No abstract

Several isotypes of protein kinase C (PKC) have been reported to be expressed in mammalian eggs, but it is unknown whether these isotypes have a common function in the egg during or within the first few hours of fertilization. Here we show that the isotypes of PKC exhibit distinct patterns of enrichment immediately after mouse egg activation. PKCalpha and gamma accumulate in the egg cortex 25 min post-activation, while only PKCalpha accumulates at the contractile ring of the forming second polar body about 1.5 h post-activation. PKCzeta exhibits some unique features that resulted in it being the focus of more extensive analysis. PKCzeta is tightly associated with the meiotic spindle as determined by detergent extraction and is closely associated with alpha-tubulin as determined by FRET analysis in the metaphase II (MII) egg. In addition, after egg activation, PKCzeta remains associated with the spindle as it transits into anaphase II and later telophase II, becoming associated with the midzone microtubules. Antibodies to the active form of PKCzeta are enriched on the spindle poles and later in development on the midzone microtubules. Active PKCzeta also is enriched in both pronuclei in the 6-h post-fertilization and in the 14-h post-fertilization embryo as well as in the nuclei of the two-cell embryo. Inhibition of PKCzeta, but not inhibition of other isotypes of PKC, results in rapid disruption of the meiotic spindle. This study suggests that PKCzeta has a role in spindle stability, while other PKC isotypes have different roles in the conversion of the egg to the zygote.

Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating this process is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase-contrast video microscopy demonstrated that in the majority of events, short protrusions (∼3 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (∼12 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data suggest that an actin-based protrusion formed at the leading edge initiates macrophage fusion.

Upon fertilization, the mammalian egg undergoes a precise series of signaling events that orchestrate its conversion into a zygote. Mouse eggs contain acentrosomal spindle poles when arrested at meiotic metaphase II. The meiotic spindle is thought to provide a scaffold that mediates spatial and temporal regulation of the signaling pathways orchestrating post-fertilization events. Many kinases have been found to be enriched at the MII meiotic spindle, such as Protein Kinase C (PKC), and are thought to have an important role in regulating signaling events initiated through fertilization. In this study phosphorylated PKC zeta (p-PKC zeta) and Glycogen Synthase Kinase 3beta (GSK3 beta) were found to be enriched at both acentrosomal spindle poles and the kinetochore region. Phosphorylated PKC zeta (p-PKC zeta) was immunopurified from MII eggs and was found to co-localize with known microtubule stabilizing components found in somatic cells, including GSK3 beta and Partition deficit protein 6 (Par6). Both fluorescence resonance energy transfer (FRET) and immunofluorescence confirmed the existence and close association of these proteins with p-PKC zeta at the meiotic spindle. When GSK3 beta is phosphorylated on ser9 its activity is inhibited and the spindle is stabilized. However, when GSK3 beta is dephosphorylated (on ser9) it becomes active and the spindle is destabilized. The mechanism by which p-PKC zeta maintains spindle organization appears to be through GSK3 beta and suggests that p-PKC zeta phosphorylates GSK3 beta on the ser9 position inactivating GSK3 beta and consequently maintaining spindle stability during meiotic metaphase arrest.

This paper presents a shape-based approach in extracting thin structures, such as lines and sheets, from three-dimensional (3D) biomedical images. Of particular interest is the capability to recover cellular structures, such as microtubule spindle fibers and plasma membranes, from laser scanning confocal microscopic (LSCM) data. Hessian-based shape methods are reviewed. A synthesized linear structure is used to evaluate the sensitivity of the multiscale filtering approach in extracting closely positioned fibers. We find that the multiscale approach tends to fuse lines together, which makes it unsuitable for visualizing mouse egg spindle fibers. Single-scale Gaussian filters, balanced between sensitivity and noise resistance, are adopted instead. In addition, through an ellipsoidal Gaussian model, the eigenvalues of the Hessian matrix are quantitatively associated with the standard deviations of the Gaussian model. Existing shape filters are simplified and applied to LSCM data. A significant improvement in extracting closely positioned thin lines is demonstrated by the resultant images. Further, the direct association of shape models and eigenvalues makes the processed images more understandable qualitatively and quantitatively.