Myosin Assembly Research Articles

Myosin supramolecular self-assembly: The crucial precursor that manipulates the covalent aggregation, emulsification and rheological properties of myosin

The transformation of molecular conformation and self-assembly properties of myosin during the heating process at different ionic strengths (0.2 M, 0.4 M and 0.6 M NaCl) and its effect on rheological behavior and emulsification properties were investigated. Under incubation temperatures between 40 °C and 50 °C, myosin underwent a supramolecular self-assembly stage dominated by noncovalent forces (hydrogen bonding, ionic bonding and hydrophobic interactions). Higher ionic strength facilitated molecular rearrangement through enhanced swelling of myosin heads and head-to-head assemblies, which contributed to enhanced ordering and homogeneity of myosin covalent aggregates (above 60 °C) and manifested itself macroscopically as enhanced gel viscoelasticity and emulsion stability. In contrast, at lower ionic strength, the tail-to-tail assemblies of myosin led to the preferential formation of covalent cross-links in the tails, which resulted in the inability of molecular rearrangement and the formation of disordered aggregates and finally led to the deterioration of the gel and the destabilization of the emulsion. In conclusion, the supramolecular self-assembly behavior of myosin, as an intermediate process in myosin’s sol–gel transition, is crucial for the orderliness of myosin assemblies, gel network strengthening, and emulsion stability. The obtained insight provides a reference for the precise implementation of quality improvement strategies for meat products.

Abstract Mo122: Investigating the Role of the Lysine Methyltransferase SMYD1 in Striated Muscle Motor Domain Folding

Properly coordinated folding and assembly of myosin into the sarcomere thick filament is critical for cardiac and skeletal muscle development, function, growth, and maintenance. The motor domain of myosin heavy chain (MHC) requires muscle-specific chaperones for proper folding, making it currently impossible to fold in non-muscle cells. Previous studies found that HSP90 and UNC45B are necessary, but not sufficient, for folding the motor domain, indicating other factors are involved. Defining the minimal set of factors for motor domain folding would allow for development of an improved expression system and accelerate the study of myosin mutations and myosin-targeted drug development. We hypothesize that the muscle-specific lysine methyltransferase SMYD1 contributes to MHC folding and stability. We found SMYD1 associates with UNC45B, HSP90, and MHC in the adult human heart. To study motor domain folding we generated a human MYH7 motor domain fused to GFP (MD::GFP). Transfection into COS-7 cells resulted in unfolded aggregates while adenoviral infection of C 2 C 12 myotubes leads to folded and functional MD::GFP. Interestingly, the folded MD::GFP contains lysine trimethylation, which may arise from the methyltransferase activity of SMYD1. When COS-7 cells were co-transfected with UNC45B and SMYD1, we observed increased total protein levels of non-functional MD::GFP without affecting RNA levels as assessed by RT-qPCR. This effect was not observed when MD::GFP was co-transfected with UNC45B or SMYD1 alone and is dependent on HSP90. We found that UNC45B and SMYD1 stabilized MD::GFP as determined by cycloheximide chase assays. We also found SMYD1 associates with MD::GFP, suggesting this stabilizing effect is due to a direct interaction. Interestingly, a catalytically inactive SMYD1 also increased MD::GFP protein levels in COS-7 cells, consistent with our observation that no significant changes in global protein methylation were observed in the presence of WT SMYD1. Our data suggests that SMYD1 stabilizes the motor domain of striated muscle myosin in conjunction with UNC45B and HSP90, in a manner that is independent of its methyltransferase activity. However, UNC45B and SMYD1 are insufficient for motor domain folding in a non-muscle cell line, suggesting that additional folding factors or proper stoichiometric ratios of chaperones/co-chaperones may be required.

Structured RhoGEF recruitment drives myosin II organization on large exocytic vesicles.

The Rho family of GTPases plays a crucial role in cellular mechanics by regulating actomyosin contractility through the parallel induction of actin and myosin assembly and function. Using exocytosis of large vesicles in the Drosophila larval salivary gland as a model, we followed the spatiotemporal regulation of Rho1, which in turn creates distinct organization patterns of actin and myosin. After vesicle fusion, low levels of activated Rho1 reach the vesicle membrane and drive actin nucleation in an uneven, spread-out pattern. Subsequently, the Rho1 activator RhoGEF2 distributes as an irregular meshwork on the vesicle membrane, activating Rho1 in a corresponding punctate pattern and driving local myosin II recruitment, resulting in vesicle constriction. Vesicle membrane buckling and subsequent crumpling occur at local sites of high myosin II concentrations. These findings indicate that distinct thresholds for activated Rho1 create a biphasic mode of actomyosin assembly, inducing anisotropic membrane crumpling during exocrine secretion.

LUZP1 regulates the maturation of contractile actomyosin bundles

Contractile actomyosin bundles play crucial roles in various physiological processes, including cell migration, morphogenesis, and muscle contraction. The intricate assembly of actomyosin bundles involves the precise alignment and fusion of myosin II filaments, yet the underlying mechanisms and factors involved in these processes remain elusive. Our study reveals that LUZP1 plays a central role in orchestrating the maturation of thick actomyosin bundles. Loss of LUZP1 caused abnormal cell morphogenesis, migration, and the ability to exert forces on the environment. Importantly, knockout of LUZP1 results in significant defects in the concatenation and persistent association of myosin II filaments, severely impairing the assembly of myosin II stacks. The disruption of these processes in LUZP1 knockout cells provides mechanistic insights into the defective assembly of thick ventral stress fibers and the associated cellular contractility abnormalities. Overall, these results significantly contribute to our understanding of the molecular mechanism involved in actomyosin bundle formation and highlight the essential role of LUZP1 in this process.

Moonwalking molecular machines: Unraveling the choreography of myosin filament assembly.

We have made tremendous progress in identifying the machines that shape the architecture of actin filaments. However, we know less about the mechanisms mediating myosin assembly at the supramolecular level. In this issue, Quintanilla et al. (https://doi.org/10.1083/jcb.202305023) provide important new insights into this process.

Local monomer levels and established filaments potentiate non-muscle myosin 2 assembly.

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet direct biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here, we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the static actin architecture plays a less clear role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin-driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes filament stacks prior to partitioning into clusters that feed higher-order networks. Together, these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology.

UNC-82/NUAK kinase is required by myosin A, but not myosin B, to assemble and function in the thick filament arms of C. elegans striated muscle.

The mechanisms that ensure proper assembly, activity, and turnover of myosin II filaments are fundamental to a diverse range of cellular processes. In Caenorhabditis elegans striated muscle, thick filaments contain two myosins that are functionally distinct and spatially segregated. Using transgenic double mutants, we demonstrate that the ability of increased myosin A expression to restore muscle structure and movement in myosin B mutants requires UNC-82/NUAK kinase activity. Myosin B function appears unaffected in the kinase-impaired unc-82(e1220) mutant: the recessive antimorphic effects on early assembly of paramyosin and myosin A in this mutant are counteracted by increased myosin B expression and exacerbated by loss of myosin B. Using chimeric myosins and motility assays, we mapped the region of myosin A that requires UNC-82 activity to a 531-amino-acid region of the coiled-coil rod. This region includes the 264-amino-acid Region 1, which is sufficient in chimeric myosins to rescue the essential filament-initiation function of myosin A, as well as two sites that interact with myosin head domains in the Interacting Heads Motif. A specific physical interaction between myosin A and UNC-82::GFP is supported by GFP labeling of ectopic myosin A filaments but not thin filaments. We hypothesize that UNC-82 regulates assembly competence of myosin A during parallel assembly in the filament arms.

Solubilization strategy of myofibrillar proteins in low-ionic media (prototype soup): The effect of high-intensity ultrasound combined with non-covalent or covalent modification of polyphenols on myosin molecular assembly

This study investigated the effect of (−)-Epigallocatechin-3-gallate (EGCG) non-covalent/covalent grafting onto myofibrillar protein (MP) by high-intensity ultrasound (HIU) on its water-solubility and filament forming behavior. The results showed that the introduction of EGCG, especially in the case of covalent grafting, could inhibit the molecular assembly of myosin and improve the MP water solubility from 2.7% to 53.1% (P < 0.05). The HIU pretreatment provided more opportunities for EGCG grafting onto the ultrasound-treated protein (UMP) by disrupting the filamentous polymerization of myosin and thus further facilitated MP dissolution. Additionally, compared with the UMP-EGCG non-covalent complexes, the covalent complexes with a yellow appearance exhibited a higher absolute zeta potential (35.9 mV) and a lower particle size (53.7 μm) (P < 0.05). Overall, the combination of HIU pretreatment and EGCG covalent modification may provide a promising method for improving the solubility and processing properties of MP in low ionic media (prototype soup).

3D cell segregation geometry and dynamics are governed by tissue surface tension regulation

Tissue morphogenesis and patterning during development involve the segregation of cell types. Segregation is driven by differential tissue surface tensions generated by cell types through controlling cell-cell contact formation by regulating adhesion and actomyosin contractility-based cellular cortical tensions. We use vertebrate tissue cell types and zebrafish germ layer progenitors as in vitro models of 3-dimensional heterotypic segregation and developed a quantitative analysis of their dynamics based on 3D time-lapse microscopy. We show that general inhibition of actomyosin contractility by the Rho kinase inhibitor Y27632 delays segregation. Cell type-specific inhibition of non-muscle myosin2 activity by overexpression of myosin assembly inhibitor S100A4 reduces tissue surface tension, manifested in decreased compaction during aggregation and inverted geometry observed during segregation. The same is observed when we express a constitutively active Rho kinase isoform to ubiquitously keep actomyosin contractility high at cell-cell and cell-medium interfaces and thus overriding the interface-specific regulation of cortical tensions. Tissue surface tension regulation can become an effective tool in tissue engineering.

Assembly and disassembly dynamics of nonmuscle myosin II control endosomal fission.

Endocytic vesicular trafficking requires merging of two lipid bilayers, but how the two lipid bilayers can come close together during fusion and fission in endocytic trafficking is not well explored. Here, we establish that knocking down nonmuscle myosin IIs (NM IIs) by small interfering RNA (siRNA) or inhibition of their activities by (-) blebbistatin causes the formation of a ring-like assembly of early endosomes (raEE). Inhibition of NM II assembly by an inhibitor of regulatory light-chain (RLC) kinase results in the formation of raEE, whereas inhibition of NM II disassembly by inhibitors of heavy chain kinases, protein kinase C (PKC) and casein kinase 2 (CK2), causes the dispersion of early endosomes. The raEEs retain EEA1, Rab7, and LAMP2 markers. Overexpression of an assembly incompetent form, RLC-AA, and disassembly incompetent form, NMHCIIB-S6A or NMHCIIA-1916A, induces such defects, respectively. Altogether, these data support that NM II assembly and disassembly dynamics participate in endocytic trafficking by regulating fission to maintain the size of early endosomes.

The effect of high‐pressure homogenization on rabbit myofibrillar proteins in dilute sodium chloride solutions: weak ionic shielding promotes the depolymerisation

SummaryThe study attempts to determine the effect of ionic strength on the structure and solubility of rabbit myofibrillar proteins (MPs) and improves MPs dissolution in low salt medium. There was a significant effect of ionic strength on the structure and dissolution characteristics of MPs in low salt medium range (0–200 mM NaCl), increased ionic strength led to MPs aggregation and reduced solubility (P &lt; 0.05), while decreased ionic strength weakened charge shielding effect, resulted in stronger electrostatic repulsion and weaker hydrophobic interaction between molecules, thus promoting MPs structural swell and separation between filaments. The modification effect of HPH on the MPs also depended on the ionic strength. Upon HPH, due to the difference in charge shielding effect and hydrophobic interactions, there was a progressive improvement in MPs solubility (from 23.51% to 85.25%) and dispersion stability at 0 mM salt medium (I = 0.0217) while limited increase in solubility at medium higher than 13 mM (from 4.39% to 7.20% at 20 mM). Decreasing ionic strength promoted the depolymerisation of HPH on MPs, inhibited the myosin assembly into filaments after HPH. The modification of ionic strength on the surface charge and hydrophobicity of MPs were further amplified, also contributed to the improved solubility and dispersion stability.

Aurora A and cortical flows promote polarization and cytokinesis by inducing asymmetric ECT-2 accumulation.

In the early Caenorhabditis elegans embryo, cell polarization and cytokinesis are interrelated yet distinct processes. Here, we sought to understand a poorly understood aspect of cleavage furrow positioning. Early C. elegans embryos deficient in the cytokinetic regulator centralspindlin form furrows, due to an inhibitory activity that depends on aster positioning relative to the polar cortices. Here, we show polar relaxation is associated with depletion of cortical ECT-2, a RhoGEF, specifically at the posterior cortex. Asymmetric ECT-2 accumulation requires intact centrosomes, Aurora A (AIR-1), and myosin-dependent cortical flows. Within a localization competent ECT-2 fragment, we identified three putative phospho-acceptor sites in the PH domain of ECT-2 that render ECT-2 responsive to inhibition by AIR-1. During both polarization and cytokinesis, our results suggest that centrosomal AIR-1 breaks symmetry via ECT-2 phosphorylation; this local inhibition of ECT-2 is amplified by myosin-driven flows that generate regional ECT-2 asymmetry. Together, these mechanisms cooperate to induce polarized assembly of cortical myosin, contributing to both embryo polarization and cytokinesis.

Beyond Chaperoning: UCS Proteins Emerge as Regulators of Myosin-Mediated Cellular Processes.

The UCS (UNC-45/CRO1/She4p) family of proteins has emerged as chaperones specific for the folding, assembly, and function of myosin. UCS proteins participate in various myosin-dependent cellular processes including myofibril organization and muscle functions, cell differentiation, striated muscle development, cytokinesis, and endocytosis. Mutations in the genes that code for UCS proteins cause serious defects in myosin-dependent cellular processes. UCS proteins that contain an N-terminal tetratricopeptide repeat (TPR) domain are called UNC-45. Vertebrates usually possess two variants of UNC-45, the ubiquitous general-cell UNC-45 (UNC-45A) and the striated muscle UNC-45 (UNC-45B), which is exclusively expressed in skeletal and cardiac muscles. Except for the TPR domain in UNC-45, UCS proteins comprise of several irregular armadillo (ARM) repeats that are organized into a central domain, a neck region, and the canonical C-terminal UCS domain that functions as the chaperoning module. With or without TPR, UCS proteins form linear oligomers that serve as scaffolds that mediate myosin folding, organization into myofibrils, repair, and motility. This chapter reviews emerging functions of these proteins with a focus on UNC-45 as a dedicated chaperone for folding, assembly, and function of myosin at protein and potentially gene levels. Recent experimental evidences strongly support UNC-45 as an absolute regulator of myosin, with each domain of the chaperone playing different but complementary roles during the folding, assembly, and function of myosin, as well as recruiting Hsp90 as a co-chaperone to optimize key steps. It is becoming increasingly clear that UNC-45 also regulates the transcription of several genes involved in myosin-dependent cellular processes.

The structural rearrangement and depolymerisation induced by high‐pressure homogenisation inhibit the thermal aggregation of myofibrillar protein

SummaryThe effects of heating on the dissolution and aggregation of high‐pressure homogenisation (HPH)‐treated myofibrillar protein (MP) in low ionic strength medium were investigated in this study. Upon heating, HPH‐treated MP (HTMP) had a greater solubility and lower turbidity than control MP (P &lt; 0.05). The rheological measurement exhibited that the thermal aggregation ability of HTMP was hampered due to lower storage modulus (G′) and loss modulus (G″), except near 50 °C. The increased G′ and G″ of HTMP at near 50 °C could be attributed to structural rearrangement induced by HPH. The analysis of proteomics indicated that HPH destroyed the cytoskeletal proteins in myofibrillar structure, made MP structure more swollen and flexible, promoted MP rearrangement and formation of oligomers with higher negative charge, which produced stronger steric hindrance and electrostatic repulsion between molecules, thus inhibiting thermally induced MP aggregation. The depolymerisation effect induced by HPH destroyed myosin head and tail portion, hampered the assembly of myosin into filaments and therefore suppressed the formation of strand‐type aggregates under heating. The in vitro digestion suggested that the depolymerisation and rearrangement effect significantly improved MP digestibility (P &lt; 0.05) regardless of whether the protein was heat treated or not.

Improved water solubility of myofibrillar proteins by ultrasound combined with glycation: A study of myosin molecular behavior

The poor water solubility of myofibrillar proteins (MPs) limits their application in food industry, and is directly related to the molecular behavior associated with myosin assembly into filaments. This study aims to explore the effect of high-intensity ultrasound (HIU) combined with nonenzymatic glycation on the solubility, structural characteristics, and filament-forming behavior of MPs in low ionic strength media. The results showed that the HIU (200–400 W) application could promote the subsequent glycation reaction between MPs and dextran (DX) and interfere with the electrostatic balance between myosin rods, suppressing the formation of filamentous myosin polymers. Glycated MPs pretreated by 400 W HIU had the highest solubility, which corresponded to the smallest particle size, highest zeta potential, and optimum storage stability (P < 0.05). Structure analysis and microscopic morphology observations suggested that the loss of the MP superhelix and the depolymerization of filamentous polymers were the main mechanisms for MP solubilization. In conclusion, HIU combined with glycation can effectively improve the water solubility of MPs by destroying or suppressing the assembly of myosin molecules.

Myosin assembly of smooth muscle: from ribbons and side polarity to a row polar helical model

After decades of debate over the structure of smooth muscle myosin filaments, it is still unclear whether they are helical, as in all other muscle types, or square in shape. In both cases bipolar building units are proposed, but the deduced cross-bridge arrangements are fundamentally different. The opposite polarity of the adjusting longitudinal rows is proposed for the helical structure, while in the case of square filaments, or myosin ribbons, only their two faces are appositively polarized. Analysis of our unpublished archival data on light meromyosin (LMM) paracrystals and myosin rod assemblies as well as the filaments themselves indicated that the rods were assembled with a 6°–7° tilt angle from the rods’ longitudinal axis, in contrast to the lack of tilt in LMM, both exhibiting a 14.3 nm myosin periodicity. Optical diffraction analysis of EM images of the rod assemblies and those of intact myosin confirmed their helical architecture characterized by 28 nm residue translations, 172 nm repeats and 516 nm pitch. A detailed helical model of these filaments was elucidated with bipolar tetramer building units made of two polar trimers. The filaments elongate at their two ends in a head-to-head manner, enabling targeted cross-bridge polarity of the adjacent rows, in the form of a unique Boerdijk–Coxeter type helix, similar to that of collagen or desmin fibers, with the covalent links replaced by a head-to-head clasp.

FAMOUS: a fast instrumental and computational pipeline for multiphoton microscopy applied to 3D imaging of muscle ultrastructure

We present a new instrumental and computational pipeline named FAMOUS: fast algorithm for three-dimensional (3D) multiphoton microscopy of biomedical structures. This pipeline rests on a MPM strategy combined with an original 3D post-processing computational approach. In the present work, FAMOUS approach is devoted to the 3D imaging of the myosin assembly of the ultrastructure of a whole striated skeletal muscle unsliced. Raw recordings of second harmonic generation (SHG) from myosin and instrumental point-spread functions (PSF) are led simultaneously all along the unsliced muscle depth. This procedure highlights a space-variant distortion of the PSF and the SHG signals, and an optical degradation of the axial resolution increasing with imaging depth resulting from the optical heterogeneity of the muscle structure. A 3D mathematical modelling of the PSF, relying on the recent FIGARO method, evaluates and models the depth-variant evolution of the optical distortions. Then, the fast image deblurring algorithm BD3MG is employed to correct those non-stationary distortions all along the sample, thanks to a sounded regularized inverse problem methodology. This leads to the pipeline called FAMOUS, whose performance are highlighted for the optimization of the axial information of myosin structure, whose dimensions are close to the axial resolution limit. For the first time, the 3D organization of the myosin in skeletal muscle is visually shown from an unsliced whole muscle, starting with a solution of optical microscopy. The axial visualization of this organization presently disclosed were never shown until now without a preliminary procedure of sample slicing and labelling. Our original solution FAMOUS delivers a new point of view of this biological structure in the 3D and especially in the optical axis. Image information theoretically expected are now revealed visually in the optical axis for the first time in a whole organ unsliced and label free.

Toll receptors remodel epithelia by directing planar-polarized Src and PI3K activity

Toll-like receptors are essential for animal development and survival, with conserved roles in innate immunity, tissue patterning, and cell behavior. The mechanisms by which Toll receptors signal to the nucleus are well characterized, but how Toll receptors generate rapid, localized signals at the cell membrane to produce acute changes in cell polarity and behavior is not known. We show that Drosophila Toll receptors direct epithelial convergent extension by inducing planar-polarized patterns of Src and PI3-kinase (PI3K) activity. Toll receptors target Src activity to specific sites at the membrane, and Src recruits PI3K to the Toll-2 complex through tyrosine phosphorylation of the Toll-2 cytoplasmic domain. Reducing Src or PI3K activity disrupts planar-polarized myosin assembly, cell intercalation, and convergent extension, whereas constitutive Src activity promotes ectopic PI3K and myosin cortical localization. These results demonstrate that Toll receptors direct cell polarity and behavior by locally mobilizing Src and PI3K activity.

Molecular cloning and expression dynamics of UNC-45B upon heat shock in the muscle of yellowtail

As a myosin chaperone, uncoordinated-45B (UNC-45B) is expressed specifically in striated muscle tissues. It has been proved that overheat shock will lead to myosin denaturation, and that UNC-45B is involved in myosin assembly. Yellowtail was chosen for this study, as an important economic aquaculture fish worldwide. To establish the heat shock response of UNC-45B in yellowtail, cDNA cloning and expression analysis were carried out. The cDNA of yellowtail unc-45b was successfully cloned, and consisted of 3051 bp encoding 930 amino acid residues (ORF 2793 bp). Based on the sequence of yellowtail unc-45b, we designed original primers and an anti-peptide antibody for our study. Yellowtail were then exposed to short-term or long-term heat shock, and UNC-45B expression levels were determined by qPCR and western blot analysis. For short-term heat shock exposure, the fish were kept in water tanks at 25 °C (control group) and 30 °C (heat shock group) for up to 30 h. The mRNA and protein expression levels of UNC-45B increased markedly after 8 h and 24 h, respectively. To investigate the effects of long-term heat shock exposure, cultured yellowtail were sampled in summer and winter at sea surface temperatures of 30 °C and 14 °C. The expression of UNC-45B in both mRNA and protein levels was significantly higher in summer than in winter. Our results show that the increase in UNC-45B expression levels can reflect heat shock response sensitively, and it is suggested that UNC-45B can repair the denatured myosin during heat shock exposure in yellowtail. Moreover, we successfully detected the protein expression of UNC-45B in aquaculture fish species in native conditions by using western blot analysis for the first time. Therefore, we assume that UNC-45B can be regarded as a biomarker to predict heat shock resistance level. This would help in aquaculture production, in the selection of juveniles able to endure heat shock.

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

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.