Intercellular Transport of Oct4 in Mammalian Cells: A Basic Principle to Expand a Stem Cell Niche?

Background: The octamer-binding transcription factor 4 (Oct4) was originally described as a marker of embryonic stem cells. Recently, the role of Oct4 as a key regulator in pluripotency was shown by its ability to reprogram somatic cells in vitro, either alone or in concert with other factors. While artificial induction of pluripotency using transcription factors is possible in mammalian cell culture, it remains unknown whether a potential natural transfer mechanism might be of functional relevance in vivo. The stem cell based regeneration of deer antlers is a unique model for rapid and complete tissue regeneration in mammals and therefore most suitable to study such mechanisms. Here, the transfer of pluripotency factors from resident stem cell niche cells to differentiated cells could recruit more stem cells and start rapid tissue regeneration. Methodology/Principal Findings: We report on the ability of STRO-1 + deer antlerogenic mesenchymal stem cells (DaMSCs) to transport Oct4 via direct cell-to-cell connections. Upon cultivation in stem cell expansion medium, we observed nuclear Oct4 expression in nearly all cells. A number of these cells exhibit Oct4 expression not only in the nucleus, but also with perinuclear localisation and within far-ranging intercellular connections. Furthermore, many cells showed intercellular connections containing both F-actin and a-tubulin and through which transport could be observed. To proof that intercellular Oct4-transfer has functional consequences in recipient cells we used a co-culture approach with STRO-1 + DaMSCs and a murine embryonic fibroblast indicator cell line (Oct4-GFP MEF). In this cell line a reporter gene (GFP) unde

Der Blogger als Markenbotschafter. Empirische Studie über die Glaubwürdigkeit aus Sicht der Konsumenten

Blogger genießen innerhalb ihrer Community ein gewisses Ansehen und werden als eine Art Vertrauensperson gesehen. Durch ihre authentische und ehrliche Berichterstattung, werden sie als glaubwürdig wahrgenommen. Aktuell unterliegt die Blogosphäre einer zunehmenden Kommerzialisierung, weil private Blogger durch Werbung auf ihrem Blog, Einnahmen erzielen. Sie veröffentlichen bezahlte Artikel oder werden als Gesicht verschiedener Kampagnen von Unternehmen eingesetzt. Diese Veränderung des Weblogs, wirft die Frage auf, ob ein Blogger glaubhaft sein kann, wenn er als Markenbotschafter fungiert. Im theoretischen Teil der Arbeit wird der bisherige Forschungsstand zu dieser Thematik erläutert. Anhand einer empirischen Untersuchung, soll geklärt werden, wie die Blogger- Community die Glaubwürdigkeit von Weblogs bewertet. Die empirische Studie zeigt, dass sich die einst zugeschriebene Glaubwürdigkeit von Weblogs verschlechtert hat. Generell werden Blogs nur eine mittelmäßige Glaubwürdigkeit zugeschrieben. Wenn Blogger, gezielt als Markenbotschafter fungieren, fällt die Glaubwürdigkeitszuschreibung negativer aus

Destabilization of YopE by the Ubiquitin-Proteasome Pathway Fine-Tunes Yop Delivery into Host Cells and Facilitates Systemic Spread of Yersinia enterocolitica in Host Lymphoid Tissue▿

Pathogenic Yersinia species inject a panel of Yop virulence proteins by type III protein secretion into host cells to modulate cellular defense responses. This enables the survival and dissemination of the bacteria in the host lymphoid tissue. We have previously shown that YopE of the Y. enterocolitica serogroup O8 is degraded in the host cell through the ubiquitin-proteasome pathway. YopE normally manipulates rearrangements of the actin cytoskeleton and triggers phagocytosis resistance. To shed light into the physiological role of YopE inactivation, we mutagenized the lysine polyubiquitin acceptor sites of YopE in the Y. enterocolitica serogroup O8 virulence plasmid. The resulting mutant strain escaped polyubiquitination and degradation of YopE and displayed increased intracellular YopE levels, which was accompanied by a pronounced cytotoxic effect on infected cells. Despite its intensified activity on cultured cells, the Yersinia mutant with stabilized YopE showed reduced dissemination into liver and spleen following enteral infection of mice. Furthermore, the accumulation of degradation-resistant YopE was accompanied by the diminished delivery of YopP and YopH into cultured, Yersinia-infected cells. A role of YopE in the regulation of Yop translocation has already been described. Our results imply that the inactivation of YopE by the proteasome could be a tool to ensure intermediate intracellular YopE levels, which may effectuate optimized Yop injection into host cells. In this regard, Y. enterocolitica O8 appears to exploit the host ubiquitin proteasome system to destabilize YopE and to fine-tune the activities of the Yop virulence arsenal on the infected host organism

Cytoskeletal elements of STRO-1⁺ DaMSCs and long-distance cell-to-cell connections (TNTs).

<p>(<b>a,b</b>) Different cell-to-cell connections between STRO-1⁺ cells. Intercellular connections are able to bridge long distances even across neighbouring cells (Phalloidin/Tubulin staining, merged images). (<b>b</b>) Thick tubes (diameter >0.4 µm, example is marked by red arrow) contain F-actin and α-tubulin, whereas thin tubes (diameter <0.4 µm, example is marked by green arrow) contain only F-actin. (<b>c</b>) Negative control, staining without Tubulin antibody. (<b>d–f</b>) Scanning electron microscope pictures (SEM) of a mixed culture of antlerogenic cells. Cells form long connections across neighbouring cells (<b>d,e</b>) and very high magnification (<b>f</b>) proves that the surface of TNTs exhibit small appendages; long-distance connections are also possible between morphologically distinguishable cell types (<b>d</b>). (<b>h–m</b>) Multichannel pictures of Phalloidin (<b>h,k</b>) and Tubulin (<b>i,l</b>) stained DaMSCs demonstrate that their intercellular connections continuously consist of F-actin but only partially of microtubules. The visible spindle apparatus (<b>i</b>, detail enlargement) point to an initiating cell division and provides evidence that the used α-tubulin antibody is also efficient in DaMSCs. (<b>j,m</b>). Merged Images.</p

Transport of Oct4 fusion protein via cytoplasmic connections.

<p>(<b>a–d</b>) Time series pictures of a transfected STRO-1⁺ DaMSC. The GFP fluorescence of the Oct4 fusion protein is visible within the cytoplasm as well as inside the membrane tubes. Membrane dilatations (gondolas) filled with Oct4 fusion proteins are moving away from the cellular body. Starting points are marked with white dashed lines. Scale bar = 20 µm.</p

Mixed culture of Oct4-GFP MEF cells and STRO-1⁺ DaMSCs.

<p>(<b>a</b>) Colony of Oct4-GFP MEF cells without any GFP expression. (<b>b</b>) Individual Oct4-GFP MEF cell after 24 hours of pre-cultivation in stem cell expansion medium on the eve of co-cultivation. (<b>c</b>) The same Oct4-GFP MEF cell after 24 hours of co-cultivation with STRO-1⁺ DaMSCs (interacting DaMSCs are outside of the display window). GFP expression (pseudo-coloured green) is visible within the cytoplasm. (<b>d</b>) Mixed culture after 96 hours of co-cultivation. More GFP⁺ cells interacting with GFP⁻ cells are visible. (<b>e</b>) Cell with distinct GFP expression after 120 hours of co-cultivation. At that time GFP⁺ cells interact continuously with GFP⁻ cells and are well integrated into the forming multilayer. (<b>f</b>) After 144 hours of co-cultivation some cells exhibit widely distributed intracellular GFP expression. (a,b,c,f = phase contrast pictures; d,e = varel contrast pictures; a–e = pictures of living cultures, f = fixed cells).</p

RT-PCR analyses of Oct4 expression in STRO-1⁺ cells.

<p>STRO-1⁺ cells derived from cultured pedicle periost (pp) of regenerating fallow deer antlers, growth zone (gz) of regenerating red deer antlers and human bone marrow (bm) from iliac crest biopsies were used as source for cDNA. PCR on these samples yielded bands representing the 3′-end of exon1 (335 bp). The bands were extracted and sequenced, demonstrating a very high homology of Oct4 of deer and human origin.</p