Fig. 2: The LLPS of ELKS1 facilitates CLASP2 accumulation at the FA vicinity.

a Schematic model showing the involvement of CLASP2 in two complexes at the MT plus end and FA vicinity for cortical MT targeting. b Overall structure of the ELKS2_CC3/LL5β_EBM complex. c Molecular details of the interface between ELKS2_CC3 and LL5β_EBM. R290 in ELKS2, corresponding to R294 in ELKS1, forms salt bridges and hydrogen bonds with LL5β_EBM, as indicated by dashed lines. d Cell imaging analysis showing the critical role of R294 for recruiting endogenous LL5β into the ELKS1 condensate. Cells were fixed and stained with anti-LL5β antibody. e Quantification of the relative fluorescence intensity (RFI) of endogenous LL5β in the ELKS1 condensate as indicated in (d). The RFI was normalized to background fluorescence intensity. The error bars represent the values in the 5%–95% range. The middle, bottom, and top of boxplots represent, respectively, the median, the 25th percentile, and the 75th percentile (n = 156 condensates from 20 cells). f, Analysis of CLASP2 accumulation at the FA vicinity in nocodazole-treated cells. HeLa cells were co-transfected with GFP-tagged CLASP2α with mCherry-tagged ELKS1 or its mutants. FA was stained by anti-Paxillin antibody. The CLASP2 distribution at the FA vicinity is further indicated by arrowheads in the line analysis. g Quantification of the percentage of FAs with CLASP2α clusters at their vicinity as shown in (e). The paxillin-stained FAs (>0.3 μm²) were selected for statistics. The error bars represent the values in the 5%-95% range. The middle, bottom, and top of boxplots represent, respectively, the median, the 25th percentile, and the 75th percentile (n = 30 cells). h Confocal imaging of Cy5-labeled LL5β_2BM in solution. The experiment was repeated three times. i In vitro co-phase separation of mCherry-ELKS1 and Cy5-labeled LL5β_2BM. The concentration of each protein was 10 μM. The experiment was repeated three times. j In vitro co-phase separation of mCherry-ELKS1, LL5β_2BM, and 405-labeled CLASP2_TOG4 or its TBM-binding deficient mutant. The concentration of each protein was 10 μM. The experiment was repeated three times. k A schematic model illustrating the cortical ELKS1/LL5β co-condensate recruiting CLASP2 via LLPS.