A high resolution view of the fly actin cytoskeleton lacking a functional WAVE complex.

The development of multicellular organisms involves a series of morphogenetic processes coordinating a highly dynamic and organized interplay between cells and their environment. Thus, the generation of forces that drive cellular and intracellular movements is prerequisite to shape single cells into tissues and organs. The actin cytoskeleton represents a highly dynamic filamentous system providing cell structure and mechanical forces to drive membrane protrusion, cell migration and vesicle trafficking. Here, we apply the structured-illumination microscopy (SIM) technique to analyse the actin cytoskeleton in fixed Drosophila Schneider (S2R+) cells, both in wild type and in cells depleted for WAVE, a major activator of Arp2/3 mediated actin polymerization. In addition, we demonstrate that live cell SIM imaging also allows the visualization of actin-driven lamellipodial membrane dynamics at high spatial resolution in S2R+ cells. Three dimensional (3D) SIM images of up to 70 µm thick Drosophila wild-type and abi-mutant egg chambers further enables us to resolve changes of actin structures in a multicellular context with an impressive lateral and axial resolution, which is not possible with conventional confocal microscopy. Thus, the combination of superresolution 3D microscopy with Drosophila genetics and cell biology allows detailed insights into the structural and molecular requirements of different actin-dependent processes.

The E8 domain confers a novel long-distance transcriptional repression activity on the E8E2C protein of high-risk human papillomavirus type 31.

Infections with high-risk human papillomaviruses (HPVs) are the major risk factor for the development of anogenital cancers. Viral E2 proteins are involved in viral DNA replication and regulation of transcription. Repression of the viral P97 promoter by E2 proteins has been implicated in the modulation of the immortalization capacity and DNA replication properties of high-risk HPVs. Analysis of the cis and trans requirements for repression of the HPV type 31 (HPV31) P97 promoter, however, revealed striking differences between the full-length E2 and the E8E2C fusion protein which were due to conserved residues W6 and K7 of the E8 domain. In contrast to E2, E8E2C completely inhibited the P97 promoter from a single promoter-distal E2 binding site. This novel long-distance repression activity of the E8 domain also enabled E8E2C to inhibit the HPV6a P2 promoter and minimal-promoter constructs containing E2 binding sites. Thus, E8E2C may represent the master repressor of viral gene expression during a high-risk HPV infection, and changes in the activity of E8E2C might contribute to the progression of high-risk HPV-induced lesions.