Transcriptionally active chromatin loops contain both 'active' and 'inactive' histone modifications that exhibit exclusivity at the level of nucleosome clusters.

Chromatin state is thought to impart regulatory function to the underlying DNA sequence. This can be established through histone modifications and chromatin organisation, but exactly how these factors relate to one another to regulate gene expression is unclear. In this study, we have used super-resolution microscopy to image the Y loops of Drosophila melanogaster primary spermatocytes, which are enormous transcriptionally active chromatin fibres, each representing single transcription units that are individually resolvable in the nuclear interior.

The anatomy of transcriptionally active chromatin loops in Drosophila primary spermatocytes using super-resolution microscopy.

While the biochemistry of gene transcription has been well studied, our understanding of how this process is organised in 3D within the intact nucleus is less well understood. Here we investigate the structure of actively transcribed chromatin and the architecture of its interaction with active RNA polymerase. For this analysis, we have used super-resolution microscopy to image the Drosophila melanogaster Y loops which represent huge, several megabases long, single transcription units.

Synthetic Antibodies Detect Distinct Cellular States of Chromosome Passenger Complex Proteins.

High performance affinity reagents are essential tools to enable biologists to profile the cellular location and composition of macromolecular complexes undergoing dynamic reorganization. To support further development of such tools, we have assembled a high-throughput phage display pipeline to generate Fab-based affinity reagents that target different dynamic forms of a large macromolecular complex, using the Chromosomal Passenger Complex (CPC), as an example. The CPC is critical for the maintenance of chromosomal and cytoskeleton processes during cell division.

Induced Arp2/3 Complex Depletion Increases FMNL2/3 Formin Expression and Filopodia Formation.

The Arp2/3 complex generates branched actin filament networks operating in cell edge protrusion and vesicle trafficking. Here we employ a conditional knockout mouse model permitting tissue- or cell-type specific deletion of the murine gene (encoding Arp3). A functional gene appeared essential for fibroblast viability and growth.

Engineering the Drosophila Genome for Developmental Biology.

The recent development of transposon and CRISPR-Cas9-based tools for manipulating the fly genome in vivo promises tremendous progress in our ability to study developmental processes. Tools for introducing tags into genes at their endogenous genomic loci facilitate imaging or biochemistry approaches at the cellular or subcellular levels. Similarly, the ability to make specific alterations to the genome sequence allows much more precise genetic control to address questions of gene function.

FlyOde - a platform for community curation and interactive visualization of dynamic gene regulatory networks in Drosophila eye development.

Motivation: Understanding the regulatory mechanisms governing eye development of the model organism Drosophila melanogaster (D. m.) requires structured knowledge of the involved genes and proteins, their interactions, and dynamic expression patterns.

Requirements for and consequences of Rac-dependent protrusion.

Small GTPases of the Rac subfamily exert multiple functions, the most prominent of which includes stimulation of dynamic actin rearrangements at the cell periphery. Frequently, these actin reorganizations cause the protrusion of leaflets of plasma membrane, so-called lamellipodia, which remain anchored at flat surfaces during forward protrusion of migrating cells, or develop into ruffles when lifting up- and backwards. Ruffling membranes are also engaged in fluid and particle uptake during pino- and phagocytosis, respectively.

Arp2/3 complex is essential for actin network treadmilling as well as for targeting of capping protein and cofilin.

Lamellipodia are sheet-like protrusions formed during migration or phagocytosis and comprise a network of actin filaments. Filament formation in this network is initiated by nucleation/branching through the actin-related protein 2/3 (Arp2/3) complex downstream of its activator, suppressor of cAMP receptor/WASP-family verprolin homologous (Scar/WAVE), but the relative relevance of Arp2/3-mediated branching versus actin filament elongation is unknown. Here we use instantaneous interference with Arp2/3 complex function in live fibroblasts with established lamellipodia.

Actin branching in the initiation and maintenance of lamellipodia.

Using correlated live-cell imaging and electron tomography we found that actin branch junctions in protruding and treadmilling lamellipodia are not concentrated at the front as previously supposed, but link actin filament subsets in which there is a continuum of distances from a junction to the filament plus ends, for up to at least 1 μm. When branch sites were observed closely spaced on the same filament their separation was commonly a multiple of the actin helical repeat of 36 nm. Image averaging of branch junctions in the tomograms yielded a model for the in vivo branch at 2.

Cofilin cooperates with fascin to disassemble filopodial actin filaments.

Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown.

Microtubules as platforms for assaying actin polymerization in vivo.

The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol.

F- and G-actin concentrations in lamellipodia of moving cells.

Cells protrude by polymerizing monomeric (G) into polymeric (F) actin at the tip of the lamellipodium. Actin filaments are depolymerized towards the rear of the lamellipodium in a treadmilling process, thereby supplementing a G-actin pool for a new round of polymerization. In this scenario the concentrations of F- and G-actin are principal parameters, but have hitherto not been directly determined.

Unravelling the structure of the lamellipodium.

Summary Pushing at the cell front is the business of lamellipodia and understanding how lamellipodia function requires knowledge of their structural organization. Analysis of extracted, critical-point-dried cells by electron microscopy has led to a current dogma that the lamellipodium pushes as a branched array of actin filaments, with a branching angle of 70 degrees , defined by the Arp2/3 complex. Comparison of different preparative methods indicates that the critical-point-drying-replica technique introduces distortions into actin networks, such that crossing filaments may appear branched.

Differentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front.

Eukaryotic cells advance in phases of protrusion, pause and withdrawal. Protrusion occurs in lamellipodia, which are composed of diagonal networks of actin filaments, and withdrawal terminates with the formation of actin bundles parallel to the cell edge. Using correlated live-cell imaging and electron microscopy, we have shown that actin filaments in protruding lamellipodia subtend angles from 15-90 degrees to the front, and that transitions from protrusion to pause are associated with a proportional increase in filaments oriented more parallel to the cell edge.