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. 2020 Jan 13;10:168. doi: 10.1038/s41598-019-56867-0

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Mlh1-FZZ exclusively localizes to the MIC. (A) Left: Schematic representation of proteolytic cleavage of full length Mlh1. The predicted product size and molecular weights are indicated. The position and size of the FZZ tag is indicated in blue. Proteolysis of the full-length Mlh1-FZZ results in β-FZZ product. Right: Expression analysis of endogenously tagged Mlh1-FZZ in comparison to the untagged controls by Western blotting in WCEs. The black arrowhead represents the β fragment (~25.5 kDa + FZZ 18 kDa). Anti-FLAG antibody was used to probe the top panel for Mlh1-FZZ detection. The bottom panel was probed with anti-Actin as a loading control. Two separate clones of Mlh1-FZZ were analysed. (B) Mlh1-FZZ localizes to MIC only during vegetative growth, starvation and conjugation. Mlh1-FZZ cells (mating type VII) were mated with untagged cells of mating type II. Nuclear events are illustrated as cartoons. Mlh1-FZZ signal was observed only in the MICs of the FZZ tagged cells and not in the untagged controls. The signal observed in both mating pairs (Mlh1-FZZ and controls) at the anlagen stage indicates mixing of cellular contents between the pairing cells. DAPI was used to stain the nuclei. The images were processed using ImageJ (version 1.50i) (https://imagej.nih.gov/ij/).