Journal: mSphere
Article Title: A genetic strategy to allow detection of F-actin by phalloidin staining in diverse fungi
doi: 10.1128/msphere.00517-25
Figure Lengend Snippet: Phenotype of actA V75I in A. nidulans . ( A ) Schematic showing the integration strategy to introduce the V75I point mutation at the native locus linked to the pyrG selectable marker ( actA V75I :pyrG ) and the strategy used to generate the isogenic control ( actA:pyrG ). ( B ) Growth of a wild-type strain (DLY26757), the isogenic control strain, actA:pyrG (DLY26755), or the actA V75I :pyrG strain (DLY26756) after 3 days of growth on the indicated media at the indicated temperatures. ( C ) Western blot showing expression of ActA in two different clones of the isogenic control strain, actA:pyrG (ActA), and in two different clones of the mutant strain, actA V75I :pyrG (ActA V75I ). ( D ) Widefield time-series of live actA:pyrG (DLY26755) and act1 V75I :pyrG (DLY26756) conidia germinating on PDA at room temperature (20–22°C). Scale bar, 20 µm. ( E ) Elongation rates of actA:pyrG (DLY26755) and act1 V75I :pyrG (DLY26756) germlings grown on PDA at room temperature ( n = 132 actA:pyrG , n = 84 actA V75I :pyrG germlings). Statistical significance calculated by two-way Student’s t -test, P = 0.0793. ( F ) act1 V75I :pyrG (DLY26756) conidia and germlings fixed and stained with phalloidin showing F-actin patches (puncta), cables (linear structures), and actomyosin ring (dashed box). The insert in F′ shows the area in the dashed box in F with the contrast adjusted to highlight the ring structure. Scale bar in F and F′ is 5 µm. ( G ) Phalloidin staining in actA:pyrG (DLY26755) germlings expressing wild-type actin shows no detectable F-actin structures. Scale bar, 5 µm.
Article Snippet: Strains were imaged at room temperature (20–22°C) on a widefield Nikon ECLIPSE Ti2 inverted microscope with a Plan Apo 20× air objective (NA 0.75, Nikon Instruments), an sCMOS pco.edge camera (Excelitas Technologies), and an X-Cite XYLIS LED Illumination System (Excelitas Technologies) controlled by NIS-Elements software (Nikon Instruments).
Techniques: Introduce, Mutagenesis, Marker, Control, Western Blot, Expressing, Clone Assay, Staining
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