Masaaki Yoshigi

Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia

For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires sphingosine 1-phosphate signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo1, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement

Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.

Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling

Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, we characterized the phenotype of fibroblasts in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. We identified differences in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. We postulate that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling.

Quantification of stretch-induced cytoskeletal remodeling in vascular endothelial cells by image processing.

Reorientation of the cell axis induced by cyclic stretching is an early response to mechanical forces in vitro. However, quantitative assay for this phenomenon has been difficult due to lack of robust methods. We hypothesized that cell orientation may be redefined by the orientation of actin fibers. We developed image processing methods to quantitate the orientation and density of actin fibers.

Stretch-induced actin remodeling requires targeting of zyxin to stress fibers and recruitment of actin regulators

Mechanical stimulation induces zyxin-dependent actin cytoskeletal reinforcement. Stretch induces MAPK activation, zyxin phosphorylation, and recruitment to actin stress fibers, independent of p130Cas. Zyxins C-terminal LIM domains are required for stretch-induced targeting to stress fibers, and zyxins N-terminus is necessary for actin remodeling.

β3 integrin-EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cells.

Filopodia formation is positively regulated by β3 integrin–EGFR cross-talk, which regulates p190RhoGAP activity and localization in normal mouse mammary gland epithelial cells.

Loss of the Serum Response Factor Cofactor, Cysteine-Rich Protein 1, Attenuates Neointima Formation in the Mouse

Objective—Cysteine-rich protein (CRP) 1 and 2 are cytoskeletal lin-11 isl-1 mec-3 (LIM)-domain proteins thought to be critical for smooth muscle differentiation. Loss of murine CRP2 does not overtly affect smooth muscle differentiation or vascular function but does exacerbate neointima formation in response to vascular injury. Because CRPs 1 and 2 are coexpressed in the vasculature, we hypothesize that CRPs 1 and 2 act redundantly in smooth muscle differentiation. Methods and Results—We generated Csrp1 (gene name for CRP1) null mice by genetic ablation of the Csrp1 gene and found that mice lacking CRP1 are viable and fertile. Smooth muscle–containing tissues from Csrp1-null mice are morphologically indistinguishable from wild-type mice and have normal contractile properties. Mice lacking CRPs 1 and 2 are viable and fertile, ruling out functional redundancy between these 2 highly related proteins as a cause for the lack of an overt phenotype in the Csrp1-null mice. Csrp1-null mice challenged by wire-induced arterial injury display reduced neointima formation, opposite to that seen in Csrp2-null mice, whereas Csrp1/Csrp2 double-null mice produce a wild-type response. Conclusion—Smooth muscle CRPs are not essential for normal smooth muscle differentiation during development, but may act antagonistically to modulate the smooth muscle response to pathophysiological stress.

Mechanical signals activate p38 MAPK pathway-dependent reinforcement of actin via mechanosensitive HspB1

Mechanical force induces protein phosphorylations, subcellular redistributions, and actin remodeling. We show that mechanical activation of the p38 MAPK pathway leads to phosphorylation of HspB1 (hsp25/27), which redistributes to cytoskeletal structures, and contributes to the actin cytoskeletal remodeling induced by mechanical stimulation.

Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelity approach to identify constituents of protein complexes

AbstractBackgroundHere we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophila melanogaster, PINCH has two known high-affinity binding partners—Integrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5—but has been postulated to bind additional proteins as well.ResultsTo purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry.ConclusionsThe iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co-purifications inherent in iGEO allow for rapid and unambiguous identification of the constituents of protein complexes, without the need for extensive follow-up experiments.

Noncanonical translation via deadenylated 3′ UTRs maintains primordial germ cells

AbstractPrimordial germ cells (PGCs) form during early embryogenesis with a supply of maternal mRNAs that contain shorter poly(A) tails. How translation of maternal mRNAs is regulated during PGC development remains elusive. Here we describe a small-molecule screen with zebrafish embryos that identified primordazine, a compound that selectively ablates PGCs. Primordazines effect on PGCs arises from translation repression through primordazine-response elements in the 3′ UTRs. Systematic dissection of primordazines mechanism of action revealed that translation of mRNAs during early embryogenesis occurs by two distinct pathways, depending on the length of their poly(A) tails. In addition to poly(A)-tail-dependent translation (PAT), early embryos perform poly(A)-tail-independent noncanonical translation (PAINT) via deadenylated 3′ UTRs. Primordazine inhibits PAINT without inhibiting PAT, an effect that was also observed in quiescent, but not proliferating, mammalian cells. These studies reveal that PAINT is an alternative form of translation in the early embryo and is indispensable for PGC maintenance.Primordazine inhibits poly(A)-tail-independent noncanonical translation (PAINT) in early zebrafish embryos and in mammalian cells under select conditions, an effect mediated by deadenylated 3ʹ UTRs that results in ablation of primordial germ cells.