Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure 1 - GFP-nonmuscle myosin IIA heavy chain and GFP-actin as functional probes in interphase NRK cells. Expressed proteins become incorporated into stress fibers and focal adhesions (A, arrows), as imaged by TIRF-M. Images of GFP-actin and Alexa-546-phalloidin staining show extensive colocalization, suggesting GFP-actin is incorporated into most if not all actin filament structures (B, orange arrows indicate equator). In addition, transfected cells grow and divide normally.
• Supplemental Figure 2 - TIRF-M time-lapse sequence of GFP-myosin in NRK cell injected with C3, a Rho inhibitor. The cell shows strong inhibition of myosin localization along the equator (arrows indicate the equator).
• Supplemental Figure 3 - Recruitment of myosin to the equator in cells treated with latrunculin B. TIRF-M of GFP-myosin shows the recruitment to the equatorial cortex despite the inhibition of cytokinesis and disassembly of actin filaments (A; asterisk and boxed region indicate the equator in a tri-polar cell). However, the myosin band begins to widen at about 10 min after anaphase onset, and eventually disperses at about 25 min after anaphase onset, indicating that F-actin may be involved maintaining myosin organization in the furrow. Time is indicated as minutes:seconds from anaphase onset. Kymograph analysis shows that myosin dots persist for minutes (B, arrows indicate equator, white rectangular box indicates the region analyzed). Supplemental Video 6 shows the corresponding movie.
• Supplemental Video 1 - TIRF-M of an NRK cell expressing GFP-nonmuscle myosin IIA heavy chain during the assembly of equatorial cortex. No directed flow of cortical myosin is detectable. Enlarged view of the equatorial region is shown to the right.
• Supplemental Video 2 - TIRF-M and the corresponding TDM of GFP-myosin in an NRK cell during the assembly of the equatorial cortex. Dynamic domains of myosin assembly/association appear throughout the cortex. Some assembly domains appear to travel across the cell cortex. The total duration of recording is 87 seconds.
• Supplemental Video 3 - Recruitment of GFP-myosin to the equator in cell treated with Y-27632. TIRF-M shows that myosin in the equatorial region is very stably associated with cortex, in contrast to dots outside the equator. The total duration of recording is 66 seconds.
• Supplemental Video 4 - Equatorial recruitment of GFP-myosin in a cell treated with ML-7. Cytokinesis is delayed. The total duration of recording is 3.75 minutes.
• Supplemental Video 5 - Equatorial recruitment of GFP-myosin in a cell treated with (-)-blebbistatin. Cortical myosin intensity shows steady increase both in and outside the equatorial region, while cytokinesis is inhibited. The total duration of recording is 34 seconds.
• Supplemental Video 6 - Equatorial recruitment of GFP-myosin in a cell treated with latrunculin B, despite the inhibition of furrow ingression. Equatorial myosin dots appear to scatter over a wide region than in control cells. The total duration of recording is 4.5 minutes.
• Supplemental Video 7 - TIRF-M of an NRK cell expressing GFP-actin during the assembly of equatorial cortex, showing dramatic fluxes of actin filaments toward the equator.
• Supplemental Video 8 - TIRF-M of an NRK cell expressing mCherry-actin during the assembly of equatorial cortex, showing dramatic fluxes of actin filaments toward the equator, as well as active ruffles of a neighboring cell. The total duration of recording is 70 seconds.
• Supplemental Video 9 - TIRF-M of NRK cells expressing mCherry-actin treated with (+)-blebbistatin (negative control, left) or (-)-blebbistatin (myosin ATPase inhibitor, right). (+)-blebbistatin has no effect on actin flux, while (-)-blebbistatin abolishes actin flux. Despite the inhibition of flux, equatorial actin concentration remains unaffected.
• Supplemental Video 10 - TIRF-M and the corresponding TDM of mCherry-actin in an NRK cell treated with (-)-blebbistatin. Equatorial band appears despite the inhibition of actin flux. Bands of de novo assembly activities are visible in the TDM image along the equator during the first half of the recording. The total duration of recording is 75 seconds.