Fig 1.
Interplay between Las17 polyproline and WCA domains (A) schematic showing domain structure of Las17. WH1 –WASP homology-1 region; W–WH2 or WASP homology 2 region; C–central region; A–acidic region; P denotes tracts of 5 proline residues. R denotes those tracts with paired arginines in close proximity. Actin polymerization assays following increased fluorescence due to incorporation of pyrene-labeled actin into filaments were carried out to assess the effect of Las17 fragments in the absence (B) and presence (C) of Arp2/3. Shown are representative experiments. Each assay has been repeated independently a minimum of three times.
Fig 2.
Analysis of truncated fragments of Las17 to determine regions required for nucleation in the absence of Arp2/3.
(A) Five fragments of Las17 were generated containing 9 (180–633), 8 (300–633), 4 (414–633), 3 (460–633) or 2 (490–633) tracts of 5 prolines, fused to the WCA region. These were used in a pyrene-actin based polymerization assay to determine the parts of the polyproline region required for actin filament nucleation and elongation. The lower graph shows the first 10 minutes of polymerization to highlight the effect of the fragments at early time points more clearly. The effect of the fragments on (B) the reduction in the lag phase relative to actin alone and (C) the relative rates of F-actin elongation were analysed from at least three independent experiments with each fragment. In statistical analysis the Las17 180–633 and 300–633 fragments show significant reductions in lag phase (p value <0.0001) and elongation (p value <0.0001) using one-way ANOVA when compared to actin alone. ** highlights significance based on Dunnetts multiple comparison test in one-way ANOVA. (D) Pyrene-actin based polymerization assay were used to compare the activity of the PP-WCA fragment (residues 300–633) with full length Las17 purified from yeast in the absence of Arp2/3.
Fig 3.
The importance of paired arginine residues for actin binding and polymerization.
(A) Microscale thermophoresis was used to measure the binding affinity of a wild type or RRRR-AAAA mutant fragment of Las17(300–422) for G-actin (50 nM). The effect of both single and double arginine pair mutations on actin polymerization was assessed in the context of the longer PP-WCA fragment in the presence (B) or absence (C) of Arp2/3. Independent assays performed ≥3 times.
Fig 4.
The effect of Las17 RRRR-AAAA expression in cells.
(A) A mutant form of las17 encoding Las17 RR349,350 AA;RR383,383 AA was integrated into the genome. Expression levels were tested using anti-Las17 antibodies. Numbers indicate size of standard markers. (B) Halo assays were used to determine latrunculin-A sensitivity of the mutant strain compared to wild-type and las17 deletion strains. Numbers indicate the concentration of latrunculin-A (mM) applied to the filter discs. (C) Rhodamine-phalloidin staining was used to determine organization of F-actin in cells. Two cells are shown for each strain. Bar = 5 μm.
Fig 5.
The effect of the Las17 RRRR-AAAA mutation on recruitment of key endocytic proteins.
Strains expressing reporters for early stages of endocytosis (Sla1-GFP) and for Arp2/3 (Arc15-mCherry) were generated. The behaviour of reporters was analysed in otherwise wild-type cells (A) or in cells harbouring an integrated mutant allele of Las17 (las17 RRRR-AAAA) (B). The profiles show fluorescence intensity over time for 3 different patches analysed. A total of 30 patches were analysed for each strain and the combined data are shown in (C). Error bars denote standard deviation. Sla1-GFP has a significantly different lifetime between the strains (**** students t-test p<0.0001). Arc15-mCherry has a significantly different lifetime between the strains (*** students t-test p = 0.0009). Mean lifetimes are given. n = 30.
Fig 6.
The role of proline tracts in the polyproline region.
(A) Pyrene assays were used to assess the possible contribution of profilin to the Las17 PP-mediated actin nucleation. Profilin was added to the assays in the presence or absence of Las17 PP and actin–A. (B) The effect of proline tract number on filament elongation was assessed by incubating actin in the presence of Las17 fragments carrying 4 (300–414) or 8 (300–536) proline tracts. Inset shows early time points to highlight effects on nucleation. (C) The length of actin filaments formed after 30 minutes polymerization in the presence of the 4- or 8-proline tract fragments was assessed by visualizing filaments using fluorescence microscopy. Multiple fields of view were recorded and the length (C-left) and number (C-right) of filaments recorded. The difference between filament lengths formed in the presence of the short or long fragment was significant (*), p value in Mann Whitney test 0.029, from 4 independent experiments. Number of filament lengths measured for each condition in each experiment ≥102. The filament numbers per unit image area were counted and normalized to actin. The graph shows fold increase in filament number for the two Las17 fragments tested. The difference in filament number is statistically significant (P = 0.0036) in a 1 way ANOVA analysis (n = 3 independent experiments). The effect of increasing proline tract number on elongation rate (D) and depolymerization rate (E) was recorded as described for fragments carrying increasing numbers of polyproline tracts. These experiments used actin at 5 μM. Fragments used: PP peptide (residues 501–528); mutant peptide (residues 501–508 with alanine for proline substitutions at residues 506,507, 525,526); ppC–which carries the C-terminal 4 proline tracts (residues 414–536); PP (full proline tract 300–536) and PP-WCA (full proline tract and the WCA region).
Fig 7.
N-terminal region of Las17 mediates localization to membranes (A) Constructs expressing full length Las17, PP-WCA or 2xWH1 fused to GFP were expressed in yeast and extracts tested for expression using antibodies against GFP. (B) Localization of the GFP constructs were assessed in cells co-expressing the endocytic site marker Sla2-mRFP. Top panels: full length Las17-GFP, middle panels PP-WCA-GFP; lower panels, 2xWH1-GFP. Bar = 2 μm. (C) Full length Las17-myc purified from yeast and the recombinant PP-WCA Las17 fragment (300–633) purified from E.coli were incubated with liposomes before centrifuging to assess binding. S-supernatant and P–pellet fractions. Size of MW standard markers are marked in kilodaltons.
Table 1.
Plasmids used in this study.