Transthyretin amyloid fibrils alter primary fibroblast structure, function and inflammatory gene expression.

Age-related wild type transthyretin amyloidosis (wtATTR) is characterized by systemic deposition of amyloidogenic fibrils of misfolded transthyretin (TTR) in the connective tissue of many organs. In the heart this leads to cardiac dysfunction, which is a significant cause of age-related heart failure. The hypothesis tested is that TTR affects cardiac fibroblasts in ways that may contribute to fibrosis. When primary cardiac fibroblasts were cultured on TTR-deposited substrates, the F-actin cytoskeleton disorganized, focal adhesion formation decreased, and nuclear shape was flattened. Fibroblasts had faster collective and single cell migration velocities on TTR-deposited substrates. Additionally, fibroblasts cultured on microposts with TTR deposition had reduced attachment and increased proliferation above untreated. Transcriptomic and proteomic analyses of fibroblasts grown on glass covered with TTR showed significant upregulation of inflammatory genes after 48 hours, indicative of progression in TTR-based diseases. Together, results suggest that TTR deposited in tissue extracellular matrix may affect both the structure, function and gene expression of cardiac fibroblasts. As therapies for wtATTR are cost-prohibitive and only slow disease progression, better understanding of cellular maladaptation may elucidate novel therapeutic targets.

Differential cellular responses to adhesive interactions with galectin-8 and fibronectin coated substrates

The mechanisms underlying the cellular response to extracellular matrices (ECM), consisting of multiple adhesive ligands, are still poorly understood. Here we address this topic by monitoring the differential cellular response to two different extracellular adhesion molecules – fibronectin, a major integrin ligand, and galectin-8, a lectin, that binds β-galactoside residues, as well as to mixtures of the two proteins. Cell spreading on galectin-8 coated substrates results in a larger projected cell area, a more extensive extension of filopodia, yet an inability to form focal adhesions and stress fibers, compared to cell spreading on fibronectin. These differences can be partially reversed by experimental manipulations of small G-proteins of the Rho family and their downstream targets, such as formins, Arp2/3 complex, and Rho kinase. We also show that the physical adhesion of cells to galectin-8 is stronger than the adhesion to fibronectin. Notably, galectin-8 and fibronectin differentially regulate cell spreading and focal adhesion formation, yet they act synergistically to upregulate the number and length of filopodia. The physiological significance of the coherent cellular response to a molecularly complex matrix is discussed.

The origin of the expressed retrotransposed gene ACTBL2 and its influence on human melanoma cells’ motility and focal adhesion formation

We have recently found that β-actin-like protein 2 (actbl2) forms complexes with gelsolin in human melanoma cells and can polymerize. Phylogenetic and bioinformatic analyses showed that actbl2 has a common origin with two non-muscle actins, which share a separate history from the muscle actins. The actin groups’ divergence started at the beginning of vertebrate evolution, and actbl2 actins are characterized by the largest number of non-conserved amino acid substitutions of all actins. We also discovered that ACTBL2 is expressed at a very low level in several melanoma cell lines, but a small subset of cells exhibited a high ACTBL2 expression. We found that clones with knocked-out ACTBL2 (CR-ACTBL2) or overexpressing actbl2 (OE-ACTBL2) differ from control cells in the invasion, focal adhesion formation, and actin polymerization ratio, as well as in the formation of lamellipodia and stress fibers. Thus, we postulate that actbl2 is the seventh actin isoform and is essential for cell motility.

α-actinin-4 drives invasiveness by regulating myosin IIB expression and myosin IIA localization

The mechanisms by which the mechanoresponsive actin crosslinking protein α-actinin-4 (ACTN4) regulates cell motility and invasiveness remains incompletely understood. Here we show that in addition to regulating protrusion dynamics and focal adhesion formation, ACTN4 transcriptionally regulates expression of non-muscle myosin IIB (NMM IIB), which is essential for mediating nuclear translocation during 3D invasion. We further demonstrate association between NMM IIA and ACTN4 at the cell front ensures retention of NMM IIA at the cell periphery. A protrusion-dependent model of confined migration recapitulating experimental observations predicts a dependence of protrusion forces on the degree of confinement and on the ratio of nucleus to matrix stiffness. Together, our results suggest that ACTN4 is a master regulator of cancer invasion that regulates invasiveness by controlling NMM IIB expression and NMM IIA localization.

Rabgap1 promotes recycling of active β1 integrins to support effective cell migration

ABSTRACTIntegrin function depends on the continuous internalization of integrins and their subsequent endosomal recycling to the plasma membrane to drive adhesion dynamics, cell migration and invasion. Here we assign a pivotal role for Rabgap1 (GAPCenA) in the recycling of endocytosed active β1 integrins to the plasma membrane. The phosphotyrosine-binding (PTB) domain of Rabgap1 binds to the membrane-proximal NPxY motif in the cytoplasmic domain of β1 integrin subunits on endosomes. Silencing Rabgap1 in mouse fibroblasts leads to the intracellular accumulation of active β1 integrins, alters focal adhesion formation, and decreases cell migration and cancer cell invasion. Functionally, Rabgap1 facilitates active β1 integrin recycling to the plasma membrane through attenuation of Rab11 activity. Taken together, our results identify Rabgap1 as an important factor for conformation-specific integrin trafficking and define the role of Rabgap1 in β1-integrin-mediated cell migration in mouse fibroblasts and breast cancer cells.

Abstract 395: The Role of Transglutaminase 2 (tg2) in Regulation of Macrophage Phenotype

Transglutaminase 2 (TG2) expression is increased in infarcted and remodeling hearts and modulates cellular responses through enzymatic effects and non-enzymatic actions. In monocytic cells, TG2 is predominantly expressed by M2 macrophages in both mice and humans. Although TG2 is considered a preserved and reliable marker of M2 macrophages, its role in regulation of macrophage phenotype remains unknown. In order to study the role of TG2 in macrophage function in homeostasis and disease, we generated mice with myeloid cell-specific loss of TG2 (MyTG2KO), and examined their response to experimental myocardial infarction (MI). Using myeloid cell reporter CSF1R EGFP mice, we found that TG2 is highly expressed in the majority of spleen, liver and lung macrophages, and is markedly upregulated in M2-like Arg1+ macrophages infiltrating the infarcted heart in contrast to infarct neutrophils. MyTG2KO mice had no baseline defects. Following MI, MyTG2KO mice and TG2 fl/fl controls had comparable chamber dilation, systolic dysfunction and infarct size, but exhibited attenuated diastolic dysfunction and reduced atrial size, reflecting lower filling pressures. Macrophages harvested from infarcted MyTG2KO mice had comparable pro-inflammatory and anti-inflammatory cytokine synthesis, but markedly increased expression of caveolin-1, the integrins a1, b1 and b3, and of the anti-fibrotic proteoglycan decorin. In vitro, we examined the effects of TG2 loss on the transcriptomic profile of bone marrow macrophages using RNAseq. Reactome and KEGG analysis showed that TG2 loss did not affect the baseline macrophage transcriptome differences. In contrast, following TGF-b stimulation, TG2 KO and WT cells had differential expression of genes regulating focal adhesion formation and the actin cytoskeleton. Consistent with the in vivo findings, TGF-b stimulation resulted in higher caveolin-1 and decorin in TG2 KO macrophages. In summary, TG2 expression is induced in M2-like macrophages infiltrating the infarcted heart and mediates diastolic dysfunction. The effects of TG2 in macrophages are not due to actions on their inflammatory profile, but may involve modulation of focal adhesion formation and cell migration, and subsequent expression of anti-fibrotic genes.

A distinct talin2 structure directs isoform specificity in cell adhesion

Integrin receptors regulate normal cellular processes such as signaling, cell migration, adhesion to the extracellular matrix, and leukocyte function. Talin recruitment to the membrane is necessary for its binding to and activation of integrin. Vertebrates have two highly conserved talin homologs that differ in their expression patterns. The F1–F3 FERM subdomains of cytoskeletal proteins resemble a cloverleaf, but in talin1, its F1 subdomain and additional F0 subdomain align more linearly with its F2 and F3 subdomains. Here, we present the talin2 crystal structure, revealing that its F0-F1 di-subdomain displays another unprecedented constellation, whereby the F0-F1-F2 adopts a new cloverleaf-like arrangement. Using multiangle light scattering (MALS), fluorescence lifetime imaging (FLIM), and FRET analyses, we found that substituting the corresponding residues in talin2 that abolish lipid binding in talin1 disrupt the binding of talin to the membrane, focal adhesion formation, and cell spreading. Our results provide the molecular details of the functions of specific talin isoforms in cell adhesion.

Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

Non-muscle actins have been studied for many decades; however, the reason for the existence of both isoforms is still unclear. Here we show, for the first time, a successful inactivation of the ACTB (CRISPR clones with inactivated ACTB, CR-ACTB) and ACTG1 (CRISPR clones with inactivated ACTG1, CR-ACTG1) genes in human melanoma cells (A375) via the RNA-guided D10A mutated Cas9 nuclease gene editing [CRISPR/Cas9(D10A)] technique. This approach allowed us to evaluate how melanoma cell motility was impacted by the lack of either β actin coded by ACTB or γ actin coded by ACTG1. First, we observed different distributions of β and γ actin in the cells, and the absence of one actin isoform was compensated for via increased expression of the other isoform. Moreover, we noted that γ actin knockout had more severe consequences on cell migration and invasion than β actin knockout. Next, we observed that the formation rate of bundled stress fibers in CR-ACTG1 cells was increased, but lamellipodial activity in these cells was impaired, compared to controls. Finally, we discovered that the formation rate of focal adhesions (FAs) and, subsequently, FA-dependent signaling were altered in both the CR-ACTB and CR-ACTG1 clones; however, a more detrimental effect was observed for γ actin-deficient cells. Our research shows that both non-muscle actins play distinctive roles in melanoma cells’ FA formation and motility.