Epigallocatechin Gallate Modulates Microglia Phenotype to Suppress Pro-Inflammatory Signalling Cues and Inhibit Phagocytosis

Microglia are crucial players in the pathogenesis of late onset Alzheimer’s Disease (AD), with evidence for both deleterious and beneficial effects. Identifying interventions to modulate microglial responsiveness, to promote Amyloid β (Aβ) clearance, disrupt plaque formation, or to dampen excessive inflammation has therapeutic potential. Bioavailable flavonoids, such as the flavan 3-ols, are of interest due to their antioxidant, metal chelating, signalling and anti-inflammatory potential. Primary microglia were treated with a series of structurally related flavanol 3-ols to assess effects on phagocytosis, cytokine release and transcriptional responses by RNA sequencing. Data indicated that the extent of hydroxylation and the presence of the galloyl moiety were strong determinants of flavan 3-ol activity. Epigallocatechin gallate (EGCG) was the most effective flavan-3-ol tested and strongly inhibited phagocytosis of Aβ independent of any metal chelating properties, suggesting a more direct modulation of microglia responsiveness. EGCG was broadly anti-inflammatory, reducing cytokine release and downregulating transcription, particularly of components of the microglia extracellular matrix such as MMP3 and SerpinB2. Collectively, this brings new insight into the actions of flavonoids on microglial responsiveness with potential implications for the therapeutic use of EGCG and structurally related flavanol-3-ols in AD

High Aspect Ratio-Nanostructured Surfaces as Biological Metamaterials

Materials patterned with high-aspect-ratio nanostructures have features on similar lengthscales to cellular components. These surfaces are an extreme topography on the cellular level and have become useful tools for perturbing and sensing the cellular environment. Motivation comes from the ability of high-aspect-ratio nanostructures to deliver cargoes into cells and tissues, access the intracellular environment, and control cell behavior. These structures directly perturb cells’ ability to sense and respond to external forces, influencing cell fate and enabling new mechanistic studies. Through careful design of their nanoscale structure, these systems act as biological metamaterials, eliciting unusual biological responses. While predominantly used to interface eukaryotic cells, there is growing interest in non-animal and prokaryotic cell interfacing. Both experimental and theoretical studies have attempted to develop a mechanistic understanding for the observed behaviors, predominantly focusing on the cell – nanostructure interface. Here, we consider how high-aspect-ratio nanostructured surfaces are used to both stimulate and sense biological systems and discuss remaining research questions

Identification of phenotype-specific networks from paired gene expression-cell shape imaging data

The morphology of breast cancer cells is often used as an indicator of tumor severity and prognosis. Additionally, morphology can be used to identify more fine-grained, molecular developments within a cancer cell, such as transcriptomic changes and signaling pathway activity. Delineating the interface between morphology and signaling is important to understand the mechanical cues that a cell processes in order to undergo epithelial-to-mesenchymal transition and consequently metastasize. However, the exact regulatory systems that define these changes remain poorly characterized. In this study, we used a network-systems approach to integrate imaging data and RNA-seq expression data. Our workflow allowed the discovery of unbiased and context-specific gene expression signatures and cell signaling subnetworks relevant to the regulation of cell shape, rather than focusing on the identification of previously known, but not always representative, pathways. By constructing a cell-shape signaling network from shape-correlated gene expression modules and their upstream regulators, we found central roles for developmental pathways such as WNT and Notch, as well as evidence for the fine control of NF-kB signaling by numerous kinase and transcriptional regulators. Further analysis of our network implicates a gene expression module enriched in the RAP1 signaling pathway as a mediator between the sensing of mechanical stimuli and regulation of NF-kB activity, with specific relevance to cell shape in breast cancer

Paxillin Mediates Sensing of Physical Cues and Regulates Directional Cell Motility by Controlling Lamellipodia Positioning

Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5–10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax−/− and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax−/− cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax−/− and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices

ZR75.1

Files are 16-bit tiffs (which means they will appear black if opened in Preview, Photoshop, or similar program). Each file is a Multi-plane tiff, containing three fluorescence channels: Channel 1 = DAPI (Sigma) Channel 2 = NF-kappaB (anti-p65; Abcam ab16502 / Alexa-488 anti-rabbit; Invitrogen) Channel 3 = DHE (dihydroethidium, hydroethidine; Sigma) File names refer to [row][column]-[field] See ReadMe file for culture and staining information

hs578T

Files are 16-bit tiffs (which means they will appear black if opened in Preview, Photoshop, or similar program). Each file is a Multi-plane tiff, containing three fluorescence channels: Channel 1 = DAPI (Sigma) Channel 2 = NF-kappaB (anti-p65; Abcam ab16502 / Alexa-488 anti-rabbit; Invitrogen) Channel 3 = DHE (dihydroethidium, hydroethidine; Sigma) File names refer to [row][column]-[field

Data from: Cell shape and the microenvironment regulate nuclear translocation of NF-kappaB in breast epithelial and tumor cells

Although a great deal is known about the signaling events that promote nuclear translocation of NF‐κB, how cellular biophysics and the microenvironment might regulate the dynamics of this pathway is poorly understood. In this study, we used high‐content image analysis and Bayesian network modeling to ask whether cell shape and context features influence NF‐κB activation using the inherent variability present in unperturbed populations of breast tumor and non‐tumor cell lines. Cell–cell contact, cell and nuclear area, and protrusiveness all contributed to variability in NF‐κB localization in the absence and presence of TNFα. Higher levels of nuclear NF‐κB were associated with mesenchymal‐like versus epithelial‐like morphologies, and RhoA‐ROCK‐myosin II signaling was critical for mediating shape‐based differences in NF‐κB localization and oscillations. Thus, mechanical factors such as cell shape and the microenvironment can influence NF‐κB signaling and may in part explain how different phenotypic outcomes can arise from the same chemical cues

HCC1954

Files are 16-bit tiffs (which means they will appear black if opened in Preview, Photoshop, or similar program). Each file is a Multi-plane tiff, containing three fluorescence channels: Channel 1 = DAPI (Sigma) Channel 2 = NF-kappaB (anti-p65; Abcam ab16502 / Alexa-488 anti-rabbit; Invitrogen) Channel 3 = DHE (dihydroethidium, hydroethidine; Sigma) File names refer to [row][column]-[field