(C) Cells were stained with antibodies against MAP2 (red; rabbit polyclonal) and vimentin (green; mouse monoclonal). of MAP2D to vimentin and tubulin by two- and three-fold, respectively. PKA-dependent phosphorylation of vimentin (Ser32 and Ser38) promoted binding of vimentin to MAP2D and increased contraction of granulosa cells with reorganization of vimentin filaments and MAP2D from the periphery into a thickened layer surrounding the nucleus and into prominent cellular extensions. Chemical disruption of vimentin filament organization increased progesterone production. Taken together, these results suggest that hCG-stimulated dephosphorylation of MAP2D at Thr256 and Thr259, phosphorylation of vimentin at Ser38 and Ser72, and the resulting enhanced binding of MAP2D to vimentin might contribute to the progesterone synthetic response required for ovulation. using recombinant MAP2D (Flynn et al., 2008) and are recognized PKA targets in other cells (Ozer and Halpain, 2000). Preovulatory granulosa cells in primary culture MYO9B on fibronectin substratum appear fibroblastic, with long bundles of F-actin (Karlsson et al., 2010). hCG promotes the PKA-dependent dephosphorylation of the actin-depolymerizing factor cofilin on Ser3, resulting in cell rounding and the appearance of spindly processes that appear neuronal-like (Karlsson et al., 2010). These events are required for the progesterone synthetic response (Karlsson et al., 2010) necessary for ovulation (Lydon et al., 1995; Robker et al., 2000). Based on the prominent role of MAP2 proteins in regulating the microtubule and microfilament cytoskeleton and, hence, cell Pafuramidine shape and function in neuronal cells, we sought to evaluate the association of MAP2D in preovulatory granulosa cells with Pafuramidine the cytoskeleton and the effect of hCG on this association. Confocal immunofluorescence microscopy results show that MAP2D partially colocalizes with the intermediate filament vimentin and microtubule cytoskeletons, but not with the microfilament cytoskeleton in untreated (naive) preovulatory granulosa cells. Binding studies show that MAP2D binds directly to vimentin and to -tubulin, and that the phosphorylation of recombinant MAP2D on Thr256 and Thr259, which mimics the phosphorylation status of MAP2D in untreated granulosa cells, reduces binding of MAP2D to vimentin two-fold and to tubulin by three-fold. Activation of the luteinizing hormone choriogonadotropin receptor, a G-protein coupled receptor that drives dephosphorylation of MAP2D on Thr256 and Thr259, promotes rapid PKA-dependent phosphorylation of vimentin on Ser38 and Ser72, a coincident increase in the binding of vimentin to MAP2D (44%), and contraction of granulosa cells with coincident reorganization of vimentin filaments and MAP2D from the Pafuramidine periphery into a thickened layer surrounding the nucleus and prominent cellular extensions. The ability of the vimentin networks to rapidly redistribute within epithelial granulosa cells in response to luteinizing hormone choriogonadotropin receptor activation is consistent with the recognized dynamic properties of intermediate filaments (reviewed in Helfand et al., 2003). Artificial disruption of vimentin filaments increased progesterone production in granulosa cells in the absence of trophic hormone by two-fold. These results suggest that in preovulatory granulosa cells, the hCG-stimulated, PKA-dependent dephosphorylation of MAP2D at Thr256 and Thr259, phosphorylation of vimentin at Ser38 and Ser72, and resulting enhanced binding of MAP2D to vimentin might contribute to the progesterone synthetic response required for ovulation. RESULTS MAP2D colocalizes with the Pafuramidine vimentin intermediate filament and microtubule cytoskeletons in granulosa cells as assessed by confocal immunofluorescence microscopy We initially sought to determine whether MAP2D, the majority of which is highly phosphorylated in naive granulosa cells (Flynn et al., 2008), colocalized with the microtubule, microfilament and/or intermediate cytoskeleton in granulosa cells. Untreated fixed granulosa cells appear flattened and fibroblastic with long bundles of phalloidin-stained F-actin (Karlsson et al., 2010). Staining with antibodies against -tubulin, vimentin [the predominant intermediate protein in granulosa cells (Albertini and Kravit, 1981)], actin and MAP2D proteins also showed that each of these proteins reached into the periphery of the cells (Fig.?1ACC). Dual staining with antibodies against -tubulin and MAP2D revealed that a portion of MAP2D localized to the microtubule cytoskeleton (Fig.?1A). Dual staining for vimentin and MAP2D demonstrated that MAP2D also appeared to colocalize to a portion of vimentin intermediate filaments especially in the peri-nuclear region (Fig.?1B, arrowheads). However, colocalization of vimentin and MAP2D in naive granulosa cells was variable (see top panel of Fig.?1D), even within granulosa cells in the same vision field. Comparisons between MAP2D and microfilaments, as determined by staining for -actin, surprisingly revealed no colocalization (Fig.?1C). These results demonstrate that MAP2D partially localizes to both the microtubule and vimentin intermediate filament cytoskeletons in granulosa.