Rac1 and Rac2 differentially regulate actin free barbed end formation downstream of the fMLP receptor

Actin assembly at the leading edge of migrating cells depends on the availability of high-affinity free barbed ends (FBE) that drive actin filament elongation and subsequent membrane protrusion. We investigated the specific mechanisms through which the Rac1 and Rac2 small guanosine triphosphatases (GTPases) generate free barbed ends in neutrophils. Using neutrophils lacking either Rac1 or Rac2 and a neutrophil permeabilization model that maintains receptor signaling to the actin cytoskeleton, we assessed the mechanisms through which these two small GTPases mediate FBE generation downstream of the formyl-methionyl-leucyl-phenylalanine receptor. We demonstrate here that uncapping of existing barbed ends is mediated through Rac1, whereas cofilin- and ARP2/3-mediated FBE generation are regulated through Rac2. This unique combination of experimental tools has allowed us to identify the relative roles of uncapping (15%), cofilin severing (10%), and ARP2/3 de novo nucleation (75%) in FBE generation and the respective roles played by Rac1 and Rac2 in mediating actin dynamics

Treponema denticola Major Outer Sheath Protein Induces Actin Assembly at Free Barbed Ends by a PIP2-Dependent Uncapping Mechanism in Fibroblasts

The major outer sheath protein (Msp) of Treponema denticola perturbs actin dynamics in fibroblasts by inducing actin reorganization, including subcortical actin filament assembly, leading to defective calcium flux, diminished integrin engagement of collagen, and retarded cell migration. Yet, its mechanisms of action are unknown. We challenged Rat-2 fibroblasts with enriched native Msp. Msp activated the small GTPases Rac1, RhoA and Ras, but not Cdc42, yet only Rac1 localized to areas of actin rearrangement. We used Rac1 dominant negative transfection and chemical inhibition of phosphatidylinositol-3 kinase (PI3K) to show that even though Rac1 activation was PI3K-dependent, neither was required for Msp-induced actin rearrangement. Actin free barbed end formation (FBE) by Msp was also PI3K-independent. Immunoblotting experiments showed that gelsolin and CapZ were released from actin filaments, whereas cofilin remained in an inactive state. Msp induced phosphatidylinositol (4,5)-bisphosphate (PIP2) formation through activation of a phosphoinositide 3-phosphatase and its recruitment to areas of actin assembly at the plasma membrane. Using a PIP2 binding peptide or lipid phosphatase inhibitor, PIP2 was shown to be required for Msp-mediated actin uncapping and FBE formation. Evidently, Msp induces actin assembly in fibroblasts by production and recruitment of PIP2 and release of the capping proteins CapZ and gelsolin from actin barbed ends

Social-biological interactions in oral disease: A \u27cells to society\u27 view

Oral diseases constitute a major worldwide public health problem, with their burden concentrating in socially disadvantaged and less affluent groups of the population, resulting in significant oral health inequalities. Biomedical and behavioural approaches have proven relatively ineffective in reducing these inequalities, and have potentially increased the health gap between social groups. Some suggest this stems from a lack of understanding of how the social and psychosocial contexts in which behavioural and biological changes occur influence oral disease. To unravel the pathways through which social factors affect oral health outcomes, a better understanding is thus needed of how the social \u27gets under the skin,\u27 or becomes embodied, to alter the biological. In this paper, we present the current knowledge on the interplay between social and biological factors in oral disease. We first provide an overview of the process of embodiment in chronic disease and then evaluate the evidence on embodiment in oral disease by reviewing published studies in this area. Results show that, in periodontal disease, income, education and perceived stress are correlated with elevated levels of stress hormones, disrupted immune biomarkers and increased allostatic load. Similarly, socioeconomic position and increased financial stress are related to increased stress hormones and cariogenic bacterial counts in dental caries. Based on these results, we propose a dynamic model depicting social-biological interactions that illustrates potential interdependencies between social and biological factors that lead to poor oral health. This work and the proposed model may aid in developing a better understanding of the causes of oral health inequalities and implicate the importance of addressing the social determinants of oral health in innovating public health interventions

How does the social “get under the gums”? The role of socio-economic position in the oral-systemic health link

OBJECTIVES: To evaluate the extent of association between systemic inflammation and periodontal disease in American adults, and to assess whether socio-economic position mediated this relationship. METHODS: We used data from the National Health and Nutrition Examination Survey (NHANES IV) (2001-2010). Systemic inflammation was defined by individual and aggregate (cumulative inflammatory load) biomarkers (C-reactive protein, white blood cell counts, neutrophil counts, and neutrophil: lymphocyte ratio). Loss of attachment and bleeding on probing were used to define periodontal disease. Poverty:income ratio and education were indicators of socio-economic position. Covariates included age, sex, ethnicity, smoking, alcohol, and attendance for dental treatment. Univariate and multivariable logistic regressions were constructed to assess the relationships of interest. RESULTS: In a total of 2296 respondents, biomarkers of systemic inflammation and cumulative inflammatory load were significantly associated with periodontal disease after adjusting for age, sex, and behavioural factors. Socio-economic position attenuated the association between markers of systemic inflammation and periodontal disease in the fully adjusted model. CONCLUSION: Socio-economic position partly explains how systemic inflammation and periodontal disease are coupled, and may thus have a significant role in the mechanisms linking oral and non-oral health conditions. It is of critical importance that the social and living conditions are taken into account when considering prevention and treatment strategies for inflammatory diseases, given what appears to be their impactful effect on disease processes

Requirements for Vav Guanine Nucleotide Exchange Factors and Rho GTPases in FcγR- and Complement-Mediated Phagocytosis

SummaryVav guanine nucleotide exchange factors (GEFs) have been implicated in cell adhesion by integrin and immune response receptors through the regulation of Rho GTPases. Here, we examine the role of Vav and Rho GTPases in phagocytosis by using primary murine macrophages. The genetic deletion of Rac1 and Rac2 prevents phagocytosis mediated by integrin and Fcγ receptors (FcγR), whereas the genetic deletion of Vav1 and Vav3 only prevents integrin-mediated phagocytosis through the complement receptor αMβ2. In addition, a Rac1/2 or Vav1/3 deficiency blocks Arp2/3 recruitment and actin polymerization at the complement-induced phagosome, indicating that these proteins regulate early steps in phagocytosis. Moreover, constitutively active Rac is able to rescue actin polymerization and complement-mediated phagocytosis in Vav-deficient macrophages. These studies indicate that Rac is critical for complement- and FcγR-mediated phagocytosis. In contrast, Vav is specifically required for complement-mediated phagocytosis, suggesting that Rac is regulated by GEFs other than Vav downstream of the FcγR

The N. gonorrhoeae Type IV Pilus Stimulates Mechanosensitive Pathways and Cytoprotection through a pilT-Dependent Mechanism

The Neisseria gonorrhoeae type IV pilus is a retractile appendage that can generate forces near 100 pN. We tested the hypothesis that type IV pilus retraction influences epithelial cell gene expression by exerting tension on the host membrane. Wild-type and retraction-defective bacteria altered the expression of an identical set of epithelial cell genes during attachment. Interestingly, pilus retraction, per se, did not regulate novel gene expression but, rather, enhanced the expression of a subset of the infection-regulated genes. This is accomplished through mitogen-activated protein kinase activation and at least one other undefined stress-activated pathway. These results can be reproduced by applying artificial force on the epithelial membrane, using a magnet and magnetic beads. Importantly, this retraction-mediated signaling increases the ability of the cell to withstand apoptotic signals triggered by infection. We conclude that pilus retraction stimulates mechanosensitive pathways that enhance the expression of stress-responsive genes and activate cytoprotective signaling. A model for the role of pilus retraction in influencing host cell survival is presented

Time-scale and other invariants of integrative mechanical behavior in living cells.

In dealing with systems as complex as the cytoskeleton, we need organizing principles or, short of that, an empirical framework into which these systems fit. We report here unexpected invariants of cytoskeletal behavior that comprise such an empirical framework. We measured elastic and frictional moduli of a variety of cell types over a wide range of time scales and using a variety of biological interventions. In all instances elastic stresses dominated at frequencies below 300 Hz, increased only weakly with frequency, and followed a power law; no characteristic time scale was evident. Frictional stresses paralleled the elastic behavior at frequencies below 10 Hz but approached a Newtonian viscous behavior at higher frequencies. Surprisingly, all data could be collapsed onto master curves, the existence of which implies that elastic and frictional stresses share a common underlying mechanism. Taken together, these findings define an unanticipated integrative framework for studying protein interactions within the complex microenvironment of the cell body, and appear to set limits on what can be predicted about integrated mechanical behavior of the matrix based solely on cytoskeletal constituents considered in isolation. Moreover, these observations are consistent with the hypothesis that the cytoskeleton of the living cell behaves as a soft glassy material, wherein cytoskeletal proteins modulate cell mechanical properties mainly by changing an effective temperature of the cytoskeletal matrix. If so, then the effective temperature becomes an easily quantified determinant of the ability of the cytoskeleton to deform, flow, and reorganize

Comparison and relative utility of inequality measurements: as applied to Scotland’s child dental health

This study compared and assessed the utility of tests of inequality on a series of very large population caries datasets. National cross-sectional caries datasets for Scotland’s 5-year-olds in 1993/94 (n = 5,078); 1995/96 (n = 6,240); 1997/98 (n = 6,584); 1999/00 (n = 6,781); 2002/03 (n = 9,747); 2003/04 (n = 10,956); 2005/06 (n = 10,945) and 2007/08 (n = 12,067) were obtained. Outcomes were based on the d3mft metric (i.e. the number of decayed, missing and filled teeth). An area-based deprivation category (DepCat) measured the subjects’ socioeconomic status (SES). Simple absolute and relative inequality, Odds Ratios and the Significant Caries Index (SIC) as advocated by the World Health Organization were calculated. The measures of complex inequality applied to data were: the Slope Index of Inequality (absolute) and a variety of relative inequality tests i.e. Gini coefficient; Relative Index of Inequality; concentration curve; Koolman and Doorslaer’s transformed Concentration Index; Receiver Operator Curve and Population Attributable Risk (PAR). Additional tests used were plots of SIC deciles (SIC10) and a Scottish Caries Inequality Metric (SCIM10). Over the period, mean d3mft improved from 3.1(95%CI 3.0–3.2) to 1.9(95%CI 1.8–1.9) and d3mft = 0% from 41.1(95%CI 39.8–42.3) to 58.3(95%CI 57.8–59.7). Absolute simple and complex inequality decreased. Relative simple and complex inequality remained comparatively stable. Our results support the use of the SII and RII to measure complex absolute and relative SES inequalities alongside additional tests of complex relative inequality such as PAR and Koolman and Doorslaer’s transformed CI. The latter two have clear interpretations which may influence policy makers. Specialised dental metrics (i.e. SIC, SIC10 and SCIM10) permit the exploration of other important inequalities not determined by SES, and could be applied to many other types of disease where ranking of morbidity is possible e.g. obesity. More generally, the approaches described may be applied to study patterns of health inequality affecting worldwide populations

Filamin-A Regulates Neutrophil Uropod Retraction through RhoA during Chemotaxis

Filamin-A (FLNa) has been shown to be a key cross-linker of actin filaments in the leading edge of a motile melanoma cell line, however its role in neutrophils undergoing chemotaxis is unknown. Using a murine transgenic model in which FLNa is selectively deleted in granulocytes, we report that, while neutrophils lacking FLNa show normal polarization and pseudopod extension, they exhibit obvious defects in uropod retraction. This uropod retraction defect was found to be a direct result of reduced FLNa mediated activation of the small GTPase RhoA and myosin mediated actin contraction in the FLNa null cells. This results in a neutrophil recruitment defect in FLNa null mice. The compensatory increase in FLNb levels that was observed in the FLNa null neutrophils may be sufficient to compensate for the lack of FLNa at the leading edge allowing for normal polarization, however this compensation is unable to regulate RhoA activated tail retraction at the rear of the cell