dictyNews

Electronic Edition

Volume 44, number 24

August 24, 2018



Please submit abstracts of your papers as soon as they have been

accepted for publication by sending them to [log in to unmask]

or by using the form at

http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.



Back issues of dictyNews, the Dicty Reference database and other

useful information is available at dictyBase - http://dictybase.org.



Follow dictyBase on twitter:

http://twitter.com/dictybase





=========

Abstracts

=========





Diacylglycerol kinase (DGKA) regulates the effect of the epilepsy and 

bipolar disorder treatment valproic acid in Dictyostelium discoideum    



Elizabeth Kelly, Devdutt Sharma, Christopher J. Wilkinson and 

Robin S.B. Williams



Centre for Biomedical Sciences, School of Biological Sciences, 

Royal Holloway University of London, Egham, TW20 0EX, UK





Disease Models and Mechanisms, in press



Valproic acid (VPA) provides a common treatment for both epilepsy and 

bipolar disorder yet common cellular mechanisms relating to both disorders 

have yet to be proposed. Here, we explore the possibility of diacylglycerol 

kinase (DGK) playing a role in regulating the effect of VPA relating to the 

treatment of both disorders, using the biomedical model Dictyostelium 

discoideum. DGK provides the first step in the phosphoinositide recycling 

pathway, implicated in seizure activity. DGK also regulates levels of 

diacylglycerol (DAG), thereby regulating protein kinase C (PKC) activity that 

is linked to bipolar disorder-related signalling. Here, we show that ablation 

of the single Dictyostelium dgkA gene results in reduced sensitivity to the 

acute effects of VPA on cell behaviour. Loss of dgkA also provides reduced 

sensitivity to VPA in extended exposure during development. To differentiate 

a potential role for this DGKA-dependent mechanism in epilepsy and bipolar 

disorder treatment, we further show that the dgkA null mutant is resistant to 

the developmental effects of a range of structurally distinct branched medium 

chain fatty acids with seizure control activity and to the bipolar disorder 

treatment lithium. Finally, we show that VPA, lithium and novel epilepsy 

treatments function through DAG regulation, where the presence of DGKA is 

necessary for compound-specific increases in DAG levels following treatment. 

Thus, these experiments suggest that, in Dictyostelium, loss of DGKA 

attenuates a common cellular effect of VPA relating to both epilepsy and 

bipolar disorder treatments and that a range of new compounds with this 

effect should be investigated as alternative therapeutic agents.

 



submitted by: Robin Williams [[log in to unmask]]

——————————————————————————————————————





High-throughput Measurement of Dictyostelium discoideum Macropinocytosis 

by Flow Cytometry. 



Thomas Williams, Robert R. Kay



MRC-Laboratory of Molecular Biology, Cambridge, UK





Journal of Visual Experiments, in press



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.





submitted by:  Thomas Williams  [[log in to unmask]]

==============================================================

[End dictyNews, volume 44, number 24