Thesis Review : Doctoral Thesis in Military Informatics (OpenPhD #openphd) - Lethal Autonomy of Weapons is Designed and/or Recessive by Nyagudi Musandu Nyagudi 18-12-2016-ISRA-eLife-24402

Doctoral Thesis Review (OpenPhD #openphd)<br

Strains used in this study.

<p>Strains used in this study.</p

Mutations in residues R374 and W391 specifically impair access to the alternative exocytic pathway.

<p>(A) Ten-fold serial dilutions of <i>chs3Δ</i> (RSY1699) or <i>chs3Δ chs6Δ AP-1Δ</i> (SPY10) cells expressing alanine-substitution mutants of <i>CHS3</i> were spotted onto synthetic complete medium -Ura + 100 ug/ml calcofluor (-Ura + CF). (B) Subcellular fractionation of wt Chs3, Chs3-W391R and Chs3-R374A expressed in <i>chs3Δ</i> and <i>chs3Δ chs6Δ AP-1Δ</i> cells on step sucrose/EDTA gradients. Total membranes from spheroplasts were separated on a step sucrose/EDTA gradient. The protein composition of fractions obtained from differential centrifugations and sucrose gradients were analyzed by SDS/PAGE and immunoblotting (PM marker: Pma1p; Golgi/EE markers: Tlg1p).</p

Mutations of D19 and E21 result in intracellular retention of Chs3p.

<p>(A) Ten-fold serial dilutions of <i>chs3Δ</i> (RSY1699) or <i>chs3Δ AP-1Δ</i> (SPY21) cells expressing N-terminal deletion mutants of <i>CHS3</i>, with and without the W391R substitution, were spotted onto synthetic complete medium -Ura plates +100 ug/ml calcofluor (-Ura + CF). (B) Ten-fold serial dilutions of <i>chs3Δ AP-1Δ</i> cells expressing alanine substitution mutants from residue 16 to residue 25 of Chs3-W391R were spotted onto - Ura + CF. (C) Subcellular fractionation of Δ15Chs3-W391R, Δ25Chs3-W391R and Chs3-D19AE21AW391R expressed in <i>chs3Δ AP-1Δ</i> cells on step sucrose/EDTA gradients. Total membranes from spheroplasts were separated on a step sucrose/EDTA gradient. The protein composition of fractions obtained from the sucrose gradients was analyzed by SDS/PAGE and immunoblotting (PM marker: Pma1p; Golgi/EE markers: Tlg1p).</p

Chs3p residues 19DEESLL24 constitute an intracellular retention signal.

<p>(A) Ten-fold serial dilutions of <i>chs3Δ chs6Δ</i> cells (TSY194) expressing point mutant alleles of <i>CHS3</i> that define 19DEESLL24 as an intracellular retention signal. Dilutions were spotted on YPD, ½ YPD, SC-Ura (-Ura) and SC-Ura +50 ug/ml calcofluor (-Ura +CF). (B) Chitin staining of TSY194 expressing the <i>chs3-L24P</i> mutant allele. (C) Ten-fold serial dilutions of TSY194 expressing alanine-scanning point mutants of Chs3p residues 17–25.</p

The DEESLL signal is required for a physical interaction between AP-1 and Chs3p.

<p>Cells of the genotype <i>chs3Δ pep4Δ prb1Δ prc1Δ APS1-TAP</i> (TSY131), expressing either wt Chs3p or Chs3-DEESLLΔ were grown overnight, converted to spheroplasts, and treated with the crosslinking agent DSP prior to lysis and purification of the AP-1 complex via a TAP tag. Samples were separated on a 10% polyacrylamide gel, which was visualized with sypro red stain prior to transfer to a PVDF membrane for evaluation by imunoblot. Shown is a western for Chs3p and a total protein stain for the AP-1 purification. Note that Chs3-DEESLLΔ does not copurify with AP-1.</p

AP-3 is required for the intracellular retention of Chs3p.

<p>(A) Ten-fold serial dilutions of wt (YPH499), <i>chs6Δ</i> (TSY49), <i>chs6Δ apl2Δ</i> (RSY3393), <i>chs6Δ apl1Δ</i> (TSY300) and <i>chs6Δ apl6Δ</i> (TSY269) cells on YPD, YPD + 50 ug/ml calcofluor (YPD + CF), synthetic complete medium (SC), and synthetic complete medium +50 ug/ml calcofluor (SC + CF). (B) An average of 5 quantitative chitin assays. Cultures were grown to saturation and cell walls were isolated by lysing 35–50 mg of cells in 6% KOH. Chitin was digested with chitinase from <i>T. viride</i> and the resulting GlcNAc was quantified. For each assay the results of 2–3 replicates of each strain were averaged and normalized against the wt level of chitin. Five separate normalized experiments were then averaged together.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Mutation of D19 and E21 impairs the <i>in vitro</i> binding of Chs3p with exomer.

<p>(A) Constructs corresponding to the full N-terminal cytosolic tail of Chs3p (Chs3 (1–170)), with and without the double substitution D19AE21A, or to the second cytosolic part of Chs3p (Chs3 (224–451)) were expressed in <i>E. coli</i> and purified as soluble GST-fusions. His-Chs5 and His-exomer complexes (expressed and purified from baculovirus infected culture cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046386#pone.0046386-Wang1" target="_blank">[31]</a>) were immobilized on Ni-NTA beads and incubated with 150 ug/ml purified GST-Chs3 fragments at RT for 30 min. Beads were washed and bound proteins were eluted with sample buffer followed by SDS-PAGE and Coomassie Blue staining. All samples shown were analyzed in the same gel. The images of each pertinent lane were cropped and reassembled. (B) The amount of each co-bound Chs3p fragment was quantified and normalized against the amount of Chs5p present in the same lane. The ratios of the amount of co-bound D19AE21A and 224–451 were normalized against the ratio of the wt pull-down, which was arbitrarily set to 1. The values from two independent experiments were averaged and graphed. Error bars represent standard deviations.</p

ALG-2 attenuates COPII dependent budding.

<p>The budding efficiency was measured from the amount of p58 and Sec22b in vesicles relative to the amount in donor membranes. HeLa cell donor membranes were incubated with rat liver cytosol, ATP and an ATP regenerating system, and wild-type recombinant ALG-2 (lanes 10–13) or ALG-2 with mutant versions of either EF-hand 1 (ALG2EF1, lane 14) or EF-hand 3 (ALG2EF3, lane 15) in the presence of either Ca²⁺ (lanes 6–16) or chelator (EGTA, lanes 17–20). As controls, membranes were incubated in the absence of either nucleotides (No NT, lane 1) or cytosol (No cyt, lane 2). As a control for the requirement of COPII proteins, we performed assays in the presence of a GTP-restricted dominant-negative form of the GTPase Sar1, Sar1 (H79G, lanes 3, 16 and 20). Budding efficiency was measured from band intensities and normalized to the amount of budding in the no Ca²⁺, no ALG-2 vesicle budding (0 Ca, lane 4) reaction. Bars indicate average +/− SD of three independent experiments.</p