Animal cell movement is effected through a combination of protrusive and contractile events. Non-muscle cells contain stress fibres – bundles of actomyosin that are the major mediators of cell contraction and that can be compared to the highly organised actomyosin arrays of muscle cells. Recent studies have defined regulatory mechanisms that control stress fibre formation, placing the ROCK protein kinase at the centre of a complex signalling network controlling actomyosin contractility and stress fibre assembly. As we uncover the details of stress fibre construction, it is becoming clear that different categories of stress fibres exist. Some of these structures are less suited for cell motility and more suited to static contraction. In keeping with this, many specialised contractile cell types use stress fibres to remodel tissues and extracellular matrix.

Eukaryotic cells produce a variety of specialized actin-rich surface protrusions. These include filopodia-thin, highly dynamic projections that help cells to sense their external environment. Filopodia consist of parallel filaments of actin, bundled by actin crosslinking proteins. The filaments are oriented with their rapidly growing "barbed" ends at the protruding tip and their slowly growing "pointed" ends at the base. Extension occurs by polymerization at the tip and is controlled by regulation of filament capping. The Rho GTPase Cdc42 is a key mediator of filopodia formation, which it regulates through binding CRIB domain-containing effectors. Cdc42 binds and activates the WASP proteins, which in turn activate the actin-nucleating complex Arp2/3. It also binds and activates IRSp53, which recruits the Ena/WASP family protein Mena to the filopodial tip and protects elongating actin filaments from capping. Previously, we identified another Rho family GTPase, Rif, as a potent stimulator of filopodial protrusion through a mechanism that does not require Cdc42. Here we characterize the differences between filopodia induced by these two small GTPases and show that the Rif effector in this pathway is the Diaphanous-related formin mDia2. Thus, Rif and Cdc42 represent two distinct routes to the induction of filopodia-producing structures with both shared and unique properties.

The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34 + cells. This pure population of immature erythroblasts can be expanded to obtain 4¥10 8 erythroblasts from 1¥10 8 PBMC after 13-14 days in culture. Upon synchronized differentiation, high levels of enucleation (80-90%) and low levels of cell death (<10%) are achieved. We compared the yield of erythroblasts obtained from PBMC, CD34+ cells or PBMC depleted of CD34 + cells and show that CD34 -cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available.

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