Cytoskeleton Dynamics

How does the contractome protein network regulate actomyosin contractility?

Non-muscle myosin II isoforms have a similar structure and function to their muscle equivalents. However, their interaction with actin serves to generate cellular forces rather than muscular contraction. During non-muscle actomyosin contractility, non-muscle myosin II uses energy from ATP hydrolysis to slide the actin filament to produce contractile force, and these forces have been implicated in multiple cell functions, such as cell adhesion, establishing cell polarity, and cell migration [1][2]. During cell division, actomyosin contractility regulates forces on the nucleus which affect DNA synthesis and chromatin organization, and is also required for formation and contraction of the mitotic spindle [3][4][5][6].

The varied functions associated with actomyosin contractility require the involvement of many proteins other than actin and myosin. Data mining of the literature has revealed the comprehensive network of proteins that regulate actomyosin contractility, termed the ‘contractome’. A total of 100 contractome proteins were identified, comprising of 97 proteins and 3 cofactors. After organizing these proteins based on their primary function, the three biggest functional groups were serine/threonine phosphorylation regulators, primarily kinases (27 proteins), scaffolding proteins (24 proteins), and regulators of actin dynamics (12 proteins).

Using a protein interaction database to probe the contractome for interactions, researchers have been able to isolate the major features of the network. The Rho family of small GTPases activates serine/threonine kinases, and this activation is propagated by self-phosphorylation. The serine/threonine kinases activate regulators of actin dynamics, myosin phosphatase, and the myosin light chain. The scaffold group of proteins act as connectors, forming a link between actin and myosin. Scaffolding proteins also bind to the serine/threonine kinases, RhoGTPases and its RhoGEF and RhoGAP regulators, and regulate the myosin heavy chain. While the contractome demonstrates the complexity of actomyosin contractility, the initial data also reveals certain common functional processes and regulatory pathways [7].

How does cofilin regulate actomyosin formation and Myosin II mediated contractility?

Along with actin filament disassembly or severing, ADF/cofilin was recently shown to carry out another important role; specifically the regulation of Myosin II mediated contractility and actomyosin formation. This was proposed to result from competitive antagonism, where myosin II must compete with cofilin for binding sites on F-actin [8].

In this study it was shown that the binding affinities of each protein are ATP dependent, with ADF/cofilin possessing a competitive advantage at cellular levels of ATP, whilst in the absence of ATP the binding affinities of each protein is similar. Importantly, a reduction in the levels of both ADF and cofilin lead to an increase in the concentration of F-actin, a finding that was attributed not to a loss in cofilin mediated F-actin severing, but rather to an increase in myosin-II dependent actin assembly via its crosslinking properties. This was confirmed with the introduction of blebbistatin which inhibited myosin II activity and subsequently lead to the disassembly of F-actin [8].

The implications for this role of ADF/cofilin may be described at the molecular level, however as shown by Wiggan O et al the consequences are clearly evident at a cellular level, with persistent membrane blebs being observed in HeLa cells depleted of the proteins [8]. As it had previously been reported that non-apoptotic blebs were produced as a means of releasing cell tension, it is probable that the observed phenotype occurred for a similar purpose, and highlights the importance of ADF/cofilin in the regulation of cortical tension and actomyosin activity [8].

Despite these findings, in some situations a cooperative relationship between ADF/cofilin and Myosin-II appears to exist. This has been described, for example, in a study investigating actomyosin ring constriction in budding yeast cells [9]. Supporting earlier findings [10][11], this study also confirmed that deletion or inhibition of the motor-domain of Myosin II (MyoI) did not completely prevent constriction, but noted that a 40% reduction in the rate of contraction was observed. This was in contrast to mutations in cofilin, or stabilization of actin filaments, which did prevent actomyosin ring constriction. Model simulations using this data indicated a role of Myosin-II in the promotion of cofilin mediated depolymerization, and it was suggested that it is the disassembly of F-actin that is the primary contributor to actomyosin ring constriction [9].

References

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2. Vicente-Manzanares M, Ma X, Adelstein RS, and Horwitz AR. Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat. Rev. Mol. Cell Biol. 2009; 10(11):778-90. [PMID: 19851336]
3. Hossain MM, Smith PG, Wu K, and Jin J. Cytoskeletal tension regulates both expression and degradation of h2-calponin in lung alveolar cells. Biochemistry 2006; 45(51):15670-83. [PMID: 17176089]
4. Kumar A, Maitra A, Sumit M, Ramaswamy S, and Shivashankar GV. Actomyosin contractility rotates the cell nucleus. Sci Rep 2014; 4:3781. [PMID: 24445418]
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7. Zaidel-Bar R, Zhenhuan G, and Luxenburg C. The contractome–a systems view of actomyosin contractility in non-muscle cells. J. Cell. Sci. 2015; 128(12):2209-17. [PMID: 26021351]
8. Wiggan O, Shaw AE, DeLuca JG, and Bamburg JR. ADF/cofilin regulates actomyosin assembly through competitive inhibition of myosin II binding to F-actin. Dev. Cell 2012; 22(3):530-43. [PMID: 22421043]
9. Mendes Pinto I, Rubinstein B, Kucharavy A, Unruh JR, and Li R. Actin depolymerization drives actomyosin ring contraction during budding yeast cytokinesis. Dev. Cell 2012; 22(6):1247-60. [PMID: 22698284]
10. Fang X, Luo J, Nishihama R, Wloka C, Dravis C, Travaglia M, Iwase M, Vallen EA, and Bi E. Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin II. J. Cell Biol. 2010; 191(7):1333-50. [PMID: 21173112]
11. Lord M, Laves E, and Pollard TD. Cytokinesis depends on the motor domains of myosin-II in fission yeast but not in budding yeast. Mol. Biol. Cell 2005; 16(11):5346-55. [PMID: 16148042]