Search

During neural tube closure (NTC), a sheet-like structure of cells folds into a tube that forms the brain and spinal cord. Neural tube defects can arise when cells fail to constrict their apical surfaces, a process dependent on Shroom3. Yet, we lack understanding how Shroom3-constriction is achieved. Mechanisms of cell constriction in the developing embryo vary by organism and stage of development. Each observed constriction mechanism has been associated with a different model, the best-established being “ratchet-like” medioapical constriction, purse-string junctional constriction, and pulsed medioapical constriction. While these models help us distinguish mechanisms by attributes we can visually observe, we need methods to describe more complex mechanisms, with precision. Here, we present a framework to characterize cell constrictions quantitatively, according to cell parameters that are readily measurable and associated with morphogenesis, including protein localization, cell polarity, cell shape descriptors, and cell rearrangement. We use partial least squares regression (PLS) to understand which parameters are associated with cellular apical area changes and apply our framework to in-vivo time-lapse imaging of Xenopus tropicalis during NTC. Using PLS, we generate a model of cell constrictions in wild-type and Shroom3 mutant cells during NTC. Our comparisons highlight possible modes of apical constriction across developmental contexts.

Localized Aggressive Periodontitis (LAP) is clinically characterized as a rapid, early onset bone loss around the first molars and incisors. Polymorphonuclear neutrophils (PMN) in LAP patients are hyperactive, generating excessive superoxide anions, cytokines, and enzymes, leading to host tissue injury. This hyper-responsive phenotype of LAP neutrophils suggests that the cells are somehow pre-activated, or primed in LAP, implicating the signaling molecules in the pathway for NADPH oxidase activation. Protein Kinase C has been identified as key molecule in the production of superoxide in neutrophils. In the first study, the role of PKC in priming of neutrophils from LAP patients was investigated. PKC activity was found to be higher in the membranes of neutrophils from LAP patients than healthy subjects, suggesting translocation of the enzyme from the cytosol to the membrane. Analysis of five PKC isoforms indicated that at rest, phosphorylated PKC β1, ξ, and δ, and from LAP subjects were expressed at higher levels than of those from healthy subjects. There were no differences in the expression of PKC isoform transcripts between healthy and LAP groups, indicating that the increase in PKC is not a result of increased transcription. Expression of p47phox phosphorylation was enhanced in neutrophils from subjects with LAP, supporting our findings that the neutrophils are indeed primed. Inhibition studies, using PKC inhibitors, demonstrated a greater inhibition of superoxide production in neutrophils from healthy subjects than those subjects with LAP. Application of classical and zeta PKC inhibitors demonstrated greater superoxide inhibition than the use of beta and delta inhibitors. In the latter half of the study, whole cell neutrophil lysates from healthy and LAP groups pre treated with RvE1 were analyzed for phosphorylated PKC isoforms. In the healthy group, RvE1 did not inhibit phosphorylation of any PKC isoforms. However, in the LAP group, RvE1 reduced the phosphorylation of PKC ô and compared to PBS-treated neutrophils. Similarly, RvEI only reduced phosphorylation of p47PiX in neutrophils from LAP subjects. Pharmacologically induced dephosphorylation with RvE1 returns LAP PMN to a normal neutrophil phenotype. RvE1 is a potential therapeutic modality for the treatment of the periodontal lesions associated with LAP.