Visualizing and quantifying adhesive signals

Understanding the structural adaptation and signaling of adhesion sites in response to mechanical stimuli requires in situ characterization of the dynamic activation of a large number of adhesion components. Here, we review high resolution live cell imaging approaches to measure forces, assembly and interaction of adhesion components, and the activation of adhesion-mediated signals. We conclude by outlining computational multiplexing as a framework for the integration of these data into comprehensive models of adhesion signaling pathways

A RhoC Biosensor Reveals Differences in the Activation Kinetics of RhoA and RhoC in Migrating Cells

RhoA and RhoC GTPases share 92% amino acid sequence identity, yet play different roles in regulating cell motility and morphology. To understand these differences, we developed and validated a biosensor of RhoC activation (RhoC FLARE). This was used together with a RhoA biosensor to compare the spatio-temporal dynamics of RhoA and RhoC activity during cell protrusion/retraction and macropinocytosis. Both GTPases were activated similarly at the cell edge, but in regions more distal from the edge RhoC showed higher activation during protrusion. The two isoforms differed markedly in the kinetics of activation. RhoC was activated concomitantly with RhoA at the cell edge, but distally, RhoC activation preceded RhoA activation, occurring before edge protrusion. During macropinocytosis, differences were observed during vesicle closure and in the area surrounding vesicle formation

A miR-155–Peli1–c-Rel pathway controls the generation and function of T follicular helper cells

MicroRNA (miRNA) deficiency impairs the generation of T follicular helper (Tfh) cells, but the contribution of individual miRNAs to this phenotype remains poorly understood. In this study, we performed deep sequencing analysis of miRNAs expressed in Tfh cells and identified a five-miRNA signature. Analyses of mutant mice deficient of these miRNAs revealed that miR-22 and miR-183/96/182 are dispensable, but miR-155 is essential for the generation and function of Tfh cells. miR-155 deficiency led to decreased proliferation specifically at the late stage of Tfh cell differentiation and reduced CD40 ligand (CD40L) expression on antigen-specific CD4+T cells. Mechanistically, miR-155 repressed the expression of Peli1, a ubiquitin ligase that promotes the degradation of the NF-κB family transcription factor c-Rel, which controls cellular proliferation and CD40L expression. Therefore, our study identifies a novel miR-155-Peli1-c-Rel pathway that specifically regulates Tfh cell generation and functionC. Xiao is a Pew Scholar in Biomedical Sciences. This study is supported by the PEW Charitable Trusts, Cancer Research Institute, Lupus Research Institute, National Institutes of Health (grants R01AI087634, R01AI089854, R56AI110403, and R56AI121155 to C. Xiao and grants R01AI103646 and R01AI108651 to L.-F. Lu), National Natural Science Foundation of China (grant 31570882 to W.-H. Liu, grant 31570883 to N. Xiao, and grant 31570911 to G. Fu), 1000 Young Talents Program of China (grant K08008 to N. Xiao), the Fundamental Research Funds for the Central Universities of the People’s Republic of China (grant 20720150065 to N. Xiao and G. Fu), the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, Information and Communications Technology, and Future Planning (grant NRF-2015R1C1A1A01052387 to S.G. Kang), and a 2016 research grant from Kangwon National University (to S.G. Kang

Molecular weight effects on chain pull-out fracture of reinforced polymeric interfaces

Using Brownian dynamics, we simulate the fracture of polymer interfaces reinforced by diblock connector chains. We find that for short chains the interface fracture toughness depends linearly on the degree of polymerization $N$ of the connector chains, while for longer chains the dependence becomes $N^{3/2}$. Based on the geometry of initial chain configuration, we propose a scaling argument that accounts for both short and long chain limits and crossover between them.Comment: 5 pages, 3 figure

FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions

Guanine-nucleotide dissociation inhibitors (GDI) are negative regulators of Rho family GTPases that sequester the GTPases away from the membrane. Here we ask how GDI-Cdc42 interaction regulates localized Cdc42 activation for cell motility. The sensitivity of cells to overexpression of Rho family pathway components led us to a new biosensor design (GDI.Cdc42 FLARE), in which Cdc42 was modified with a FRET ‘binding antenna’ that selectively reported Cdc42 binding to endogenous GDI. Similar antennae could also report GDI-Rac1 and GDI-RhoA interaction. Through computational multiplexing and simultaneous imaging, we determined the spatiotemporal dynamics of GDI-Cdc42 interaction and Cdc42 activation during cell protrusion and retraction. This revealed a remarkably tight coordination of GTPase release and activation on a time scale of 10 seconds, suggesting that GDI-Cdc42 interactions are a critical component in the spatiotemporal regulation of Cdc42 activity, and not merely a mechanism for global sequestration of an inactivated pool of signaling molecules

Study of fracture and stress-induced morphological instabilities in polymeric materials

We study the phenomena of fracture in polymers at the molecular and continuum level. At a molecular level, we study the failure of polymer/polymer interfaces. Our main focus is on a specific mode of failure known as chain pull-out fracture, which is common to weak adhesive junctions, and polymer blends and mixtures. In the case of the interface between incompatible polymers, reinforcement is achieved by adding a block copolymer to the interface. We introduce a microscopic model based on Brownian dynamics to investigate the effect of the polymerization index N, of the block connector chain, on fracture toughness of such reinforced polymeric junctions. We consider the mushroom regime, where connector chains are grafted with low surface density, for the case of large pulling velocity. We find that for short chains the interface fracture toughness depends linearly on the polymerization index N of the connector chains, while for longer chains the dependence becomes N 3/2. We propose a scaling argument, based on the geometry of the initial configuration, that accounts for both short and long chains and the crossover between them.At the continuum level, we study the pattern selection mechanism of finger-like crack growth phenomena in gradient driven growth problems in general, and the structure of stress-induced morphological instabilities in crazing of polymer glasses in particular. We simulate solidification in a narrow channel through the use of a phase-field model with an adaptive grid. By tuning a dimensionless parameter, the Peclet number, we show a continuous crossover from a free dendrite at high Peclet numbers to anisotropic viscous fingering at low Peclet numbers. At low Peclet numbers we find good agreement between our results, theoretical predictions, and experiment, providing the first quantitative test of solvability theory for anisotropic viscous fingers. For high undercoolings, we find new phenomena, a solid forger which satisfies stability and thermodynamic criterion. We further provide an analytical form for the shape of these fingers, based on local models of solidification, which fits our numerical results from simulation. Later we study the growth of crazes in polymer glasses by deriving the equations of motion of plastic flow at the craze tip, and the steady-state velocity profile of this flow. By developing a phenomenological model, we solve the full time-dependent equations of motion of this highly non-linear phenomena. Our simulation produces the steady-state cellular pattern observed in experiments. We further show that polymer glasses with lower yield stress produce cellular patterns with sharper tips and more cells, indicating instabilities with smaller wavelengths

Solidification of a supercooled liquid in a narrow channel

We simulate solidification in a narrow channel through the use of a phase-field model with an adaptive grid. In different regimes, we find that the solid can grow in fingerlike steady-state shapes, or become unstable, exhibiting unsteady growth. At low melt undercoolings, we find good agreement between our results, theoretical predictions, and experiment. For high undercoolings, we report evidence for a new stable steady-state finger shape which exists in experimentally accessible ranges for typical material