Normal but not altered mucins activate neutrophils

Interactions between leucocytes and their surroundings are mediated through oligosaccharide epitopes, some of which are also expressed on ocular mucins. Neutrophils represent the majority of immune cells in the proinflammatory environment of the ocular surface during sleep. We have tested whether changes in mucin glycosylation, as occur in dry eyes, influence the phenotype and activation of neutrophils. Peripheral blood leucocytes were circulated over equal concentration mats of ocular surface mucins purified from normal volunteers and dry-eye patients, and in sequence over normal and pathological mucins in all combinations. Non-adherent cells were tagged with monoclonal fluorescent antibodies to leucocyte determinants and analysed by flow cytometry. Oxidative burst, assessed with dihydrorhodamine, was followed in cells and supernatant. At a speed similar to that of leucocyte traffic in the retina, normal mucins caused a decrease in neutrophil cathepsin G fluorescence, a decrease that was not observed with mucins from patients with Meibomian gland disease or Sjögren syndrome. No effect was detected at a higher flow. Supernatant and cells collected after circulation over normal mucin showed increased rhodamine fluorescence, indicative of oxidative burst. Fluorescence could also be observed in intact cells adherent to dry-eye mucins. Non-adherent cells could be activated with phorbol 12-myristate 13-acetate after flow over any mucin or combination of mucins. Differences in neutrophil activation after exposure to normal and pathological mucins highlight reciprocal influences at the interface between local and systemic immunity.     

An Equatorial Contractile Mechanism Drives Cell Elongation but not Cell Division

Cell shape changes and proliferation are two fundamental strategies for morphogenesis in animal development. During embryogenesis of the simple chordate Ciona intestinalis, elongation of individual notochord cells constitutes a crucial stage of notochord growth, which contributes to the establishment of the larval body plan. The mechanism of cell elongation is elusive. Here we show that although notochord cells do not divide, they use a cytokinesis-like actomyosin mechanism to drive cell elongation. The actomyosin network forming at the equator of each notochord cell includes phosphorylated myosin regulatory light chain, α-actinin, cofilin, tropomyosin, and talin. We demonstrate that cofilin and α-actinin are two crucial components for cell elongation. Cortical flow contributes to the assembly of the actomyosin ring. Similar to cytokinetic cells, membrane blebs that cause local contractions form at the basal cortex next to the equator and participate in force generation. We present a model in which the cooperation of equatorial actomyosin ring-based constriction and bleb-associated contractions at the basal cortex promotes cell elongation. Our results demonstrate that a cytokinesis-like contractile mechanism is co-opted in a completely different developmental scenario to achieve cell shape change instead of cell division. We discuss the occurrences of actomyosin rings aside from cell division, suggesting that circumferential contraction is an evolutionally conserved mechanism to drive cell or tissue elongation.     

Cell transformation by kFGF requires secretion but not glycosylation

The Kfgf gene, which encodes a member of the fibroblast growth factor family, was originally discovered by assaying human tumor DNA for dominantly transforming oncogenes. The 22-kD kFGF product contains a single site for asparagine-linked glycosylation and an amino-terminal signal peptide for vectorial synthesis into the endoplasmic reticulum and eventual secretion. To determine whether these features are necessary for transformation, we have constructed mutants of kFGF that are impaired for glycosylation or secretion. All mutants retained the ability to induce DNA synthesis when added to quiescent cells, and the absence of glycosylation had no appreciable effect on the transformation efficiency on NIH3T3 cells. In contrast, mutants of kFGF that remain in the cytoplasm or are retained in the secretory pathway, through addition of a KDEL motif, score negative in standard transformation assays. Since transformation by either the glycosylated or unglycosylated form of kFGF can be reversed by addition of suramin, the data imply that secretion of kFGF, or surface localization of the ligand/receptor complex, is a prerequisite for transformation.     

Diatom-Derived Polyunsaturated Aldehydes Activate Cell Death in Human Cancer Cell Lines but Not Normal Cells

Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs) that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1) and Fas Associated Death Domain (FADD) leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP). The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.     

Normal cells, but not cancer cells, survive severe Plk1 depletion

We previously reported the phenotype of depletion of polo-like kinase 1 (Plk1) using RNA interference (RNAi) and showed that p53 is stabilized in Plk1-depleted cancer cells. In this study, we further analyzed the Plk1 depletion-induced phenotype in both cancer cells and primary cells. The vector-based RNAi approach was used to evaluate the role of the p53 pathway in Plk1 depletion-induced apoptosis in cancer cells with different p53 backgrounds. Although DNA damage and cell death can occur independently of p53, p53-deficient cancer cells were much more sensitive to Plk1 depletion than cancer cells with functional p53. Next, the lentivirus-based RNAi approach was used to generate a series of Plk1 hypomorphs. In HeLa cells, two weak hypomorphs showed only slight G2/M arrest, a medium hypomorph arrested with 4N DNA content, followed later by apoptosis, and a strong Plk1 hypomorph underwent serious mitotic catastrophe. In well-synchronized HeLa cells, a medium level of Plk1 depletion caused a 2-h delay of mitotic progression, and a high degree of Plk1 depletion significantly delayed mitotic entry and completely blocked cells at mitosis. In striking contrast, normal hTERT-RPE1 and MCF10A cells were much less sensitive to Plk1 depletion than HeLa cells; no apparent cell proliferation defect or cell cycle arrest was observed after Plk1 depletion in these cells. Therefore, these data further support suggestions that Plk1 may be a feasible cancer therapy target.     

The Bacterial Peptide Pheromone Plantaricin A Permeabilizes Cancerous, but not Normal, Rat Pituitary Cells and Differentiates between the Outer and Inner Membrane Leaflet

Plantaricin A (PlnA) is a 26-mer peptide pheromone with membrane-permeabilizing, strain-specific antibacterial activity, produced by Lactobacillus plantarum C11. We investigated the membrane-permeabilizing effects of PlnA on cultured cancerous and normal rat anterior pituitary cells using patch-clamp techniques and microfluorometry (fura-2). Cancerous cells displayed massive permeabilization within 5 s after exposure to 10–100 μm PlnA. The membrane depolarized to nearly 0 mV, and the membrane resistance decreased to a mere fraction of the initial value after less than 1 min. In outside-out membrane patches, 10 μm PlnA induced membrane currents reversing at 0 mV, which is compatible with an unspecific conductance increase. The d and l forms of the peptide had similar potency, indicating a nonchiral mechanism for the membrane-permeabilizing effect. Surprisingly, inside-out patches were insensitive to 1 mm PlnA. Primary cultures of normal rat anterior pituitary cells were also insensitive to the peptide. Thus, PlnA differentiates between plasma membranes and membrane leaflets. Microfluorometric recordings of [Ca²⁺] _i and cytosolic concentration of fluorochrome verified the rapid permeabilizing effect of PlnA on cancerous cells and the insensitivity of normal pituitary cells.     

Single Cell Analysis of a Bacterial Sender-Receiver System

Monitoring gene expression dynamics on the single cell level provides important information on cellular heterogeneity and stochasticity, and potentially allows for more accurate quantitation of gene expression processes. We here study bacterial senders and receivers genetically engineered with components of the quorum sensing system derived from Aliivibrio fischeri on the single cell level using microfluidics-based bacterial chemostats and fluorescence video microscopy. We track large numbers of bacteria over extended periods of time, which allows us to determine bacterial lineages and filter out subpopulations within a heterogeneous population. We quantitatively determine the dynamic gene expression response of receiver bacteria to varying amounts of the quorum sensing inducer N-3-oxo-C6-homoserine lactone (AHL). From this we construct AHL response curves and characterize gene expression dynamics of whole bacterial populations by investigating the statistical distribution of gene expression activity over time. The bacteria are found to display heterogeneous induction behavior within the population. We therefore also characterize gene expression in a homogeneous bacterial subpopulation by focusing on single cell trajectories derived only from bacteria with similar induction behavior. The response at the single cell level is found to be more cooperative than that obtained for the heterogeneous total population. For the analysis of systems containing both AHL senders and receiver cells, we utilize the receiver cells as ‘bacterial sensors’ for AHL. Based on a simple gene expression model and the response curves obtained in receiver-only experiments, the effective AHL concentration established by the senders and their ‘sending power’ is determined.