Journal: Current Biology

Article Title: An unconventional TOG domain is required for CLASP localization

doi: 10.1016/j.cub.2023.07.009

Figure Lengend Snippet: The CTD of CLASPs folds like a TOG domain (A) Schematic of the Hs CLASP1 protein domain delimitations. (B) Alignment and secondary structure comparison of the CTDs from Hs CLASP1, Hs CLASP2, and Ce CLS-2, and of the TOG2 of Hs CLASP1. The secondary structure of Hs CLASP1 TOG2 is assigned from the crystal structure and that of Hs CLASP1 CTD from the AlphaFold2 model. (C and D) AlphaFold2 3D structure prediction of Hs CLASP1 CTD, color-coded by either (C) HEAT repeats (HRs) A–F or (D) amino acid conservation. Right: zoom on the C-terminal-conserved patch highlighting the R1481 residue. Amino acid conservation is color-coded (red, high conservation, yellow, low conservation). (E) Still images of spindle in C. elegans zygotes expressing Ce CLS-2-GFP (orange) or Ce CLS-2 ΔCTD :: Hs CTD::GFP (blue) upon depletion of endogenous CLS-2. Scale bars, 5 μm. (F) Quantification of mean WT Ce CLS-2-GFP (orange) or Ce CLS-2 ΔCTD :: Hs CTD::GFP (blue) along the mitotic spindle upon depletion of endogenous CLS-2. Error bars, SEM. Sample sizes (n metaphase plate) are indicated on the graph. (G) Quantification of embryonic viability in the indicated conditions. Sample sizes (n embryos) are on the left of each bar plot. See also Figure S1 .

Article Snippet: Immunoprecipitations of EGFP-tagged Hs CLASP1 CTD wild-type or R1481A were performed in quadruplicate using GFP-Trap Magnetic Agarose beads (ChromoTek, gtma-20) after 24 hours of expression of the fusion proteins in HEK293T cells, according to the manufacturer’s protocol.

Techniques: Comparison, Residue, Expressing

Journal: Current Biology

Article Title: An unconventional TOG domain is required for CLASP localization

doi: 10.1016/j.cub.2023.07.009

Figure Lengend Snippet: A conserved arginine in the CTD of CLASPs is essential for their proper sub-cellular localizations (A–F) Immunofluorescence images of DLD-1 cells transiently expressing wild-type (WT) or R1481A EGFP- Hs CLASP1, and stained for (A) the trans -Golgi marker TGN46, (C) the focal adhesion marker paxillin, or (E) the centromeric marker ACA (bottom right: zoom on one kinetochore pair; scale bars, 1 μm). Scale bars, 5 μm. White arrowheads indicate centrosomes (localized with an anti-pericentrin antibody, not shown). Quantification of mean intensity of WT or R1481A EGFP- Hs CLASP1 at (B) the Golgi, (D) the cell cortex (CMSCs), and (F) kinetochores. Error bars: SD for (B) and (D), SEM for (F). Sample sizes (n areas, N kinetochores) are indicated on each graph. Unpaired t tests, p < 0.0001. (G) Still images of C. elegans zygotes expressing WT or R970A Ce CLS-2-GFP and the chromosomal marker mCherry-H2B. Scale bars, 5 μm. (H) Quantification of WT or R970A Ce CLS-2-GFP along the mitotic spindle. Error bars, SD. Sample sizes (n metaphase plate) are indicated on each graph. See also Figure S2 .

Article Snippet: Immunoprecipitations of EGFP-tagged Hs CLASP1 CTD wild-type or R1481A were performed in quadruplicate using GFP-Trap Magnetic Agarose beads (ChromoTek, gtma-20) after 24 hours of expression of the fusion proteins in HEK293T cells, according to the manufacturer’s protocol.

Techniques: Immunofluorescence, Expressing, Staining, Marker

Journal: Current Biology

Article Title: An unconventional TOG domain is required for CLASP localization

doi: 10.1016/j.cub.2023.07.009

Figure Lengend Snippet: Identification of Hs CLASP1 CTD interacting proteins (A) Schematic of the strategy used to compare WT or R1481A Hs CLASP1 CTD protein partners using EGFP immunoprecipitation followed by mass-spectrometric identification. (B) Result of LC-MS/MS analysis from four independent experiments. Volcano plot of −log 10 p values against log 2 fold change (R1481A versus WT). Proteins significantly enriched in the WT but not the R1481A mutant (log 2 enrichment > 1, p < 0.05) are indicated in pink, with selected proteins highlighted. (C) Result from two independent yeast-two-hybrid screens from a human fibroblast or a human placental library, respectively. The predicted biological score (PBS) of each interaction is ranked from A (very high confidence) to D (moderate confidence). Proteins are highlighted as significantly enriched (pink) or not enriched (gray) in the WT compared with the R1481A mutant (identified in the proteomic analysis). Proteins not found in the proteomic analysis are displayed in black. (D) Venn diagram displaying the overlaps between hits from the fibroblast and placenta Y2H screens, and the results from the mass spectrometry analysis (only known CLASP interactors, or proteins also identified in at least one of the Y2H screen, are included). See also Figure S3 and .

Article Snippet: Immunoprecipitations of EGFP-tagged Hs CLASP1 CTD wild-type or R1481A were performed in quadruplicate using GFP-Trap Magnetic Agarose beads (ChromoTek, gtma-20) after 24 hours of expression of the fusion proteins in HEK293T cells, according to the manufacturer’s protocol.

Techniques: Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Mass Spectrometry

Journal: Current Biology

Article Title: An unconventional TOG domain is required for CLASP localization

doi: 10.1016/j.cub.2023.07.009

Figure Lengend Snippet:

Article Snippet: Immunoprecipitations of EGFP-tagged Hs CLASP1 CTD wild-type or R1481A were performed in quadruplicate using GFP-Trap Magnetic Agarose beads (ChromoTek, gtma-20) after 24 hours of expression of the fusion proteins in HEK293T cells, according to the manufacturer’s protocol.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Western Blot, Expressing, Luciferase, Variant Assay, Mutagenesis, Software