Cell-cycle-dependent regulation of cell motility and determination of the role of Rac1 P.S. Walmod, a, * R. Hartmann-Petersen, a,1 S. Prag, a,2 E.L. Lepekhin, a,3 C. Ro ¨pke, b V. Berezin, a and E. Bock a a Protein Laboratory, Institute of Molecular Pathology, University of Copenhagen, Copenhagen, Denmark b Department of Medical Anatomy, University of Copenhagen, Copenhagen, Denmark Received 24 November 2003 Abstract To study cell motility in different phases of the cell cycle, time-lapse recording by computer-assisted microscopy of unsynchronised cells from three mammalian cell lines (L929, BT4Cn, HeLa) was used for the determination of the displacements of individual cells. The displacements were used for calculation of three key parameters describing cell motility: speed, persistence time and rate of diffusion. All investigated cell lines demonstrated a lower cell displacement in the G2 phase than in the G1/S phases. This was caused by a decrease in speed and/or persistence time. The decrease in motility was accompanied by changes in morphology reflecting the larger volume of cells in G2 than in G1. Furthermore, L-cells and HeLa-cells appeared to be less adherent in the G2 phase. Transfection of L-cells with constitutively active Rac1 led to a general increase in the speed and rate of diffusion in G2 to levels comparable to those of control cells in G1. In contrast, transfection with dominant-negative Rac1 reduced cell speed and resulted in cellular displacements, which were identical in G1 and G2. These observations indicate that migration of cultured cells is regulated in a cell-cycle-dependent manner, and that an enhancement of Rac1 activity is sufficient for a delay of the reduced cell displacement otherwise seen in G2. D 2004 Elsevier Inc. All rights reserved. Keywords: Actin; Cell cycle; Cell migration; Cell morphology; Cell motility; Cytoskeleton; GTPase; Rac1; Tubulin Introduction Regulated migration of mammalian cells plays a key role in several physiological processes. Morphogenesis during embryonic development is dependent on the movement of sheets of interacting cells as well as of the migration of single cells [1], and cell motility is also an integral part of wound healing and inflammation [2,3]. Tumour growth occurs as the result of deregulated cell cycle progression, but invasion and metastasis in addition require degradation of tissue by extracellular proteases and subsequent migra- tion of the malignant cells [4]. The cell cycle is traditionally divided into the G1 (gap1) phase, the S phase (synthesis phase during which the DNA is replicated), the G2 phase and the M phase (mitosis). Inter- estingly, the regulation of cell cycle progression, expression of extracellular proteases and cell motility share many signal transduction pathways. Thus, the mitogen-activated protein, MAP, -kinase pathway, which is activated by growth factors and integrin-mediated cell – substrate attachment, is one of the key regulators of cell proliferation [5]. Many extracellu- lar matrix metalloproteinases, MMPs, are regulated by the transcription factors c-Jun and c-Fos downstream of the MAP-kinase pathway [6], and the MAP-kinase pathway has also been demonstrated to regulate cell motility through the phosphorylation of the myosin light chain kinase, MLCK, by the MAP kinases termed the extracellular sig- nal-regulated kinases, ERK1 and ERK2 [7,8]. 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.01.011 * Corresponding author. Protein Laboratory, Panum Institute, Univer- sity of Copenhagen, Blegdamsvej 3C, Bld. 6.2, DK-2200 Copenhagen N, Denmark. Fax: +45-35-36-01-16. E-mail address: psw@plab.ku.dk (P.S. Walmod). 1 Present address: MRC Human Genetics Unit, Western General Hospital, Edinburgh, Scotland, UK. 2 Present address: Randall Centre, King’s College London, Guy’s Campus, London, UK. 3 Present address: Adherex Technologies Inc., Ottawa, Ontario, Canada. www.elsevier.com/locate/yexcr Experimental Cell Research 295 (2004) 407 – 420