The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis

Abstract

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51's localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8(+) cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.

Figure 1.

The novel protein Rng8 is involved in cytokinesis. (A) Rng8 localizes to the contractile ring (arrowheads), cables (arrows), and cytoplasmic puncta (asterisks). (B) Rng8 depends on actin filaments to localize. Cells were treated with DMSO or 100 µM Lat-A for 10 min at 25°C. (C) Time course (in minutes) of contractile ring formation in cells expressing Rng8-mEGFP Sad1-mCherry (Video 1). Time 0 marks SPB separation. The arrows and arrowhead indicate the Rng8 meshwork and the compact ring, respectively. In this and other figures, the cell boundary of some cells is marked with broken lines. (D) Time of appearance of Rng8 meshwork and the compact ring after SPB separation. Error bars indicate 1 SD. (E) Cytokinesis defects in rng8Δ cells. DIC images are shown. Arrows indicate examples of the aberrant septa. (F) Quantification of abnormal septating cells (see Materials and methods) as shown in E. n > 400 cells for each of three independent experiments. (G) Condensation of Rlc1 nodes into a compact ring (arrowheads) is delayed in rng8Δ at 23°C. Time 0 is the appearance of Rlc1 nodes. (H) Times of contractile ring assembly, maturation, and constriction in wt and rng8Δ cells (n > 25 cells for each) at 23°C. *, P < 0.001 compared with wt from two-tailed t test in this and other graphs. (I and J) Synthetic genetic interaction between rng8Δ and myo2-E1 and their septation indexes at 25°C. Bars, 5 µm.

Figure 2.

Rng9 is a binding partner of Rng8. (A) Rng8 and Rng9 coIP with each other (see Materials and methods). (B) Rng8 and Rng9 are interdependent for localization. Bar, 5 µm. (C) FRAP analysis of Rng8 (left) and Rng9 (right) in the contractile ring. Curve fit is shown in red, SD in gray. (D and E) Numbers of Rng8 and Rng9 molecules globally in the whole cell (D) and locally in the contractile ring (E). Error bars indicate 1 SD.

Figure 3.

Rng8 and Rng9 are critical for Myo51 localization. (A) Colocalization of Myo51 and Rng8 in the contractile ring (arrow), cables (arrowheads), and puncta (asterisks). The imperfect colocalization on the cables is likely caused by the highly dynamic nature and movement of the structure. (B) Myo51 localization is essentially abolished without either Rng8 or Rng9. Arrows indicate weak signals left at the contractile ring in some cells. (C) Localization of Rng8 and Rng9 in myo51Δ. Bars, 5 µm. (D) Rng8 and Rng9 coIP with Myo51. Broken lines indicate that intervening lanes have been spliced out. (E) FRAP analysis of Myo51 in the contractile ring. Curve fit is shown in red, SD in gray. (F) Numbers of Myo51 molecules in the whole cells and in the contractile ring. The fluorescence intensity of Myo51 was compared with that of Rng8. Error bars indicate 1 SD.

Figure 4.

The rod region in Myo51 tail determines Myo51 localization. (A) Schematics of Myo51 domains and truncations constructed. (B) Localizations of FL and truncated Myo51. (C and D) Micrographs and quantification of localization of headless Myo51(753–1,471) to the contractile ring in rng8Δ. (E) CoIP assays between Myo51 FL or truncations and Rng9. Arrowheads mark the Myo51 bands with the expected sizes. (F) The Myo51-Myo52 chimera M2IQ2-CC1-GTD2 localizes similarly to FL Myo51 in wt and myo51Δ cells. Cell boundaries are marked with broken lines. Bars, 5 µm.

Figure 5.

Myo51 is involved in both early and late stages of cytokinesis. (A and B) Time courses (A) and quantifications (B) of contractile ring assembly in myo51Δ (Video 3) and two headless myo51 mutants using Rlc1 as the node and ring marker at 23°C. Arrows indicate the compact rings. Time 0 marks node appearance. Cell boundaries are indicated by broken lines. *, P < 0.001 compared with wt from a two-tailed t test. (C–F) Movement of Rlc1 nodes during contractile ring assembly in wt and myo51Δ at 23°C. (C) Time courses of node condensation from the boxed regions on the left. Red arrows mark nodes moving toward the center of the broad bands of nodes whereas green arrows indicate nodes moving toward cell tips for a certain time. (D–F) n = ∼30 nodes for each strain. (D) Histograms of angles of node displacements. Angles between directions of node displacements during ∼3 min and the long cell axis. (E) Node displacements during ∼3 min. (F) Distribution of instantaneous speed of node movements during ∼3 min (n > 2,000 speeds). (G) Synthetic interactions between rng8Δ, rng9Δ, or myo51 mutations and myp2Δ. Cells were grown at 36°C for 8 h before imaging. (H) myo51Δ myp2Δ cells are defective in ring stability/anchoring and disassembly during ring constriction. Arrows indicate distorted contractile ring, and the arrowhead indicates a contractile ring uncoupled from the invaginated membrane. See Video 4. Bars, 5 µm.

Figure 6.

Myo51 regulates actin structure and dynamics during cytokinesis. Actin was labeled with Lifeact-mGFP. (A) Time courses showing actin meshwork during contractile ring assembly (Video 5). Cell boundaries are indicated by broken lines. (B and C) Actin cable morphology (B) and orientation (C) during cytokinesis and interphase. Cells were treated with 100 µM Arp2/3 inhibitor CK666 for 5 min before imaging. Red and yellow arrows indicate misoriented and curved cables during cytokinesis and interphase, respectively. (C) Quantification of misoriented actin cables (see Materials and methods). Error bars indicate 1 SD. *, P < 0.001 compared with wt from a two-tailed t test. (D and E) Actin in the contractile ring is more dynamic in myo51Δ. Cells were preincubated with 100 µM CK666 for 5 min and imaged immediately after adding 4 µM Lat-A at time 0. (D and E) Micrographs (D) and fluorescence curves (E; mean ± SD) showing the fluorescence decay in the contractile ring before ring constriction. Bars, 5 µm.

Figure 7.

The Rng8–Rng9 complex promotes Myo51 clustering. (A) Myo51 self-interaction does not depend on Rng8 or Rng9 in coIP assays of cell extracts from diploid cells at different salt concentrations. Both copies of Myo51 are the truncation without the GTD domain. The broken lines indicate that intervening lanes have been spliced out. (B) Single focal plan showing Cdc12 speckles, Myo51 speckles, and Myo51 puncta imaged at the same settings. Examples of brighter puncta and dimmer speckles are marked by an arrowhead and arrows, respectively. Cell boundaries are indicated by broken lines. (C) Histograms showing the molecule numbers in Myo51 puncta (rng8⁺) and speckles (rng8Δ), and in Rng8 and Rng9 puncta with mean and SD indicated. The intensities were compared with those of Cdc12 speckles (see Materials and methods). (D) Myo51 punctum formation depends on Rng8. The arrowheads, arrows, and asterisks indicate the puncta, speckles, and the contractile ring, respectively. (E) Punctum formation of the Myo51-Myo52 chimera M2IQ2-CC1-GTD2 depends on Rng8. The arrowheads and arrow indicate the puncta and a cable, respectively. Bars, 5 µm.

Figure 8.

Rng8 is required for continuous movement of Myo51 puncta on actin tracks. (A–D) Movements of Myo51 puncta on actin tracks in wt cells. (A) Time course of the continuous movement of a punctum (arrowheads) in a cell expressing both Lifeact-mCherry (left) and Myo51-3GFP (right). See Video 6. Cell boundaries are indicated by broken lines. (B) Kymograph showing the movement of a punctum over 3.6 s. Images were collected with 0.1-s intervals. (C) Dot plot of travel distances of Myo51 puncta. This experiment was completed once (n = 16). (D) Distribution of Myo51 instantaneous speed (µm/s). n = 28 from five puncta. (E) Myo51 punctum movement depends on actin cables. Red arrows mark a punctum moving continuously and directionally in a for3⁺ cell, and the green arrows mark two puncta staying still in for3Δ. Images were collected with 0.2-s intervals. (F–H) Continuous movement of Myo51 puncta depends on Rng8. (F) Time course showing that Myo51-3GFP speckles (top) in a rng8Δ cell do not move continuously on actin tracks (bottom). Arrows mark speckles that are on actin tracks at one point and disappear at the next time point. (G) Time course showing the movement of Myo51-3YFP puncta (in rng8⁺; green arrowheads) or Myo51 speckles (in rng8Δ). Speckles in rng8Δ either diffuse out of plane (lost track, red arrows) or diffuse around but do not move continuously (yellow arrows). See Video 7. (H) Percentage of Myo51 puncta or speckles with continuous movement, which is defined as movements that are traceable and showing unidirectional movement for ≥0.3 s in an 8-s movie with 0.1-s intervals. (I) Time courses of the nuclear displacement toward the cell tip in two cells expressing Myo51N-GFP-Nup146 after MBC treatment (Video 8). The left cell shows the best focal plane and the right shows one maximum projection. Cells were grown in EMM5S for ∼24 h and then treated with MBC for 15 min. The broken lines in E, G, and I are reference lines to aid comparisons of movements. (J) Nuclear displacement by Myo51N-GFP-Nup146 in rng8⁺ or rng8Δ cells 1.5 h after MBC treatment. (K) Quantification of cells with tip-localized nuclei in cells expressing Myo51N-GFP-Nup146 or Myo52N-GFP-Nup146. Definition of tip localization is shown on the top. More than 150 cells in each experiment (n ≥ 3) for each strain. Error bars indicate 1 SD. Bars, 5 µm.

Figure 9.

Rng8 and Rng9 form higher-order complexes. (A) SDS-PAGE of purified 3FLAG-Rng8 and Rng9-13Myc from a myo51Δ strain. (B) Purified Rng8 and Rng9 form a large protein complex revealed by gel filtration. Elution of the purified Rng8–Rng9 complex mixed with standard proteins from a calibrated gel filtration column is shown. The peaks of two standard proteins are indicated. (C) Western blotting using anti-FLAG or anti-Myc antibodies to detect Rng8 or Rng9 from the elution fractions 1–20 (1 ml/fraction) and the sample before gel filtration (arrowhead). Broken lines indicate that intervening lanes have been spliced out. (D) Distribution of sedimentation coefficients of the Rng8–Rng9 complex at two protein concentrations, assuming a continuous c(S) distribution. Molecular weight for individual peaks are indicated. (E) Possible mechanism for the regulation of Myo51 by the Rng8–Rng9 complex in vivo. (top) Myo51 forms dimers without Rng8 and/or Rng9, and it easily detaches from actin filaments/bundles due to its low duty ratio (Clayton et al., 2010). (bottom) The Rng8–Rng9 complexes cluster multiple Myo51 dimers, and the motor heads cooperate to maintain the attachment and move continuously on actin filaments/bundles. This motor–track interaction may help to stabilize/organize actin filaments/bundles.