Novel invadopodia components revealed by differential proteomic analysis

When highly invasive cancer cells are cultured on an extracellular matrix substrate, they extend proteolytically active membrane protrusions, termed invadopodia, from their ventral surface into the underlying matrix. Our understanding of the molecular composition of invadopodia has rapidly advanced in the last few years, but is far from complete.To accelerate component discovery, we resorted to a proteomics approach by applying DIfference Gel Electrophoresis (DIGE) to compare invadopodia-enriched sub-cellular fractions with cytosol and cell body membrane fractions and the whole cell lysate. The fractionation procedure was validated through step-by-step monitoring of the enrichment in typical actin-related invadopodia-associated proteins. After statistical analysis, 129 protein spots were selected for peptide mass fingerprinting analysis; of these 76 were successfully identified and found to correspond to 58 proteins belonging to different functional classes including aerobic glycolysis and other metabolic pathways, protein synthesis, degradation and folding, cytoskeletal components and membrane-associated proteins.Finally, validation of a number of identified proteins was carried out by a combination of immuno-blotting on cell fractions and immunofluorescence localization at invadopodia. These results reveal newly identified components of invadopodia and open further avenues to the molecular study of invasive growth behavior of cancer cells.     

Class III β-tubulin,a novel biomarker in the human melanocyte lineage

It is generally thought that class III β-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III β-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III β-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.     

Restoration of Botshol (The Netherlands) by reduction of external nutrient load: recovery of a characean community,dominated by Chara connivens

Till about 1965 a rich characean community occurred in the shallow peat lake Botshol with six species of which the rare Nitellopsis obtusa and Chara major dominated at many sites. In the period 1980–1988 characean biomass strongly decreased and only two species, Chara globularis and C. connivens, remained in small populations at a few localities. Of the macrophyte Najas marina also some small populations remained, while the aquatic moss Fontinalis antipyretica and the filamentous alga Vaucheria dichotoma predominated at many sites. These phenomena may have been due to eutrophication by the input of polluted water. This process of impoverishment was stopped by restoration measures in 1989, resulting in a lower phosphorus concentration (ca 25 µg l^-1) and a higher water transparency. Immediately after these measures the Characeae community increased in abundance and number of species. During the summer of 1990, and especially 1991, a spectacular growth occurred of Chara connivens. C. connivens was often accompanied by C. major. Other species with scattered occurrence were C. aculeolata, C. aspera, C. contraria and C. globularis. The reasons for the shift in dominance from Nitellopsis obtusa to Chara connivens are discussed. From growth experiments evidence was obtained that neither the recent higher chloride level, nor the lowered phosphate concentration were the main factors for the domination of Chara connivens.     

Endothelium-derived stromal cells contribute to hematopoietic bone marrow niche formation

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Analysis of CRISPR‐Cas9 screens identifies genetic dependencies in melanoma

Targeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. CRISPR‐Cas9 genetic screens provide a genome‐wide approach to uncover novel genetic dependencies that might serve as therapeutic targets. Here, we analyzed recently reported CRISPR‐Cas9 screens comparing data from 28 melanoma cell lines and 313 cell lines of other tumor types in order to identify fitness genes related to melanoma. We found an average of 1,494 fitness genes in each melanoma cell line. We identified 33 genes, inactivation of which specifically reduced the fitness of melanoma. This set of tumor type‐specific genes includes established melanoma fitness genes as well as many genes that have not previously been associated with melanoma growth. Several genes encode proteins that can be targeted using available inhibitors. We verified that genetic inactivation of DUSP4 and PPP2R2A reduces the proliferation of melanoma cells. DUSP4 encodes an inhibitor of ERK, suggesting that further activation of MAPK signaling activity through its loss is selectively deleterious to melanoma cells. Collectively, these data present a resource of genetic dependencies in melanoma that may be explored as potential therapeutic targets.