dictyNews

Electronic Edition

Volume 44, number 16

June 8, 2018



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Abstracts

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CRISPR/Cas9 mediated targeting of multiple genes in Dictyostelium



Ryoya Sekine, Takefumi Kawata & Tetsuya Muramoto



Department of Biology, Faculty of Science, Toho University, 2-2-1 

Miyama, Funabashi, Chiba, 274-8510, Japan





Scientific Reports



CRISPR/Cas9 has emerged in various organisms as a powerful 

technology for targeted gene knockout; however, no reports of editing 

the Dictyostelium genome efficiently using this system are available. 

We describe here the application of CRISPR/Cas9-mediated gene 

modification in Dictyostelium. The endogenous tRNA-processing 

system for expressing sgRNA was approximately 10 times more 

effective than the commonly used U6 promoter. The resulting sgRNA 

affected the sub-nuclear localisation of Cas9, indicating that the 

expression level of sgRNA was sufficiently high to form Cas9 and 

sgRNA complexes within the nucleus. The all-in-one vector containing 

Cas9 and sgRNA was transiently expressed to generate mutants in 

five PI3K genes. Mutation detective PCR revealed the mutagenesis 

frequency of the individual genes to be between 72.9% and 100%. We 

confirmed that all five targeting loci in the four independent clones had 

insertion/deletion mutations in their target sites. Thus, we show that the 

CRISPR/Cas9 system can be used in Dictyostelium cells to enable 

efficient genome editing of multiple genes. Since this system utilises 

transient expression of the all-in-onevector, it has the advantage that 

the drug resistance cassette is not integrated into the genome and 

simple vector construction, involving annealing two oligo-DNAs.





submitted by:  Tetsuya Muramoto [[log in to unmask]]

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Oscillatory switches of dorso-ventral polarity in cells confined between 

two surfaces



Jonne Helenius, Mary Ecke, Daniel J. Müller, and Günther Gerisch



Biophysical Journal, accepted



To manoeuvre in a 3-dimensional space, migrating cells need to 

accommodate to multiple surfaces. In particular, phagocytes have to 

explore their environment in the search for particles to be ingested. To 

examine how cells decide between competing surfaces, we exposed 

single cells of Dictyostelium to a defined 3-dimensional space by 

confining them between two planar surfaces, those of a cover glass and 

of a wedged microcantilever. These cells form propagating waves of 

filamentous actin and PIP3 on their ventral substrate-attached surface. 

The dynamics of wave formation in the confined cells was explored using 

two-focus fluorescence imaging. When waves formed on one substrate, 

wave formation on the other substrate was efficiently suppressed. The 

propensity for wave formation switched between the opposing cell 

surfaces with periods of 2 to 5 minutes by one of two modes: (1) a rolling 

mode involving the slipping of a wave along the non-attached plasma 

membrane, and (2) de novo initiation of waves on the previously blank 

cell surface. These data provide evidence for a cell-autonomous oscillator 

that switches dorso-ventral polarity in a cell simultaneously exposed to 

multiple substrate surfaces.





submitted by:  Günther Gerisch [[log in to unmask]]

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The Effect of Overexpressed DdRabS on Development, Cell Death, Vesicular 

Trafficking,and the Secretion of Lysosomal Glycosidase Enzymes



Azure Yarbrough, Katherine Maringer, Entsar J. Saheb, Sanaa Jawed, 

and John Bush 





Biology (Basel)



Rab GTPases are essential regulators of many cellular processes and play 

an important role in downstream signaling vital to proper cell function. We 

sought to elucidate the role of novel D. discoideum GTPase RabS. Cell lines 

over-expressing DdRabS and expressing DdRabS N137I (dominant negative 

(DN)) proteins were generated, and it was determined that DdRabS localized 

to endosomes, ER-Golgi membranes, and the contractile vacuole system. It 

appeared to function in vesicular trafficking, and the secretion of lysosomal 

enzymes. Interestingly, microscopic analysis of GFP-tagged DdRabS (DN) 

cells showed differential localization to lysosomes and endosomes compared 

to GFP-tagged DdRabS overexpressing cells. Both cell lines over-secreted 

lysosomal glycosidase enzymes, especially beta-glucosidase. Furthermore, 

DdRabS overexpressing cells were defective in aggregation due to decreased 

cell–cell cohesion and sensitivity to cAMP, leading to abnormal chemotactic 

migration, the inability to complete development, and increased induced cell 

death. These data support a role for DdRabS in trafficking along the vesicular 

and biosynthetic pathways. We hypothesize that overexpression of DdRabS 

may interfere with GTP activation of related proteins essential for normal 

development resulting in a cascade of defects throughout these processes.





submitted by:  Azure Yarbrough  [[log in to unmask]]

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[End dictyNews, volume 44, number 16]