Charakterisierung des Myb-Transkriptionsfaktors p42POP: Eine Verbindung des Mikrofilamentsystems zur Transkription

Die cytoskelettalen Proteine Aktin und Profilin sind in morphologische Prozesse und in die Generierung einer funktionellen und strukturellen Zellpolarität involviert. Zudem besitzen beide Proteine Aufgaben im Zellkern und scheinen u.a. bei der RNA-Prozessierung und der Transkription beteiligt zu sein. Mit p42POP wurde ein neuer Ligand für Profilin und G-Aktin identifiziert, der Homologie zu Transkriptionsfaktoren der Myb-Familie aufweist. Durch den Nachweis der spezifischen Interaktion mit einem DNA-Erkennungsmotiv der Myb-Transkriptionsfaktoren und der Charakterisierung der DNA-Bindungsdomäne konnte p42POP als erstes Protein in Vertebraten mit einem einzigen funktionellen Myb-Repeat identifiziert werden. Eine transkriptionsaktivierende Wirkung wurde einem sauren Bereich zugewiesen, dessen Aktivität durch C-terminal gelegene Bereiche beeinflusst wird. In diesem Zusammenhang stellt die durch ein Leuzin-Zipper Motiv vermittelte Dimerisierung von p42POP einen möglichen Regulationsmechanismus dar. Die Lokalisation der Bindungsstellen für Profilin und Aktin gibt Hinweise auf eine mögliche Funktion dieser Interaktionen bei der p42POP-vermittelten Genaktivierung: Das Bindungsmotiv für Aktin überlappt mit der Myb-Domäne und könnte die Interaktion mit DNA modulieren. Prolinreiche Motive, die den Leuzin-Zipper flankieren, vermitteln die Wechselwirkung mit Profilin, wodurch möglicherweise die Dimerisierung beeinflusst wird. Eine Regulation dieser Wechselwirkung konnte zum einen auf die potentielle Phosphorylierungsstelle Threonin 273 in p42POP, zum anderen auf eine Phosphorylierung von Profilin I an Serin 138 zurückgeführt werden.The cytoskeletal proteins actin and profilin are involved in morphological processes and in the establishment of cell polarity. Both proteins also possess functions in the nucleus and seem to participate in RNA-processing and transcription. The protein p42POP, identified as a new ligand for profilin and actin, reveals homology to Myb-transcription factors. The specific interaction with a DNA recognition motif of the Myb-transcription factors and the characterisation of the DNA-binding domain leads to the identification of p42POP as the first mammalian Myb-protein comprising only one functional Myb-repeat. Transcriptional activation was assigned to an acidic region, which activity is modulated by C-terminal regions. Coherently, the dimerisation of p42POP mediated by a leucine zipper could be involved in the regulation. The localisation of the binding domain for profilin and actin might help to elucidate the function of these interactions in the context of p42POP mediated gene activation: The binding motif for actin overlaps with the Myb-domain and could thus modulate the binding to DNA. Proline-rich regions flanking the leucine zipper motif mediate the interaction with profilin, which therefore could influence dimerisation of p42POP. This interaction could be regulated by the putative phosphorylation of threonine 273 of p42POP or by the PKC-mediated phosphorylation of serine 138 of profilin I

Extraction of protein profiles from primary neurons using active contour models and wavelets

AbstractThe function of complex networks in the nervous system relies on the proper formation of neuronal contacts and their remodeling. To decipher the molecular mechanisms underlying these processes, it is essential to establish unbiased automated tools allowing the correlation of neurite morphology and the subcellular distribution of molecules by quantitative means.We developed NeuronAnalyzer2D, a plugin for ImageJ, which allows the extraction of neuronal cell morphologies from two dimensional high resolution images, and in particular their correlation with protein profiles determined by indirect immunostaining of primary neurons. The prominent feature of our approach is the ability to extract subcellular distributions of distinct biomolecules along neurites. To extract the complete areas of neurons, required for this analysis, we employ active contours with a new distance based energy. For locating the structural parts of neurons and various morphological parameters we adopt a wavelet based approach. The presented approach is able to extract distinctive profiles of several proteins and reports detailed morphology measurements on neurites.We compare the detected neurons from NeuronAnalyzer2D with those obtained by NeuriteTracer and Vaa3D-Neuron, two popular tools for automatic neurite tracing. The distinctive profiles extracted for several proteins, for example, of the mRNA binding protein ZBP1, and a comparative evaluation of the neuron segmentation results proves the high quality of the quantitative data and proves its practical utility for biomedical analyses

Musashi–1—A Stemness RBP for Cancer Therapy?

The RNA–binding protein Musashi–1 (MSI1) promotes stemness during development and cancer. By controlling target mRNA turnover and translation, MSI1 is implicated in the regulation of cancer hallmarks such as cell cycle or Notch signaling. Thereby, the protein enhanced cancer growth and therapy resistance to standard regimes. Due to its specific expression pattern and diverse functions, MSI1 represents an interesting target for cancer therapy in the future. In this review we summarize previous findings on MSI1′s implications in developmental processes of other organisms. We revisit MSI1′s expression in a set of solid cancers, describe mechanistic details and implications in MSI1 associated cancer hallmark pathways and highlight current research in drug development identifying the first MSI1–directed inhibitors with anti–tumor activity

Keratins Mediate Localization of Hemidesmosomes and Repress Cell Motility

The keratin (K)–hemidesmosome (HD) interaction is crucial for cell-matrix adhesion and migration in several epithelia, including the epidermis. Mutations in constituent proteins cause severe blistering skin disorders by disrupting the adhesion complex. Despite extensive studies, the role of keratins in HD assembly and maintenance is only partially understood. Here we address this issue in keratinocytes in which all keratins are depleted by genome engineering. Unexpectedly, such keratinocytes maintain many characteristics of their normal counterparts. However, the absence of the entire keratin cytoskeleton leads to loss of plectin from the hemidesmosomal plaque and scattering of the HD transmembrane core along the basement membrane zone. To investigate the functional consequences, we performed migration and adhesion assays. These revealed that, in the absence of keratins, keratinocytes adhere much faster to extracellular matrix substrates and migrate approximately two times faster compared with wild-type cells. Reexpression of the single keratin pair K5 and K14 fully reversed the above phenotype. Our data uncover a role of keratins, which to our knowledge is previously unreported, in the maintenance of HDs upstream of plectin, with implications for epidermal homeostasis and pathogenesis. They support the view that the downregulation of keratins observed during epithelial–mesenchymal transition supports the migratory and invasive behavior of tumor cells

Spatial regulation of beta-actin translation by Src-dependent phosphorylation of ZBP1

Localization of beta-actin messenger RNA to sites of active actin polymerization modulates cell migration during embryogenesis, differentiation and possibly carcinogenesis. This localization requires the oncofetal protein ZBP1 (Zipcode binding protein 1), which binds to a conserved 54-nucleotide element in the 3\u27-untranslated region of the beta-actin mRNA known as the \u27zipcode\u27. ZBP1 promotes translocation of the beta-actin transcript to actin-rich protrusions in primary fibroblasts and neurons. It is not known how the ZBP1-RNA complex achieves asymmetric protein sorting by localizing beta-actin mRNA. Here we show that chicken ZBP1 modulates the translation of beta-actin mRNA. ZBP1 associates with the beta-actin transcript in the nucleus and prevents premature translation in the cytoplasm by blocking translation initiation. Translation only occurs when the ZBP1-RNA complex reaches its destination at the periphery of the cell. At the endpoint of mRNA transport, the protein kinase Src promotes translation by phosphorylating a key tyrosine residue in ZBP1 that is required for binding to RNA. These sequential events provide both temporal and spatial control over beta-actin mRNA translation, which is important for cell migration and neurite outgrowth