Deficiency of the 15-kDa selenoprotein led to cytoskeleton remodeling and non-apoptotic membrane blebbing through a RhoA/ROCK pathway

Abstract

The 15-kDa selenoprotein (Sep15) has been implicated in etiology of some types of cancer. Herein, inducible RNAi cell lines were established and cell morphology and motility were analyzed. The majority of Sep15-deficient cells (>95%) formed membrane blebs in a dynamic manner. Blebbing cells transformed cell morphology from a normal flat spindle shape to a spherical morphology. In blebbing cells, actin fibers moved to the cell periphery, covering and obscuring visualization of α-tubulin. Bleb formation was suppressed by the inhibitors of Rho-associated protein kinase (ROCK), RhoA or myosin light chain (MLC), restoring blebbing cells to wild-type morphology. RhoA activation and phosphorylation of myosin phosphatase target subunit 1 was induced by Sep15 knockdown. Sep15-deficient cells were non-apoptotic, and displayed a distinct relative localization of F-actin and α-tubulin from typical apoptotic blebbing cells. Our data suggest that Sep15 in Chang Liver cells regulates the pathway that antagonizes RhoA/ROCK/MLC-dependent non-apoptotic bleb formation.

KIF 13A‐regulated RhoB plasma membrane localization governs membrane blebbing and blebby amoeboid cell migration

“…This is in line with our finding that RhoA and RhoB controlled different aspects of 3D amoeboid leukaemia cell migration, with RhoA being important for cell rear retraction, while RhoB controlled membrane blebbing. This contrasts with previous studies that have suggested a role for RhoA in cell membrane blebbing (Gadea et al , ; Godin & Ferguson, ; Cartier‐Michaud et al , ; Tozluoglu et al , ; Takahara et al , ; Bang et al , ; Aoki et al , ). However, in most of these studies, experimental methods (e.g.…”

“…However, in most of these studies, experimental methods (e.g. non-specific drug-based inhibition) did not provide clear indications as to which Rho family member is responsible for effects and in other cases, blebbing function was inferred from gross cell morphology rather than direct visualization of blebbing per se (Gadea et al, 2007;Pinner & Sahai, 2008;Godin & Ferguson, 2010;Cartier-Michaud et al, 2012;Tozluoglu et al, 2013;Takahara et al, 2014;Bang et al, 2015;Aoki et al, 2016). In any case, our finding that depletion of RhoB impaired membrane blebbing in four distinct cell models of different origin shows that RhoB is critical for membrane blebbing under a variety of conditions.…”

Selenophosphate synthetase 1 is an essential protein with roles in regulation of redox homoeostasis in mammals

“…When the cells grew to a confluent state, each clone was transferred to a single well in a 24-well plate and allowed to expand. The cells were then analyzed for the expression of mouse Sps1 , mouse Glrx1 , and human Glrx1 by quantitative PCR (qPCR) as previously described [21]. Briefly, total RNA was isolated from each cell line using TRIzol®.…”

“…The vector (pcDNA4/TO.mSPS1Rescue) was transfected into F9/shSPS1 cells and the transfected cells were selected in the presence of 400 μg/ml Zeocin ™ . Cells were then analyzed by qPCR for the expression of endogenous mouse Sps1 , mouse Glrx1 , and rescue mouse Sps1 as previously described [21]. Briefly, total RNA was isolated from each cell line, first-strand cDNA was synthesized, and the cDNA was subjected to PCR using the following specific primer sets: for endogenous mouse Sps1 , mSPS1-F and mSPS1-R; for mouse Glrx1 , mGlrx1-F and mGlrx1-R; for rescue mouse Sps1 , mSPS1 (Rescue)-F and mSPS1 (Rescue)-R; and for mouse Actb , ACTB-F and ACTB-R.…”

“…Cell invasion assays were performed as previously described [21] using a Matrigel®-coated invasion chamber with an 8.0 μm pore size filter following the manufacturer’s protocol with minor modifications including the final concentration of cells (2 × 10 4 cells/mL) and the incubation time (10 h).…”