Imp2p forms actin-dependent clusters and imparts stiffness to the contractile ring

FIGURE 1:. Imp2p impacts the stiffness of the constricting contractile ring. (A) Confocal fluorescence micrographs of fields of wild-type (left) and ∆imp2 (right) cells. Asterisks, multiseptated cells. Dotted line, cell outline. (B) Graph of the ring offset measured in still images. n = 50 cells for each genotype. Inset, confocal micrographs of a representative wild-type mEGFP-Myo2p cell stained with FB28 that has a ring with a 2% offset. Dotted line, cell outline. (C) Time-lapse montages of wild-type and ∆imp2 cells showing the position of the ring over time. Magenta arrowheads, position of ring at the time of assembly. Blue arrowheads, position of the ring at the end of cytokinesis (wild type) or maximum displacement (∆imp2). (D) Graph of the change in the percentage of ring offset measured after ring sliding in time-lapse movies. n = 21 cells for each genotype. (E) Time-lapse montages of wild-type (top) and ∆imp2 (bottom) cells before and after laser ablation. Green arrowheads, sites of ablation. (F) Graph of the recoil displacement of severed tips in wild-type and ∆imp2 cells. Gray shaded box, equations representing our framework. (G) Kymographs of constricting contractile ring aligned…

FIGURE 2:. Severed contractile rings of ∆imp2 cells can heal. (A) Recoil and healing of typical severed wild-type (top) and ∆imp2 (bottom) cells where the visible severed tip recoils out of the imaging plane. Top row, inverted gray LUT. Bottom row, Fire LUT. Green arrowheads, sites of ablation. (B) Graph showing the ability of a severed wild-type or ∆imp2 contractile ring to heal after ablation vs. maximum displacement A. (C) Graph of the recoil displacement (5 s rolling average) for a single severed tip of a wild-type or ∆imp2 contractile ring showing recoil and healing. Dotted lines, time at which healing begins. Tracking of the severed tips of a wild-type ring show the progressive creeping motion until the tips joined across the gap. Tracking of the severed tips of the ∆imp2 ring shows initial progressive creeping motion of the tip until tracking appearance of a strand ∼170 s after ablation.

FIGURE 3:. Imp2p can cluster in the contractile ring. (A) Plots of the distribution of the number of polypeptides per ring for cytokinesis proteins in wild-type, ∆imp2, and Cdc15p-depleted cells. Resulting actin monomer values from strains containing the mEGFP-Lifeact construct were scaled to reflect that the Lifeact construct labels 6% of polymerized actin (Malla et al., 2022). Differences between the distributions of polypeptides were determined by a standard least squares model; p values stated on each plot. (B) Examples of SMLM of Imp2p-mEos3.2 (left) and mEos3.2-Imp2p (right) in cells with contractile rings at different stage of constriction. (C) Representative image of a cropped contractile ring in a cell expressing mEos3.2-Imp2p. (D) Four representative cropped protein clusters from contractile rings of cells expressing either N- or C-terminal tagged Imp2p or Cdc15p (left). Protein clusters are color coded for density. Graphs of RDD (right) and radii in nanometers (top right of each graph). (E) Overlaid CDF plots of mEos3.2-Imp2p and mEos3.2-Cdc15p (left) and Imp2p-mEos3.2 and Cdc15p-mEos3.2 (right) in protein clusters of the contractile ring. Dotted line, squared distance away from…

FIGURE 4:. Imp2p localization at the plasma membrane depends on actin. (A) Time-lapse montages of cells coexpressing Cdc15p-HALO*TMR and Imp2p-mEGFP after the addition of 5 µM LatA. Arrow, perinuclear cluster of Imp2p. Dotted circular outline, nucleus. (B) Time-lapse montages of strains expressing Imp2p-mEGFP after the addition of 10 µM LatA. Arrow, perinuclear cluster of Imp2p. Arrowheads, speckles of Imp2p. Dotted circular outline, nucleus.