9Zellbiologie aktuell · 30. Jahrgang · Ausgabe 3/2004

Generation of directed movements atthe cellular and molecular levels andtheir physiological functionsMartin Bähler

One of the fascinating observations that canbe made by viewing living cells under a lightmicroscope is that cells are able to move on asubstrate and/or change their morphology.Also within cells, directed movements of orga-nelles and chromosomes can be seen. Withthe help of modern techniques in fluorescencemicroscopy directed movements of complexesof molecules and even single molecules can bevisualized in living cells. How these directedmovements are generated and how they arecontrolled is the subject of current research. Itis already well established that the dynamicorganization of the actin cytoskeleton andactin-dependent motor molecules contributeto a number of directed movements of cellsand within cells.

Dynamic organization and functionsof the actin cytoskeletonA large number of cellular activities such ase.g. cell migration, cell adhesion, cytokinesis,polarized cell growth and membrane traffi-cking are dependent on the actin cytoskele-ton. To understand the molecular mechanismsunderlying these actin- dependent processes,it is necessary to elucidate how the organisa-tion and dynamics of the actin cytoskeletonare regulated. The actin cytoskeleton consistsof a number of differently arranged arrays ofactin filaments (Fig. 1). Actin filaments arepolymerized from G-actin molecules in lamel-lipodia and filopodia at the cell periphery. The-se newly formed actin filaments are highlydynamic and turn-over relatively fast. It waspostulated that all actin filaments that are

found in cells have their origin in the lamelli-podia and small membrane ruffles (Small etal., 1998). Actin filaments are arranged in net-works (in lamellipodia), parallel bundles (infilopodia), antiparallel bundles (in stressfibers), peripheral convex and concave bundlesand geodesic arrays. Different sets of proteinsthat interact with G-actin or actin filamentscontrol the formation of functionally differentsubsets of actin filament arrays and endowthe actin cytoskeleton with different func-tions. Recently, we identified a novel subset ofloose actin filament arrays through regulatedassociation with the protein SWAP-70 (Hilpe-lä et al., 2003)(Fig. 2) . These loose actin fila-ment arrays are commonly located behindprotruding lamellipodia and membrane ruffles(Figs. 1 and 2). Lamellipodial protrusion andassociation of SWAP-70 with these loose actinfilament arrays are both dependent on phos-phoinositide 3-kinase activity. Currently, weare investigating how these actin filamentsare generated and what their function is.Current work addresses also the dynamics offocal contacts, cellular structures that med-iate the adhesion to the extracellular matrix.Focal contacts consist of multi-protein com-plexes that are connected to the actin cyto-skeleton (Fig. 1). They control cell migrationand morphogenesis and transduce signalsacross the cell membrane in both directions.These signals regulate the formation of extra-cellular matrix, cell division, cell proliferationand cell death. We are analysing the role(s) ofa newly discovered signaling molecule that isa component of focal contacts. An additional regulated actin-dependent pro-cess that we are investigating is macropinocy-tosis. Macropinocytosis allows cells to take upextracellular fluids and antigens (Fig. 1). Den-dritic cells of the immune system take up anti-gens by macropinocytosis (West et al., 2004).The internalized antigen is then processed andloaded on to MHC molecules and transportedback to the plasma membrane. The MHC-anti-gen complexes are presented on the dendriticcell surface to T-cells that then become acti-vated. Our aim is to unravel by which compo-

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Fig. 1: Schematic representation of the cellular organisation of the actin cytoskeleton. The front of a spreading or moving cell, the lamellipodium, consist of a dense network of actin filaments. Fine, finger-likeprotrusions extending beyond the leading edge are termed filopodia and contain parallel bundles of actinfilaments. Behind the extending lamellipodium in the so called lamella, a specific subset of loose actinfilament arrays can be visualized by staining of the protein SWAP-70 (see also Fig. 2). Stress fibers arethick contractile bundles of antiparallel actin filaments and myosin II that traverse the cell. They often terminate in focal contacts, a multiprotein complex linking the actin filaments to the extracellular matrix(enlargement on the right). Cells take up extracellular fluids by an actin-dependent process called macro-pinocytosis (enlargement on the left). Circular membane ruffles rich in actin filaments harbor upon closurefluid-filled intracellular vesicles, called macropinosomes. Substances taken up by macropinocytosis aresorted and processed by membrane fusion and fission events. Vesicles can be propelled through the cytosolby actin comet tails. A loose network of actin filaments can be found throughout the cell.

10 Zellbiologie aktuell · 30. Jahrgang · Ausgabe 3/2004

nents and mechanisms successive steps inmacropinocytosis like circular ruffle forma-tion, closure and pinching off of macropinoso-mes, „maturation“ and centripetal movementof macropinosomes are initiated and coordi-nated.

Myosin motor moleculesMyosin molecules represent a specific class ofactin-binding proteins. They convert chemicalenergy liberated by ATP-hydrolysis into direc-ted mechanical force along actin filaments. Alleukaryotic cells – not only muscle cells –express a number of different myosin molecu-

les. Based on sequence homology, myosinshave been subdivided into 18 classes (Berg etal., 2000). All myosins have in common a headdomain that contains a nucleotide- and actin-binding site, a light chain binding domain anda tail domain (Fig. 3). The light chain bindingdomains of different myosins differ in length,as they consist of a variable number of IQ-motifs that represent binding sites for theCa2+-sensor calmodulin or calmodulin-relat-ed EF-hand protein light chains. Myosinsmove by amplifying small structural changesin the core motor (head) domain via a leverarm rotation of the light chain binding

domain. The step size of myosins dependslinearly on the length of the lever arm (lightchain binding domain). However, myosin stepsizes are not only controlled by lever armlength, but also by substantial differences inthe degree of lever arm rotation (Köhler et al.,2003). Lever arm rotations between 30o and90o have been deduced for different myosins.The lever arm (light chain binding domain) isalso critically involved in determining thedirection of movement of a myosin along thepolar actin filament. Depending on the orien-tation of the light chain binding domain, amyosin moves towards the plus- or the minus-end of the actin filament (Tsiavaliaris et al.,2004). In addition to step size and direction ofmovement, myosins differ in several othermotor properties relevant to their particularphysiological functions such as e.g. speed ofmovement, amount of force produced, proces-sivity and regulation of motor activity. Myo-sins are called processive, when they are ableto take a number of successive steps along theactin filament without dissociating. As myo-sins go through their ATPase cycle they adoptdifferent nucleotide-binding states (no nucle-otide, ATP, ADP.Pi, ADP) that exhibit differentaffinities for F-actin. Many myosins, includingmuscle myosin, are not able to move conti-nously along actin filaments, because theyspend a large fraction of their ATPase cycletime in nucleotide-binding states that exhibitonly a weak affinity for F-actin. To achievecontinous movement of actin filaments, anensemble of these myosins is needed, so thatat any given time at least one of them is hol-ding on to the actin filament. If for example amyosin spends only 5% of the total cyclingtime attached to F-actin, at least 20 myosinsare needed so that at any given time one isholding on to F-actin. Members of class Vmyosins were the first myosins for which pro-cessive movement was demonstrated. In classV myosins the tail domains dimerize to form a

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Fig. 2: SWAP-70 co-localizes with a subset of loose actin filament arrays. Double fluorescence images of actin filaments (red) and SWAP-70 (green) in a B16/F1mouse melanoma cell.

Fig. 3: Schematic representation of selected myosin molecules. Myosins contain a head domain (orange),a light chain binding domain of variable length and different numbers of associated light chains (red) anda tail domain. Tail domains of class V myosins dimerize giving rise to a double-headed myosin. Class I andIX myosins are single-headed, because their tail domains do not form dimers. These tail domains containmotifs also found in other proteins (indicated in different colors).

11Zellbiologie aktuell · 30. Jahrgang · Ausgabe 3/2004

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myosin with two motor domains that „walk“processively in a hand over hand mechanismalong F-actin (Tyska and Mooseker, 2003). Thesingle-headed class IX myosin, called myosin9b (previously also named myr 5), is a single-headed myosin (Fig. 4). Although it has only asingle motor domain, it has been reported tobe a processive motor (Post et al., 2002, Inoueet al., 2002). Compared to other myosins,myosin 9b carries a large extension at the N-terminus of the head domain and a largeinsertion within its head domain (Fig. 4). Wehypothesized that the extension influencesnucleotide-binding and the insertion repre-sents a second actin-binding site tetheringmyosin 9b to actin. These ideas are currentlyunder investigation. At the moment, the direc-tionality of myosin 9b movement is controver-sial as it has been reported to be both a plus-and a minus-end directed motor (Inoue et al.,2002; O’Connell and Mooseker, 2003). We arecurrently trying to resolve this controversy.Myosin 9b is not only an actin-dependentmotor molecule, but exhibits also signalingactivity (Fig. 4). The tail domain of myosin 9bnegatively regulates the activity of the smallG-protein RhoC, that controls the cellularactin organization on to which the motordomain depends for its function (Reinhard etal., 1995; Müller et al., 1997; Graf et al.,2000). Active RhoC promotes cell adhesion tothe extracellular matrix and cell contractility(Burridge and Wennerberg, 2004). We specu-late that myosin 9b is targeted to regions ofactin polymerization in extending lamellipodiaby its motor domain. The myosin 9b tail

domain will thus keep the concentration ofactive Rho low in this region of the cell andthereby might prevent inappropriate adhesionand/or retraction and allow for continuousmovement of cells. In the future, we will try toverify this hypothesis.

References:Berg JS, Powell BC, Cheney RE (2001) A millennialmyosin census. Mol Biol Cell. 12(4):780-94.

Burridge, K. and Wennerberg, K. (2004) Rho and Ractake center stage. Cell 116: 167-179.

Graf, B., Bähler, M., Hilpelä, P., Böwe, C., and Adam, T.(2000)Functional role for the class IX myosin myr5 in epithe-lial cell infection by Shigella flexneri. Cell. Microbiol. 2:601-616.

Hilpelä, P., Oberbanscheidt, P., Hahne, P., Hund, M., Kal-hammer, G., Small, J.V., and Bähler, M. (2003)SWAP-70 identifies a transitional subset of actin fila-ments in motile cells. Mol. Biol. Cell 14:3242-3253.

Inoue, A., Saito, J., Ikebe, R. and Ikebe, M. (2002) Myo-sin IXb is a single-headed minus-end-directed proces-sive motor. Nature Cell Biol. 4: 302-306.

Köhler, D., Ruff, C., Meyhöfer, E., and Bähler, M. (2003)Different degrees of lever arm rotation control myosinstep size. J. Cell Biol. 161: 237-241.

Müller, R.T., Honnert, U., Reinhard, J. and Bähler, M.(1997) The myosin myr 5 acts as a RhoGAP in vivo.Essential role of arginine 1695. Mol. Biol. Cell 8:2039-2053.

O‘Connell, C.B. and Mooseker M.S. (2003) Native Myo-sin-IXb is a plus-, not a minus-end-directed motor.Nature Cell Biol. 5: 171-172

Post, P.L., Tyska, M.J., O‘Connell, C.B., Johung, K., Hay-ward, A. and Mooseker, M.S. (2002) Myosin-IXb Is aSingle-headed and Processive Motor J. Biol. Chem.277: 11679-11683.

Reinhard, J., Scheel, A.A., Diekmann, D., Hall, A., Rup-pert, C. and Bähler, M. (1995). A novel type of myosinimplicated in signalling by rho family GTPases. EMBO J.14: 697-704.

Small, J.V., Rottner, K., Kaverina, L., and Anderson, K.I.(1998) Assembling an actin cytoskeleton for cellattachment and movement. Biochim. Biophys. Acta1404: 271-281.

Tyska MJ, Mooseker MS. Myosin-V motility: theselevers were made for walking. Trends Cell Biol. 2003Sep;13(9):447-51.

Tsiavaliaris G, Fujita-Becker S, Manstein DJ. Molecularengineering of a backwards-moving myosin motor.Nature. 2004 Feb 5;427(6974):558-61.

West, M.A., Wallin, R.P.A., Matthews, S.P., Svensson,H.G., Zaru, R., Ljunggren, H.-G., Prescott, A.R. andWatts, C. (2004) Enhanced dendritic cell antigen cap-ture via Toll-like receptor-induced actin remodeling.Science 305: 1153-1157.

Anschrift des Verfassers:

Martin BählerInstitute for General Zoology and GeneticsWestfälische Wilhelms-UniversitySchlossplatz 548149 MünsterGermanyE-mail: baehler@uni-muenster.de

Fig. 4: Schematic representation of myosin 9b (myr 5). The motor domain produces directed movementsalong actin filaments and the GTPase-activating protein domain (GAP) negatively regulates the monome-ric GTPase Rho that in its active GTP-bound conformation resides in the membrane.