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Abstract
BackgroundMyosin II recruitment to the equatorial cortex is one of the earliest events in establishment of the cytokinetic contractile ring. In Drosophila S2 cells, we previously showed that myosin II is recruited to the furrow independently of F-actin, and that Rho1 and Rok are essential for this recruitment [1]. Rok phosphorylates several cellular proteins, including the myosin regulatory light chain (RLC).Methodology/Principal FindingsHere we express phosphorylation state mimic constructs of the RLC in S2 cells to examine the role of RLC phosphorylation involving Rok in the localization of myosin. Phosphorylation of the RLC is required for myosin localization to the equatorial cortex during mitosis, and the essential role of Rok in this localization and for cytokinesis is to maintain phosphorylation of the RLC. The ability to regulate the RLC phosphorylation state spatio-temporally is not essential for the myosin localization. Furthermore, the essential role of Citron in cytokinesis is not phosphorylation of the RLC.Conclusions/SignificanceWe conclude that the Rho1 pathway leading to myosin localization to the future cytokinetic furrow is relatively straightforward, where only Rok is needed, and it is only needed to maintain phosphorylation of the myosin RLC.
Highlights
Cytokinesis involves the formation of a myosin II containing contractile ring at the furrow of dividing cells
E-mail: jspudich@stanford. edu regulatory light chain (RLC) phosphorylation is required for myosin II recruitment to the cleavage furrow While phospho-RLC has been detected in the cytokinetic furrows of mammalian cells [22,23], to our knowledge its presence has not been verified in the furrows of dividing Drosophila cells
In the presence of endogenous RLC, the mutant proteins will co-assemble with the endogenous RLCs onto myosin heavy chains resulting in myosin II filaments that contain a mixture of wildtype and mutant RLCs
Summary
Cytokinesis involves the formation of a myosin II containing contractile ring at the furrow of dividing cells The placement of this contractile ring is controlled by the small GTPase Rho1/ RhoA [2,3] which stimulates both actin filament formation at the furrow by localized activation of formin proteins [4] and ring contraction by activating Rho kinase (Rok) and Citron kinase, which can phosphorylate the myosin II regulatory light chain (RLC) [5,6,7]. Rok directly phosphorylates myosin II RLC at threonine and serine in mammalian cells [8] (T20 and S21 in Drosophila [9]) and suppresses its dephosphorylation by inactivating the myosin phosphatase [10]. We conclude that the Rho pathway leading to myosin localization to the future cytokinetic furrow is relatively straightforward, where only Rok is needed, and it is only needed to maintain phosphorylation of the myosin RLC
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