared to the corresponding mock-transfected control cells. NHE1 expression was verified by Western blotting, cariporide (HOE642) was used to inhibit NHE1 activity, cell stiffness was determined by atomic force microscopy, and F-actin was visualized by phalloidin-staining. Migration on, and invasion of, native and glutaraldehyde-fixed collagen I substrates were analyzed using time-lapse video microscopy and Boyden-chamber assays, respectively. MMP secretion and activity were detected by Western blot and zymography, respectively. MMP activity was inhibited with NNGH. Results The cortical, but not the bulk stiffness, was significantly higher in NHE1 overexpressing cells. This increase in cortical stiffness was accompanied by a reorganization of the cortical cytoskeleton, i.e. a condensation of F-actin underneath and along the plasma membrane. However, it was not affected by NHE1 inhibition. Nevertheless, actin dynamics is required for cell invasion as demonstrated with the application of cytochalasin D. NHE1 overexpression was associated with an elevated MMP3 secretion and an increase in the invasion of a native matrix. This increase in invasiveness could be antagonized by the MMP inhibitor NNGH. Transmigration through a glutaraldehyde-fixed, indigestible substrate was not affected by NHE1 overexpression. Conclusion NHE1, as a structural element and independently of its transport activity, contributes to the organization of the cortical F-actin meshwork and thus impacts cortical stiffness. Since NHE1 overexpression stimulates MMP3 secretion but does not change transmigration through a fixed substrate, MV3 cell invasion of a native substrate depends on MMP activity rather than on a modifiable cortical stiffness.

scholarly journals Serum Response Factor (SRF)-cofilin-actin signaling axis modulates mitochondrial dynamics

2012 ◽  
Vol 109 (38) ◽  
pp. E2523-E2532 ◽  
Author(s):  
Henning Beck ◽  
Kevin Flynn ◽  
Katrin S. Lindenberg ◽  
Heinz Schwarz ◽  
Frank Bradke ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Serum Response Factor ◽  
Mitochondrial Dynamics ◽  
Actin Dynamics ◽  
Response Factor ◽  
Signaling Axis ◽  
Mitochondrial Number ◽  
Mitochondrial Networks ◽  
The Impact ◽  
Serum Response

Aberrant mitochondrial function, morphology, and transport are main features of neurodegenerative diseases. To date, mitochondrial transport within neurons is thought to rely mainly on microtubules, whereas actin might mediate short-range movements and mitochondrial anchoring. Here, we analyzed the impact of actin on neuronal mitochondrial size and localization. F-actin enhanced mitochondrial size and mitochondrial number in neurites and growth cones. In contrast, raising G-actin resulted in mitochondrial fragmentation and decreased mitochondrial abundance. Cellular F-actin/G-actin levels also regulate serum response factor (SRF)-mediated gene regulation, suggesting a possible link between SRF and mitochondrial dynamics. Indeed, SRF-deficient neurons display neurodegenerative hallmarks of mitochondria, including disrupted morphology, fragmentation, and impaired mitochondrial motility, as well as ATP energy metabolism. Conversely, constitutively active SRF-VP16 induced formation of mitochondrial networks and rescued huntingtin (HTT)-impaired mitochondrial dynamics. Finally, SRF and actin dynamics are connected via the actin severing protein cofilin and its slingshot phosphatase to modulate neuronal mitochondrial dynamics. In summary, our data suggest that the SRF-cofilin-actin signaling axis modulates neuronal mitochondrial function.

scholarly journals The Cytoskeleton-associated PDZ-LIM Protein, ALP, Acts on Serum Response Factor Activity to Regulate Muscle Differentiation

2007 ◽  
Vol 18 (5) ◽  
pp. 1723-1733 ◽  
Author(s):  
Pascal Pomiès ◽  
Mohammad Pashmforoush ◽  
Cristina Vegezzi ◽  
Kenneth R. Chien ◽  
Charles Auffray ◽  
...  
Keyword(s):  
Serum Response Factor ◽  
Critical Role ◽  
Muscle Differentiation ◽  
Actin Dynamics ◽  
Response Factor ◽  
Cytoskeletal Regulation ◽  
Expression Construct ◽  
Filament Bundles ◽  
Serum Response

In this report, an antisense RNA strategy has allowed us to show that disruption of ALP expression affects the expression of the muscle transcription factors myogenin and MyoD, resulting in the inhibition of muscle differentiation. Introduction of a MyoD expression construct into ALP-antisense cells is sufficient to restore the capacity of the cells to differentiate, illustrating that ALP function occurs upstream of MyoD. It is known that MyoD is under the control of serum response factor (SRF), a transcriptional regulator whose activity is modulated by actin dynamics. A dramatic reduction of actin filament bundles is observed in ALP-antisense cells and treatment of these cells with the actin-stabilizing drug jasplakinolide stimulates SRF activity and restores the capacity of the cells to differentiate. Furthermore, we show that modulation of ALP expression influences SRF activity, the level of its coactivator, MAL, and muscle differentiation. Collectively, these results suggest a critical role of ALP on muscle differentiation, likely via cytoskeletal regulation of SRF.

scholarly journals Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding

2016 ◽  
Vol 36 (10) ◽  
pp. 1526-1539 ◽  
Author(s):  
Julia Weissbach ◽  
Franziska Schikora ◽  
Anja Weber ◽  
Michael Kessels ◽  
Guido Posern
Keyword(s):  
Serum Response Factor ◽  
De Novo ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Serum Stimulation ◽  
Competitive Mechanism ◽  
Related Transcription Factor ◽  
Simultaneous Inhibition ◽  
Serum Response

The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin–MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actinin vitro. The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, andde novoformation of the G-actin–RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation.

scholarly journals Docosahexaenoic fatty acid-containing phospholipids affect plasma membrane susceptibility to disruption by bacterial toxin-induced macroapertures

2020 ◽  
Author(s):  
Meng-Chen Tsai ◽  
Lucile Fleuriot ◽  
Sébastien Janel ◽  
David Gonzalez-Rodriguez ◽  
Camille Morel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Large Scale ◽  
Umbilical Vein ◽  
Critical Role ◽  
Membrane Dynamics ◽  
Bacterial Toxin ◽  
Rhoa Gtpase ◽  
Docosahexaenoic Fatty Acid ◽  
Umbilical Vein Endothelial Cells ◽  
The Impact

AbstractMetabolic studies and animal knockout models point to the critical role of polyunsaturated docosahexaenoic acid (22:6, DHA)-containing phospholipids (PLs) in physiology. Here, we study the impact of DHA-PLs on the dynamics of transendothelial cell macroapertures (TEMs) tunnels triggered by the RhoA GTPase inhibitory exotoxin C3 from Clostridium botulinum. Through lipidomic analyses, we show that primary human umbilical vein endothelial cells (HUVECs) subjected to DHA-diet undergo a 6-fold DHA-PLs enrichment in plasma membrane at the expense of monounsaturated OA-PLs. In contrast, OA-diet had almost no effect on PLs composition. Consequently, DHA treatment increases the nucleation rate of TEMs by 2-fold that we ascribe to a reduction of cell thickness. We reveal that the global transcellular area of cells remains conserved through a reduction of the width and lifetime of TEMs. Altogether, we reveal a homeostasis between plasma membrane DHA-PLs content and large-scale membrane dynamics.

scholarly journals Runx2 Represses Myocardin-Mediated Differentiation and Facilitates Osteogenic Conversion of Vascular Smooth Muscle Cells

2007 ◽  
Vol 28 (3) ◽  
pp. 1147-1160 ◽  
Author(s):  
Toru Tanaka ◽  
Hiroko Sato ◽  
Hiroshi Doi ◽  
Carolina A. Yoshida ◽  
Takehisa Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Molecular Mechanisms ◽  
Serum Response Factor ◽  
Critical Role ◽  
Muscle Cells ◽  
Tightly Coupled

ABSTRACT Phenotypic plasticity and the switching of vascular smooth muscle cells (SMCs) play a critical role in atherosclerosis. Although Runx2, a key osteogenic transcription factor, is expressed in atherosclerotic plaques, the molecular mechanisms by which Runx2 regulates SMC differentiation remain unclear. Here we demonstrated that Runx2 repressed SMC differentiation induced by myocardin, which acts as a coactivator for serum response factor (SRF). Myocardin-mediated induction of SMC gene expression was enhanced in mouse embryonic fibroblasts derived from Runx2 null mice compared to wild-type mice. Forced expression of Runx2 decreased the expression of SMC genes and promoted osteogenic gene expression, whereas the reduction of Runx2 expression by small interfering RNA enhanced SMC differentiation in human aortic SMCs. Runx2 interacted with SRF and interfered with the formation of the SRF/myocardin ternary complex. Thus, this study provides the first evidence that Runx2 inhibits SRF-dependent transcription, as a corepressor independent of its DNA binding. We propose that Runx2 plays a pivotal role in osteogenic conversion tightly coupled with repression of the SMC phenotype in atherosclerotic lesions.

scholarly journals Lamina-associated polypeptide 2α is required for intranuclear MRTF-A activity

2021 ◽  
Author(s):  
Ekaterina Sidorenko ◽  
Maria Sokolova ◽  
Antti Pennanen ◽  
Salla Kyheroinen ◽  
Guido Posern ◽  
...  
Keyword(s):  
Target Genes ◽  
Serum Response Factor ◽  
Actin Dynamics ◽  
Chromatin Binding ◽  
Binding Partner ◽  
Related Transcription Factor ◽  
Direct Binding ◽  
Efficient Expression ◽  
Serum Response

Myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), regulates the expression of many cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Here we describe a novel mechanism to regulate MRTF-A activity within the nucleus by showing that lamina-associated polypeptide 2α (Lap2α), the nucleoplasmic isoform of Lap2, is a direct binding partner of MRTF-A, and required for the efficient expression of MRTF-A/SRF target genes. Mechanistically, Lap2α is not required for MRTF-A nuclear localization, unlike most other MRTF-A regulators, but is required for binding of MRTF-A to its target genes. This regulatory step takes place prior to MRTF-A chromatin binding, because Lap2α neither interacts with, nor specifically influences active histone marks on MRTF-A/SRF target genes. Phenotypically, Lap2α is required for serum-induced cell migration, and deregulated MRTF-A activity may also contribute to muscle and proliferation phenotypes associated with loss of Lap2α. Our studies therefore add another regulatory layer to the control of MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2α in transcriptional regulation.

Proline residues in two tightly coupled helices of the sulphate transporter, SHST1, are important for sulphate transport

2001 ◽  
Vol 356 (2) ◽  
pp. 589-594 ◽  
Author(s):  
Megan C. SHELDEN ◽  
Patrick LOUGHLIN ◽  
M. Louise TIERNEY ◽  
Susan M. HOWITT
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Transmembrane Helices ◽  
Transport Activity ◽  
Post Translational Modification ◽  
Sulphate Transport ◽  
Stylosanthes Hamata ◽  
Tightly Coupled

The sulphate transporter SHST1, from Stylosanthes hamata, features three tightly coupled transmembrane helices which include proline residues that are conserved in most related transporters. We used site-directed mutagenesis and expression of the mutant transporters in yeast to test whether these proline residues are important for function. Four proline residues were replaced by both alanine and leucine. Only one of these proline residues, Pro-144, was essential for sulphate transport. However, mutation of either Pro-133 or Pro-160 resulted in a severe decrease in sulphate transport activity; this was due more to a decrease in transport activity than to a decrease in the amount of mutant SHST1 in the plasma membrane. These results suggest that all three proline residues are important for transport, and that the conformation of the three tightly coupled helices may play a critical role in sulphate transport. We also show that SHST1 undergoes a post-translational modification that is required for trafficking to the plasma membrane.

Actin-mediated gene expression in neurons: the MRTF-SRF connection

2010 ◽  
Vol 391 (6) ◽  
Author(s):  
Bernd Knöll
Keyword(s):  
Gene Expression ◽  
Nervous System ◽  
Serum Response Factor ◽  
Actin Dynamics ◽  
Response Factor ◽  
Related Transcription Factor ◽  
Gene Regulatory ◽  
Regulatory Loop ◽  
Serum Response ◽  
Prime Target

Abstract The traditional view of cellular actin is a rather autarkic cytoskeletal framework function confined to the cytoplasm. However, there is now evidence that alterations in actin dynamics are sensed by the nucleus and subsequently modulate gene expression. In communicating with the nucleus, cytoplasmic, and most likely also nucleus-resident actin, provides a further (gene) regulatory loop to cell motility. A transcription module composed of MRTF (myocardin-related transcription factor) and SRF (serum response factor) emerges as prime target of such actin signaling. Here, I focus on the nervous system, where the actin-MRTF-SRF entity governs multiple aspects of neuronal motility.

scholarly journals Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking

2006 ◽  
Vol 398 (1) ◽  
pp. 97-105 ◽  
Author(s):  
Sanchita Mukherjee ◽  
Laura Kallay ◽  
Christopher L. Brett ◽  
Rajini Rao
Keyword(s):  
Plasma Membrane ◽  
Mutational Analysis ◽  
Vesicle Trafficking ◽  
Critical Role ◽  
Physiological Role ◽  
Carboxypeptidase Y ◽  
Loss Of Function ◽  
Transmembrane Segments ◽  
Tightly Coupled ◽  
Trafficking Defects

Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has ident-ified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These appear distinct from well-studied mammalian plasma membrane isoforms (NHE1–NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link between ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu355 in ion transport is consistent with the invariance of this residue in all NHEs. Surprisingly, residues specifically conserved in the intracellular isoforms (such as Phe357 and Tyr361) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing unexpected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the importance of the H10 loop of the NHE family, highlights the differences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis.

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