Filamin A promotes efficient migration and phagocytosis of neutrophil-like HL-60 cells.

The primary defense machinery to combat inflammation involves neutrophil granulocytes which in order to execute their functions rely on the efficiency of different cellular mechanisms including adhesion, spreading, migration in different environments, and phagocytosis. These functions require an accurately regulated actin network as well as the activation and adjustment of various signaling pathways. Mammalian filamins (FLNs) comprise three highly homologous large actin-binding proteins that are obvious candidates to control these processes as FLNs have been described to play a role in migration, spreading and adhesion in a variety of different cell types.

EB1 contributes to proper front-to-back polarity in neutrophil-like HL-60 cells.

Directed migration of leukocytes towards a chemotactic source is largely dependent on coordinated actin cytoskeleton functions that provide the driving forces at the cell front and enable contractility at the rear. In contrast to the force-generating properties of the actin cytoskeleton, the microtubule network assumes a regulatory function in balancing front-to-back polarity. In migrating neutrophils, microtubules are mostly concentrated at the cell rear, and previously published work suggested that microtubules are stabilized and kept in place by a mechanism involving Cdc42, WASP, CD11b, and the end-binding protein 1 (EB1).

Balanced cortical stiffness is important for efficient migration of Dictyostelium cells in confined environments.

Dictyostelium discoideum cells resemble in many aspects human leukocytes and serve as a model to study actin cytoskeleton dynamics and cell migration of highly motile cells. Dictyostelium cells deficient in the actin-binding protein filamin (ddFLN) showed a surprisingly subtle change in phenotype with no or only minor effects in single cell motility. These findings were in contrast to the strong actin-crosslinking activities measured for filamin in vitro.

Aberrant spindle dynamics and cytokinesis in Dictyostelium discoideum cells that lack glycogen synthase kinase 3.

Eukaryotic cell division requires the co-ordinated assembly and disassembly of the mitotic spindle, accurate chromosome segregation and temporal control of cytokinesis to generate two daughter cells. While the absolute details of these processes differ between organisms, there are evolutionarily conserved core components common to all eukaryotic cells, whose identification will reveal the key processes that control cell division. Glycogen synthase kinase 3 (GSK-3) is a major protein kinase found throughout the eukaryotes and regulates many processes, including cell differentiation, growth, motility and apoptosis.

A non-mitotic CENP-E homolog in Dictyostelium discoideum with slow motor activity.

Kinesins are ATP-dependent molecular motors that mediate unidirectional intracellular transport along microtubules. Dictyostelium discoideum has 13 different kinesin isoforms including two members of the kinesin-7 family, Kif4 and Kif11. While Kif4 is structurally and functionally related to centromere-associated CENP-E proteins involved in the transport of chromosomes to the poles during mitosis, the function of the unusually short CENP-E variant Kif11 is unclear.

Live cell-imaging techniques for analyses of microtubules in Dictyostelium.

Dictyostelium amoebae provide a popular model system for analyses of cell and cytoskeletal dynamics. Yet, the sensitivity of Dictyostelium cells to phototoxic effects, their rapid cell movement, and the extraordinary motility of their microtubule system are specific challenges for live cell imaging. The protocols outlined in this chapter are optimized to minimize these challenges, using Dictyostelium cells expressing green fluorescent tubulin or microtubule plus-end markers such as TACC.

Analysis of Dictyostelium TACC reveals differential interactions with CP224 and unusual dynamics of Dictyostelium microtubules.

We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores.

Dictyostelium Sun1 is a dynamic membrane protein of both nuclear membranes and required for centrosomal association with clustered centromeres.

Centrosomal attachment to nuclei is crucial for proper mitosis and nuclear positioning in various organisms, and generally involves Sun-family proteins located at the inner nuclear envelope. There is still no common scheme for the outer nuclear membrane proteins interacting with Sun1 in centrosome/nucleus attachment. Here we propose a model in which Sun1 mediates a physical link between centrosomes and clustered centromeres through both nuclear membranes in Dictyostelium.

Identification and cell cycle-dependent localization of nine novel, genuine centrosomal components in Dictyostelium discoideum.

The centrosome is the main microtubule-organizing center and constitutes the largest protein complex in a eukaryotic cell. The Dictyostelium centrosome is an established model for acentriolar centrosomes and it consists of a layered core structure surrounded by a so-called corona, which harbors microtubule nucleation complexes. We have identified 34 new centrosomal candidate proteins through mass spectrometrical analysis of the proteome of isolated Dictyostelium centrosomes.