Filamins are essential, evolutionarily conserved, modular, multidomain, actin-binding proteins that organize the actin cytoskeleton and maintain extracellular matrix connections by anchoring actin ...filaments to transmembrane receptors. By cross-linking and anchoring actin filaments, filamins stabilize the plasma membrane, provide cellular cortical rigidity, and contribute to the mechanical stability of the plasma membrane and the cell cortex. In addition to binding actin, filamins interact with more than 90 other binding partners including intracellular signaling molecules, receptors, ion channels, transcription factors, and cytoskeletal and adhesion proteins. Thus, filamins scaffold a wide range of signaling pathways and are implicated in the regulation of a diverse array of cellular functions including motility, maintenance of cell shape, and differentiation. Here, we review emerging structural and functional evidence that filamins are mechanosensors and/or mechanotransducers playing essential roles in helping cells detect and respond to physical forces in their local environment.
The interactions between actin networks and cell membrane are immensely important for eukaryotic cell functions including cell shape changes, motility, polarity establishment, and adhesion. ...Actin-binding proteins are known to compete and cooperate using a finite amount of actin monomers to form distinct actin networks. How actin-bundling protein fascin and actin-branching protein Arp2/3 complex compete to remodel membranes is not entirely clear. To investigate fascin- and Arp2/3-mediated actin network remodeling, we applied a reconstitution approach encapsulating bundled and dendritic actin networks inside giant unilamellar vesicles (GUVs). Independently reconstituted, membrane-bound Arp2/3 nucleation forms an actin cortex in GUVs, whereas fascin mediates formation of actin bundles that protrude out of GUVs. Coencapsulating both fascin and Arp2/3 complex leads to polarized dendritic aggregates and significantly reduces membrane protrusions, irrespective of whether the dendritic network is membrane bound or not. However, reducing Arp2/3 complex while increasing fascin restores membrane protrusion. Such changes in network assembly and the subsequent interplay with membrane can be attributed to competition between fascin and Arp2/3 complex to utilize a finite pool of actin.
Fibroblasts produce matrix, regulate inflammation, mediate reparative processes, and serve as pluripotent mesenchymal cells. Analyzing digested normal human skin by single-cell RNA sequencing, we ...explored different fibroblast populations. T-distributed stochastic neighbor embedding and clustering of single-cell RNA sequencing data from six biopsy samples showed two major fibroblast populations, defined by distinct genes, including SFRP2 and FMO1, expressed exclusively by these two major fibroblast populations. Further subpopulations were defined within each of the SFRP2 and FMO1 populations and five minor fibroblast populations, each expressing discrete genes: CRABP1, COL11A1, FMO2, PRG4, or C2ORF40. Immunofluorescent staining confirmed that SFRP2 and FMO1 define cell types of dramatically different morphology. SFRP2+ fibroblasts were small, elongated, and distributed between collagen bundles. FMO1+ fibroblasts were larger and distributed in both interstitial and perivascular locations. Differential gene expression by SFRP2+, FMO1+, and COL11A1+ fibroblasts suggests roles in matrix deposition, inflammatory cell retention, and connective tissue cell differentiation, respectively.
Calcification of vessels is strongly associated with atherosclerosis and leads to coronary artery disease (CAD) and myocardial infarction (MI). Genome-wide association studies (GWAS) revealed several ...genes that are associated with and contribute to CAD/MI as well as coronary artery calcification (CAC); however, the underlying mechanisms are unknown. PHACTR1, which encodes phosphatase and actin regulator 1, is among these risk genes. The aim of this study was to functionally test whether Phactr1 regulates calcification in vitro using murine embryonic stem cell (mESC)-derived smooth muscle cells (SMCs).
Phactr1 was stably up- or down-regulated in mESCs. These mESCs were differentiated into SMCs, and calcification was enhanced using osteogenic medium. Calcium phosphate deposits were detected and quantified.
RT-PCR analysis demonstrated that gene expression of Phactr1 correlated with increased calcification in mESC-derived SMCs as well as primary human aortic SMCs. Down-regulation of Phactr1 decreased calcification. Decreased expression of the osteogenic marker osteopontin confirmed this finding at the molecular level. By contrast, overexpression of Phactr1 in calcifying mESC-derived SMCs enhanced mineralization.
Taken together, we demonstrated that PHACTR1 gene expression increases with the progression of calcification and that regulation of PHACTR1 in SMCs modulates the severity of vascular calcification.
•PHACTR1 was identified as risk gene for coronary artery calcification by GWAS.•A direct functional link between PHACTR1 and calcification was so far missing.•Here we established and validated a functional calcification in-vitro assay.•The gene expression of PHACTR1 increases with the progression of calcification.•Down/up-regulation of PHACTR1 expression modulates the severity of calcification.
Cervical artery dissection (CeAD), a mural hematoma in a carotid or vertebral artery, is a major cause of ischemic stroke in young adults although relatively uncommon in the general population ...(incidence of 2.6/100,000 per year). Minor cervical traumas, infection, migraine and hypertension are putative risk factors, and inverse associations with obesity and hypercholesterolemia are described. No confirmed genetic susceptibility factors have been identified using candidate gene approaches. We performed genome-wide association studies (GWAS) in 1,393 CeAD cases and 14,416 controls. The rs9349379G allele (PHACTR1) was associated with lower CeAD risk (odds ratio (OR) = 0.75, 95% confidence interval (CI) = 0.69-0.82; P = 4.46 × 10(-10)), with confirmation in independent follow-up samples (659 CeAD cases and 2,648 controls; P = 3.91 × 10(-3); combined P = 1.00 × 10(-11)). The rs9349379G allele was previously shown to be associated with lower risk of migraine and increased risk of myocardial infarction. Deciphering the mechanisms underlying this pleiotropy might provide important information on the biological underpinnings of these disabling conditions.
Molecular dynamics simulations hold the promise of providing an atomic-level description of protein folding that cannot easily be obtained from experiments. Here, we examine the extent to which the ...molecular mechanics force field used in such simulations might influence the observed folding pathways. To that end, we performed equilibrium simulations of a fast-folding variant of the villin headpiece using four different force fields. In each simulation, we observed a large number of transitions between the unfolded and folded states, and in all four cases, both the rate of folding and the structure of the native state were in good agreement with experiments. We found, however, that the folding mechanism and the properties of the unfolded state depend substantially on the choice of force field. We thus conclude that although it is important to match a single, experimentally determined structure and folding rate, this does not ensure that a given simulation will provide a unique and correct description of the full free-energy surface and the mechanism of folding.
Eukaryotic cells generate a diversity of actin filament networks in a common cytoplasm to optimally perform functions such as cell motility, cell adhesion, endocytosis and cytokinesis. Each of these ...networks maintains precise mechanical and dynamic properties by autonomously controlling the composition of its interacting proteins and spatial organization of its actin filaments. In this review, we discuss the chemical and physical mechanisms that target distinct sets of actin-binding proteins to distinct actin filament populations after nucleation, resulting in the assembly of actin filament networks that are optimized for specific functions.
Cytokinesis can fail during normal postnatal liver development, leading to polyploid hepatocytes. We investigated whether inhibiting cytokinesis in the liver slows tumor growth without compromising ...the health of normal hepatocytes. We inhibited cytokinesis in cancer cells by knocking down ANLN, a cytoskeletal scaffolding protein that regulates cytokinesis and might promote tumorigenesis, in mice with liver disease.
We analyzed clinical and gene expression data from The Cancer Genome Atlas, Oncomine, PrognoScan, and a hepatocellular carcinoma (HCC) tissue microarray. We knocked down ANLN with small interfering RNAs (siRNAs) in H2.35 liver cells and performed image analyses of cells undergoing cytokinesis. siRNAs were delivered to LAP-MYC mice, which develop hepatoblastoma, using lipid nanoparticles. H2.35 cells with knockdown of ANLN or control cells were injected into FRG mice, which develop chronic liver damage, and tumor growth was monitored. We also developed mice with inducible expression of transgenes encoding small hairpin RNAs (shRNAs) against Anln messenger RNA and studied liver tumorigenesis after administration of diethylnitrosamine and carbon tetrachloride. siRNAs against Anln messenger RNA were conjugated to N-acetylgalactosamine to reduce toxicity and increase hepatocyte tropism; their effects were studied in mouse models of liver cancer and regeneration.
Levels of ANLN messenger RNA were increased in human HCC tissues compared to non-tumor liver tissues. siRNA knockdown of ANLN blocked cytokinesis in H2.35 liver cells. Administration of siRNA against ANLN increased survival times of LAP-MYC mice, compared to mice given a control siRNA. H2.35 liver cells with shRNA knockdown of ANLN formed tumors more slowly in FRG mice than control H2.35 cells. Mice with inducible expression of shRNAs against Anln mRNA developed fewer liver tumors after administration of diethylnitrosamine and carbon tetrachloride than control mice. Knockdown of ANLN did not affect liver regeneration after acute and chronic liver injuries.
Knockdown of ANLN in liver cells blocks cytokinesis and inhibits development of liver tumors in mice. Agents that inhibit ANLN in the liver might be effective for prevention or treatment of HCC.
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(Macro)autophagy is a fundamental degradation process for macromolecules and organelles of vital importance for cell and tissue homeostasis. Autophagy research has gained a strong momentum in recent ...years because of its relevance to cancer, neurodegenerative diseases, muscular dystrophy, lipid storage disorders, development, ageing and innate immunity. Autophagy has traditionally been thought of as a bulk degradation process that is mobilized upon nutritional starvation to replenish the cell with building blocks and keep up with the energy demand. This view has recently changed dramatically following an array of papers describing various forms of selective autophagy. A main driving force has been the discovery of specific autophagy receptors that sequester cargo into forming autophagosomes (phagophores). At the heart of this selectivity lies the LC3-interacting region (LIR) motif, which ensures the targeting of autophagy receptors to LC3 (or other ATG8 family proteins) anchored in the phagophore membrane. LIR-containing proteins include cargo receptors, members of the basal autophagy apparatus, proteins associated with vesicles and of their transport, Rab GTPase-activating proteins (GAPs) and specific signaling proteins that are degraded by selective autophagy. Here, we comment on these new insights and focus on the interactions of LIR-containing proteins with members of the ATG8 protein family.
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