High-throughput measurement of dictyostelium discoideum macropinocytosis by flow cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment. Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria. This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.</p

A polycystin-type transient receptor potential (Trp) channel that is activated by ATP.

ATP and ADP are ancient extra-cellular signalling molecules that in Dictyostelium amoebae cause rapid, transient increases in cytosolic calcium due to an influx through the plasma membrane. This response is independent of hetero-trimeric G-proteins, the putative IP3 receptor IplA and all P2X channels. We show, unexpectedly, that it is abolished in mutants of the polycystin-type transient receptor potential channel, TrpP. Responses to the chemoattractants cyclic-AMP and folic acid are unaffected in TrpP mutants. We report that the DIF morphogens, cyclic-di-GMP, GABA, glutamate and adenosine all induce strong cytoplasmic calcium responses, likewise independently of TrpP. Thus, TrpP is dedicated to purinergic signalling. ATP treatment causes cell blebbing within seconds but this does not require TrpP, implicating a separate purinergic receptor. We could detect no effect of ATP on chemotaxis and TrpP mutants grow, chemotax and develop almost normally in standard conditions. No gating ligand is known for the human homologue of TrpP, polycystin-2, which causes polycystic kidney disease. Our results now show that TrpP mediates purinergic signalling in Dictyostelium and is directly or indirectly gated by ATP.Medical Research Council (U105115237 to RRK

My 2,000 best films: parallel phenotyping of Dictyostelium development

Developmental phenotyping of 2,000 Dictyostelium mutants using parallel time-lapse filming

Cell-Fate Choice in Dictyostelium: Intrinsic Biases Modulate Sensitivity to DIF Signaling

AbstractCell fate in Dictyostelium development depends on intrinsic differences between cells, dating from their growth period, and on cell interactions occurring during development. We have sought for a mechanism linking these two influences on cell fate. First, we confirmed earlier work showing that the vegetative differences are biases, not commitments, since cells that are stalky-biased when developed with one partner are sporey with another. Then we tested the idea that these biases operate by modulating the sensitivity of cells to the signals controlling cell fate during development. Cells grown without glucose are stalky-biased when developed with cells grown with glucose. We find, using monolayer culture conditions, that they are more sensitive to each of the stalk-inducing signals, DIFs 1–3. Mixing experiments show that this bias is a cell-intrinsic property. Cells initiating development early in the cell cycle are stalky compared to those initiating development later in the cycle. Likewise, they are more sensitive to DIF-1. Assays of standard markers for prestalk and prespore cell differentiation reveal similar differences in DIF-1 sensitivity between biased cells; DIF-1 dechlorinase (an early prestalk cell marker enzyme) behaves in a consistent manner. We propose that cell-fate biases are manifest as differences in sensitivity to DIF

Amplification of pip3 signalling by macropinocytic cups

In a role distinct from and perhaps more ancient than that in signal transduction, PIP3 and Ras help to spatially organize the actin cytoskeleton into macropinocytic cups. These large endocytic structures are extended by actin polymerization from the cell surface and have at their core an intense patch of active Ras and PIP3, around which actin polymerizes, creating cup-shaped projections. We hypothesize that active Ras and PIP3 self-amplify within macropinocytic cups, in a way that depends on the structural integrity of the cup. Signalling that triggers macropinocytosis may therefore be amplified downstream in a way that depends on macropinocytosis. This argument provides a context for recent findings that signalling to Akt (an effector of PIP3) is sensitive to cytoskeletal and macropinocytic inhibitors.</p

Protecting State Constitutional Rights from Unconstitutional Conditions

The unconstitutional conditions doctrine limits the ability of governments to force individuals to choose between retaining a right and enjoying a government benefit. The doctrine has primarily remained a creature of federal law, with neither courts nor commentators focusing on the potentially important role of state doctrines of unconstitutional conditions. This omission has become especially significant during the COVID-19 pandemic, as actions by state and local governments have presented unconstitutional conditions questions in a range of novel contexts. The overruling of Roe v. Wade and the resulting focus on state constitutional rights to abortion will offer additional new settings for state unconstitutional conditions analysis. As attention turns to distinctive state constitutional rights — in the context of COVID-19 disputes, abortion litigation, and more generally — state courts should develop their own state doctrines of unconstitutional conditions, rather than simply reverting to federal unconstitutional conditions analysis. Three reasons in particular drive this doctrinal claim. First, the unconstitutional conditions doctrine helps to define the scope and weight of a constitutional right. A state court that ignores the unconstitutional conditions doctrine when considering the constitutionality of a state statute or regulation risks undermining the very nature of the right. Second, uncritically adopting federal doctrine ignores the state’s distinctive legal framework, interests, and history, all of which might lead to a deviation from federal law. With respect to the topics on which unconstitutional conditions litigation typically focuses, such as licenses and permits, the federal-state disparities are especially stark. Third, robust legal development in our federal system depends in part upon the interplay of different institutional interpreters. When state courts and federal courts engage in independent interpretative activity, they create the possibility of dialogue and mutual learning. This interpretive interplay enhances federal doctrine, as well as doctrinal development in other states. Given the gaps and inconsistencies in the unconstitutional conditions doctrine, such interjurisdictional enlightenment is especially needed in this area. After explaining why states should develop their own doctrines of unconstitutional conditions, we suggest the relevant considerations that should guide states in formulating their doctrines

Effect of hydrofracking fluid on colloid transport in the unsaturated zone

Hydraulic fracturing is expanding rapidly in the US to meet increasing energy demand and requires high volumes of hydrofracking fluid to displace natural gas from shale. Accidental spills and deliberate land application of hydrofracking fluids, which return to the surface during hydrofracking, are common causes of environmental contamination. Since the chemistry of hydrofracking fluids favors transport of colloids and mineral particles through rock cracks, it may also facilitate transport of in situ colloids and associated pollutants in unsaturated soils. We investigated this by subsequently injecting deionized water and flowback fluid at increasing flow rates into unsaturated sand columns containing colloids. Colloid retention and mobilization was measured in the column effluent and visualized in situ with bright field microscopy. While &lt;5% of initial colloids were released by flushing with deionized water, 32–36% were released by flushing with flowback fluid in two distinct breakthrough peaks. These peaks resulted from 1) surface tension reduction and steric repulsion and 2) slow kinetic disaggregation of colloid flocs. Increasing the flow rate of the flowback fluid mobilized an additional 36% of colloids, due to the expansion of water filled pore space. This study suggests that hydrofracking fluid may also indirectly contaminate groundwater by remobilizing existing colloidal pollutants