How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response

Actin-crosslinking proteins organize actin filaments into dynamic and complex subcellular scaffolds that orchestrate important mechanical functions, including cell motility and adhesion. Recent mutation studies have shown that individual crosslinking proteins often play seemingly non-essential roles, leading to the hypothesis that they have considerable redundancy in function. We report live-cell, in vitro, and theoretical studies testing the mechanical role of the two ubiquitous actin-crosslinking proteins, alpha-actinin and fascin, which co-localize to stress fibers and the basis of filopodia. Using live-cell particle tracking microrheology, we show that the addition of alpha-actinin and fascin elicits a cell mechanical response that is significantly greater than that originated by alpha-actinin or fascin alone. These live-cell measurements are supported by quantitative rheological measurements with reconstituted actin filament networks containing pure proteins that show that alpha-actinin and fascin can work in concert to generate enhanced cell stiffness. Computational simulations using finite element modeling qualitatively reproduce and explain the functional synergy of alpha-actinin and fascin. These findings highlight the cooperative activity of fascin and alpha-actinin and provide a strong rationale that an evolutionary advantage might be conferred by the cooperative action of multiple actin-crosslinking proteins with overlapping but non-identical biochemical properties. Thus the combination of structural proteins with similar function can provide the cell with unique properties that are required for biologically optimal responses.     

Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions.

Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.     

Ascorbate enhances the toxicity of the photodynamic action of Verteporfin in HL-60 cells

As a reducing agent, ascorbate serves as an antioxidant. However, its reducing function can in some settings initiate an oxidation cascade, i.e., seem to be a "pro-oxidant." This dichotomy also seems to hold when ascorbate is present during photosensitization. Ascorbate can react with singlet oxygen, producing hydrogen peroxide. Thus, if ascorbate is present during photosensitization the formation of highly diffusible hydrogen peroxide could enhance the toxicity of the photodynamic action. On the other hand, ascorbate could decrease toxicity by converting highly reactive singlet oxygen to less reactive hydrogen peroxide, which can be removed via peroxide-removing systems such as glutathione and catalase. To test the influence of ascorbate on photodynamic treatment we incubated leukemia cells (HL-60 and U937) with ascorbate and a photosensitizer (Verteporfin; VP) and examined ascorbic acid monoanion uptake, levels of glutathione, changes in membrane permeability, cell growth, and toxicity. Accumulation of VP was similar in each cell line. Under our experimental conditions, HL-60 cells were found to accumulate less ascorbate and have lower levels of intracellular GSH compared to U937 cells. Without added ascorbate, HL-60 cells were more sensitive to VP and light treatment than U937 cells. When cells were exposed to VP and light, ascorbate acted as an antioxidant in U937 cells, whereas it was a pro-oxidant for HL-60 cells. One possible mechanism to explain these observations is that HL-60 cells express myeloperoxidase activity, whereas in U937 cells it is below the detection limit. Inhibition of myeloperoxidase activity with 4-aminobenzoic acid hydrazide (4-ABAH) had minimal influence on the phototoxicity of VP in HL-60 cells in the absence of ascorbate. However, 4-ABAH decreased the toxicity of ascorbate on HL-60 cells during VP photosensitization, but had no affect on ascorbate toxicity in U937 cells. These data demonstrate that ascorbate increases hydrogen peroxide production by VP and light. This hydrogen peroxide activates myeloperoxidase, producing toxic oxidants. These observations suggest that in some settings, ascorbate may enhance the toxicity of photodynamic action.     

Dendritic cells infected with vpr-positive human immunodeficiency virus type 1 induce CD8+ T-cell apoptosis via upregulation of tumor necrosis factor alpha

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) plays a crucial role in viral replication and pathogenesis by inducing cell cycle arrest, apoptosis, translocation of preintegration complex, potentiation of glucocorticoid action, impairment of dendritic cell (DC) maturation, and T-cell activation. Recent studies involving the direct effects of Vpr on DCs and T cells indicated that HIV-1 containing Vpr selectively impairs phenotypic maturation, cytokine network, and antigen presentation in DCs and dysregulates costimulatory molecules and cytokine production in T cells. Here, we have further investigated the indirect effect of HIV-1 Vpr(+) virus-infected DCs on the bystander CD8(+) T-cell population. Our results indicate that HIV-1 Vpr(+) virus-infected DCs dysregulate CD8(+) T-cell proliferation and induce apoptosis. Vpr-containing virus-infected DC-mediated CD8(+) T-cell killing occurred in part through enhanced tumor necrosis factor alpha production by infected DCs and subsequent induction of death receptor signaling and activation of the caspase 8-dependent pathway in CD8(+) T cells. Collectively, these results provide evidence that Vpr could be one of the important contributors to the host immune escape by HIV-1 through its ability to dysregulate both directly and indirectly the DC biology and T-cell functions.     

Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice

While reports suggest a single dose of senolytics may improve vasomotor function, the structural and functional impact of long‐term senolytic treatment is unknown. To determine whether long‐term senolytic treatment improves vasomotor function, vascular stiffness, and intimal plaque size and composition in aged or hypercholesterolemic mice with established disease. Senolytic treatment (intermittent treatment with Dasatinib + Quercetin via oral gavage) resulted in significant reductions in senescent cell markers (TAF⁺ cells) in the medial layer of aorta from aged and hypercholesterolemic mice, but not in intimal atherosclerotic plaques. While senolytic treatment significantly improved vasomotor function (isolated organ chamber baths) in both groups of mice, this was due to increases in nitric oxide bioavailability in aged mice and increases in sensitivity to NO donors in hypercholesterolemic mice. Genetic clearance of senescent cells in aged normocholesterolemic INK‐ATTAC mice phenocopied changes elicited by D+Q. Senolytics tended to reduce aortic calcification (alizarin red) and osteogenic signaling (qRT–PCR, immunohistochemistry) in aged mice, but both were significantly reduced by senolytic treatment in hypercholesterolemic mice. Intimal plaque fibrosis (picrosirius red) was not changed appreciably by chronic senolytic treatment. This is the first study to demonstrate that chronic clearance of senescent cells improves established vascular phenotypes associated with aging and chronic hypercholesterolemia, and may be a viable therapeutic intervention to reduce morbidity and mortality from cardiovascular diseases.     

Cdc42-induced actin filaments are protected from capping protein.

Each actin filament has a pointed and a barbed end, however, filament elongation occurs primarily at the barbed end. Capping proteins, by binding to the barbed end, can terminate this elongation. The rate of capping depends on the concentration of capping protein [1], and thus, if capping terminates elongation, the length of filaments should vary inversely with the concentration of capping protein. In cell extracts, such as those derived from neutrophils, new actin filaments can be nucleated by addition of GTPgammaS-activated Cdc42 (a small GTPase of the Rho family). To determine whether elongation of these filaments is terminated by capping, we manipulated the concentration of capping protein, the major calcium-independent capping protein in neutrophils, and observed the effects on filament lengths. Depletion of 70% of the capping protein from extracts increased the mean length of filaments elongated from spectrin-actin seeds (very short actin filaments with free barbed ends) but did not increase the mean length of filaments induced by Cdc42. Furthermore, doubling the concentration of capping protein in cell extracts by adding pure capping protein did not decrease the mean length of filaments induced by Cdc42. These results suggest that the barbed ends of Cdc42-induced filaments are protected from capping by capping protein.     

OpenAWSEM with Open3SPN2: A fast,flexible, and accessible framework for large-scale coarse-grained biomolecular simulations

We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM’s Custom Forces software framework. By utilizing GPUs, we achieve around a 30-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a single CPU core. We showcase the benefits of OpenMM’s Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen’s early Nobel prize winning experiments by using OpenMM’s Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology.     

Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosis

Multiple ankyrin repeat motif-containing proteins play an important role in protein-protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s).