Actomyosin Ring Formation and Tension Generation in Eukaryotic Cytokinesis  Thomas H. Cheffings, Nigel J. Burroughs, Mohan K. Balasubramanian  Current Biology  Volume 26, Issue 15, Pages R719-R737 (August 2016) DOI: 10.1016/j.cub.2016.06.071 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Order and dependencies of protein recruitment to cytokinesis nodes. After the onset of mitosis, six main proteins are recruited to Mid1p-containing interphase nodes. The order of this recruitment is depicted here, with the black arrows indicating the dependence of node localisation on specific proteins (e.g. Myo2p localisation depends on Rng2p, etc). After the arrival of the formin Cdc12p, actin begins to appear around the cell middle, and the nodes begin to condense on the membrane into a ring. Current Biology 2016 26, R719-R737DOI: (10.1016/j.cub.2016.06.071) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 The three proposed mechanisms for actomyosin ring formation in S. pombe. (A) The search, capture, pull and release (SCPR) mechanism, where cytokinesis nodes containing the formin Cdc12p nucleate actin filaments, which are captured by the myosins in other nodes. The myosins then pull these nodes together, to gradually condense the node network. Occasionally, some lagging nodes that don’t incorporate into the ring are seen. (B) The formin spot mechanism, where actin is nucleated from a single formin spot, which then gradually extends around the circumference of the cell to make a ring. This mechanism may be responsible for ring formation in cells that cannot form nodes, or where the SCPR pathway is not adequate to form a ring (e.g. in spherical mutants or in protoplasts). (C) Ring formation by the incorporation of nonmedially nucleated cables into the ring. This mechanism likely works alongside SCPR, as it is does not seem to be robust enough to work in the absence of SCPR. However, these cables may play a role in aiding compaction of the actin ring. Current Biology 2016 26, R719-R737DOI: (10.1016/j.cub.2016.06.071) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Actomyosin ring formation in budding yeast and metazoan cells. (A) In budding yeast, septins act as a scaffold for the initial recruitment of myosin to the bud site, which is determined by the division site of the previous cell cycle. As the bud begins to grow, the septin ring splits into an hourglass shape. Once mitosis has been completed, actin cables also accumulate at the division site to form the actomyosin ring. Finally, signalling from the mitotic exit network (MEN) triggers the septins to reorganise into a double-ring structure, and the process of ring constriction begins. (B) In metazoan cells, signalling from the central spindle and from astral microtubules leads to the formation of a band of active RhoA on the membrane at the cell mid-plane. A combination of cable transport and de novo nucleation leads to the accumulation of actin at the division site, along with myosin. This then condenses into a ring and initiates furrow ingression. If the central spindle is moved at this point, the ring will disassemble, and a new band of active RhoA will be reformed around the new location of the central spindle, starting the process again. Current Biology 2016 26, R719-R737DOI: (10.1016/j.cub.2016.06.071) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 In vitro contraction of reconstituted actomyosin bundles. In each panel, the upper actin filament is orientated with its barbed end to the left, and pointed end on the right. (A) By considering the simplest possible contractile actomyosin system, consisting of two actin filaments crosslinked by a single myosin cluster, it can be argued that actin–myosin interactions do not necessarily lead to a net contraction. Myosin crosslinking between parallel filaments generates no relative sliding, while antiparallel filaments are just as likely to undergo expansion or contraction, in the absence of other mechanisms. (B) If the myosins do not all have the same walking speed (possibly due to varying numbers of myosin motors per cluster), then it is possible that slower motors could act as ‘brakes’ between the faster motors, leading to regions of compression and extension along the lengths of the actin filaments between the motors. The actin filament will be more resistant to extensional forces than compressive forces, the latter of which can result in filament buckling, generating a net shortening of individual filaments. (C) If myosin heads remain attached to actin filaments when they reach the barbed end, a net contraction between parallel filaments can be generated if the myosin continues to walk along the second filament. However, this would also increase the propensity for expansion between antiparallel filaments, so this effect may cancel itself out. Current Biology 2016 26, R719-R737DOI: (10.1016/j.cub.2016.06.071) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Models of in vivo myosin-dependent ring constriction. (A) It has been proposed that actin treadmilling and crosslinking are sufficient to facilitate contraction in random actomyosin arrays. Treadmilling tends to counteract the processive effect of myosin, meaning the relative motion of the two filaments shown is reduced compared with the system without treadmilling. However, passive crosslinking of these filaments to the surrounding actin network generates contraction in the local environment. Red actin monomers are included as reference points to better depict the actin dynamics. The upper actin filament at each timepoint has its barbed end on the left. (B) Another model of ring constriction proposes that the turnover of ring components, combined with anchoring of actin filaments to the membrane, are suitable mechanisms for generating contractility. Actin filaments are anchored at their barbed ends by formins, meaning that only their pointed ends are available for actomyosin interaction. Turnover of formin and actin filaments, via unbinding from the ring and severing by cofilin, helps to bias the system towards contractile arrangements of actin and myosin (see Figure 3A). Actin crosslinking can also occur, but simulations suggest it is not important for this mechanism to generate contraction. The brown background in (B) represents the plasma membrane beneath the ring, where the formins are anchored. Current Biology 2016 26, R719-R737DOI: (10.1016/j.cub.2016.06.071) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Constriction dependent on actin depolymerisation or end-tracking crosslinkers. (A) For two antiparallel actin filaments, if actin depolymerisation causes a crosslinking protein bound near the pointed end to lose its attachment, then it is possible that it will be able to reattach to the actin filament and exert a force to contract the two filaments. While in the case of antiparallel filaments there is still a net expansion, this is reduced by the presence of the end-tracking crosslinker, i.e. in the second timepoint the filament overlap has decreased from four monomers to two, but the end-tracking crosslinker increases this to three monomers in the fourth timepoint. (B) Between parallel filaments, the action of end-tracking crosslinkers can generate a net contraction, as illustrated. In (B), each red arrow denotes an entire cycle of depolymerisation and reattachment, as depicted in (A) for antiparallel filaments. (C) In S. cerevisiae, actin filaments are thought to undergo minimal turnover, so the mechanisms depicted in (A) and (B) will not contribute to constriction. Instead, it is thought that actin-severing proteins such as cofilin facilitate this method of constriction. If a cofilin severs a filament near a crosslinker at the pointed end, then fluctuations in the crosslinker and the severed filament could bring them back into contact, leading to a net contraction. In (A–C) red actin monomers are included as reference points to better depict the actin dynamics. Current Biology 2016 26, R719-R737DOI: (10.1016/j.cub.2016.06.071) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 Entropic force mechanism for contracting overlapping microtubules. (A) For a system of two overlapping microtubules, two diffusible crosslinkers bound within the overlap region can adopt one, three or six possible configurations as the overlap increases from two to four dimers. (B) Since all these arrangements have the same enthalpy, the probability of the system having a given overlap length is proportional to the number of available configurations with that overlap. For a system with 10 crosslinkers between two microtubules, each of which is 40 tubulin dimers in length, there is a high probability that the system will occupy a configuration of near maximal overlap. If the system is perturbed to a state with a reduced overlap length, random one-dimensional diffusion of one microtubule along the other will return the system to a configuration with a maximal overlap. Current Biology 2016 26, R719-R737DOI: (10.1016/j.cub.2016.06.071) Copyright © 2016 Elsevier Ltd Terms and Conditions