Quantitative analysis of transient and sustained transforming growth factor-β signaling dynamics

Mathematical modeling and experimental analyses reveal that TGF-β ligand depletion has an important role in converting short-term graded signaling responses to long-term switch-like responses

Multiomic Analysis Reveals Disruption of Cholesterol Homeostasis by Cannabidiol in Human Cell Lines

The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines. CBD treatment leads to alterations in the abundance of metabolites, mRNA transcripts, and proteins associated with activation of cholesterol biosynthesis, transport, and storage. We found that CBD rapidly incorporates into cellular membranes, alters cholesterol accessibility, and disrupts cholesterol-dependent membrane properties. Sustained treatment with high concentrations of CBD induces apoptosis in a dose-dependent manner. CBD-induced apoptosis is rescued by inhibition of cholesterol synthesis and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use. &nbsp;</p

The development of a novel high throughput computational tool for studying individual and collective cellular migration.

Understanding how cells migrate individually and collectively during development and cancer metastasis can be significantly aided by a computation tool to accurately measure not only cellular migration speed, but also migration direction and changes in migration direction in a temporal and spatial manner. We have developed such a tool for cell migration researchers, named Pathfinder, which is capable of simultaneously measuring the migration speed, migration direction, and changes in migration directions of thousands of cells both instantaneously and over long periods of time from fluorescence microscopy data. Additionally, we demonstrate how the Pathfinder software can be used to quantify collective cell migration. The novel capability of the Pathfinder software to measure the changes in migration direction of large populations of cells in a spatiotemporal manner will aid cellular migration research by providing a robust method for determining the mechanisms of cellular guidance during individual and collective cell migration

The Pathfinder cell motility program uniquely incorporates measurements of cellular position, speed, direction, and persistence.

<p>Although several cellular motility programs are available to measure cellular motility in terms of position and speed, only the Pathfinder program additionally reports both cellular migration direction and cellular migration persistence.</p

Measuring individual cellular behavior with speed and migration persistence reveals cell type and ligand specific cellular migration behavior.

<p>Cellular tracks of low density cells are displayed for treatments of either Mock, TGFβ or EGF for MDA-MB-231 cells (<b>A</b>) or HaCaT cells (<b>B</b>). Calibration bars represent 150 µm. <b>C</b>) Neither TGF-Beta nor EGF stimulation affects migration persistence in MDA-MB-231cells (top). In contrast, both treatments affect cellular speed, but with different induction kinetics (bottom). <b>D</b>) In HaCaT cells, both ligand treatments affect migration persistence and cellular speed (top and bottom, respectively). However, EGF stimulates migration persistence with earlier kinetics than that of TGFβ (top), and EGF is a poor stimulator of migration speed (bottom, right). Each condition represents greater than 1000 cells.</p

Overlapping Intervals Suppress Angular Noise in Cellular Migration.

<p><b>A</b>) A schematic representation of a cellular track illustrates how increasing interval size results in the calculation of a cellular migration direction that is resistant to track vibration noise. <b>B</b>) The average absolute angle of deflection as a function of time for TGFβ treated HaCaT H2B mCherry cells for an interval size of one frame shows strong scattering of measurements. Upon increasing interval size, such scattering is suppressed. <b>C</b>) Such scattering can be also measured by measuring the standard deviation in the absolute angle of deflection, which can be suppressed in a similar manner by increasing interval size. Data represents greater than 1000 cells for each plot.</p

Partners in crime: the TGFβ and MAPK pathways in cancer progression

Abstract The TGFβ and Ras-MAPK pathways play critical roles in cell development and cell cycle regulation, as well as in tumor formation and metastasis. In the absence of cellular transformation, these pathways operate in opposition to one another, where TGFβ maintains an undifferentiated cell state and suppresses proliferation, while Ras-MAPK pathways promote proliferation, survival and differentiation. However, in colorectal and pancreatic cancers, the opposing pathways' mechanisms are simultaneously activated in order to promote cancer progression and metastasis. Here, we highlight the roles of the TGFβ and Ras-MAPK pathways in normal and malignant states, and provide an explanation for how the concomitant activation of these pathways drives tumor biology. Finally, we survey potential therapeutic targets in these pathways.</p

Angular measurements can be used to quantify collective migration behavior.

<p><b>A</b>) Cellular tracks of confluent monolayers of HaCaT (top) and MDA-MB-231 cells (bottom) in the presence and absence of EGF stimulation. Calibration bar represents 150 µm. <b>B</b>) Confluent monolayers of HaCaT cells in the presence and absence of EGF stimulation were quantified for their collective migration behavior by calculating the average standard deviation of the angle of trajectory (also called the paired random migration index (PRMI Θ_Tajectory) amongst nearest neighboring cells. Random pairing was used to determine whether the observed behavior was local or global amongst the population. <b>C</b>) The same quantification was conducted for MDA-MB-231 cells. <b>D</b>) Cellular tracks of epithelial sheets of HaCaT cells in the presence and absence of EGF stimulation. Calibration bar represents 150 µm. <b>E</b>) Inspection of the spatial distribution of collective migration behavior reveals that EGF stimulation elicits collective migration that propagates away from the leading edge. Double asterisks indicate a <i>p</i> value<0.01.</p