The WAVE Regulatory Complex Is Required to Balance Protrusion and Adhesion in Migration

YesCells migrating over 2D substrates are required to polymerise actin at the leading edge to form lamellipodia protrusions and nascent adhesions to anchor the protrusion to the substrate. The major actin nucleator in lamellipodia formation is the Arp2/3 complex, which is activated by the WAVE regulatory complex (WRC). Using inducible Nckap1 floxed mouse embryonic fibroblasts (MEFs), we confirm that the WRC is required for lamellipodia formation, and importantly, for generating the retrograde flow of actin from the leading cell edge. The loss of NCKAP1 also affects cell spreading and focal adhesion dynamics. In the absence of lamellipodium, cells can become elongated and move with a single thin pseudopod, which appears devoid of N-WASP. This phenotype was more prevalent on collagen than fibronectin, where we observed an increase in migratory speed. Thus, 2D cell migration on collagen is less dependent on branched actin

A unique talin homologue with a villin headpiece-like domain is required for multicellular morphogenesis in Dictyostelium

AbstractMolecules involved in the interaction between the extracellular matrix, cell membrane and cytoskeleton are of central importance in morphogenesis. Talin is a large cytoskeletal protein with a modular structure consisting of an amino-terminal membrane-interacting domain, with sequence similarities to members of the band 4.1 family, and a carboxy-terminal region containing F-actin-binding and vinculin-binding domains [1,2]. It also interacts with the cytoplasmic tail of β integrins which, on the external face of the membrane, bind to extracellular matrix proteins [3]. The possible roles of talin in multicellular morphogenesis in development remain largely unexplored. In Dictyostelium, a eukaryotic microorganism capable of multicellular morphogenesis, a talin homologue (TALA) has previously been identified and shown to play an important role in cell-to-substrate adhesion and maintenance of normal elastic properties of the cell [4–6]. Here, we describe a second talin homologue (TALB) that is required for multicellular morphogenesis in the development of Dictyostelium. Unlike any other talin characterised to date, it contains an additional carboxy-terminal domain homologous to the villin headpiece

WASP restricts active Rac to maintain cells' front-rear polarization

YesEfficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.Cancer Research UK grants A15672, A24450, and multidisciplinary grant A20017

Curved Tails in Polymerization-Based Bacterial Motility

The curved actin ``comet-tail'' of the bacterium Listeria monocytogenes is a visually striking signature of actin polymerization-based motility. Similar actin tails are associated with Shigella flexneri, spotted-fever Rickettsiae, the Vaccinia virus, and vesicles and microspheres in related in vitro systems. We show that the torque required to produce the curvature in the tail can arise from randomly placed actin filaments pushing the bacterium or particle. We find that the curvature magnitude determines the number of actively pushing filaments, independent of viscosity and of the molecular details of force generation. The variation of the curvature with time can be used to infer the dynamics of actin filaments at the bacterial surface.Comment: 8 pages, 2 figures, Latex2

Atomic Force Microscopy of height fluctuations of fibroblast cells

We investigated the nanometer scale height fluctuations of 3T3 fibroblast cells with the atomic force microscope (AFM) under physiological conditions. Correlation between these fluctuations and lateral cellular motility can be observed. Fluctuations measured on leading edges appear to be predominantly related to actin polymerization-depolymerization processes. We found fast (5 Hz) pulsatory behavior with 1--2 nm amplitude on a cell with low motility showing emphasized structure of stress fibres. Myosin driven contractions of stress fibres are thought to induce this pulsation.Comment: 6 pages, 5 figures, 1 tabl

Phosphorylation of phospholipase-γ l, profilin and tyrosine

No abstract available

Actin-Based Protrusions: Promoters or Inhibitors of Cancer Invasion?

In a recent issue of Cell, Silva and colleagues reported the identification of CYFIP1, a member of the actin-assembly-promoting Scar/WAVE complex, as an invasion suppressor in epithelial cancers. This study challenges ideas about the role of actin in cancer invasion

Actin-bundling proteins in cancer progression at a glance

Cells use their cytoskeletons to move, polarise, divide and maintain organisation within multicellular tissues. Actin is a highly conserved essential building block of the cytoskeleton that forms cables and struts, which are constantly remodelled by more than 100 different actin-binding proteins. The initiation of new actin filaments and their subsequent organisation is a key step in the development of specialised cellular structures, such as filopodia (spike-like protrusions), lamellipodia (sheet-like protrusions), stress fibers (elastic contractile bundles), microvilli (finger-like surface protrusions) and invadopodia (invasive cell feet) (see Table 1 for a more complete list). Whereas the cytoskeleton is important in normal cellular function, it can be subverted in cancer cells and contributes to changes in cell growth, stiffness, movement and invasiveness. We hereby give an overview of the role of actin-filament bundling in cellular structures and discuss how alterations in the activity or expression patterns of actin-bundling proteins could be linked to cancer initiation or progression