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Figure 2 | Cell Division

Figure 2

From: The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence

Figure 2

Interdependence of the subunits of the Tof1/Csm3/Mrc1 complex for their chromatin binding. After partial spheroplasting by LongLife™ Zymolyase®, all GFP strains are subjected to soluble proteins washing via TritonX-100 detergent treatment. In all experiments where chromatin binding of Mrc1 is studied, cells are treated with 0.5% w/v of detergent. When Tof1 or Csm3 are studied, 3.0% w/v of detergent is used. Cells are paraformaldehyde fixed and subjected to fluorescent microscopy analysis to detect the position of GFP-tagged proteins. 2.5 μg/ml DAPI staining is used for all of the probes to visualize the position of DNA. The obtained GFP and DAPI signals are analyzed for co-localizations. The match of obtained DAPI and GFP signals of TritonX-100 treated MRC1-GFP (A.a), TOF1-GFP (B.a) and CSM3-GFP (C.a) is indicative of their chromatin bound state. When csm3 or tof1 genes are deleted (TOF1-GFP; csm3∆ (B.b) and CSM3-GFP; tof1∆ (C.b)), the reciprocal binding partner is not detected on chromatin. After TritonX-100 wash of the soluble proteins from TOF1-GFP; mrc1∆ (B.c) and CSM3-GFP; mrc1∆ (C.c) strains, the GFP signals coincide with DAPI signals. Both MRC1-GFP; tof1∆ (A.b) and MRC1-GFP; csm3∆ (A.c) strains, when treated with TritonX-100, demonstrate lack of GFP signal. GFP - Filter set 38HE (Zeiss); DAPI - Filter set 01 (Zeiss); BF – bright field.

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