8 research outputs found
Involvement of Rho GAP GRAF1 in maintenance of epithelial phenotype
<p>Adhesion of epithelial cell to each other and to extracellular matrix, as well as cell migration ability and cytoskeleton organization undergo significant alterations in the course of neoplastic transformation, but regulatory mechanisms involved in these processes are not fully understood. Here, we studied the role of a Rho GAP protein GRAF1 (<i>G</i>TPase <i>R</i>egulator <i>A</i>ssociated with <i>F</i>ocal adhesion kinase-<i>1</i>) in the regulation of the epithelial phenotype in cells of breast derived, non-malignant, MCF10A cell line. GRAF1 was shown to be localized to cell-cell junctions, and its depletion resulted in accelerated cell migration velocity, elongation of the cells and cell colonies, impaired monolayer integrity and significant disruption of desmosomes with a loss of associated keratin filaments. These processes were accompanied by formation of larger focal adhesions, an increased number of contractile actin stress fibers, reduction in epithelial markers and increase in mesenchymal markers such as epithelial-mesenchymal transition (EMT)-specific transcription factors Snail-1 and Snail-2, as well as N-cadherin, and vimentin. Moreover, unlike control cells, GRAF1 knocked-down cells demonstrated anchorage-independent growth in soft agar. GRAF1 expression in several highly invasive breast cancer cell lines was low, as compared to the non-malignant MCF10A cells, while overexpressing of GRAF1 in the malignant BT-549 cell line led to a decrease of mesenchymal markers, especially the Snail-1 and 2. Altogether, our analysis suggests that GRAF1 plays a role in the maintenance of normal epithelial phenotype and its depletion leads to an EMT-like process that might be involved in neoplastic transformation.</p
Identification of Tmem10/opalin as an oligodendrocyte enriched gene using expression profiling combined with genetic cell ablation
Oligodendrocytes form an insulating multilamellar structure
of compact myelin around axons, which allows efficient
and rapid propagation of action potentials. However, little
is known about the molecular mechanisms operating at the
onset of myelination and during maintenance of the myelin
sheath in the adult. Here we use a genetic cell ablation
approach combined with Affymetrix GeneChip microarrays
to identify a number of oligodendrocyte-enriched genes that
may play a key role in myelination. One of the ââoligogenesââ
we cloned using this approach is Tmem10/Opalin, which
encodes for a novel transmembrane glycoprotein. In situ
hybridization and RT-PCR analysis revealed that Tmem10
is selectively expressed by oligodendrocytes and that its
expression is induced during their differentiation. Developmental
immunofluorescence analysis demonstrated that
Tmem10 starts to be expressed in the white matter tracks
of the cerebellum and the corpus callosum at the onset of
myelination after the appearance of other myelin genes
such as MBP. In contrast to the spinal cord and brain,
Tmem10 was not detected in myelinating Schwann cells,
indicating that it is a CNS-specific myelin protein. In
mature oligodendrocytes, Tmem10 was present at the cell
soma and processes, as well as along myelinated internodes,
where it was occasionally concentrated at the paranodes.
In myelinating spinal cord cultures, Tmem10 was
detected in MBP-positive cellular processes that were
aligned with underlying axons before myelination commenced.
These results suggest a possible role of Tmem10
in oligodendrocyte differentiation and CNS myelination.This work was supported by grants from the
Dr. Miriam and Sheldon G. Adelson Medical Research
Foundation
Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies
Biocompatibility of Tungsten Disulfide Inorganic Nanotubes and Fullerene-Like Nanoparticles with Salivary Gland Cells
Neuronal release of d-serine: a physiological pathway controlling extracellular d-serine concentration
d-Serine is thought to be a glia-derived transmitter that activates N-methyl d-aspartate receptors (NMDARs) in the brain. Here, we investigate the pathways for d-serine release using primary cultures, brain slices, and in vivo microdialysis. In contrast with the notion that d-serine is exclusively released from astrocytes, we found that d-serine is released by neuronal depolarization both in vitro and in vivo. Veratridine (50 ÎŒM) or depolarization by 40 mM KCl elicits a significant release of endogenous d-serine from primary neuronal cultures. Controls with astrocyte cultures indicate that glial cells are insensitive to veratridine, but release d-serine mainly by the opening of volume-regulated anion channels. In cortical slices perfused with veratridine, endogenous d-serine release is 10-fold higher than glutamate receptor-evoked release. Release of d-serine from slices does not require internal or external Ca2+, suggesting a nonvesicular release mechanism. To confirm the neuronal origin of d-serine, we selectively loaded neurons in cortical slices with d-[3H]serine or applied d-alanine, which specifically releases d-serine from neurons. Depolarization with veratridine promotes d-serine release in vivo monitored by high temporal resolution microdialysis of the striatum. Our data indicate that the neuronal pool of d-serine plays a major role in d-serine dynamics, with implications for the regulation of NMDAR transmission. Rosenberg, D., Kartvelishvily, E., Shleper, M., Klinker, C. M. C., Bowser, M. T., Wolosker, H. Neuronal release of d-serine: a physiological pathway controlling extracellular d-serine concentration
Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis
Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe(-/-) mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received Zr-89-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe(-/-) mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP immune cell interactions significantly decreased over the disease progression. Zr-89-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosi