Diphosphorylated but not monophosphorylated myosin II regulatory light chain localizes to the midzone without its heavy chain during cytokinesis

Abstract

Myosin II is activated by the monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). Its ATPase activity is further enhanced by MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC). As these phosphorylated MRLCs are colocalized with their heavy chains at the contractile ring in dividing cells, we believe that the phosphorylated MRLC acts as a subunit of the activated myosin II during cytokinesis. However, the distinct role(s) of 1P- and 2P-MRLC during cytokinesis has not been elucidated. In this study, a monoclonal antibody (4F12) specific for 2P-MRLC was raised and used to examine the roles of 2P-MRLC in cultured mammalian cells. Our confocal microscopic observations using 4F12 revealed that 2P-MRLC localized to the contractile ring, and, unexpectedly, to the midzone also. Interestingly, 2P-MRLC did not colocalize with 1P-MRLC, myosin II heavy chain, and F-actin at the midzone. These results suggest that 2P-MRLC has a role different from that of 1P-MRLC at the midzone, and is not a subunit of myosin II.

A complex of p190RhoGAP and anillin modulates RhoGTP and the cytokinetic furrow in human cells

“…Both antibodies recognize cytokinetic furrows, and have different staining patterns as anti-pSer19 MLC II antibody stains phosphorylated MLC II (pMLC II) localized only to the contracting membrane, whereas anti-pThr18/Ser19 antibody also stains pMLC localized to the midzone (Kondo et al, 2012). Cells were stained with anti-pSer19 MLC II antibody, the intensity of staining at cytokinetic furrows was quantified (areas quantified are outlined in supplementary material Fig.…”

Aurora B but Not Rho/MLCK Signaling Is Required for Localization of Diphosphorylated Myosin II Regulatory Light Chain to the Midzone in Cytokinesis

“…We have previously reported that an anti-2P-MRLC antibody, 4F12, stains the centrosomal region, contractile ring, midzone, and midbody during cytokinesis in various mammalian cultured cells [14]. In this study, we further investigated the specificity of this antibody at the immunofluorescence level by attempting to absorb its antigen (2P-MRLC).…”

“…Indirect immunofluorescence observations with a conventional light microscope showed that 1P- and 2P-MRLC localize to the contractile ring in dividing cells [11]–[13]. Using confocal microscopy, we have previously shown that 2P-MRLC, but not 1P-MRLC, also localizes to the midzone in cultured mammalian cells [14]. Interestingly, MHC was not observed at the midzone, suggesting that 2P-MRLC plays a unique role different from its role as a subunit of myosin II.…”

Phosphorylation of myosin II regulatory light chain by ZIP kinase is responsible for cleavage furrow ingression during cell division in mammalian cultured cells

“…Immunostaining was performed as previously described [16] except for the use of Vectashield as a mounting reagent. Images were captured using a LSM 700 confocal microscope (Carl Zeiss).…”

“…The motor activity of myosin II is a driving force of the cleavage furrow ingression through the actomyosin contraction [13]. Many studies report that phosphorylated MRLC localizes at the contractile ring [14,15,16]. It was reported that a nonphosphorylatable form of MRLC delays myosin II/actin turnover at the contractile ring [17], slows furrow ingression [18], and induces multinucleation [19,20], suggesting a major role of myosin II activation by MRLC phosphorylation in cytokinesis.…”