The N-terminus of the long MLCK induces a disruption in normal spindle morphology and metaphase arrest

Abstract

We have shown previously that only the long myosin light chain kinase (MLCK), which is the predominant MLCK isoform expressed in nonmuscle cells, localizes to the cleavage furrow. To further examine the in vivo localization of the long MLCK in HeLa cells and the mechanisms responsible for kinase targeting during the cell cycle, we examined the distribution of the endogenous kinase and constructed green fluorescent protein (GFP) fusions of long HeLa MLCK truncations. A GFP fusion containing the N-terminal IgG domain and the five DXR motifs localized to stress fibers during interphase and the cleavage furrow during mitosis. Although individual fusions of the five DXRs and IgG domain both independently localized to stress fibers, only the five DXRs demonstrated a cortical localization in mitotic cells. Thus, robust targeting of the long MLCK to the cleavage furrow required the five DXRs and additional sequences from the IgG domain. Expression of the IgG domain alone or with five DXRs increased the number of multinucleate cells tenfold, whereas expression of the five DXRs or GFP had no effect. Furthermore, expression of the IgG domain alone or with five DXRs disrupted normal spindle morphology during mitosis. Extended astral microtubules and increased bundling of kinetochore microtubules, and spindle pole fragmentation were detected in mitotic cells. These microtubule defects were associated with abnormalities in metaphase chromosome alignment and a subsequent metaphase arrest caused by activation of the spindle assembly checkpoint at the kinetochores of mono-oriented chromosomes. Together, these results suggest that MLCK has an unexpected regulatory function during mitosis.

Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix?

“…Abundant evidence has accumulated that actin and myosin (including activated (phosphorylated) myosin) are present in spindles in general and kinetochore fibers in particular (reviews in Forer et al 2003;Woolner and Bement 2009;Dulyaninova et al 2004;Weber et al 2004;Fabian et al 2007b;Fabian and Forer 2007;Woolner et al 2008;Vilmos et al 2009), as are proteins that interact in actin-myosin function such as titin (Fabian et al 2007a), Band 4.1 (Krauss et al 1997), zyxin (Hirota et al 2000), myosin light chain kinase (Dulyaninova et al 2004), and moesin (Vilmos et al 2009). Actin and myosin interact with microtubules in a variety of other motile situations (e.g., Rodriguez et al 2003;Weber et al 2004;Pizon et al 2005;Woolner and Bement 2009) so it would not be surprising were they to interact with spindle microtubules.…”

Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery

“…Myosin II and actin filaments have long been known to assemble at the membrane surface (DeBiasio et al, 1988;McKenna et al, 1989;Rhee et al, 1994) and to interact with both L-MLCK and S-MLCK (Blue et al, 2002;Dulyaninova et al, 2004;Poperechnaya et al, 2000;Stull et al, 1998). The additional F-actin-binding and -bundling sequences in the unique L-MLCK N-terminus apparently increase the affinity of L-MLCK, relative to S-MLCK, for actin filaments (Smith et al, 2002;Yang et al, 2006).…”

“…Plasmids encoding Flag-tagged wild-type and KD murine 220 kDa L-MLCK and Flag-tagged rabbit 130 kDa S-MLCK were gifts from Patricia Gallagher (Indiana University School of Medicine, IN) (Blue et al, 2002). Constructs encoding EGFP-tagged N-terminal fragments of human L-MLCK (amino acids 2-867 and 2-1024) and EGFP-tagged rabbit five DXR actin-binding domains (Smith et al, 2002) were provided by Anne R. Bresnick (Albert Einstein College of Medicine, Bronx, NY) (Dulyaninova et al, 2004).…”

“…Transfection methods have been described (Takizawa et al, 2006). Briefly, COS7 cells were transfected using Effectene Transfection Reagent (Qiagen) for 24 hours, except in experiments with L-MLCK N-terminal sequences, which were lethal to cells transfected for longer than 16 hours (Dulyaninova et al, 2004). A549 cells at 60% confluence were transfected twice with 10 nM dsRNA and Lipofectamine 2000, as recommended (Invitrogen), and grown for 4 days.…”

Myosin-IIA Heavy-Chain Phosphorylation Regulates the Motility of MDA-MB-231 Carcinoma Cells

“…The membrane was stained with Ponceau S and autoradiographed, and 32 P-labeled protein bands were excised and blocked for 30 min at 37°C in 0.5% PVP-40 in 100 mM acetic acid. Tryptic digests and phosphoamino analysis and two-dimensional phosphopeptide mapping were performed as described previously (Dulyaninova et al, 2004).…”