Petr Folk

Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces.

Tumor cell invasion is the most critical step of metastasis. Determination of the mode of invasion within the particular tumor is critical for effective cancer treatment. Protease-independent amoeboid mode of invasion has been described in carcinoma cells and more recently in sarcoma cells on treatment with protease inhibitors. To analyze invasive behavior, we compared highly metastatic sarcoma cells with parental nonmetastatic cells. The metastatic cells exhibited a functional up-regulation of Rho/ROCK signaling and, similarly to carcinoma cells, an amoeboid mode of invasion. Using confocal and traction force microscopy, we showed that an up-regulation of Rho/ROCK signaling leads to increased cytoskeletal dynamics, myosin light chain localization, and increased tractions at the leading edge of the cells and that all of these contributed to increased cell invasiveness in a three-dimensional collagen matrix. We conclude that cells of mesenchymal origin can use the amoeboid nonmesenchymal mode of invasion as their primary invading mechanism and show the dependence of ROCK-mediated amoeboid mode of invasion on the increased capacity of cells to generate force. (Mol Cancer Res 2008;6(9):1410–20)

Transcriptional coregulator SNW/SKIP: the concealed tie of dissimilar pathways.

Eukaryotic gene expression requires that all the steps of messenger RNA production are regulated in concert to integrate the diverse inputs cells receive. We discuss the functioning of SNW/SKIP, an essential spliceosomal component and transcriptional coregulator, which may provide regulatory coupling of transcription initiation and splicing. SNW/SKIP potentiates the activity of important transcription factors, such as vitamin D receptor, CBF1 (RBP-Jκ), Smad2/3, and MyoD. It synergizes with Ski in overcoming pRb-mediated cell cycle arrest, and it is targeted by the viral transactivators EBNA2 and E7. SNW/SKIP may aid in conformational transition of the gene expression machine through its avidity to nuclear matrix fractions or by recruiting foldases such as the prolyl isomerase PPIL1. The extensive list of SNW/SKIP partners, its unique primary structure, conserved from yeast to humans, and its essential character suggest a distinct function of general importance.

The structure of invadopodia in a complex 3D environment.

Invadopodia and podosomes have been intensively studied because of their involvement in the degradation of extracellular matrix. As both structures have been studied mostly on thin matrices, their commonly reported shapes and characteristics may differ from those in vivo. To assess the morphology of invadopodia in a complex 3D environment, we observed invadopodial formation in cells grown on a dense matrix based on cell-free dermis. We have found that invadopodia differ in morphology when cells grown on the dermis-based matrix and thin substrates are compared. The cells grown on the dermis-based matrix display invadopodia which are formed by a thick protruding base rich in F-actin, phospho-paxillin, phospho-cortactin and phosphotyrosine signal, from which numerous thin filaments protrude into the matrix. The protruding filaments are composed of an F-actin core and are free of phospho-paxillin and phospho-cortactin but capped by phosphotyrosine signal. Furthermore, we found that a matrix-degrading activity is localized to the base of invadopodia and not along the matrix-penetrating protrusions. Our description of invadopodial structures on a dermis-based matrix should greatly aid the development of new criteria for the identification of invadopodia in vivo, and opens up the possibility of studying the invadopodia-related signaling in a more physiological environment.

Secondary structure is required for 3′ splice site recognition in yeast

Higher order RNA structures can mask splicing signals, loop out exons, or constitute riboswitches all of which contributes to the complexity of splicing regulation. We identified a G to A substitution between branch point (BP) and 3′ splice site (3′ss) of Saccharomyces cerevisiae COF1 intron, which dramatically impaired its splicing. RNA structure prediction and in-line probing showed that this mutation disrupted a stem in the BP-3′ss region. Analyses of various COF1 intron modifications revealed that the secondary structure brought about the reduction of BP to 3′ss distance and masked potential 3′ss. We demonstrated the same structural requisite for the splicing of UBC13 intron. Moreover, RNAfold predicted stable structures for almost all distant BP introns in S. cerevisiae and for selected examples in several other Saccharomycotina species. The employment of intramolecular structure to localize 3′ss for the second splicing step suggests the existence of pre-mRNA structure-based mechanism of 3′ss recognition.

Prp45 affects Prp22 partition in spliceosomal complexes and splicing efficiency of non-consensus substrates†

Human transcription co‐regulator SNW1/SKIP is implicated in the regulation of both transcription elongation and alternative splicing. Prp45, the SNW/SKIP ortholog in yeast, is assumed to be essential for pre‐mRNA processing. Here, we characterize prp45(1–169), a temperature sensitive allele of PRP45, which at permissive temperature elicits cell division defects and hypersensitivity to microtubule inhibitors. Using a synthetic lethality screen, we found that prp45(1–169) genetically interacts with alleles of NTC members SYF1, CLF1/SYF3, NTC20, and CEF1, and 2nd step splicing factors SLU7, PRP17, PRP18, and PRP22. Cwc2‐associated spliceosomal complexes purified from prp45(1–169) cells showed decreased stoichiometry of Prp22, suggesting its deranged interaction with the spliceosome. In vivo splicing assays in prp45(1–169) cells revealed that branch point mutants accumulated more pre‐mRNA whereas 5′ and 3′ splice site mutants showed elevated levels of lariat‐exon intermediate as compared to wild‐type cells. Splicing of canonical intron was unimpeded. Notably, the expression of Prp45(119–379) in prp45(1–169) cells restored Prp22 partition in the Cwc2‐pulldowns and rescued temperature sensitivity and splicing phenotype of prp45(1–169) strain. Our data suggest that Prp45 contributes, in part through its interaction with the 2nd step‐proofreading helicase Prp22, to splicing efficiency of substrates non‐conforming to the consensus. J. Cell. Biochem. 106: 139–151, 2009.

The homolog of chromatin binding protein Bx42 identified in Dictyostelium.

We identified in Dictyostelium a gene snwA containing a region of similarity to SH2 domains of higher eukaryotes. snwA is homologous to a novel human gene SNW1 and to Bx42 from Drosophila melanogaster, a gene coding for a chromatin binding protein responsive to 20-OH-ecdysone. snwA has one mRNA transcript of an approximate size of 2.5 kb.

Cyclophilins of a novel subfamily interact with SNW/SKIP coregulator in Dictyostelium discoideum and Schizosaccharomyces pombe

We screened the Dictyostelium discoideum two-hybrid cDNA library with the SNW/SKIP transcription coregulator SnwA and identified a novel cyclophilin CypE. Independently, the Schizosaccharomyces pombe cDNA library was screened with the SnwA ortholog Snw1 and the ortholog of CypE (named Cyp2) was found. Both cyclophilins bind the respective SNW protein in their autologous systems. The interaction was localized to the N-terminal part of SnwA as well as of Snw1. CypE was confirmed in vitro to be a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase. Remarkably, both SNW proteins bind the cyclophilins in a cyclosporin A independent manner, possibly serving as adaptors for these novel isomerases. These results are the first characterization of the members of a novel cyclophilin subfamily, which includes the human CGI-124/PPIL1 protein.

Cbf11 and Cbf12, the fission yeast CSL proteins, play opposing roles in cell adhesion and coordination of cell and nuclear division

The CSL (CBF1/RBP-Jkappa/Suppressor of Hairless/LAG-1) family is comprised of transcription factors essential for metazoan development, mostly due to their involvement in the Notch receptor signaling pathway. Recently, we identified two novel classes of CSL genes in the genomes of several fungal species, organisms lacking the Notch pathway. In this study, we characterized experimentally cbf11+ and cbf12+, the two CSL genes of Schizosaccharomyces pombe, in order to elucidate the CSL function in fungi. We provide evidence supporting their identity as genuine CSL genes. Both cbf11+ and cbf12+ are non-essential; they have distinct expression profiles and code for nuclear proteins with transcription activation potential. Significantly, we demonstrated that Cbf11 recognizes specifically the canonical CSL response element GTGA/GGAA in vitro. The deletion of cbf11+ is associated with growth phenotypes and altered colony morphology. Furthermore, we found that Cbf11 and Cbf12 play opposite roles in cell adhesion, nuclear and cell division and their coordination. Disturbed balance of the two CSL proteins leads to cell separation defects (sep phenotype), cut phenotype, and high-frequency diploidization in heterothallic strains. Our data show that CSL proteins operate in an organism predating the Notch pathway, which should be of relevance to the understanding of (Notch-independent) CSL functions in metazoans.

Fungal CSL transcription factors

BackgroundThe CSL (C BF1/RBP-Jκ/S uppressor of Hairless/L AG-1) transcription factor family members are well-known components of the transmembrane receptor Notch signaling pathway, which plays a critical role in metazoan development. They function as context-dependent activators or repressors of transcription of their responsive genes, the promoters of which harbor the GTG(G/A)GAA consensus elements. Recently, several studies described Notch-independent activities of the CSL proteins.ResultsWe have identified putative CSL genes in several fungal species, showing that this family is not confined to metazoans. We have analyzed their sequence conservation and identified the presence of well-defined domains typical of genuine CSL proteins. Furthermore, we have shown that the candidate fungal protein sequences contain highly conserved regions known to be required for sequence-specific DNA binding in their metazoan counterparts. The phylogenetic analysis of the newly identified fungal CSL proteins revealed the existence of two distinct classes, both of which are present in all the species studied.ConclusionOur findings support the evolutionary origin of the CSL transcription factor family in the last common ancestor of fungi and metazoans. We hypothesize that the ancestral CSL function involved DNA binding and Notch-independent regulation of transcription and that this function may still be shared, to a certain degree, by the present CSL family members from both fungi and metazoans.

Molecular characterization of a calmodulin‐like Dictyostelium protein CalB

A gene named calB was cloned and characterized in Dictyostelium. A relationship to calmodulin (CaM) is suggested by sequence identity (50%), similar exon–intron structure and cross‐reactivity with anti‐CaM sera. The level of calB mRNA is developmentally regulated with maxima during aggregation and in spores. CalB null cells grow normally, develop and produce viable spores. We demonstrated the capacity of tagged CalB to bind Ca2+ using the 45Ca2+ overlay assay and showed that its mobility on SDS–PAGE is dependent on Ca2+/EGTA pretreatment.