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Tropomyosins Are Present in Lamellipodia of Motile Cells Louise Hillberga,1, Li-Sophie Zhao Rathjea,1, Maria Nyakern-Meazza( A, Brian Helfandb, Robert D

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Tropomyosins Are Present in Lamellipodia of Motile Cells Louise Hillberga,1, Li-Sophie Zhao Rathjea,1, Maria Nyakern-Meazza( A, Brian Helfandb, Robert D ARTICLE IN PRESS European Journal of Cell Biology 85 (2006) 399–409 www.elsevier.de/ejcb Tropomyosins are present in lamellipodia of motile cells Louise Hillberga,1, Li-Sophie Zhao Rathjea,1, Maria Nyakern-Meazza( a, Brian Helfandb, Robert D. Goldmanb, Clarence E. Schuttc, Uno Lindberga,Ã aDepartment of Cell Biology, The Wenner-Gren Institute, Stockholm University, SE-10691 Stockholm, Sweden bDepartment of Cell and Molecular Biology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611-3008, USA cDepartment of Chemistry, Princeton University, NJ 08544, USA Received 10 October 2005; received in revised form 21 December 2005; accepted 21 December 2005 Abstract This paper shows that high-molecular-weight tropomyosins (TMs), as well as shorter isoforms of this protein, are present in significant amounts in lamellipodia and filopodia of spreading normal and transformed cells. The presence of TM in these locales was ascertained by staining of cells with antibodies reacting with endogenous TMs and through the expression of hemaglutinin- and green fluorescent protein-tagged TM isoforms. The observations are contrary to recent reports suggesting the absence of TMs in regions,where polymerization of actin takes place, and indicate that the view of the role of TM in the formation of actin filaments needs to be significantly revised. r 2006 Elsevier GmbH. All rights reserved. Keywords: Arp2/3; Actin; Lamellipodia; Tropomyosin; VASP Introduction ization at the advancing cell edge is responsible for the formation of lamellipodial actin filaments (Wang, 1985), The actin microfilament system is involved in a and subsequent investigations have corroborated this multitude of processes essential for life. It is an energy- view and added detailed information about the dynamic transducing, force-generating system, contributing to the assembly/disassembly of the actin filament system shape and integrity of the mammalian cell, in addition to (Danuser and Waterman-Storer, 2003; Ponti et al., providing cells with a system required for dynamic motile 2003, 2004; Theriot and Mitchison, 1992). activities. Cells migrating on a solid substratum form Several protein complexes regulating actin polymer- lamellipodia and filopodia in the direction of movement. ization have been identified. One is the actin-nucleating These cell surface protrusions are built of actin filaments Arp2/3 complex. This consists of seven polypeptides with associated proteins (microfilaments). Photobleach- that are regulated by the Wiskott–Aldrich syndrome ing lamellipodia and filopodia of cells microinjected with protein (WASP), neural (N)-WASP and WASP family labeled actin has provided evidence that actin polymer- verprolin-homologous (WAVE) proteins known to relay signals from Cdc42 and Rac to the actin-nucleation machinery – the Arp2/3 complex. In this complex, ÃCorresponding author. E-mail address: [email protected] (U. Lindberg). growth of the nucleated actin filament occurs at the free 1Louise Hillberg and Li-Sophie Zhao Rathje contributed equally to (+)-end (barbed end) (Pollard and Borisy, 2003; Stradal the present investigation. et al., 2004). A second type of actin polymerizing protein 0171-9335/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2005.12.005 ARTICLE IN PRESS 400 L. Hillberg et al. / European Journal of Cell Biology 85 (2006) 399–409 is mDia, which belongs to the formin family of proteins In vitro, TM inhibits Arp2/3-dependent actin filament (Pring et al., 2003; Zigmond, 2004). Both of these actin branching (Blanchoin et al., 2001) and severing by filament assembly systems are present in lamellipodia cofilin (Ono and Ono, 2002). However, the extent to and therefore may contribute in varying degrees to the which Arp2/3-dependent branching and severing by formation of the 2–4 mm zone of densely packed, highly cofilin regulates actin filament assembly in vivo remains ordered microfilaments constituting the interior of unclear. The presence of TM in cellular domains lamellipodia (Hoglund et al., 1980; Small et al., 1982). containing these regulatory factors, such as the lamelli- A third type of actin assembly factor is Spire (Quinlan podium, could also modulate actin filament assembly. et al., 2005). This protein contains four formin We recently reported that TM can interact with even homology regions, nucleates actin filament assembly, the smallest gelsolin:actin complexes to cause the and remains associated with the (À)-end of the actin dissociation of the gelsolin and facilitating the annealing filament. It is expressed preferentially in the brain and its of the released product into long filaments (Nyakern- function is linked to vesicle transport. Meazza et al., 2002). Longer gelsolin-capped oligomers An important step in the control of actin filament of actin are also annealed into long filaments (Ishikawa formation in cells involves actin filament (+)-end- et al., 1989). Thus, TM might be involved in controlling capping proteins such as CP (Mejillano et al., 2004), the accessibility of the (+)-end of actin filaments for and the gelsolin family of proteins (Silacci et al., 2004). polymerization (uncapping the barbed end), and it could Increased actin filament formation in growth factor- also act as an assembly factor in the formation of stable stimulated cells requires removal of such capping actin filaments in the cell. There is evidence for the latter proteins. In Arp2/3-dependent actin filament formation, function in budding yeast (Evangelista et al., 2002). branching and de novo nucleation of filaments would Gelsolin is known to sever actin filaments and to bind also contribute to the appearance of increased numbers with high affinity to the barbed end of actin filaments in of actin filament growth points, as would severing of vitro. It is distributed throughout the cell, even into the existing filaments, by proteins like cofilin (Chan et al., most distal parts of the advancing lamellipodium 1998). The addition of actin monomers onto free (Hartwig et al., 1989, 1990), where the addition of actin filament (+)-ends created in this fashion is thought to monomers to growing actin filaments takes place be involved in ‘‘pushing’’ the cell membrane forward (Wang, 1985). It is still unclear, however, whether during the protrusion of lamellipodia and filopodia. gelsolin or other capping proteins (e.g. CP (Bear et al., Formins, on the other hand, are thought to remain at 2002)) control filament (+)-ends in the cell. In addition, the plus end of actin filaments and operate by inserting it has been shown that gelsolin at increasing concentra- actin monomers onto the actin filament (+)-end tions binds to TM-decorated actin filaments. At satura- (Higashida et al., 2004; Kappe et al., 2003; Pring tion a 2:2:14 ratio of gelsolin:TM:actin was reached. The et al., 2003). With both mechanisms, unpolymerized results indicated that gelsolin might also play a role in actin in the form of profilin-actin appears to serve as the controlling the activity of microfilaments in non-muscle precursor (Hajkova et al., 2000; Grenklo et al., 2003; cells (Nyakern-Meazza et al., 2002). Romero et al., 2004). These findings and the observation that TM plays an Among the many proteins involved in the regulation important role in the formation of actin cables in of the microfilament system in cells are tropomyosin budding yeast prompted the investigation to determine (TM) and gelsolin. TM is a major cell protein, whether TM is present in regions with active actin constituting more that 1% of the total protein of non- polymerization, such as the lamellipodia and filopodia muscle cells (Lin et al., 1985). In skeletal muscle, TM of mammalian cells. and troponin confer calcium sensitivity to the actomyo- sin-dependent force generation system. However, TMs are also present in non-muscle cells, but their function Materials and methods with respect to the microfilament system remains unknown. In mammalian non-muscle cells, there are Plasmid constructs numerous isoforms produced from multiple promoters and by alternative splicing of four TM genes. These Vectors containing HA-tagged rat non-muscle TM isoforms display a diversity of sorting within cells (for cDNA for 7 different isoforms were kindly provided by reviews see Carlier et al., 1997; Gunning et al., 1998; Dr. M. Gimona (Gimona et al., 1995). This collection Pittenger et al., 1994). TM is comprised of two a-helical included DNA sequences coding for high-molecular- chains, which form an elongated coil-coil dimer weight TM-1, TM-2, TM-3, and low-molecular-weight molecule with 5–7 actin-binding sites, depending on TM-4, TM-5(NM1), TM-5a, and TM-5b. the length of the particular isoform. TM molecules, one GFP-tagged constructs were made by PCR amplify- on each side of the actin helix are bound to each other ing TM cDNA from the HA-tagged constructs and end-to-end providing structural stability to the filament. cloning into pEGFP-C2 (Clontech). TM cDNA start ARTICLE IN PRESS L. Hillberg et al. / European Journal of Cell Biology 85 (2006) 399–409 401 codons were removed during the polymerase chain at 37 1C for 15 min and further incubated at room reaction. Each TM fusion protein carried the GFP temperature with 4% formaldehyde and 0.1% Triton sequence at its amino terminus. Constructs were verified X-100 for 5 min, unless otherwise stated. Slides were by DNA sequencing. rinsed with PBS several times after which they were The expression of the constructs was tested by SDS- incubated with primary antibodies for 60 min at room PAGE and Western blot analysis of cell extracts. For temperature and then rinsed again with PBS. Incubation this, antibodies to hemagglutinin and GFP were used, with secondary antibodies and labeled phalloidin was respectively. In each case the analysis showed one band carried out for 30–45 min. Slides were rinsed 3 times in of the expected size and no abrogated forms of the PBS and mounted in Prolong Antifade (Molecular tagged TMs.
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