The Rho GTP exchange factor Lfc promotes spindle assembly in early mitosis

Abstract

Rho GTPases regulate reorganization of actin and microtubule cytoskeletal structures during both interphase and mitosis. The timing and subcellular compartment in which Rho GTPases are activated is controlled by the large family of Rho GTP exchange factors (RhoGEFs). Here, we show that the microtubule-associated RhoGEF Lfc is required for the formation of the mitotic spindle during prophase/prometaphase. The inability of cells to assemble a functioning spindle after Lfc inhibition resulted in a delay in mitosis and an accumulation of prometaphase cells. Inhibition of Lfc's primary target Rho GTPase during prophase/prometaphase, or expression of a catalytically inactive mutant of Lfc, also prevented normal spindle assembly and resulted in delays in mitotic progression. Coinjection of constitutively active Rho GTPase rescued the spindle defects caused by Lfc inhibition, suggesting the requirement of RhoGTP in regulating spindle assembly. Lastly, we implicate mDia1 as an important effector of Lfc signaling. These findings demonstrate a role for Lfc, Rho, and mDia1 during mitosis.

Fig. 1.

Injection of anti-Lfc antibodies into prophase cells. (A) Cells injected with control IgG at prophase, fixed 30 min postinjection, and stained with anti-tubulin antibody (green) and DAPI (blue). (B) Cells injected at prophase with anti-Lfc antibody (1.2 μg/μl), fixed 30 min after injection, and stained with DAPI (blue, Top) and anti-tubulin antibody (green, Middle). (Bottom) The merged image is shown. (C) Rat-2 fibroblasts were injected with anti-Lfc antibody and fixed 24 h postinjection. Only one interphase cell was injected per field of view. Cells were then scored on the basis of chromosome organization defects such as satellite or micronuclei, or binucleated and linked. Two hundred cells of each injection were counted. Tubulin is shown in green. (D) Prometaphase, metaphase, or anaphase Rat-2 cells were injected with anti-Lfc antibody, fixed 10 min postinjection, and stained with DAPI (blue, Top), anti-tubulin antibody (green, Middle). (Bottom) The merged image is shown. Anaphase-injected cells typically had progressed to telophase by the time of fixation. (E) Control IgG-injected or anti-Lfc-injected mitotic Rat-2 cells stained by using anti-tubulin antibody (red), CREST serum (green), and DNA (blue). (Right) High magnification images of microtubules and kinetochores in the vicinity of the chromatin. (F) Prophase Rat-2 cells were microinjected with either control IgG or anti-Lfc antibody. We acquired 3- to 4-μm sections, and the images were rotated on the axis corresponding to the long axis of the spindle (shown by red lines). The angle of rotation was then determined by measuring the degree of rotation of each spindle pole from the plane of the attachment surface. In control IgG-injected cells, spindles do not rotate significantly from the plane of attachment. However, in anti-Lfc-injected cells, the spindle poles were often significantly rotated. (All scale bars: 10 μm.)

Fig. 2.

Rho GTPase activity is important in normal spindle assembly in Rat-2, XLK-1, MDCK, and Ptk-1 cells. (A) Uninjected or PBS prophase-injected Rat-2 cells fixed 30 min postinjection and stained with DAPI (blue, Left) and anti-tubulin antibody (red, Center). (Right) The merge image is shown. (B) Rat-2 cells injected with 20 μg/ml (Left) or 40 μg/ml (Right) C3 transferase, fixed 30 min postinjection and stained with DAPI (blue, Left) and anti-tubulin antibody (red, Center). (Right) The merged image is shown. (C) Rat-2 cells injected with C3 transferase (20 μg/ml) during G₂, prophase, prometaphase, and metaphase. Cells injected during G₂ were fixed 4–6 h postinjection. Cells injected at prophase, prometaphase, and metaphase were fixed 10 min postinjection. The cells were stained with DAPI (blue, Left), and anti-tubulin antibodies (green, Center). (Right) The merged imaged is shown. (D) Xlk-1 cells were injected at prophase with either PBS (Left) or 20 μg/ml C3 toxin (Right), fixed 45 min postinjection, and stained with anti-tubulin antibody (green), and DAPI (blue). (E) MDCK cells were injected at prophase with either PBS (Left) or C3 toxin (Right), fixed 30 min postinjection, and stained as in D. (F) Ptk-1 cells were injected at prophase with PBS (Left) or C3 toxin, fixed 45 min postinjection, and stained as in D. (G) Prophase HeLa cells were injected with 20 μg/ml C3 toxin, fixed 30 min postinjection, and stained as in D. (All scale bars: 10 μm.)

Fig. 3.

Lfc regulates spindle assembly via Rho and mDial. (A) Rat-2 cells injected with anti-Lfc antibody and recombinant His-tagged RhoAL63 at prophase, fixed 30 min after injection, and stained with anti-tubulin antibody (green) and DAPI (blue). The final concentration of anti-Lfc antibody was adjusted to be the same as injection of anti-Lfc antibody alone in Fig. 2 (1.2 μg/μl), and the final concentration of RhoAL63 was 0.5 μg/μl. (B–D) Rat-2 cells injected with anti-Lfc antibody and recombinant GST-tagged RhoA G14V/T37Y/C20R (B), G14V/E40L (C), G14V/F39A (D)at prophase, fixed 30 min after injection, and stained as in A. (C) Rat-2 cells injected with anti-Lfc antibody and recombinant GST-tagged RhoA G14V/E40L (0.5 μg/μl) at prophase, fixed 30 min after injection, and stained as in A. (D) Rat-2 cells injected with anti-Lfc antibody and recombinant GST-tagged RhoA G14V/F39A (0.5 μg/μl) at prophase, fixed 30 min after injection, and stained as in A. (E) Cells were fixed 30 min after injection of the following: control IgG antibody, anti-Lfc antibody, 20 μg/ml C3 transferase, anti-Lfc antibody and RhoAL63, anti-Lfc antibody and RhoAG14V/E40L, anti-Lfc antibody and RhoAG14V/T37Y/C20R, or anti-Lfc antibody and RhoAG14V/F39A. Values represent the percentage of mitotic cells with defects in spindle and/or chromatin organization. A minimum of 50 injected cells were counted in each experiment. (F) Prophase Rat-2 cells microinjected with anti-mDia1 antibody (1 μg/μl) and fixed 30 min after injection. Tubulin is shown in green; DNA is shown in blue. (G–I) Prophase Rat-2 cells microinjected with F1F2 (G), D1 (H), or KA3 (I) recombinant protein (all at ≈1.0 μg/μl), fixed 30 min following injection, and stained as in F. Tubulin is shown in green; DNA is shown in blue. (Insets) Chromatin bridges linking two daughter nuclei. (J) Quantification of the degree of spindle rotation in anti-mDia1 antibody and D1 protein-injected cells. Each data point represents the angle of rotation of one spindle pole from the attachment plane. (K) Anti-mDia1-injected mitotic Rat-2 cell stained by using anti-tubulin antibody (red), CREST serum (green), and DNA (blue). (Right) High magnification images of microtubules and kinetochores in the vicinity of the chromatin. (L) Quantification of experiments shown in F–I. Values represent the percentage of mitotic cells with defects in spindle and/or chromatin organization. A minimum of 50 injected cells were counted in each experiment. (All scale bars: 10 μm.)