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. 2020 Oct 7:8:575226.
doi: 10.3389/fcell.2020.575226. eCollection 2020.

Animal Cell Cytokinesis: The Rho-Dependent Actomyosin-Anilloseptin Contractile Ring as a Membrane Microdomain Gathering, Compressing, and Sorting Machine

Sabrya C Carim  1 , Amel Kechad  1 , Gilles R X Hickson  1   2
Affiliations

Animal Cell Cytokinesis: The Rho-Dependent Actomyosin-Anilloseptin Contractile Ring as a Membrane Microdomain Gathering, Compressing, and Sorting Machine

Sabrya C Carim et al. Front Cell Dev Biol. .

Abstract

Cytokinesis is the last step of cell division that partitions the cellular organelles and cytoplasm of one cell into two. In animal cells, cytokinesis requires Rho-GTPase-dependent assembly of F-actin and myosin II (actomyosin) to form an equatorial contractile ring (CR) that bisects the cell. Despite 50 years of research, the precise mechanisms of CR assembly, tension generation and closure remain elusive. This hypothesis article considers a holistic view of the CR that, in addition to actomyosin, includes another Rho-dependent cytoskeletal sub-network containing the scaffold protein, Anillin, and septin filaments (collectively termed anillo-septin). We synthesize evidence from our prior work in Drosophila S2 cells that actomyosin and anillo-septin form separable networks that are independently anchored to the plasma membrane. This latter realization leads to a simple conceptual model in which CR assembly and closure depend upon the micro-management of the membrane microdomains to which actomyosin and anillo-septin sub-networks are attached. During CR assembly, actomyosin contractility gathers and compresses its underlying membrane microdomain attachment sites. These microdomains resist this compression, which builds tension. During CR closure, membrane microdomains are transferred from the actomyosin sub-network to the anillo-septin sub-network, with which they flow out of the CR as it advances. This relative outflow of membrane microdomains regulates tension, reduces the circumference of the CR and promotes actomyosin disassembly all at the same time. According to this hypothesis, the metazoan CR can be viewed as a membrane microdomain gathering, compressing and sorting machine that intrinsically buffers its own tension through coordination of actomyosin contractility and anillo-septin-membrane relative outflow, all controlled by Rho. Central to this model is the abandonment of the dogmatic view that the plasma membrane is always readily deformable by the underlying cytoskeleton. Rather, the membrane resists compression to build tension. The notion that the CR might generate tension through resistance to compression of its own membrane microdomain attachment sites, can account for numerous otherwise puzzling observations and warrants further investigation using multiple systems and methods.

Keywords: anillin; contractile ring mechanism; contractile ring tension; cytokinesis; membrane cytoskeleton; membrane microdomains; rho (Rho GTPase); septin.

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Figures

FIGURE 1
FIGURE 1
The Rho-dependent network controlling the contractile ring. The Rho GTPase is the master activator of cytokinesis. Rho-GTP activates multiple essential effectors during cytokinesis: Rho kinase, Citron kinase, the formin Diaphanous and the multi-domain scaffold protein, Anillin. Anillin is the master organizer of cytokinesis that can bind many other components of the Rho network and the plasma membrane. Within the contractile ring, Anillin organizes two Rho-dependent cytoskeletal sub-networks: actomyosin and anillo-septin.
FIGURE 2
FIGURE 2
Actomyosin and anillo-septin are independently anchored to the plasma membrane and can laterally separate from one another. (A) Rho1 drives assembly of anillo-septin structures directly at the equatorial plasma membrane in the presence of the F-actin inhibitor, Latrunculin A. Maximum intensity projection of a fixed S2 cell expressing Anillin-GFP, stained for endogenous septin (Peanut) and DNA. Similar behavior of anillo-septin is observed upon additional inhibition of myosin activation (not shown). (B) Actin-independent assembly of anillo-septin sequesters the plasma membrane. Time-lapse frames of close-ups of the cell cortex of an S2 cell co-expressing Anillin-GFP (green) and mCherry-tubulin (red) treated with Latrunculin A and progressing through anaphase. The nascent anillo-septin structures reorient from parallel to perpendicular to the cell surface as they envelop themselves in plasma membrane. This envelopment and reorientation suggest that, as the anillo-septin complexes bind and sequester the membrane microdomains, the resulting tubular structures can deform the membrane to make it fit their shape. Time is shown as h:min:s from anaphase, scale bar, 2 μm. (C) Anillin-mCherry (mCh, green) shedding from the late contractile ring/nascent midbody ring (indicated by the white arrowhead) of a Drosophila S2 cell co-expressing the F-actin probe Lifeact-GFP (red), which does not shed). Split channels of the dashed boxed region are shown magnified below. (D) Shed material contains Anillin and Peanut. Fixed cells expressing Anillin-GFP (green), mCh-tubulin (red) and stained for endogenous Peanut (cyan) and DNA (Hoechst, blue). (E) The Anillin C-terminus (Anillin-ΔN) and the septin, Peanut, are co-recruited to the CR. Fixed Drosophila S2 cell expressing Anillin-ΔN-GFP, stained for endogenous Peanut and DNA (Hoechst, blue). (F) Drosophila S2 cell during early furrowing co-expressing myosin-GFP (green) and either Anillin-mCherry (top) or the Anillin C-terminus (Anillin-ΔN-mCherry, red, bottom). Intensity profiles along a line drawn between the asterisks are shown and white dashed boxed regions are shown magnified on the right. While Anillin-mCherry localization closely follows that of myosin-GFP, Anillin-ΔN-mCherry appears in puncta that extrude outward toward the cell exterior. (G) Frames from a time-lapse sequence of an S2 cell co-expressing myosin-GFP (green) and Anillin-ΔN-mCh (red) and depleted of endogenous Anillin. Anillin-ΔN (anillo-septin) forms punctate structures that herniate the membrane outward, whilst myosin (actomyosin) undergoes back-and-forth, lateral oscillations beneath these distinct structures. This clearly shows that actomyosin and anillo-septin are separate from one another (at least when the Anillin N-terminus is missing) and that they are independently anchored to the plasma membrane. The 00:06:08 and 00:22:00 time points are shown magnified on the right. Times are h:min:s from anaphase. (A) adapted and (B) reproduced from © 2008 Hickson and O’Farrell originally published in Journal of Cell Biology: https://doi.org/10.1083/jcb.200709005 which is available under a Creative Commons License (Attribution–Non-commercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/) and for which we retain copyright (Hickson and O’Farrell, 2008b). (C,D) adapted from© 2013 El-Amine et al., originally published in Journal of Cell Biology: https://doi.org/10.1083/jcb.201305053, which is available under a Creative Commons License (Attribution–Non-commercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/) and for which we retain copyright (El Amine et al., 2013). (E–G) are adapted from Kechad et al. (2012), originally published in Current Biology with permission from Elsevier (publisher) license number: 4844230307942 (obtained on June 08th, 2020) https://doi.org/10.1016/j.cub.2011.11.062.
FIGURE 3
FIGURE 3
Two-stage model for CR assembly and closure. (A) Cartoons of the conventional view of cytokinesis in the “perpendicular-to-the-ring” axis, showing how the architecture of the spindle establishes a gradient of centralspindlin/RhoGEF that peaks at the equator and at the cell center to promote cortical flow during CR assembly and closure. (B) 90° rotations of the dotted lines in panel (A) showing the “around-the-ring” axis. (C) Cartoons in the “around-the-ring” axis to illustrate the proposed concept of how membrane microdomain attachment sites of the CR oppose contractility to generate tension during CR assembly, and regulate tension during CR closure/disassembly. At a certain density of microdomain compaction, which represents a certain tension threshold, the CR becomes “mature” and shifts from the assembly stage to the disassembly stage, which involves relative outflow of microdomains in the “perpendicular-to-the-ring” axis. (D) More detailed views of the boxed regions in panel (C), showing the hypothetical arrangement of actomyosin, anillo-septin and their associated membrane microdomains. Note: The “actomyosin sub-network” comprises numerous components and membrane anchors (not least for F-actin nucleating formins and for myosin II motors), but it is purposefully depicted as one entity here for clarity. Similarly, the stoichiometry and scaling of depicted components are for illustrative purposes only.
FIGURE 4
FIGURE 4
Hypothetical steps in the contraction-coupled anillo-septin membrane sorting model for CR closure, viewed down the spindle/“perpendicular-to-the-ring” axis. (A) Actomyosin elements anchored to microdomains 1 and 5 contract in the circumferential, “around-the-ring” axis, compressing the intervening membrane microdomains. Anillin is connected to the membrane either indirectly via actomyosin (microdomains 1, 3, and 5), or directly as anillo-septin (microdomains 2 and 4), but not via both mechanisms at the same time. (B,C) Continued contraction advances microdomains 1 and 5 toward the viewer, while the intervening microdomains stay behind (relative outflow). Microdomains 2 and 4 are the latest additions to a growing anillo-septin filament that projects back away from the viewer. Continued contraction pulls microdomains 1 and 5 closer together squeezing them forward, past the intervening microdomains. (C,D) Contraction of actomyosin anchored to microdomains 1 and 5, also compresses the intervening actomyosin (reducing its tension) that is anchored to microdomain 3. This in turn allows Rho/Anillin to displace actomyosin (which depolymerizes) and assemble Anillo-septin on microdomain 3, thereby extending the growing anillo-septin filament. (E,F) Further contraction pulls microdomains 1 and 5 both closer together and closer to the viewer, past the end of the nascent anillo-septin filament, thereby equilibrating the tension and reducing the circumference of the CR. Thus, the CR closes by contraction-coupled membrane sorting, in which membrane enters and advances the ring with actomyosin but stays behind and exits with anillo-septin. Thus, it disassembles in a bifurcated fashion: actomyosin depolymerization and anillo-septin-membrane outflow.
FIGURE 5
FIGURE 5
Different viewing perspectives of the contraction-coupled membrane sorting model for CR closure. (A) Cartoon showing the proposed relative inflow/outflow of actomyosin/anillo-septin and the concept of closure by bifurcated disassembly, in which the CR sheds components both by depolymerization (actomyosin) and by membrane-anchored outflow (anillo-septin). (B) Flattened image viewed down the spindle axis showing both the “around-the-ring” and the “perpendicular-to-the-ring” axes. Arrows depict the relative flow of actomyosin-bound microdomains into the ring (blue) and anillo-septin-bound microdomains out of the ring (purple) as it advances in the “perpendicular-to-the-ring” axis and shrinks in the “around-the-ring” axis. (C) Close-up view of the magenta boxed region in panel (B), showing the proposed inflow of actomyosin-bound membrane microdomains, and switching events, as they transfer to nascent anillo-septin filaments that emanate back into the flanks of the furrow. The membrane microdomains are numbered to be able to follow their shifting positions in the time sequence.
FIGURE 6
FIGURE 6
How the proposed model can account for phenotypes observed upon loss of anillo-septin or its coupling to actomyosin. (A) Anillin-depleted CR. Actomyosin assembly and contractility persists and is able to gather and compress its own membrane microdomains. However, in the absence of anillo-septin to facilitate membrane microdomain outflow, CR closure stalls because the membrane microdomains impede it and actomyosin cannot disassemble. Tension mounts within the CR to above the usual threshold and this leads to furrow instability and oscillations in the “perpendicular-to-the-ring” axis. (B) Deletion of the Anillin N-terminus uncouples anillo-septin from actomyosin. Anillo-septin sequesters membrane, but in a manner that is no longer coupled to actomyosin contractility and at patches of cortex that already trail the CR. Disconnected from Anillin, actomyosin contracts without the means to regulate tension or its own disassembly because it cannot appropriately hand over membrane microdomains to anillo-septin for them to be removed, again leading to stalled CR closure, furrow instability and lateral oscillations.
FIGURE 7
FIGURE 7
The Rho-dependent network drives cytokinetic progression from CR assembly, through closure/disassembly and to midbody ring assembly.
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