scholarly journals Cell-substrate adhesion drives Scar/WAVE activation and phosphorylation, which controls pseudopod lifetime

2019 ◽  
Author(s):  
Shashi Prakash Singh ◽  
Peter A. Thomason ◽  
Sergio Lilla ◽  
Matthias Schaks ◽  
Qing Tang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Extracellular Signals ◽  
Rich Domain ◽  
Substrate Adhesion ◽  
Cell Substrate ◽  
Cell Substrate Adhesion ◽  
Wave Complex

AbstractThe Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE’s proline-rich domain is polyphosphorylated after the complex is activated. Treatments that stop activation block phosphorylation in both Dictyostelium and mammalian cells. This implies that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether a protrusion is initiated. Unexpectedly, activation-dependent phosphorylation is not promoted by chemotactic signalling, or by signal-dependent kinases such as ERKs, but is greatly stimulated by cell:substrate adhesion. Scar/WAVE that has been mutated to be either unphosphorylatable or phosphomimetic is activated normally, and rescues the phenotype of scar− cells, demonstrating that phosphorylation is dispensible for activation and actin regulation. However, pseudopods and patches of Scar/WAVE complex recruitment last substantially longer in unphosphorylatable mutants, altering cell polarisation and the efficiency of migration. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE’s activity, and that phosphorylation acts as a timer, restricting pseudopod lifetime by promoting Scar/WAVE turnover.

scholarly journals Extracellular Signalling Modulates Scar/WAVE Complex Activity through Abi Phosphorylation

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3485
Author(s):  
Shashi Prakash Singh ◽  
Peter A. Thomason ◽  
Robert H. Insall
Keyword(s):  
Cell Migration ◽  
Environmental Cues ◽  
Complex Activity ◽  
Extracellular Signals ◽  
Substrate Adhesion ◽  
Cell Substrate ◽  
Environmental Responses ◽  
Cell Substrate Adhesion ◽  
Extracellular Signalling ◽  
Wave Complex

The lamellipodia and pseudopodia of migrating cells are produced and maintained by the Scar/WAVE complex. Thus, actin-based cell migration is largely controlled through regulation of Scar/WAVE. Here, we report that the Abi subunit—but not Scar—is phosphorylated in response to extracellular signalling in Dictyostelium cells. Like Scar, Abi is phosphorylated after the complex has been activated, implying that Abi phosphorylation modulates pseudopodia, rather than causing new ones to be made. Consistent with this, Scar complex mutants that cannot bind Rac are also not phosphorylated. Several environmental cues also affect Abi phosphorylation—cell-substrate adhesion promotes it and increased extracellular osmolarity diminishes it. Both unphosphorylatable and phosphomimetic Abi efficiently rescue the chemotaxis of Abi KO cells and pseudopodia formation, confirming that Abi phosphorylation is not required for activation or inactivation of the Scar/WAVE complex. However, pseudopodia and Scar patches in the cells with unphosphorylatable Abi protrude for longer, altering pseudopod dynamics and cell speed. Dictyostelium, in which Scar and Abi are both unphosphorylatable, can still form pseudopods, but migrate substantially faster. We conclude that extracellular signals and environmental responses modulate cell migration by tuning the behaviour of the Scar/WAVE complex after it has been activated.

scholarly journals Extracellular signalling modulates Scar/WAVE complex activity through Abi phosphorylation

2021 ◽  
Author(s):  
Shashi Prakash Singh ◽  
Peter Thomason ◽  
Robert Insall
Keyword(s):  
Cell Migration ◽  
Environmental Cues ◽  
Complex Activity ◽  
Extracellular Signals ◽  
Substrate Adhesion ◽  
Cell Substrate ◽  
Environmental Responses ◽  
Cell Substrate Adhesion ◽  
Extracellular Signalling ◽  
Wave Complex

The lamellipodia and pseudopodia of migrating cells are produced and maintained by the Scar/WAVE complex. Thus, actin-based cell migration is largely controlled through regulation of Scar/WAVE. Here we report that the Abi subunit - not Scar/WAVE - is phosphorylated in response to extracellular signalling. Like Scar, Abi is phosphorylated after the complex has been activated, implying that Abi phosphorylation modulates pseudopodia, rather than causing new ones to be made. Consistent with this, Scar/WAVE complex mutants that cannot bind Rac are also not phosphorylated. Several environmental cues also affect Abi phosphorylation - cell-substrate adhesion promotes it and increased extracellular osmolarity diminishes it. Both unphosphorylatable and phosphomimetic Abi efficiently rescue the chemotaxis of Abi KO cells and pseudopodia formation, confirming that Abi phosphorylation is not required for activation or inactivation of the Scar/WAVE complex. However, pseudopodia and Scar/WAVE patches in the cells with unphosphorylatable Abi protrude for longer, altering pseudopod dynamics and cell speed. Cells in which Scar and Abi are both unphosphorylatable can still form pseudopods, but migrate substantially faster. We conclude that extracellular signals and environmental responses modulate cell migration by tuning the behaviour of the Scar/WAVE complex after it has been activated.

scholarly journals Regulation of cell-substrate adhesion by proteoglycans immobilized on extracellular substrates

1989 ◽  
Vol 264 (14) ◽  
pp. 8012-8018 ◽  
Author(s):  
M Yamagata ◽  
S Suzuki ◽  
S K Akiyama ◽  
K M Yamada ◽  
K Kimata
Keyword(s):  
Substrate Adhesion ◽  
Cell Substrate ◽  
Cell Substrate Adhesion

scholarly journals A GTPase controls cell-substrate adhesion in Xenopus XTC fibroblasts.

1992 ◽  
Vol 118 (5) ◽  
pp. 1235-1244 ◽  
Author(s):  
M H Symons ◽  
T J Mitchison
Keyword(s):  
Control Cell ◽  
Response To Treatment ◽  
Nucleotide Analogs ◽  
Cell Contractility ◽  
Cell Rounding ◽  
Substrate Adhesion ◽  
Cell Substrate ◽  
Adhesive Interactions ◽  
Cell Substrate Adhesion ◽  
Gamma S

Cell-substrate adhesion is crucial at various stages of development and for the maintenance of normal tissues. Little is known about the regulation of these adhesive interactions. To investigate the role of GTPases in the control of cell morphology and cell-substrate adhesion we have injected guanine nucleotide analogs into Xenopus XTC fibroblasts. Injection of GTP gamma S inhibited ruffling and increased spreading, suggesting an increase in adhesion. To further investigate this, we made use of GRGDSP, a peptide which inhibits binding of integrins to vitronectin and fibronectin. XTC fibroblasts injected with non-hydrolyzable analogs of GTP took much more time to round up than mock-injected cells in response to treatment with GRGDSP, while GDP beta S-injected cells rounded up in less time than controls. Injection with GTP gamma S did not inhibit cell rounding induced by trypsin however, showing that cell contractility is not significantly affected by the activation of GTPases. These data provide evidence for the existence of a GTPase which can control cell-substrate adhesion from the cytoplasm. Treatment of XTC fibroblasts with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate reduced cell spreading and accelerated cell rounding in response to GRGDSP, which is essentially opposite to the effect exerted by non-hydrolyzable GTP analogs. These results suggest the existence of at least two distinct pathways controlling cell-substrate adhesion in XTC fibroblasts, one depending on a GTPase and another one involving protein kinase C.

scholarly journals Identification of a new protein localized at sites of cell-substrate adhesion.

1986 ◽  
Vol 103 (5) ◽  
pp. 1679-1687 ◽  
Author(s):  
M C Beckerle
Keyword(s):  
Immunoblot Analysis ◽  
Rabbit Serum ◽  
Major Protein ◽  
Dynamic Interactions ◽  
Substrate Adhesion ◽  
Cell Substrate ◽  
Filament Bundles ◽  
Cell Substrate Adhesion ◽  
Adhesion Plaque ◽  
New Protein

A new protein found at sites of cell-substrate adhesion has been identified by analysis of a nonimmune rabbit serum. By indirect immunofluorescence this serum stains focal contacts (adhesion plaques) and the associated termini of actin filament bundles in cultured chicken cells. Western immunoblot analysis of total chick embryo fibroblast protein demonstrated an 82-kD polypeptide to be the major protein recognized by the unfractionated serum. This 82-kD protein is immunologically distinct from other known adhesion plaque proteins such as vinculin, talin, alpha-actinin, and fimbrin. Antibody affinity-purified against the electrophoretically isolated, nitrocellulose-bound 82-kD protein retained the ability to stain the area of the adhesion plaque, which confirms that the 82-kD protein is indeed a constituent of the focal contact. The 82-kD polypeptide has a basic isoelectric point relative to actin and fibronectin, and it appears to be very low in abundance. The 82-kD protein is ubiquitous in chicken embryo tissues. However, it appears to be more abundant in fibroblasts and smooth muscle than in brain or liver. Intermediate levels of the protein were detected in skeletal and cardiac muscle. The subcellular distribution of the 82-kD protein raises the possibility that this polypeptide is involved in linking actin filaments to the plasma membrane at sites of substrate attachment or regulating these dynamic interactions.

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