Integrin-linked kinase can facilitate syncytialization and hormonal differentiation of the human trophoblast-derived BeWo cell line

<p>Abstract</p> <p>Background</p> <p>In the fusion pathway of trophoblast differentiation, stem villous cytotrophoblast cells proliferate and daughter cells differentiate and fuse with existing syncytiotrophoblast to maintain the multi-nucleated layer. Integrin-linked kinase (ILK) is highly expressed in 1st and 2nd trimester villous cytotrophoblast cells, yet barely detectable in syncytiotrophoblast, thus we examined the potential role of ILK in aiding trophoblast fusion.</p> <p>Methods</p> <p>The temporal/spatial expression and activity of ILK were determined in BeWo cells undergoing syncytialization by immunoblot and immunofluorescence analyses. BeWo cells were also transfected with pEGFP expression vectors containing wildtype or two mutant ILK cDNA constructs. The incidence of cell fusion in transfected cells grown under syncytialization conditions was then scored by the presence or absence of E-cadherin immunostaining. Beta-hCG expression in transfected cells, a marker of syncytiotrophoblast hormonal differentiation, was also similarly assessed.</p> <p>Results</p> <p>ILK catalytic activity increased and ILK began to increasingly localize to BeWo cell nuclei during syncytialization in correlation with increased pAkt and Snail protein expression. Syncytialization was also significantly elevated (p < 0.05) in BeWo cells expressing constitutively active (ca)-ILK vs cells containing empty vector or dn-ILK. Furthermore, cytoplasmic Beta-hCG expression markedly increased (p < 0.05) in cells expressing wt- and ca-ILK.</p> <p>Conclusion</p> <p>ILK-facilitated syncytialization is dependent, at least in part, on ILK catalytic activity while hormonal differentiation appears dependent on both ILK-associated protein interactions and catalytic activity. This study demonstrates that ILK plays a novel role in BeWo syncytialization and differentiation, perhaps through an ILK-Akt-Snail pathway, and implicates ILK in the same process in villous cytotrophoblasts in vivo.</p

Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Abstract: Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways

Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore

AbstractThe permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) — sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix

Polyhydroxybutyrate targets mammalian mitochondria and increases permeability of plasmalemmal and mitochondrial membranes.

Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle

In Situ Investigation of Mammalian Inorganic Polyphosphate Localization Using Novel Selective Fluorescent Probes JC-D7 and JC-D8

Inorganic polyphosphate (polyP) is a polymer composed of many orthophosphates linked together by phosphoanhydride bonds. Recent studies demonstrate that in addition to its important role in the function of microorganisms, polyP plays multiple important roles in the pathological and physiological function of higher eukaryotes, including mammalians. However, due to the dramatically lower abundance of polyP in mammalian cells when comparing to microorganisms, its investigation poses an experimental challenge. Here, we present the identification of novel fluorescent probes that allow for specific labeling of synthetic polyP in vitro as well as endogenous polyP in living cells. These probes demonstrate high selectivity for the labeling of polyP that was not sensitive to a number of ubiquitous organic polyphosphates, notably RNA. Use of these probes allowed us to demonstrate the real time detection of polyP release from lysosomes in live cells. Furthermore, we have been able to detect the increased levels of polyP in cells with Parkinson&apos;s disease related mutations.1113sciescopu

Fluorescein-conjugated PHB (Fluo-PHB) is preferentially distributed in the mitochondria of HeLa cells.

<p>A) 1.8 ng/ml fluo-PHB was added to HeLa cells. Times indicated in the panels correspond to acquisition points after fluo-PHB was added. Scale bar 20 µm. B) HeLa were loaded with 25 nM TMRM and with 1.8 ng/ml of fluo-PHB. Scale bar 10 µm. C) Kinetics of distribution of fluo-PHB in the mitochondrial, nuclear and in the extracellular regions. Scale bar 20 µm. D) Chemical structure of fluo-PHB (poly([R]-3-hydroxybutyrate is shown).</p

Fluo-PHB but not fluorescein or fluo-DB induces mitochondrial membrane depolarization.

<p>HeLa cell loaded with 25/ml of fluorescent probes and imaged with a confocal microscope over time. Left column shows TMRM fluorescence in mitochondria before treatment. Second and third columns show TMRM and fluorescein after addition of the probes. Fluorescein did not distribute inside the cells, while fluo-DB did not show preferential mitochondrial localization. Note that neither fluorescein nor fluo-DB affected mitochondrial membrane potential, which was decreased only in the presence of fluo-PHB. This is represented in the graphs that are in the left column that show TMRM fluorescence in arbitrary units (AU) collected from the mitochondrial regions of the intact cells as a function of time. Scale bar 20 µm.</p

Investigation of the mitochondrial morphology following the addition of fluo-PHB and CCCP.

<p>A) fluo-PHB induced mitochondrial membrane depolarization occurs prior to mitochondrial swelling. HeLa cells were transiently transfected with GFP and then loaded with TMRM and fluo-PHB. Arrows point at the region which lost membrane potential but the shape of mitochondria was not changed compared to polarized mitochondria. Images were collected immediately after loading and at 400 s after addition of the Fluo-PHB; B) experimental conditions are similar to those shown in panel A) except in the lower panel the cells were imaged immediately following the addition of 10 µM CCCP. C) HeLa transiently transfected with pMito-GFP were treated with fluo-PHB (18 ng/ml) and after 400 s these cells were treated with ferutinin (25 µM) showing typical swelling of the mitochondria. Scale bar is 20 µm.</p

CSA delays fluo-PHB induced mitochondrial membrane depolarization.

<p>A) HeLa cells were loaded with 25 nM TMRM and treated (red trace, n = 12) or not (black trace, n = 20) with CSA (1 µM); this was followed by the addition of fluo-PHB (18 ng/ml). Cells were imaged with a laser confocal microscope. Traces show TMRM intensity collected from the mitochondrial regions. B) fluorescence intensity ratios in the presence of fluo-PHB between nuclear and mitochondrial regions of HeLa cells treated with CSA and non-treated control cells. (n = 40 for each group of cells);</p