A Talin Fragment As An Actin Trap Visualizing Actin Flow in Chemotaxis, Endocytosis, and Cytokinesis Igor Weber, Jens Niewo ¨ hner, Aiping Du, Ursula Ro ¨ hrig, and Gu ¨ nther Gerisch * Max-Planck-Institut fu ¨ r Biochemie, D-82152 Martinsried, Germany A C-terminal 63-kDa fragment of talin A from Dictyostelium discoideum forms a slowly dissociating complex with F-actin in vitro. This talin fragment (TalC63) has been tagged with GFP and used as a trap for actin filaments in chemotactic cell movement, endocytosis, and mitotic cell division. TalC63 efficiently sequesters actin filaments in vivo. Its translocation reflects the direction and efficiency of an actin flow. Along the body of a migrating Dictyostelium cell, this flow is directed from the front to the tail. If during chemotaxis one or two new fronts are induced, the flow is always directed away from these fronts. The flow thus reflects the re-programming of cell polarity in response to changing gradients of chemoat- tractant. In endocytosis, the fluorescent complexes are translocated to the base of a phagocytic or macropinocytic cup. During mitosis, the complexes of F-actin with TalC63 accumulate within the midzone of anaphase cells. If TalC63 is strongly expressed, the entire cleavage furrow is filled out by sequestered actin filaments, and cytokinesis is severely impaired. These cells are considered to mimic the phenotype of mutants deficient in the shredding of actin filaments that normally occurs in the mid-zone of a dividing cell. Cell Motil. Cytoskeleton 53: 136 –149, 2002. © 2002 Wiley-Liss, Inc. Key words: actin flow; cleavage furrow; Dictyostelium; green fluorescent protein; mitosis INTRODUCTION Actin distribution in a living cell reflects a momen- tary state resulting from the spatial patterns of actin polymerization, F-actin translocation, and disassembly. There is ample evidence for the polymerization of actin at the leading edge of a motile cell [for review see Small et al., 2002], and for a retrograde flow of actin associated with cell motility [reviewed by Fukui, 1993; Grebecki, 1994; Mitchison and Cramer, 1996]. In fibroblasts, the actin flow ends in the perinuclear area; in small cells, such as neutrophils and Dictyostelium cells, the flow proceeds down to the tail region. Directed actin flows are also intrinsic to cytokinesis, and they are essential for cell-surface capping [Bray and White, 1988]. Direct and indirect methods have been used to probe for actin flows. Evidence for a retrograde flow in cell migration and cytokinesis has been provided by the translocation of crosslinked proteins on the cell surface [Koppel et al., 1982; Weber et al., 1999]. These proteins exist in a diffusible state and in a state coupled to the sub-membraneous actin layer. The transmembrane cou- pling is promoted by crosslinking antibodies, lectins, or beads, and is necessary for the retrograde-directed trans- location of membrane proteins [Sheetz et al., 1989; Schmidt et al., 1993; Felsenfeld et al., 1996]. In an adherent cell, adhesion proteins couple to the actin flow and thus transmit force onto the substrate, driving the cell forward [Harris, 1994]. Abbreviations used: GFP = green fluorescent protein; TalC63 = 63 kDa C-terminal talin A fragment. Contract grant sponsor: Deutsche Forschungsgemeinschaft; Contract grant number: SFB 413/A8. *Correspondence to: Gu ¨nther Gerisch, Max-Planck-Institut fu ¨r Bio- chemie, D-82152 Martinsried, Germany. E-mail: gerisch@biochem.mpg.de Received 21 March 2002; Accepted 8 May 2002 Published online 12 August 2002 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/cm.10065 Cell Motility and the Cytoskeleton 53:136 –149 (2002) © 2002 Wiley-Liss, Inc.