Tropomodulins: life at the slow end

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Tropomodulins: life at the slow end Robert S. Fischer and Velia M. Fowler Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA

Dynamic exchange of actin monomers at filament ends is crucial for the functional architecture of many cytoskeletal-dependent processes. Recent evidence indicates that tropomodulins (Tmods), a conserved family of actin-capping proteins that bind to the pointed (slow-growing) end of actin filaments, regulate a variety of actin structures, including dynamic actin networks found in some motile cells. Actin structures that are more stable, such as sarcomeric thin filaments, require capping by Tmods to specify filament lengths and to provide filament stability. Here, we discuss the functional differences between the capping of pointed and barbed ends within the context of these actin-filament systems, and how Tmods uniquely contribute to their regulation and organization. Regulation of actin-filament organization and dynamics in eukaryotic cells is important for a wide variety of biological events. In some cases, the architecture of actin filaments remains very stable and regular in organization, whereas in others it must turn over rapidly to produce motility [1,2]. These seemingly disparate structural and functional properties are achieved through the dynamic regulation of filament polymerization and depolymerization in cells. Because polymerization and depolymerization can occur only at filament ends, the regulation of free filament ends controls the dynamics and organization of filaments. Actin filaments have an intrinsic polarity – each with a fastgrowing (barbed) end and a slow-growing (pointed) end. To regulate the dynamics at these ends, capping proteins have evolved that specifically bind to either the barbed or the pointed ends of the filament, where they block the association and dissociation of monomers. Barbed ends, for which actin monomers have relatively high association and dissociation rate-constants, are regulated by proteins such as gelsolin and capping protein (CP) [3,4]. When active, these proteins cap the barbed ends tightly, with dissociation constant (Kd) values of , 5 nM. Pointed ends, for which monomers have significantly lower association and dissociation rate-constants, are capped by either the Arp2/3 complex or tropomodulins (Tmods) [1,3]. These proteins cap pointed ends comparatively weakly, with Kd values of , 100– 200 nM [5– 8]. However, the binding of either of these proteins to pointed ends can be highly enhanced by factors such as WASp and actin filaments, for Arp2/3 [9], or tropomyosin, for Tmod [7]. When potentiated by such factors, capping proteins of the pointed end cap with high affinity, with measured Kd values of Corresponding author: Robert S. Fischer ([email protected]).

, 0.01– 0.05 nM [8,10]. High-affinity binding of both Arp2/3 and Tmod to pointed ends of filaments can be downregulated to a low-affinity state by changes in the pointed ends that they occupy. In the case of Arp2/3, nucleotide hydrolysis and phosphate release by the pointedend actin subunits decreases its affinity for the pointed end by , 40-fold [5]. In the case of Tmod, changes in the registration or occupancy of tropomyosin at the pointed end can produce the low-affinity state [11,12] (Box 1). Given the greater polymerization and depolymerization rates at the barbed end, the kinetics of free barbed ends dominate the polymerization dynamics of actin in vitro [3]. In vivo, these differences in polymerization rates, relative to the affinities of their respective capping protein, probably create distinct functionalities within actin structures (Box 1). The barbed end, with its rapid polymerization and high affinity for capping protein, provides a mechanism for rapid filament-assembly and consequent changes in cell structure that can be regulated ‘on’ or ‘off ’. Meanwhile, the pointed end, with its slower polymerization and potential for weaker capping by Tmods, serves different structural functions, perhaps acting more as a rheostat, to fine tune actin structures. By learning about where and how Tmod proteins function, we can learn more about how the actin cytoskeleton uses the dynamics of the slow end to mediate specific remodeling processes and biological events associated with the actin cytoskeleton. Here, we discuss how Tmod capping regulates the organization of both stable and dynamic actin structures in cells. The Tmod family Genes encoding Tmod are present in a variety of metazoan species, from Drosophila melanogaster to Homo sapiens [13 –15]. In vertebrates, there are four canonical isoforms that are conserved across species. The genes for these isoforms all encode proteins of ,40 kDa, with two major domains – an N-terminal unstructured domain and a C-terminal folded domain consisting of five leucine-rich repeats (Figure 1). There are also two additional longer relatives of the canonical Tmods known as leiomodins or Lmods [14]. Tmods are found in a variety of tissues, some of which are listed in Table 1. While Tmods are generally expressed in many differentiated cell types, no Tmod isoforms have been found in fibroblasts. Tissue expression of Tmod2 and Tmod4 are highly restricted to neuronal and skeletal muscle tissues, respectively [14]. Conversely, Tmod1 and Tmod3 are expressed in many tissues and cell types [14,15]. The observed tissue specificity is intriguing because the isoforms are all ,60% identical and 70% similar in amino acid sequence [14,15]. Tmod2 and Tmod3

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Box 1. Actin capping at barbed and pointed ends: are they functionally different? Because of the intrinsic differences between polymerization and depolymerization rates of actin at the barbed and pointed ends of actin filaments, capping of the two ends might result in different effects, despite biochemical similarities between the caps. As an example, we consider capping protein (CP) at the barbed end and Tmod at the pointed end. In the case of CP, the dissociation constant (Kd) is , 0.1 – 0.8 nM, and CP has an on-rate of 3.0 mM21s21, which causes barbed ends to be rapidly capped at physiological levels of CP [21,55]. In fact, this predicts that nascent free ends would have a half-life of only , 0.1 s, during which time the end could polymerize , 300 nm [21,55]. Conversely, CP has a slow off-rate (, 4 £ 1024 s21), such that the half-life for a capped end is , 28 min. Thus, once barbed ends have been capped by CP they are essentially ‘off’ with respect to their actin dynamics. The binding of phosphatidylinositol (4,5)-bisphosphate (PIP2) to CP lowers the affinity, perhaps 100 to 1000-fold [53], such that, at saturating levels of PIP2, CP has essentially no effect on the polymerization of actin [55]. In this low-affinity state, with an estimated off-rate of 0.015 s21, the contribution of CP capping is minimal in the context of the rapid polymerization kinetics of barbed ends (actin monomer on-rate , 10 –11 mM21s21; [3]). Hence, when CP is inhibited by PIP2 it equates to an ‘on’ state with respect to actin dynamics. Other barbed-end capping proteins, such as gelsolin, can likewise be downregulated in their affinity for barbed ends by PIP2 binding [84] (see Figure I). A different situation occurs with the capping of pointed-ends, where high- or low-affinity states can exist for Tmod. When filaments are copolymerized with saturating amounts of tropomyosin, Tmod binding to the pointed ends is in a high-affinity state. In this state, Tmod caps

Barbed end

have a Kd value of 0.05 nM and a low off-rate of , 5 £ 1024 s21 [8], similar to CP at the barbed end. The low-affinity binding state for Tmod can be achieved in one of two situations. First, the actin filament might have no tropomyosin, which creates a low-affinity site for Tmod that caps these sites with a Kd of , 100 –200 nM [6,8]. Alternatively, for filaments that are copolymerized with tropomyosin, when the Tmod comes off, loss or gain of actin monomers from this end converts it to a low-affinity site, because the N-terminus of tropomyosin is no longer aligned with the actin subunits at the end of the filament [8]. In this low-affinity state, the Tmod has an off-rate of , 3 s21 (calculated from the Kd, assuming a diffusion-limited on-rate, although the actual off-rate could be lower [8]). Although this represents a relatively weak cap, at physiological concentrations (e.g. , 0.5 mM in endothelial cells [38]) transient capping by Tmod is still able to lower dynamics of pointed ends in cells because of the intrinsically slower kinetics of polymerization and depolymerization at pointed ends3. Furthermore, transient capping by Tmod in this state decreases the polymerization of pointed ends by slowing monomer addition, which allows for increased nucleotide hydrolysis, which in turn further increases the critical concentration for addition at such pointed ends [8]. Thus, by capping in both a high- and a low-affinity state, Tmod can act as a ‘rheostat’ for the slower dynamics of pointed ends, depending on the time the filament spends in the two states. One might expect that the tight binding-state would probably be kinetically favored, but this could depend on factors such as the local tropomyosin concentration, free actin monomer levels or relative abundance of Tmod versus pointed ends.

Pointed end Low affinity site

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Figure I.

TM Actin capping

TM

are even more similar to one another, with 76% identity and 83% similarity [15].

LLR Actin capping

LLR

PP

LMOD

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Figure 1. Domain structure of the Tmod family proteins. In the canonical 40-kDa Tmod isoforms, represented in this diagram by the domain structure of Tmod1, there is a tightly folded C-terminal domain that contains a series of leucine-rich repeats (LRR) [80], whereas the N-terminal region is unstructured and binds to tropomyosin (TM) [18,19]. An extended a-helix associated with the C-terminal end of the LRRs and a small C-terminal extension (shaded) contain a capping activity at the actin pointed end that is independent of tropomyosin [6]. The TM domain also caps pointed ends of actin filaments with a weaker affinity on its own, but this activity is dramatically enhanced by tropomyosin [6]. The TM region is sufficient and necessary to bind to both muscle and nonmuscle tropomyosins [17,18]. The Lmod genes encode proteins of approximately 64 kDa that contain an additional domain at the C-terminus not found in the canonical Tmod proteins, as well as an insertion of variable length (slashes; [14]). Although the function of this C-terminal extension is not clear, it contains one or two long polyproline motifs (PP), suggesting potential interactions with Src-homology 3 (SH3) domains [81]. At the C-terminus, there is a conserved novel domain of unknown function found only in the Lmods (LMOD). http://ticb.trends.com

Biochemical properties of the Tmods Tmods specifically bind to pointed ends of actin filaments and not to their sides or barbed ends [7,16]. All of the canonical Tmods cap pure (Mg2þ) ATP– actin with an affinity of 100– 300 nM [6]. In addition, Tmods bind to the N-termini of tropomyosin molecules with an affinity of 0.2 –1 mM, using the unstructured domain near the N-terminus of Tmod [17 – 19] (Figure 1). Early data suggested that the binding activities of tropomyosin and actin were completely distinct, with the actin-capping activity found in the C-terminal portion [17,20]. However, recent experiments dissecting the capping activity of Tmod1 indicate that the actin-capping activity is the result of two distinct regions within the Tmod1 molecule [6] (Figure 1). The unstructured tropomyosin-binding region at the N-terminus has an actin pointed-end-capping activity that is dramatically upregulated by tropomyosin coating of

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Table 1. Tmod isoform tissue distribution and in vivo functiona,b Isoform

Tissue/Cell Type

Actin Structure

Function

Refs

Tmod1 (E)

Striated muscle Erythrocytesc Lens cellsc, neurons Neurons Endothelia, epithelia Skeletal muscle Smooth muscle Cardiac muscle Neurons Striated muscle

Sarcomeres, costameres Membrane skeleton Membrane skeleton Membrane skeleton, (?) Lamellipodia, cell-cell contacts, (?) Sarcomere Thin filaments (?) Membrane skeleton (?) Sarcomeres

Length regulation, filament stability Length regulation, filament stability (?) Synaptic plasticity Polarized cell migration Length regulation (?) (?) Asymmetric cell division Length regulation

[13,20,22,42,85] [16,69,70] [65,73,78] [34,78] [14,15,38] [13 –15] [14] [14] [76,77] [41]

Tmod2 (N) Tmod3 (U) Tmod4 (Sk) Lmod1 (SM) Lmod2 (C) Sanpodo a

Question marks indicate where the data are unclear or unknown. Despite functional similarity in vitro, Tmod isoforms have distinct and conserved expression patterns in vivo with different biological functions. Gene names are given with original nomenclature in parentheses based on the tissue in which the isoform was first discovered. Although some isoforms, such as Tmod1 and Tmod3 and the Drosophila melanogaster homolog, sanpodo, are widely expressed, only a few examples of the tissues expressing them are listed. Isoforms are conserved across the vertebrate species studied thus far, such that Tmod1 of Homo sapiens is much more similar to that of Gallus domesticus than to other Homo sapiens Tmod isoforms (e.g. Tmod3). c Note that Tmod1 is present in mammalian lens cells and erythrocytes, whereas Tmod4 is present in these chick cells [65]. b

the actin filament, allowing Tmod to cap tropomyosin – actin filaments with an affinity of #0.05 nM [6– 8]. Despite the high affinity of this capping activity, it is difficult to achieve saturation of all actin – tropomyosin filaments even with high concentrations of Tmod, possibly because of variability in the configuration of tropomyosin at pointed ends [8] (Box 1). Alteration of the critical concentration of the pointed end (see below) is not observed with tropomyosin-regulated capping [8]. The second region is found near the C-terminus (Figure 1), [6]. This tropomyosin-independent cappingdomain caps pure actin with nearly the same affinity (Kd , 400 nM) as full-length Tmod1 (Kd , 100 nM). The C-terminal portion of Tmod, like the full-length molecule, can nucleate the assembly of actin filaments, albeit weakly. Although the biological significance of Tmod-mediated nucleation is unknown, this activity suggests that the C-terminal capping-domain might bind across two monomers at the pointed end, similar to CP at the barbed end [6,21]. An interesting consequence of tropomyosinindependent capping by Tmod is that, by decreasing the rate of monomer addition, it increases the critical concentration for the polymerization of pointed ends, because conversion of ADP-PI – actin to ADP– actin occurs at pointed ends of the filament [8]. Because of this effect, it is possible that the low-affinity pointed-end capping of Tmod could inhibit polymerization more efficiently than depolymerization, as the dissociation constant for ADP– actin at pointed ends is greater than for ADP-Pi –actin. Under conditions in which low-affinity binding occurs, increased Tmod concentrations can thus favor depolymerization of filaments [22]. The relevance of these in vitro data to processes in different cell models is not yet clear. However, the existence of high profilin (an actin-monomer-binding protein) concentrations in cells could accentuate such depolymerizing effects of Tmod, because the pool of profilin – actin complexes is essentially unavailable for the polymerization of pointed ends [3]. Functions of tropomyosin at the pointed end Tropomyosins are a family of proteins that can stabilize actin filaments by copolymerizing along their lengths [23]. http://ticb.trends.com

Even in the absence of Tmod binding, copolymerization of tropomyosin with actin filaments has profound effects on the dynamics of actin pointed ends. For example, tropomyosin binding inhibits ADF (actin depolymerization factor)/cofilin-induced pointed-end depolymerization and filament severing [24,25]. Tropomyosin further stabilizes actin filaments by lowering the off-rate constant of pointed ends, thereby inhibiting their depolymerization and effectively decreasing their critical concentration [26]. Tropomyosin also favors self-annealing of filaments even in the presence of a-actinin, an actin crosslinking protein [27], or gelsolin, a barbed-end capping and severing protein [28]. The binding of tropomyosin to actin filaments also inhibits the nucleation activity of Arp2/3 [23,29]. There are more than 20 distinct vertebrate isoforms of tropomyosin, generated by alternative mRNA splicing of several genes [30]. Based on their molecular masses, these isoforms are generally divided into groups of long and short species. The N-termini of long and short tropomyosin isoforms are encoded by alternative first exons 1a and 1b [31]. Tmods bind to the N-termini of both long and short tropomyosins [18,32]. Most Tmod isoforms display differences in their tropomyosin preference, depending on which first exon is incorporated into the tropomyosin [18,33,34]. For example, Tmod1 binds better to short tropomyosins containing exon 1b than to long tropomyosins containing exon 1a [18,33]. This is interesting because tropomyosins are known to sort differentially to specific actin structures [35 –37], which suggests that association of tropomyosin with specific actin structures could preferentially target some Tmod isoforms [13,38]. For example, expression of the hTm5 (NM1) tropomyosin isoform induces increased stress-fiber formation and recruitment of other tropomyosins to these structures, whereas expression of the TmBr3 tropomyosin isoform induces the formation of lamellipodia and decreased formation of stress fibers [37]. It would be interesting to know whether these effects, on cell morphology and actin structure, are mediated by differential recruitment of Tmod isoform(s) expressed in the same cells. The effects of the two proteins on the dynamics of actin filament pointed-ends are probably mutually synergistic because Tmod can stabilize tropomyosin on actin filaments [7].

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The regulation of pointed ends in stable actin structures with dynamic subunits The in vivo regulation of actin structures by pointed ends is perhaps best understood in striated muscle. Here, the regular arrangement of thin filaments in sarcomeres enables the direct visualization of populations of pointed ends on actin filaments, because they are aligned in parallel with their barbed ends at the Z-disc and their pointed ends extending to the middle of the sarcomere. The actin filaments in sarcomeres (thin filaments) are capped at their barbed ends by CapZ (muscle CP) and at their pointed ends by Tmod1 (or Tmod4) and are tightly regulated in length [11,39]. Although the sarcomere structure itself persists for many days, the subunits within the structure are considerably more dynamic [11,22,40]. In cultured myocytes, actin incorporates at both filament ends, whereas the length of the filaments remains constant [22]. Because the lifetime of the filaments is relatively long (. 20 h), the dynamic capping of both ends allows incorporation of actin subunits and thus can contribute to a steady-state regulation of filament length. However, the barbed ends in these actin filaments are on average twofold less active in incorporation and exchange of actin monomers than the pointed ends in the same sarcomeres [22]. Interestingly, the off-rate measured for CP is nearly identical to that of the Tmod in the highaffinity state (Box 1). Given the , 10-fold higher rateconstants for actin polymerization at barbed versus pointed ends, these data suggest that, in muscle sarcomeres, there are many fewer free barbed ends at any time than free pointed ends. This could be explained by a higher local concentration of CapZ at the barbed ends, thereby decreasing the proportion of time a given barbed end is uncapped. A low number of free barbed ends at the z-disc is consistent with the fact that length regulation of thin filaments in sarcomeres is insensitive to treatment with cytochalasin D, which specifically blocks assembly of actin filaments at barbed ends [22]. In contrast to the situation at the barbed end, the length of actin filaments in sarcomeres is dynamically regulated by the extent of Tmod capping at the pointed end [20,22].

(1)

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Overexpression of Tmod in striated muscles causes shortening of thin filaments [22,41,42]. Conversely, inhibition of Tmod expression or function causes lengthening of thin filaments [20,42]. Quantitative fluorescence recovery after photobleaching (FRAP) of GFP – Tmod in cardiac muscle cells yields an estimated off-rate of 1.2 £ 1023 s21 [43], which is very similar to an off-rate of 0.5 £ 1023 s21 calculated from in vitro experiments with tropomyosincoated actin filaments [8]. It is perplexing that this Tmod off-rate at the pointed end of thin filaments is essentially equal to the CapZ off-rate at the barbed-end of these filaments, because modulation of the pointed-end dynamics by Tmod alters the length of thin filament, whereas modulation of the barbed-end dynamics by cytochalasin D does not [22]. To reconcile these observations, we speculate that the off-rate for Tmod measured by FRAP actually represents an average of a high-affinity state with a lower offrate and a low-affinity state with a higher off-rate. To regulate lengths of thin filaments, Tmod might alternately adopt high- and low-affinity binding states, depending on the position of tropomyosin relative to the end of the filament [11] (Box 1). Modulation of actinfilament length then can occur in the low-affinity state because Tmod transiently caps the pointed end competing for monomer addition. Because the high-affinity state will be preferentially stabilized, the low-affinity state probably represents the minority of filament ends at any given time. However, all the filament ends might transiently adopt the low-affinity state as Tmod stochastically falls off the end, allowing loss or gain of either actin monomers or tropomyosin molecules from the ends (Figure 2). As mentioned above, the effect of Tmod on the critical concentration at the pointed end might block actin assembly more efficiently than disassembly, therefore, increased capping by Tmod in the low-affinity state might actually favor depolymerization, and thus shorter filaments. Although there is evidence to argue against a simple vernier mechanism [11], such evidence does not take into account the effects of low-affinity capping by Tmod. These effects could be enhanced by other factors maintaining an increased local concentration of Tmod. Indeed, Tmod binds to the scaffold

(4)

High affinity cap

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Low affinity cap

Low affinity cap TRENDS in Cell Biology

Figure 2. Model for Tmod modulation of thin-filament length in sarcomeres using a modified vernier mechanism. (1) In sarcomeres, Tmod caps most of the pointed ends with high affinity. At low frequency, Tmod comes off these filaments. If Tmod rebinds, no change occurs; however, if actin monomers add to the pointed end while the Tmod is off, this creates a low-affinity site, because the tropomyosin no longer extends to the pointed end (2). Tropomyosin exchanges preferentially at pointed ends of thin filaments [82]. When tropomyosin comes off the filament (3), low-affinity capping by Tmod still allows depolymerization to occur but can inhibit elongation by alteration of the critical concentration of pointed ends at these ends [8]. Depolymerization is allowed as long as the terminal tropomyosin does not register with the end of the filament. When tropomyosin and actin are in register at the end of thin filaments (4), high-affinity capping stabilizes this new shorter filament length. For clarity, the tropomyosin molecules have not been drawn to scale, with respect to the actin subunits. http://ticb.trends.com

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protein nebulin in an epitope found at the center of the sarcomere [44]. Nebulin is a large molecule found in striated muscle that extends the length of thin filaments [45]. Further support for the two-state model of Tmod capping in length regulation has come recently from experiments in which cardiomyocytes are microinjected with a monoclonal antibody that inhibits endogenous binding of Tmod to tropomyosin. Because the antibody would be expected to convert all Tmod capping into the lowaffinity state, the model predicts that thin filaments would get shorter, as was indeed observed [46]. Furthermore, the two-state model predicts that if the tropomyosin-independent (low-affinity) actin capping by Tmod is inhibited, thin filament lengths would increase. In fact, this is true for cells microinjected with a function-blocking monoclonal antibody to the C-terminal actin-capping domain [20]. Consequently, in muscle sarcomeres, the barbed ends are capped with sufficient frequency to be effectively silent in the process of length regulation, whereas pointed-end capping by Tmod can be modulated to achieve different lengths of thin filaments. Thus, from the perspective of single actin filaments in the sarcomere, barbed-end capping acts as an ‘on/off ’ switch, whereas pointed-end capping acts as an adjustable ‘rheostat’, modulating loss or gain of subunits from the pointed end (Box 1). The capping of pointed ends in dynamic actin networks Protrusion of the leading edge of cell lamellipodia by the rapid assembly and disassembly of underlying actin filaments has been the focus of intense study in recent years. Rapid polymerization from nascent free barbed ends provides force to push the membrane forward [47]. Nucleation of new filament barbed-ends is accomplished primarily by the activation of the Arp2/3 complex [2]. Additional creation of free barbed ends can also occur by stimulated uncapping of barbed ends by gelsolin [48] or by filament severing caused by cofilin [49]. To achieve maximum cellmigration velocity over time, there might be optimum rates for the generation of free barbed ends and polymerization. This optimum polymerization rate is high enough to provide sufficient force for forward membrane protrusion, yet low enough to limit filament length and maintain a dense branched network [50– 52]. To accomplish this balance, most of the barbed ends are rapidly capped by CP and other barbed-end capping proteins [51,53,54]. Because the off-rate of CP from barbed-ends is extremely slow, relative to the polymerization of the filaments in the network (the half-life of a capped barbed end is predicted to be , 28 min [55]), capped filaments are essentially ‘off ’ in the timescale of the rapid turnover of the lamellipodia actin network [2] (Figure 3a). Equally important to the mechanism of rapidly treadmilling networks of actin filaments is the depolymerization of filaments from their pointed ends. In fact, depolymerization of pointed ends appears to be the ratelimiting step in the treadmilling cycle [56]. Disassembly from pointed ends can be enhanced by several factors. Rapid hydrolysis of ATP bound to actin monomers within the filament, followed by slow release of inorganic phosphate, occurs as filaments age, such that the pointed end is more likely to contain ADP– actin [2]. Occupation of http://ticb.trends.com

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ADP– actin at pointed ends favors disassembly over assembly by increasing the critical concentration of pointed ends by approximately threefold [57]. Furthermore, within the filament, ADP– actin favors binding of ADF/cofilin proteins, which enhance disassembly from pointed ends [58]. ADF/cofilin also severs the filaments, thus creating more pointed ends for disassembly [58]. Tmod was previously presumed not to be involved in such actin structures, partly because of the observation that expression patterns of known Tmods were limited to differentiated, nonmotile cells (Table 1). Recently, an isoform of Tmod (Tmod3) has been identified that is expressed in endothelial cells [38]. In these cells, Tmod acts as a negative regulator of cell migration in vitro and leads to depolarization of cells. Increased Tmod3 levels lead to decreased levels of free pointed ends, as well as fewer free barbed ends and F-actin in the lamellipodia, and lower mean rates of cell migration. Lower Tmod3 levels promote the opposite effect (i.e. more free pointed ends together with increased free barbed ends and F-actin in the lamellipodia and faster cell migration [38]). Although this relationship between Tmod levels and total amount of F-actin is similar to the muscle system, here it is counterintuitive because the pointed ends in the lamellipodial network are thought to be rapidly disassembling, and thus stabilization of pointed ends might have been expected to increase F-actin levels. Nevertheless, the current treadmilling-network model allows several reasonable interpretations of the observations (Figure 3). One attractive possibility is that the capping of pointed ends by Tmod antagonizes depolymerization of ADF/cofilin, interfering with the normal cycle of treadmilling [57]. If depolymerization is indeed the rate-limiting step, this would be expected to lower the local supply of monomers [57,59], which could decrease rates of nucleation and polymerization of new filaments. However, no differences in the supply of local monomers in the lamellipodia were observed when Tmod levels were modulated [38]. Unfortunately, methods to detect and quantitate monomers in situ might not identify pools of monomer bound to either profilin or thymosin b4, which comprise the major source of monomers in motile cells [3]. Thus, it is possible that increased capping of pointed ends reduces one or both of these pools, thereby inhibiting polymerization at the barbed end and cell migration. Of course, other mechanisms can explain the observed effects of Tmod in motile cells and are not mutually exclusive. Capping of pointed ends by Tmod could antagonize the function of Arp2/3 directly by preventing the capture of pointed ends or by competitively binding to Arp2/3 activators, thereby lowering the rate of nucleation in lamellipodia. However, so far the capture of pointed-ends by Arp2/3 in vitro has been reported only in one study [60]. Tmod is more likely to affect the function of Arp2/3 indirectly. For example, by increasing the lifetime of filaments, Tmod could lead to increased proportions of ADP– actin subunits within filaments (Figure 3). This would favor decreased nucleation by Arp2/3 that preferentially nucleates branches from ATP–actin [61]. Decreased nucleation by Arp2/3 would then result in fewer barbed ends and F-actin as observed. Although this model can

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(b)

(c) Key: CP

ATP-actin

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ADP-actin

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Figure 3. Models of capping functions of Tmod pointed-ends in lamellipodial actin networks. (a) At the leading edge, actin networks are initiated and maintained in an active ‘treadmilling’ state by polymerization of barbed ends (top) and depolymerization of pointed ends (bottom) [2,62,83]. Actin polymerizes onto free barbed end as ATP– actin. New barbed ends are created by activated Arp2/3, which nucleates new filaments from the sides of existing filaments. As they polymerize, CP caps some barbed ends. This stops polymerization at these ends, but allows continued polymerization at the remaining free barbed ends. Within the filaments, subsequent rapid hydrolysis of ATP, bound to actin subunits, and slow phosphate release occurs as filaments age, creating ADP– bound subunits within the polymer. ADP subunits within the filaments then permit enhanced severing by cofilin and branch release by Arp2/3. Low levels of Tmod capping might slow depolymerization of pointed ends by transiently capping pointed ends, increasing the percentage of ADP-bound subunits at filament pointed-ends. As monomers are released from pointed ends, the nucleotide is exchanged for ATP and they are recycled into the polymerizing pool (arrow). In motile cells expressing Tmod, multiple effects on the treadmilling process can be hypothesized. (b) At higher levels of Tmod pointed-end capping (1), increased filament lifetimes lead to greater stretches of ADP-bound subunits within the polymer (2). Branch release occurs normally, but released pointed ends would be transiently capped by Tmod3. Because Arp2/3 might have a preference for ATP –actin filaments [61], this could lead to decreased amounts of new branching/nucleation (3). Cofilin can sever the filaments with ADP –actin, but again Tmod3 transiently caps these filaments (4). With decreased filament turnover and decreased Arp2/3-mediated nucleation, fewer new barbed ends are created, whereas CP levels presumably remain the same, resulting in fewer free barbed ends at any given time (5). (c) Alternatively, Tmod3 capping could help stabilize tropomyosin along filaments. When tropomyosin extends to the pointed end of the filament, high-affinity capping by Tmod3 occurs (1). Stabilization of tropomyosin on filaments inhibits Arp2/3 function (2), as well as cofilin-mediated filament severing (3) [23]. High-affinity capping, coupled with tropomyosin binding along the filament, could allow some filaments to escape turnover (4). These tropomyosin-actin filaments could anneal with other actin filaments released from branches, providing a population of stable filaments to form the base of the lamella [2,62].

explain the data, it is noteworthy that the amount of ADP –actin in the lamellipodia, which is unknown, is possibly low, given the slow rate of phosphate release and rapid turnover of the network [3,57]. In some cells, lamellipodia might be enriched with the nonmuscle isoform of tropomyosin, TmBr3 [37]. This suggests an attractive mechanism for the function of Tmod3 in lamellipodia (Figure 3). In lamellipodia, Tmod3 might cap a population of TmBr3 – actin filaments in the high-affinity state (Box 1). Although the role of such a high-affinity pointed-end cap is not known, it might allow selected filaments to persist and form the base of the lamella [2,62]. At appropriate levels, this function would be helpful to cell migration, by increasing the stability of the lamella structure and allowing better adhesion [62,63]. Under conditions of high Tmod3 levels, increased amounts of tropomyosin could be stabilized in lamellipodia, which http://ticb.trends.com

would inhibit both Arp2/3 and cofilin activities, as described in Figure 3 [24,25,29]. This would lead to the observed decrease in free barbed ends in lamellipodia and decreased rates of cell migration in cells overexpressing Tmod3 [38]. However, some cell types display a paucity of tropomyosin isoforms in their lamellipodia [64], which indicates that there are differences in the composition of lamellipodial actin networks between different cell-types. Given the depolymerization requirements for treadmilling of the leading-edge network, what could be the in vivo significance of Tmod capping? Negative regulation of cell migration, dependent on upregulated Tmod3 expression upon cell differentiation, is one possibility. Indeed, expression of Tmod and its assembly into cytoskeletal structures is dramatically increased upon terminal differentiation in other cell types [13,39,65]. In motile cells, induced expression of Tmod3 could inhibit inappropriate

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cell migration in environments where local hormone signals would continue to stimulate Arp2/3 activators, as a byproduct of nonmotogenic-signaling pathways. Future studies in transgenic mice will provide better insights into the biological function of the Tmod3 isoform. Pointed-end capping in the spectrin– actin membrane skeleton Although some form of a spectrin–actin-based cytoskeleton on the cytoplasmic surface of plasma membranes is thought to be common to nearly all higher eukaryotic cell types, it has been best studied in erythrocytes [66,69]. In human erythrocytes, the membrane skeleton is optimally ordered to maintain the characteristic cell shape and must persist in the absence of protein turnover, because the cells are enucleated and survive 120 days without protein synthesis. As such, the membrane skeleton in these cells must be extraordinarily stable. In fact, the concentration of total G-actin in erythrocytes is equivalent to the critical concentration for barbed ends, suggesting that barbed ends are in equilibrium with the soluble pool of monomers, whereas all the pointed ends are possibly tightly capped [67,68]. In these cells, Tmod1 exists at a concentration of 1.1 mM, and therefore caps the ends of all of the 40-nm-long tropomyosin-coated actin filaments [69]. Because of the low concentration of monomers and the high-affinity state of the filaments for Tmod, pointed ends are essentially ‘off ’, and filaments are tightly restricted in length [69]. In fact, erythrocytes from Tmod1 knockout mice are abnormally fragile underscoring the importance of this tight regulation for erythrocyte function [70]. Meanwhile, barbed ends are essentially free to polymerize until the free monomer reaches the critical concentration for barbed ends [67], although low-affinity capping by adducin might still occur [71,72]. However, there is no direct evidence for static versus dynamic capping in this system. Careful analysis of actin dynamics in living intact erythrocytes has not been reported, perhaps because of the inherent difficulties of introducing fluorescently labeled proteins into small enucleated erythrocytes. Thus, it is possible that in the erythrocyte membrane skeleton the actin filaments experience dynamic exchange of actin subunits at barbed ends. In other cell systems, the organization of actin and dynamics of the membrane skeleton have not been well characterized. In the vertebrate lens, the membrane skeleton is biochemically similar to that of erythrocytes but organized differently [65,73,74]. In these cells, Tmod expressed upon differentiation is thought to reorganize the cytoskeleton, perhaps by enhancing the recruitment of tropomyosin to the membrane skeleton and adherens junctions [65,75]. It will be interesting to determine what role the capping of pointed ends might have in the regulation of polarized-cell adherens junctions, because this structure is not as ordered as the muscle sarcomere or the membrane skeleton of erythrocytes but is more stable than the actin network of lamellipodia. Concluding remarks With regards to the role of the pointed-end dynamics in various systems, several questions remain unanswered. http://ticb.trends.com

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For instance, because both Tmod and ADF/cofilin act at pointed ends to achieve opposing effects, is there direct competition at particular individual ends, or are the processes mutually exclusive for a single filament? How does myosin binding affect the ability of Tmod to cap pointed ends, in myofibrils or in other structures? Are there other regulatory mechanisms, such as protein phosphorylation, to control the capping of Tmod? Cell culture model systems expressing Tmods have a large percentage ($ 50%) of soluble endogenous Tmod. This suggests that either highaffinity binding sites are limiting or the soluble fraction represents a downregulated form of the protein. Alternatively, this could indicate other binding partners of Tmods. If such alternative partners exist, they might affect the capping of pointed ends, providing an additional regulatory step. Regulation of pointed ends has important consequences for higher-order functions in multicellular organisms. For example, in mice, deletion of the gene encoding Tmod1 results in embryonic lethality caused by abnormal cardiac development and hemolytic anemia [70]. Deletion of the Drosophila Tmod isoform, Sanpodo, results in abnormal neuronal development, caused by defects in asymmetric cell division [76,77]. In mammals, the neuronal isoform of Tmod, Tmod2, might be involved in long-term potentiation and synaptic plasticity [78]. This is particularly interesting as this process is dependent on dynamic changes in F-actin architecture, mediated at least in part by the inactivation of ADF/cofilin [79]. To understand how actin dynamics contribute to these higher-order functions, we will need a more detailed picture of the functional interactions within regulatory network of actin, including the capping of pointed ends. Examination of Tmods in various actin-filament systems will shed light on how the regulation of pointed ends differs from that of barbed ends, and how regulation at the slow end of actin filaments can contribute to diverse cellular functions in motile and nonmotile cells. Acknowledgements We thank Ryan Littlefield for helpful discussions. This work was supported by NIH grant GM34225. This is TSRI manuscript number 15975-CB.

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