Background : Cofilin, a key regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 by LIM-kinases and is reactivated by dephosphorylation by a family of protein phosphatases, termed Slingshot (SSH).

The interaction of astral microtubules with cortical actin networks is essential for the correct orientation of the mitotic spindle; however, little is known about how the cortical actin organization is regulated during mitosis. LIM kinase-1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. LIMK1 activity increases during mitosis. Here we show that mitotic LIMK1 activation is critical for accurate spindle orientation in mammalian cells. Knockdown of LIMK1 suppressed a mitosis-specific increase in cofilin phosphorylation and caused unusual cofilin localization in the cell cortex in metaphase, instability of cortical actin organization and astral microtubules, irregular rotation and misorientation of the spindle, and a delay in anaphase onset. Similar results were obtained by treating the cells with a LIMK1 inhibitor peptide or latrunculin A or by overexpressing a non-phosphorylatable cofilin(S3A) mutant. Furthermore, localization of LGN (a protein containing the repetitive Leu-Gly-Asn tripeptide motifs), an important regulator of spindle orientation, in the crescent-shaped cortical regions was perturbed in LIMK1 knockdown cells. Our results suggest that LIMK1-mediated cofilin phosphorylation is required for accurate spindle orientation by stabilizing cortical actin networks during mitosis.The accurate establishment of the positioning and orientation of the mitotic spindle is essential for cells to determine the axis and plane of cell division and, consequently, to specify the sizes, positions, and fates of daughter cells after mitosis. Proper spindle positioning and orientation are fundamental for both asymmetric and symmetric cell division. Asymmetric cell division produces two unequal daughter cells and is crucial for a number of biological processes including embryogenesis, neurogenesis, tissue remodeling, and stem cell differentiation (1-3). On the other hand, most cells undergo symmetric cell division that is important to give rise to two equal daughter cells. Positioning of the mitotic spindle is regulated by the interaction of astral microtubules that emanate from two spindle poles with specific regions of the cell cortex. A number of proteins have been proposed to mediate this interaction, including microtubule plus-end-associated proteins (such as EB1 and APC), the dynein-dynactin motor protein complex, and other bridging proteins that bind to the actin cytoskeleton or other components in the cell cortex (4 -7). During asymmetric cell division, the spindle orientation is controlled by a set of conserved "spindle-positioning proteins," such as Inscuteable, Partner of Inscuteable (Pins), 2 a G protein ␣ subunit, and the Par3-Par6-aPKC complex, which asymmetrically localize in the cell cortex (1-3). Recent studies have shown that during symmetric cell division of cultured mammalian cells, integrin-mediated cell adhesion guides the proper spindle orientation parallel to the substrate plane through the regulation of the cortical actin cytoskeleton (...