INTRODUCTIONIn order to crawl forward, a cell must protrude the leadingregions of its margin over the substratum and eithersimultaneously or asynchronously retract its trailing regions.To understand how the cell does this, we need to know howprotrusions are formed. Protrusions can take the form offilopodia, lamellipodia or blebs, and lamellipodia mayrepresent an intermediate state between filopodia and blebs.The leading margin of cells has long been known to be a siteof actin filament assembly (Svitkina et al., 1986; Wang,1985), and numerous studies have demonstrated that thepolymerisation of actin can drive protrusion (see reviews byCondeelis, 1993; Mitchison and Cramer, 1996; Mogilner andOster, 1996; Small et al., 1993).In view of this evidence that actin polymerisation isimportant for protrusion and motility, it might be expected thatoverexpression of actin would increase cell motility. ThemRNA and protein for the β-actin isoform are known tolocalise to the cell periphery and this isoform is therefore morelikely than γ-actin to affect motility (Hoock et al., 1991; Hilland Gunning, 1993; Kislauskis et al., 1993; Kislauskis et al.,1997). However, although overexpression of β-actin has beenshown to have a marked effect on myoblast morphology(Schevzov et al., 1992), its effect on cell motility has notpreviously been investigated. Here we have used theDRIMAPS (Digitally Recorded Interference Microscopy withAutomatic Phase Shifting) system of computer-assistedmicrointerferometry (Dunn and Zicha, 1995) to analyse thespeed, spreading and rates of protrusion and retraction ofmyoblasts overexpressing β-actin. This was complemented byfluorescence microscopy to localise β-actin and polymerisedactin in fixed cells. In order to investigate further the role ofactin polymerisation, we analysed the locomotion of myoblaststhat overexpressed β*-actin, a mutant β-actin (G244D) that isknown to be defective in its ability to polymerise in vitro(Leavitt and Kakunaga, 1980; Millonig et al., 1988; Taniguchiet al., 1988). We also measured how the expression levels ofseveral actin binding proteins were affected by overexpressionof β- or β*-actin; these include three that are known to1367Overexpression of β-actin is known to alter cellmorphology, though its effect on cell motility has not beendocumented previously. Here we show that overexpressingβ-actin in myoblasts has striking effects on motility,increasing cell speed to almost double that of control cells.This occurs by increasing the areas of protrusion andretraction and is accompanied by raised levels of β-actin inthe newly protruded regions. These regions of the cellmargin, however, show decreased levels of polymerisedactin, indicating that protrusion can outpace the rate ofactin polymerisation in these cells. Moreover, theexpression of β*-actin (a G244D mutant, which showsdefective polymerisation in vitro) is equally effective atincreasing speed and protrusion. Concomitant changes inactin binding proteins show no evidence of a consistentmechanism for increasing the rate of actin polymerisationin these actin overexpressing cells. The increase in motilityis confined to poorly spread cells in both cases and theexcess motility can be abolished by blocking myosinfunction with butanedione monoxime (BDM). Our observations on normal myoblasts are consistentwith the view that they protrude by the assembly and crosslinking of actin filaments. In contrast, the additionalmotility shown by cells overexpressing β-actin appearsnot to result from an increase in the rate of actinpolymerisation but to depend on myosin function. Thissuggests that the additional protrusion arises from adifferent mechanism. We discuss the possibility that it isrelated to retraction-induced protrusion in fibroblasts. Inthis phenomenon, a wave of increased protrusion follows asudden collapse in cell spreading. This view could explainwhy it is only the additional motility that depends onspreading, and has implications for understanding thedifferences in locomotion that distinguish tissue cells fromhighly invasive cell types such as leucocytes and malignantcells.Key words: Cell movement, Cell polarity, Cell adhesion, Interferencemicroscopy, MicrofilamentSUMMARYSpecific changes to the mechanism of cell locomotioninduced by overexpression of β-actinMichelle Peckham1,*, Gaynor Miller1, Claire Wells2, Daniel Zicha3and Graham A. Dunn41School of Biomedical Sciences, Worsley Building, University of Leeds, Leeds LS2 9JT, UK2Ludwig Institute for Cancer Research, Royal Free and University College Medical School Branch, Courtauld Building, 91 Ridinghouse Street,London, W1P 8BT, USA3Imperial Cancer Research Fund, Light Microscopy, 44 Lincoln’s Inn, London, WC2A 3PX, USA4MRC Muscle and Cell Motility Unit, The Randall Centre, New Hunt’s House, Guy’s Campus, London SE1 1UL, UK*Author for correspondence (e-mail: [email protected])Accepted 20 January 2001Journal of Cell Science 114, 000-000 © The Company of Biologists LtdRESEARCH ARTICLE1368influence actin polymerisation: β-thymosin, profilin and ADF/cofilin. The role of myosin in protrusion is more problematical. Thelarge family of myosin isoforms presents a complex array ofsingle and double-headed motor proteins that must havecountless interactions, motor functions and transport functionswithin the moving cell. The best studied of these functions isthe interaction of aggregates of non-muscle myosin II withactin filaments in meshworks to produce contraction. Oneresult of a generalised contraction of the actomyosin meshworkpervading the cell body is to set up a pressure gradient thattends to force fluid flow towards weaker regions of the actincortex. The blebbing mode of protrusion is thought to occur inthis way (Keller and Eggli, 1998; Stossel et al., 1999) and canbe prevented by counterbalancing the internal hydrostaticpressure of the cell using reverse osmotic pressure (Harris,1973). It is known that actin polymerisation is not needed forthe initial formation of blebs (Cunningham, 1995; Stossel etal., 1999) but that filaments form only when the bleb isbeginning to collapse. Blebbing tends to be the main mode of locomotion in rapidlymoving, invasive types of cells that show poor adhesion to thesubstratum. Most normal tissue cells, on the other hand, moveby extending lamellipodia. Despite the evidence that myosin isinvolved in the protrusion of blebs, there is little evidence thatit is needed for other types of protrusion.