Figure 1. Repositioning of the vimentin... | Rockefeller University Press

Figure 1.

$Repositioning of the vimentin network by rapalog-induced recruitment of kinesin motors. (A) A scheme of rapalog-induced heterodimerization constructs. A truncated motor domain of P. patens kinesin-14b (ppKin14, amino acids 861–1321) is fused to the FRB domain, along with a GCN4 leucine zipper for tetramerization and a BFP tag for detection. A fragment of human KIF5A (amino acids 1–560) is fused to an HA-tagged dimeric motor domain and the FRB domain. The FKBP domain and mCherry were fused to the C terminus of vimentin. Flexible glycine-serine linkers separate protein domains. (B and C) Schemes illustrating vimentin repositioning triggered by rapalog-induced recruitment of kinesins at the level of a single microtubule (B) and in whole cells (C). Without rapalog, Vim-mCh-FKBP does not interact with either FRB-BFP-GCN4-ppKin14 or HA-KIF5A-FRB. Upon rapalog addition, FRB and FKBP heterodimerize, triggering motor attachment to vimentin filaments and their movement along microtubules. Minus-end-directed kinesins trigger vimentin clustering around the MTOC, whereas plus end–directed kinesins cause the formation of small peripheral clusters and a large perinuclear one. (D and E) Representative fluorescence images of COS-7 cells co-transfected with Vim-mCh-FKBP and FRB fusions of the indicated motors, with or without 1 h of rapalog treatment. The top panels show untreated cells, whereas the bottom panels display rapalog-treated cells. Transfected cells are outlined with dashed lines, and non-transfected cells serve as controls. Anti-vimentin and anti-HA antibodies detect total vimentin and HA-KIF5A-FRB, respectively. (F) Quantifications of the fraction of the cell area occupied by vimentin in untransfected and transfected cells with either minus- or plus-end motor constructs, with and without rapalog treatment. (G–I) Mean fluorescence intensities of Vim-mCh-FKBP intensity (G), FRB-BFP-GCN4-ppKin14 (H), and HA-KIF5A-FRB, detected with anti-HA antibody (I) per cell, normalized to untransfected cells. In (F–I), n = 19–34 cells per condition across three independent experiments. Plots indicate mean ± SD, with individual cell measurements shown as dots. ns, not significant; ∗, P < 0.05; ∗∗∗∗, P < 0.0001. Statistical significance was assessed using the Mann–Whitney t test for (H) and (I), while the Kruskal–Wallis test followed by Dunn’s multiple comparisons test was applied for (F) and (G). (J andL) Live-cell imaging reveals the morphology of the vimentin network immediately before and at 10, 20, and 60 min after rapalog addition. Cells co-transfected with ppKin14 (J) or KIF5A constructs (L) show vimentin reorganization over time. (K and M) Cells shown in J and L, fixed and stained with antibodies against vimentin 60 min after rapalog treatment.$

Repositioning of the vimentin network by rapalog-induced recruitment of kinesin motors. (A) A scheme of rapalog-induced heterodimerization constructs. A truncated motor domain of P. patens kinesin-14b (ppKin14, amino acids 861–1321) is fused to the FRB domain, along with a GCN4 leucine zipper for tetramerization and a BFP tag for detection. A fragment of human KIF5A (amino acids 1–560) is fused to an HA-tagged dimeric motor domain and the FRB domain. The FKBP domain and mCherry were fused to the C terminus of vimentin. Flexible glycine-serine linkers separate protein domains. (B and C) Schemes illustrating vimentin repositioning triggered by rapalog-induced recruitment of kinesins at the level of a single microtubule (B) and in whole cells (C). Without rapalog, Vim-mCh-FKBP does not interact with either FRB-BFP-GCN4-ppKin14 or HA-KIF5A-FRB. Upon rapalog addition, FRB and FKBP heterodimerize, triggering motor attachment to vimentin filaments and their movement along microtubules. Minus-end-directed kinesins trigger vimentin clustering around the MTOC, whereas plus end–directed kinesins cause the formation of small peripheral clusters and a large perinuclear one. (D and E) Representative fluorescence images of COS-7 cells co-transfected with Vim-mCh-FKBP and FRB fusions of the indicated motors, with or without 1 h of rapalog treatment. The top panels show untreated cells, whereas the bottom panels display rapalog-treated cells. Transfected cells are outlined with dashed lines, and non-transfected cells serve as controls. Anti-vimentin and anti-HA antibodies detect total vimentin and HA-KIF5A-FRB, respectively. (F) Quantifications of the fraction of the cell area occupied by vimentin in untransfected and transfected cells with either minus- or plus-end motor constructs, with and without rapalog treatment. (G–I) Mean fluorescence intensities of Vim-mCh-FKBP intensity (G), FRB-BFP-GCN4-ppKin14 (H), and HA-KIF5A-FRB, detected with anti-HA antibody (I) per cell, normalized to untransfected cells. In (F–I), n = 19–34 cells per condition across three independent experiments. Plots indicate mean ± SD, with individual cell measurements shown as dots. ns, not significant; ∗, P < 0.05; ∗∗∗∗, P < 0.0001. Statistical significance was assessed using the Mann–Whitney t test for (H) and (I), while the Kruskal–Wallis test followed by Dunn’s multiple comparisons test was applied for (F) and (G). (J andL) Live-cell imaging reveals the morphology of the vimentin network immediately before and at 10, 20, and 60 min after rapalog addition. Cells co-transfected with ppKin14 (J) or KIF5A constructs (L) show vimentin reorganization over time. (K and M) Cells shown in J and L, fixed and stained with antibodies against vimentin 60 min after rapalog treatment.

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