γ-Actin plays a key role in endothelial cell motility and neovessel maintenance

Abstract

BackgroundAngiogenesis plays a crucial role in development, wound healing as well as tumour growth and metastasis. Although the general implication of the cytoskeleton in angiogenesis has been partially unravelled, little is known about the specific role of actin isoforms in this process. Herein, we aimed at deciphering the function of γ-actin in angiogenesis.MethodsLocalization of β- and γ-actin in vascular endothelial cells was investigated by co-immunofluorescence staining using monoclonal antibodies, followed by the functional analysis of γ-actin using siRNA. The impact of γ-actin knockdown on the random motility and morphological differentiation of endothelial cells into vascular networks was investigated by timelapse videomicroscopy while the effect on chemotaxis was assessed using modified Boyden chambers. The implication of VE-cadherin, VEGFR-2 and ROCK signalling was then examined by Western blotting and using pharmacological inhibitors.ResultsThe two main cytoplasmic isoforms of actin strongly co-localized in vascular endothelial cells, albeit with some degree of spatial preference. While β-actin knockdown was not achievable without major cytotoxicity, γ-actin knockdown did not alter the viability of endothelial cells. Timelapse videomicroscopy experiments revealed that γ-actin knockdown cells were able to initiate morphological differentiation into capillary-like tubes but were unable to maintain these structures, which rapidly regressed. This vascular regression was associated with altered regulation of VE-cadherin expression. Interestingly, knocking down γ-actin expression had no effect on endothelial cell adhesion to various substrates but significantly decreased their motility and migration. This anti-migratory effect was associated with an accumulation of thick actin stress fibres, large focal adhesions and increased phosphorylation of myosin regulatory light chain, suggesting activation of the ROCK signalling pathway. Incubation with ROCK inhibitors, H-1152 and Y-27632, completely rescued the motility phenotype induced by γ-actin knockdown but only partially restored the angiogenic potential of endothelial cells.ConclusionsOur study thus demonstrates for the first time that β-actin is essential for endothelial cell survival and γ-actin plays a crucial role in angiogenesis, through both ROCK-dependent and -independent mechanisms. This provides new insights into the role of the actin cytoskeleton in angiogenesis and may open new therapeutic avenues for the treatment of angiogenesis-related disorders.Electronic supplementary materialThe online version of this article (doi:10.1186/s13221-014-0027-2) contains supplementary material, which is available to authorized users.

Gamma-actin is involved in regulating centrosome function and mitotic progression in cancer cells

“…Previous studies from our group have demonstrated a role for g-actin in cell polarity and motility 21 and angiogenesis, 22 as well as resistance to microtubule-targeted agents. 23 Microtubule-targeted agents such as the anti-cancer drug paclitaxel, induce mitotic arrest and subsequent cell death.…”

Divergent roles of β‐ and γ‐actin isoforms during spread of vaccinia virus

“…This suggests that under knockdown conditions, cells overexpress one isoform to compensate for loss of the other. This has not been observed for CYA isoforms in other studies, either using the same cocktail of isoform-specific siRNA (Dugina et al, 2009) or using only one of those sequences (Pasquier et al, 2015). This could be due to their use of different cell types, varying doses of siRNA or incubation times, and/or the use of only a single CYA-specific siRNA in one of the studies.…”

“…These results are consistent with an absolute, albeit dosage-sensitive, requirement for b-CYA in VACV actin nucleation and actin-based motility. Previous studies have also demonstrated significant morphological and functional changes in various cell types with incomplete knockdown of CYA isoforms, such as taking on a less or a more contractile phenotype, or an increase/ decrease in lamellipodial structures, stress fibres, and cell motility (Dugina et al, 2009;Pasquier et al, 2015;Shum, Pasquier, Po'uha, O'Neill, Chaponnier, Gunning, & Kavallaris, 2011).…”

“…For example, while b-CYA localises to stress fibres and cellcell contacts, g-CYA is abundant in lamellipodial structures and is important for cell motility (Dugina et al, 2009). Studies have identified isoform-specific functions during multiple cellular processes (Artman et al, 2014;Hajkova, Nyman, Lindberg, & Karlsson, 2000;Joseph et al, 2014;Pasquier et al, 2015), as well as distinct requirements in the replication cycle of viruses like coronaviruses (Wang, Fang, Xiao, Chen, Tam, & Liu, 2009) and the classical swine fever virus (He, Ling, Liao, Li, Han, Zhao, . .…”

“…The muscle isoforms exhibit tissue specific expression, while the band g-cytoplasmic actins (CYA) are the most abundant in nonmuscle cells (Rubenstein, 1990) and often exist in 2:1 ratio in epithelial cell lines like HeLa and chicken embryo fibroblasts (Khaitlina, 2007), the cell types commonly used to study VACV in vitro and cell types commonly targeted by orthopoxviruses during pathogenesis (Johnston and McFadden, 2004;McFadden, 2005;Smith and Kotwal, 2002;Werden, Rahman, & McFadden, 2008). Despite their surprisingly similar sequence homologies [only differing by four amino acids; (Vandekerckhove and Weber, 1978a)], recent reports have revealed that band g-CYA have different functions and display distinct subcellular localization patterns (Artman, Dormoy-Raclet, von Roretz, & Gallouzi, 2014;Dugina, Zwaenepoel, Gabbiani, Clement, & Chaponnier, 2009;Joseph, Srivastava, & Pfister, 2014;Pasquier, Tuset, Sinnappan, Carnell, Macmillan, & Kavallaris, 2015). b-actin is predominantly found in basal areas of the cell, cell-cell contacts and stress fibres, while g-CYA is more apically abundant, and concentrated at peripheral structures such as lamellipodia and the leading edge of motile cells (Chaponnier and Gabbiani, 2004;Dugina et al, 2009].…”