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Kidney International, Vol. 41 (1992), pp. 665—670

Moesin, a new cytoskeletal protein and constituent of filopodia: Its role in cellular functions

HEINZ FURTHMAYR, WOLFGANG LANKES, and MANUEL AMIEVA

Department of Pathology, Stanford University Medical Center, Stanford, California, USA

Cell motility, cell attachment and cell-cell interaction arecrombie et al [8, 9], to the analysis of specialized cell attach- basic cellular processes that are of fundamental importancement sites with interference reflection microscopy [10], by during early development, injury and repair responses or in theimmunoelectron and light microscopy, of cell shape by scan- progression of tumors to metastatic disease. Cell movementsning electron microscopy [11, 12], it is clear that much has been often occur over large distances and the cells utilize trackslearned, but these complex processes are still not understood. (substrates) that are frequently modified, such as addition of galactose to laminin by cell surface galactosyl transferase [1] or Moesin is a member of a new family of cytoskeletal proteins proteolysis [2]. The invasion of developing tissues by other cell We have recently cloned and sequenced the complete cDNA types, for example, mesenchymal cells migrating into the ure-of a cellular protein with binding activity for heparin and teral bud [3], presumably involves an invasive phase thatheparan sulfate [13, 14]. We have termed this protein moesin includes: cell-matrix and cell-cell recognition phenomena, cell(membrane organizing extension spike protein, pronounced locomotion, and tissue degradation; a positioning phase of the[moe.ez.in]). It is a 78 kd molecule that neither contains a signal cell, in which stable interactions are formed that presumablysequence, nor a transmembrane domain, suggesting that it is have been intiated as a result of specific and appropriatenot incorporated into the plasma membrane in the classical cell-matrix and cell-cell contacts; and finally, a phase in whichway. Its amino acid sequence is unique, but it shares sequence the cells grow, differentiate and become polarized. Short-termidentity with three other proteins: 72% with ezrin [15—18], 37% movements often occur in development during epithelial-mes-with an N-terminal region of band 4.1 [19], and 23% with a enchymal transformations and cell rearrangements in the earlydomain of talin [20]. The structural relationship with these three embryo. These cells change their morphology from that of acytoskeletal proteins suggests that moesin is a cytoskeletal quiescent epithelium (polarized, stable contacts at the basalcomponent (Fig. 1). The structure contains clustered basic side and between cells) to a motile cell that extricates itself fromresidues. Such clusters have been found in other proteins that the epithelial cell layer [4]. These cells change shape, developbind heparin, such as some of the clotting factors [21]. They are cell processes such as filopodia, microspikes, blebs, etc., andalso seen in proteins that translocate into the nucleus (such as eventually begin to migrate [5]. It is tempting to speculate thatbFGF, [22]). This raises the question regarding the cellular the cellular protrusions in these situations serve to probe thelocalization of moesin. environment and/or the extracellular matrix (ECM), for attach- ment sites and other ligands [6]. In support of this idea are a Cellular localization of nmesin number of examples from different cell systems, in which We prepared polyclonal antibodies to moesin and ezrin, since receptors have been found to be concentrated in filopodia [7].the original monoclonal antibody [14] exhibited poor tissue and These complex developmentally regulated phenomena alsocell staining. Our preliminary data indicate that cells in culture seem to signal transitions of cells from one state to another andcannot be stained in immunofluorescence experiments with the are transient in nature. antibodies to moesin or ezrin, unless the cells are permeabi- Another way of looking at these phenomena is to establishlized. We have used primary cultures of endothelial (rat liver, cells in culture and to analyze their behavior in response tohuman skin, mouse brain, human aorta), calf smooth muscle obviously different conditions, such as tissue culture plastic,cells, human keratinocytes, rat astrocytes, human blood lym- growth media, and various stimuli:chemotactic factors, growthphocytes and granulocytes, and rat liver parenchymal, Kupfer factors, biological or other substrates, cytokines, etc. A fairlyand Ito cells for these studies. We have also used cell lines large body of information has been compiled of these attempts(HL6O, A43 1, T and B lymphocytes, intestinal and renal tubular to not only establish often highly differentiated cells in culture,cell lines: T84, HT-29, MDCK, LLC-PK1) as well as virus- but also to understand some of the principal mechanismstransformed mouse endothelial and rat glioma cell lines (RT-2). controlling cell behavior. Beginning with the description ofIn methanol or acetone fixed and permeabilized cells, a remark- various cellular elements involved in cell movement by Aber-able staining pattern is observed: in most of all cells grown in tissue culture the antibodies to moesin and/or ezrin visualize slender cell processes that vary in length depending on the cell © 1992 by the International Society of Nephrology type and the culture conditions. In calcium depleted cultures of

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Fig. 1.Secondary structure prediction based onthe amino acid sequence of protein 4.1, ezrin, moesin and talin. These proteins are members of a new family and share a homologous N-terminal domain that has been postulated to mediate the interaction with binding sites on the cytoplasmic face of the plasma membrane. The central rod-like domain has an a-helical structure and may be involved in binding to other cytoskeletal coipoin II proteins. The C-terminal small domains are mentrane rttnt]èfbfl structurally different from each other, with the (protein) deciage binding affachrnerit s4e exception of moesin and ezrin, which are closely related.

LLC-PK1 cells, for instance, they can be several cell diametersthese structures of vastly differing morphology are not at all in length [23].Inaddition to these slender structures, otherclear and their molecular structure is largely unknown. types of cell processes are stained (Fig. 2). They vary somewhat It is known from many different studies that the elaboration from one cell type to another. From the staining patterns it isof lamellopodia or the so-called leading edge is an extremely clear that moesin and/or ezrin are located within these cellularrapid event that can be clearly related to cell movement [6] projections. Frequently, moesin and ezrin are co-localized inand/or cell attachment [25]. But non-moving cells also exhibit the same cells and the same filopodia. There are, however, aconsiderable surface activity. This activity refers to the protru- few examples of cells that, within our limits of detection,sion of microspikes, blebs, filopodia and related surface struc- contain only one or the other protein, for example, humantures. This surface activity is not only localized to the part of granulocytes or rat liver cells contain moesin only, while humanthe cell in contact with the substrate, but it is seen over the T84 cells or chicken erythrocytes contain only ezrin. entire surface of the cell. Significantly, it is also seen in cells In trying to define the role of moesin, we were intrigued bythat grow in suspension. Cells are thus able to produce a variety the complexity and relative lack of clear cut definitions of theof different and rapidly "assembled" surface structures. These various terms that have been used in the literature to describehave also been termed "motile organs" [20]. The relationship of cell behavior and cell morphology. We have compiled namesthe lamellipod, a broad sheet of cytoplasm that forms the here for surface extensions found in the literature (referencesleading edge of moving cells, devoid of cell organelles and have not been included): composed almost exclusively of actin, to the other types of cell protrusions is also not clear. It is interesting, however, that the filopodia, microvilli, brushborder, microspikes, pro- thickness of the lamellopodia is the same as the diameter of jections, protrusions, lamellopodia, rosette, lobopo- filopodia. These other cell protrusions are frequently said to dia, invadopodia, extensions, uropod, pseudopod, contain actin, but the data are not precise on this point [12]. trailing edge, leading edge, ruffling edge, retraction Other molecules, such as myosin, tubulin and a host of addi- fiber, podosome, peripheral hyaline blebs, blebs, mi- tional proteins clearly play a role in motility, but they seem to crocolliculi, ruffle, cytekinoplast, actinoplast, be excluded from filopodia [26]. Changes in cell surface mor- Although each of these descriptive terms refers to structuresphology during the cell cycle are striking in Chinese hamster observed in specific cells and under specific conditions, they areovary cells [11] and certain types of protrusions predominate also used interchangeably to describe similar cellular struc-over others. Filopodial activity can be stimulated by cytokines, tures. This has been done often in the face of clear cut evidencesuch as the autocrine motility factor (AMF) [27] or scatter of discrete differences among these surface structures. Onefactor [28]. case in point is the term microvillus, which has definitive Lamellopodia, filopodia and other cell surface protrusions meaning in the context of the highly organized brush border ofclearly mark active areas of the cell surface. This is in contrast the intestinal or renal tubular epithelium with its particularto stable cell-substrate and cell-cell adhesions described as content of fimbrin, actin, villin, etc. [24]. But the term is alsoclose contacts, focal adhesions, ECM adhesions, or adherens used for other cell protrusions found on many different celljunctions. While the former, in particular filopodia, micro- types that never produce a highly organized brush border, andspikes, and other membraneous structures are rapidly forming that presumably do not contain the same set of cytoskeletaland retracting, the stable contact sites usually do not change proteins. There are obvious differences in the appearance ofunless the cell is stimulated to alter its present state, such as, to these extensions of the cell surface in different cells as demon-move [9]. It has been said that filopodia and other highly active strated with antibodies to moesin. The relationship betweenareas of the cell surface are domains of the cell in which actin Furthmayr et a!: Moesin S667

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Fig.2. Cellular localization of moesin. LLC-PK1 (a,b) and calf aortic smooth cells (c,d) were washed with phosphate buffer, treated with acetone at —20°C and stained with rabbit anti-moesin and secondary FITC-labelled antibodies. In a, the cells were counter stained with propidium iodide; in d double-staining was done with anti-vimentin and Texas red-labelled secondary antibodies. Moesin (green fluorescence) is located in cell surface projections that vary in shape, dimension and number. polymerization and depolymerization takes place [29]. Thissurface, where they occur, mark future ruffles and focal contact might suggest that actin polymerization is responsible for thesites [25]. Since moesin is localized in all of the various surface growth and the protrusion of the plasma membrane; electronprojections, it is reasonable to postulate that it may be involved microscopic evidence suggests, in fact, that organized microfil-in the initial protrusion, and also in the later events of convert- ament bundles are found in at least some, but not all filopodiaing a transient into a more permanent structure. However, [12]. These data do not necessarily address the questionmoesin could also serve other functions. whether actin polymerization provides the required protrusive Abercrombie et al. observed flow of the plasma membrane force rather than utilizing a previously formed structure. Bio-away from the leading edge and in a direction opposite to the physical arguments indicate that the speed at which filopodiamoving cell [8, 91.Theysuggested in essence that membrane is extend and disappear is not consistent with a process of actinadsorbed in the back of the cell and then transported forward to polymerization and depolymerization because of diffusionalthe leading edge of the cell, These membrane transfer cycles limitations [30]. According to this idea, actin polymerizationwould enable the cell to move similar to a tank. A later and filamentous organization does not push the filopodia out-modification of this idea suggested endocytosis as the principal wards, but rather forms the more rigid supportive structuremechanism by which a moving cell could bring membrane later on that allows filopodia to keep their shape. Protrusionmaterial to the leading edge [6]. The endocytic vesicles move could be achieved by other forces, such as an osmotic force [26,through the cytoplasm and are inserted into the plasma mem- 30]. This would predict that cell surface structures lacking actinbrane by exocytosis at the leading front end of the motile cell. and possibly other cytoskeletal elements exist. It has beenThis "tank" analogy is inadequate, however, since, while the suggested that some of the protrusions or areas of the cellentire tank tread is carried along in its cycle, only some 668 Furthinayret al: Moesin

Fig. 3. Tentativestructural rolefor moesin in microspikes.Moesin(red) in the form of monomer or dimer interacts with binding sites on the inner surface of the plasma membrane. Such binding sites are provided by integrins, CAMs and possibly other structures. This organization may be sufficient for allowing the formation of transient filopodia or microspikes. "Maturation" may entail stabilization by additional interactions with actin and other cytoskeletal elements.

membrane components (the lipids and selected 'circulating' Function of filopodia and the relationship of moesin and/or proteins) participate in endocytosis. Interestingly, endocytosis ezrin to these structures is just as active in non-motile cells as it is in motile cells. The major difference is that exocytosis in the non-motile cell is not We have observed that many of the cell surface protrusions directed and that new membrane is returned randomly over thelabelled with antibodies to moesin are not stained with antibod- entire surface of the cells [61. The filopodial activity we haveies to other cytoskeletal proteins: actin, tubulin, intermediate observed in many of our cultured cells could be a reflection offilament proteins, band 4.1, fodrin (spectrin). This has also been this kind of activity. This may provide the basis for yet anotherobserved in migrating cells [1] expressing galactosyltransferase working hypothesis to explain the function of moesin: it couldin filopodia. First of all this suggests that these processes are be bound to receptors, it may serve as a tag for recyclingstructurally heterogeneous, and secondly, that many of them receptors, and travel with the endocytic vesicle. Filopodialmay represent transient structures that are constantly forming activity can be influenced by changes in pH, osmolarity,at a fairly rapid rate, and apparently do not require filamentous cytokines, and other factors such as contact between cells oractin for stabilization. The cell, upon stimulation, may use its certain extracellular matrix components [31]. Filopodia havefilopodial activity to assemble and concentrate receptors in another potentially critical function, namely, to contain in-certain domains of the plasma membrane. This process may creased numbers of certain receptors relative to the remainderallow attachment of one or more of such "arms" to the of the cell surface. substrate with sufficient avidity to initiate the assembly of more Stable adhesion sites are domains in the plasma membrane atpermanent or more structurally stable structures. It may then which integrins and other ECM receptors interact with cyto-be only the latter filopodia which contain actin filaments. skeletal proteins [32, 331: in focal adhesions talin provides theTrinkaus has made the distinction between unstructured and link via vinculin and alpha actinin with actin filaments; instructured protrusions [31]. Although a number of arguments desmosomes, intermediate filaments interact with proteins ofand hypotheses have been put forward to explain protrusion of the plasma membrane [34]; in adherens junctions of the liver,membrane in molecular terms, there is no agreement. These talin is absent, but it can be found in myotendineous junctionsarguments have been discussed by Trinkaus [31] and Oster [30]. and in the motor synapse. These data suggest that a variety ofOne of the critical questions addressed in the present article structural components are required for cells to generate dif-deals with the possibility of moesin and/or ezrin to organize and ferent stable connections. Filopodia and other cell protrusionsstabilize membrane receptors in such active and transient are usually not involved in making stable contacts. Filopodiamembrane regions. According to this idea, moesin interaction are said to contain receptors for extracellular ligands in signif-with the membrane and possibly with itself would precede the icantly (10- to 20-fold) higher concentrations in comparison topolymerization of actin and other cytoskeletal assemblies (Fig. other domains of the plasma membrane [7, 27]. In certain3). It thus marks "unstable" regions of the membrane, or very systems, such as in activated neutrophils, chemotactic recep-"active" regions, including those of endothelial cells that are tors, Fe receptors, or proteases are in fact concentrated on theactive in endocytosis/transcytosis. leading edge of the migrating cell as is moesin. Similar obser- The following points are supportive evidence for this idea. vations exist in dictyostelium after a chemotactic stimulus of I) Cells placed in culture respond quickly with the elabora- cAMP has been given [35]. tion of cell protrusions [36—38]; there may be a need for de novo Furthmayr et a!: Moesin 669 synthesis and/or transcription, if the particular cell type (suchtures. This could be consistent with their putative role in as a smooth muscle cell) does not make moesin/ezrin. migration and tissue organization. In the adult rat or mouse, 2) Many of these delicate moesin/ezrin-containing cell pro-moesin antibodies react predominantely with endothelial cells cesses cannot be stained in immunofluorescence experimentsin different vascular beds. This could be related to a particular with antibodies to a number of cytoskeletal proteins, includingcellular activity of these cells, such as a high endocytotic or fodrin, band 4.1, talin, intermediate filaments, actin, tubulin. transcytotic rate. However, co-localization in the adult animal 3) Processes are generated by the cells randomly, at leastis seen, for instance, in the brush border of some renal tubular initially, and they are not restricted to cell-matrix interactionsegments and in glomeruli. Furthermore, there are tissues or [36—40]. cell types in the adult that cannot be stained at all with either of 4) It has been suggested that the presence of microspikes at athe sera in tissues: smooth muscle cells in the intestine or in the particular location of a lamellopodium or ruffling edge markswall of blood vessels or some specialized endothelial cells. future contacts, in which stress fibers terminate [25, 291. 5) Some of the cellular processes containing moesin are very Regulation of the putative interaction of moesin with the long and thin, such as in calcium-depleted LLC-PK1 cells plasma membrane (compare with Fig. 2). They are 20 to 100 nm in diameter and When unstimulated A43 1 cells are treated with EGF, there is they contact neighboring cells. "Filopodia" in these cellsa rapid response of the cells within 30 seconds consisting of apparently do not contain actin and, therefore, they may not beruffling, the extension of filopodia and microspikes [32, 37]. The involved in moving, pulling or attracting cells to each other.response peaks between two and five minutes, and by 10 They have been termed retraction fibers. Why they exist andminutes the ruffling subsides, and this response is followed by why the cells apparently remain in contact with each otherretraction of the cells [compare with 37]. The early response remains unknown. In contrast, the same morphological appear-correlates with an increase in the phosphorylation of ezrin and ance under different conditions may suggest involvement in themoesin, but there was no indication for changes in transcription well-known phenomenon of contact inhibition of growth. or translation. It has been shown by Bretscher and Gould et a! 6) In culture, all cells appear to express moesin and/or ezrin.[16, 17] that ezrin is phosphorylated at tyrosine and threonine This finding is consistent with the filopodial activity observedresidues. Our preliminary data indicate a similar response with for every cell type, regardless of shape or whether the cells arerespect to moesin, It is tempting to speculate that the phosphor- attached to the culture dish. ylation of these proteins is related to the early and rapid 7) Filopodial activity is initiated by cells in culture verymorphological change, namely the extension of the filopodia, rapidly (within seconds) in response to chemotactic stimuli,and that the translocation of moesin/ezrin into the newly formed growth factors, TPA, etc. Ruffling ceases also fairly rapidly,filopodia is regulated by phosphorylation events. when cells are placed on certain substrata. We thus believe that moesin is involved in basic cellular Acknowledgments processes that are linked to cell recognition, motility, invasion, Our research data discussed in this article were supported by grants differentiation and cell growth. The rapidly assembled cellfrom the US Public Health Service (HF), a fellowship from the surface structures may be akin to environmental sensors in aBoehringer-Inge!heim Foundation (WL) and a stipend from Stanford's very general sense. Activity of this sort is controlled either byMSTP (MA). We also acknowledge collaborative efforts by J.F. Roll at the environment (permissive environment activates, non-per- UCSF. missive environment switches filopodial activity oft), or by an intrinsic gain or loss of mobility controlled in part by expression Reprint requests to Heinz Furthmayr, Department of Pathology, Stanford University Medical Center, Stanford, Cahfornia 94305-5324, of the moesin and/or ezrin gene or by regulatory events. One USA. would predict that mutation or loss of one or both of these genes could result in lethal conditions, because of their importance in References translating developmentally regulated events into appropriate cell behavior. 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