The formin mDia2 stabilizes microtubules independently of its actin nucleation activity

A critical microtubule (MT) polarization event in cell migration is the Rho/mDia-dependent stabilization of a subset of MTs oriented toward the direction of migration. Although mDia nucleates actin filaments, it is unclear whether this or a separate activity of mDia underlies MT stabilization. We generated two actin mutants (K853A and I704A) in a constitutively active version of mDia2 containing formin homology domains 1 and 2 (FH1FH2) and found that they still induced stable MTs and bound to the MT TIP proteins EB1 and APC, which have also been implicated in MT stabilization. A dimerization-impaired mutant of mDia2 (W630A) also generated stable MTs in cells. We examined whether FH1FH2mDia2 had direct activity on MTs in vitro and found that it bound directly to MTs, stabilized MTs against cold- and dilution-induced disassembly, and reduced the rates of growth and shortening during MT assembly and disassembly, respectively. These results indicate that mDia2 has a novel MT stabilization activity that is separate from its actin nucleation activity.     

The formin mDia regulates GSK3beta through novel PKCs to promote microtubule stabilization but not MTOC reorientation in migrating fibroblasts

In migrating cells, external signals polarize the microtubule (MT) cytoskeleton by stimulating the formation of oriented, stabilized MTs and inducing the reorientation of the MT organizing center (MTOC). Glycogen synthase kinase 3beta (GSK3beta) has been implicated in each of these processes, although whether it regulates both processes in a single system and how its activity is regulated are unclear. We examined these issues in wound-edge, serum-starved NIH 3T3 fibroblasts where MT stabilization and MTOC reorientation are triggered by lysophosphatidic acid (LPA), but are regulated independently by distinct Rho GTPase-signaling pathways. In the absence of other treatments, the GSK3beta inhibitors, LiCl or SB216763, induced the formation of stable MTs, but not MTOC reorientation, in starved fibroblasts. Overexpression of GSK3beta in starved fibroblasts inhibited LPA-induced stable MTs without inhibiting MTOC reorientation. Analysis of factors involved in stable MT formation (Rho, mDia, and EB1) showed that GSK3beta functioned upstream of EB1, but downstream of Rho-mDia. mDia was both necessary and sufficient for inducing stable MTs and for up-regulating GSK3beta phosphorylation on Ser9, an inhibitory site. mDia appears to regulate GSK3beta through novel class PKCs because PKC inhibitors and dominant negative constructs of novel PKC isoforms prevented phosphorylation of GSK3beta Ser9 and stable MT formation. Novel PKCs also interacted with mDia in vivo and in vitro. These results identify a new activity for the formin mDia in regulating GSK3beta through novel PKCs and implicate novel PKCs as new factors in the MT stabilization pathway.     

Microtubule-dependent transport and organization of sarcomeric myosin during skeletal muscle differentiation

It has been proposed that microtubules (MTs) participate in skeletal muscle cell differentiation. However, it is still unclear how this happens. To examine whether MTs could participate directly in the organization of thick and thin filaments into sarcomeres, we observed the concomitant reorganization and dynamics of MTs with the behavior of sarcomeric actin and myosin by time-lapse confocal microscopy. Using green fluorescent protein (GFP)-EB1 protein to label MT plus ends, we determined that MTs become organized into antiparallel arrays along fusing myotubes. Their dynamics and orientation was found to be different across the thickness of the myotubes. We observed fast movements of Dsred-myosin along GFP-MTs. Comparison of GFP-EB1 and Dsred-myosin dynamics revealed that myosin moved toward MT plus ends. Immuno-electron microscopy experiments confirmed that myosin was actually associated with MTs in myotubes. Finally, we confirmed that MTs were required for the stabilization of myosin-containing elements prior to incorporation into mature sarcomeres. Collectively, our results strongly suggest that MTs become organized into a scaffold that provides directional cues for the positioning and organization of myosin filaments during sarcomere formation.     

EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration

Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. We investigated whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functioned as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevented microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, was sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibited fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration.     

Role of CLASP2 in microtubule stabilization and the regulation of persistent motility

In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts.     

Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization

In migrating adherent cells such as fibroblasts and endothelial cells, the microtubule-organizing center (MTOC) reorients toward the leading edge [1-3]. MTOC reorientation repositions the Golgi toward the front of the cell [1] and contributes to directional migration [4]. The mechanism of MTOC reorientation and its relation to the formation of stabilized microtubules (MTs) in the leading edge, which occurs concomitantly with MTOC reorientation [3], is unknown. We show that serum and the serum lipid, lysophosphatidic acid (LPA), increased Cdc42 GTP levels and triggered MTOC reorientation in serum-starved wounded monolayers of 3T3 fibroblasts. Cdc42, but not Rho or Rac, was both sufficient and necessary for LPA-stimulated MTOC reorientation. MTOC reorientation was independent of Cdc42-induced changes in actin and was not blocked by cytochalasin D. Inhibition of dynein or dynactin blocked LPA- and Cdc42-stimulated MTOC reorientation. LPA also stimulates a Rho/mDia pathway that selectively stabilizes MTs in the leading edge [5, 6]; however, activators and inhibitors of MTOC reorientation and MT stabilization showed that each response was regulated independently. These results establish an LPA/Cdc42 signaling pathway that regulates MTOC reorientation in a dynein-dependent manner. MTOC reorientation and MT stabilization both act to polarize the MT array in migrating cells, yet these processes act independently and are regulated by separate Rho family GTPase-signaling pathways.     

mDia mediates Rho-regulated formation and orientation of stable microtubules

Rho-GTPase stabilizes microtubules that are oriented towards the leading edge in serum-starved 3T3 fibroblasts through an unknown mechanism. We used a Rho-effector domain screen to identify mDia as a downstream Rho effector involved in microtubule stabilization. Constitutively active mDia or activation of endogenous mDia with the mDia-autoinhibitory domain stimulated the formation of stable microtubules that were capped and oriented towards the wound edge. mDia co-localized with stable microtubules when overexpressed and associated with microtubules in vitro. Rho kinase was not necessary for the formation of stable microtubules. Our results show that mDia is sufficient to generate and orient stable microtubules, and indicate that Dia-related formins are part of a conserved pathway that regulates the dynamics of microtubule ends.     

Cap Z(36/32), a barbed end actin-capping protein, is a component of the Z-line of skeletal muscle

Various biological activities have been attributed to actin-capping proteins based on their in vitro effects on actin filaments. However, there is little direct evidence for their in vivo activities. In this paper, we show that Cap Z(36/32), a barbed end, actin-capping protein isolated from muscle (Casella, J. F., D. J. Maack, and S. Lin, 1986, J. Biol. Chem., 261:10915-10921) is localized to the barbed ends of actin filaments by electron microscopy and to the Z-line of chicken skeletal muscle by indirect immunofluorescence and electron microscopy. Since actin filaments associate with the Z-line at their barbed ends, these findings suggest that Cap Z(36/32) may play a role in regulating length, orienting, or attaching actin filaments to Z-discs.     

Novel roles of formin mDia2 in lamellipodia and filopodia formation in motile cells

Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (ΔGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of ΔGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that ΔGBD-mDia2–induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia.     

The search and prime hypothesis for growth cone turning

In the fields of axonal and dendritic guidance, there is now a significant accumulation of knowledge of how extracellular signaling molecules activate their cognate growth cone receptors. Relatively little is known about the subsequent activation of intracellular signaling pathways and actin reorganization, and very little is known about how microtubules (MTs) reorganize during growth cone turning. I hypothesize that dynamic MTs are required in order to catalyze the polarized actin assembly necessary for growth cone turning, that MTs and actin filaments promote each other's assembly through positive feedback, that MT stability is enhanced further through the formation of membrane-associated MT attachment sites, and that these MT stabilization events subsequently accelerate axonal/dendritic shaft formation.     

Tropomyosin regulates elongation by formin at the fast-growing end of the actin filament

The balance between dynamic and stable actin filaments is essential for the regulation of cellular functions including the determination of cell shape and polarity, cell migration, and cytokinesis. Proteins that regulate polymerization at the filament ends and filament stability confer specificity to actin filament structure and cellular function. The dynamics of the barbed, fast-growing end of the filament are controlled in space and time by both positive and negative regulators of actin polymerization. Capping proteins inhibit the addition and loss of subunits, whereas other proteins, including formins, bind at the barbed end and allow filament growth. In this work, we show that tropomyosin regulates dynamics at the barbed end. Tropomyosin binds to constructs of FRL1 and mDia2 that contain the FH2 domain and modulates formin-dependent capping of the barbed end by relieving inhibition of elongation by FRL1-FH1FH2, mDia1-FH2, and mDia2-FH2 in an isoform-dependent fashion. In this role, tropomyosin functions as an activator of formin. Tropomyosin also inhibits the binding of FRL1-FH1FH2 to the sides of actin filaments independent of the isoform. In contrast, tropomyosin does not affect the ability of capping protein to block the barbed end. We suggest that tropomyosin and formin act together to ensure the formation of unbranched actin filaments, protected from severing, that could be capped in stable cellular structures. This role, in addition to its cooperative control of myosin function, establishes tropomyosin as a universal regulator of the multifaceted actin cytoskeleton.     

TIP maker and TIP marker; EB1 as a master controller of microtubule plus ends

The EB1 protein is a member of the exciting and enigmatic family of microtubule (MT) tip-tracking proteins. EB1 acts as an exquisite marker of dynamic MT plus ends in some cases, whereas in others EB1 is thought to directly dictate the behavior of the plus ends. How EB1 differentiates between these two roles remains unclear; however, a growing list of interactions between EB1 and other MT binding proteins suggests there may be a single mechanism. Adding another layer of complexity to these interactions, two studies published in this issue implicate EB1 in cross-talk between mitotic MTs and between MTs and actin filaments (Goshima et al., p. 229; Wu et al., p. 201). These results raise the possibility that EB1 is a central player in MT-based transport, and that the activity of MT-binding proteins depends on their ability or inability to interact with EB1.     

Rho Guanosine Triphosphatase Mediates the Selective Stabilization of Microtubules Induced by Lysophosphatidic Acid

The asymmetric distribution of stable, posttranslationally modified microtubules (MTs) contributes to the polarization of many cell types, yet the factors controlling the formation of these MTs are not known. We have found that lysophosphatidic acid (LPA) is a major serum factor responsible for rapidly generating stable, detyrosinated (Glu) MTs in serum-starved 3T3 cells. Using C3 toxin and val14 rho we showed that rho was both necessary and sufficient for the induction of Glu MTs by LPA and serum. Unlike previously described factors that induce MT stability, rho induced the stabilization of only a subset of the MTs and, in wound-edge cells, these stable MTs were appropriately oriented toward the leading edge of the cell. LPA had little effect on individual parameters of MT dynamics, but did induce long states of pause in a subset of MTs near the edge of the cell. Rho stimulation of MT stability was independent of actin stress fiber formation. These results identify rho as a novel regulator of the MT cytoskeleton that selectively stabilizes MTs during cell polarization by acting as a switch between dynamic and stable states of MTs rather than as a modulator of MT assembly and disassembly.     

Tropomodulins: life at the slow end

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.     

Mechanistic differences in actin bundling activity of two mammalian formins, FRL1 and mDia2

Formin proteins are regulators of actin dynamics, mediating assembly of unbranched actin filaments. These multidomain proteins are defined by the presence of a Formin Homology 2 (FH2) domain. Previous work has shown that FH2 domains bind to filament barbed ends and move processively at the barbed end as the filament elongates. Here we report that two FH2 domains, from mammalian FRL1 and mDia2, also bundle filaments, whereas the FH2 domain from mDia1 cannot under similar conditions. The FH2 domain alone is sufficient for bundling. Bundled filaments made by either FRL1 or mDia2 are in both parallel and anti-parallel orientations. A novel property that might contribute to bundling is the ability of the dimeric FH2 domains from both FRL1 and mDia2 to dissociate and recombine. This property is not observed for mDia1. A difference between FRL1 and mDia2 is that FRL1-mediated bundling is competitive with barbed end binding, whereas mDia2-mediated bundling is not. Mutation of a highly conserved isoleucine residue in the FH2 domain does not inhibit bundling by either FRL1 or mDia2, but inhibits barbed end activities. However, the severity of this mutation varies between formins. For mDia1 and mDia2, the mutation strongly inhibits all effects of barbed end binding, but affects FRL1 much less strongly. Furthermore, our results suggest that the Ile mutation affects processivity. Taken together, our data suggest that the bundling activities of FRL1 and mDia2, while producing phenotypically similar bundles, differ in mechanistic detail.     

Identification of residues within tropomodulin-1 responsible for its localization at the pointed ends of the actin filaments in cardiac myocytes

Tropomodulin is a tropomyosin-dependent actin filament capping protein involved in the structural formation of thin filaments and in the regulation of their lengths through its localization at the pointed ends of actin filaments. The disordered N-terminal domain of tropomodulin contains three functional sites: two tropomyosin-binding and one tropomyosin-dependent actin-capping sites. The C-terminal half of tropomodulin consists of one compact domain containing a tropomyosin-independent actin-capping site. Here we determined the structural properties of tropomodulin-1 that affect its roles in cardiomyocytes. To explore the significance of individual tropomyosin-binding sites, GFP-tropomodulin-1 with single mutations that destroy each tropomyosin-binding site was expressed in cardiomyocytes. We demonstrated that both sites are necessary for the optimal localization of tropomodulin-1 at thin filament pointed ends, with site 2 acting as the major determinant. To investigate the functional properties of the tropomodulin C-terminal domain, truncated versions of GFP-tropomodulin-1 were expressed in cardiomyocytes. We discovered that the leucine-rich repeat (LRR) fold and the C-terminal helix are required for its proper targeting to the pointed ends. To investigate the structural significance of the LRR fold, we generated three mutations within the C-terminal domain (V232D, F263D, and L313D). Our results show that these mutations affect both tropomyosin-independent actin-capping activity and pointed end localization, most likely by changing local conformations of either loops or side chains of the surfaces involved in the interactions of the LRR domain. Studying the influence of these mutations individually, we concluded that, in addition to the tropomyosin-independent actin-capping site, there appears to be another regulatory site within the tropomodulin C-terminal domain.     

Stabilization of overlapping microtubules by fission yeast CLASP

Many microtubule (MT) structures contain dynamic MTs that are bundled and stabilized in overlapping arrays. CLASPs are conserved MT-binding proteins implicated in the regulation of MT plus ends. Here, we show that the Schizosaccharomyces pombe CLASP, cls1p/peg1p, mediates the stabilization of overlapping MTs within the mitotic spindle and interphase bundles. cls1p localizes to these regions but not to interphase MT plus ends. Inactivation of cls1p leads to the rapid depolymerization of spindle midzone MTs. cls1p also stabilizes a subset of MTs within interphase bundles. cls1p prevents disassembly of the entire microtubule, while still allowing for plus-end growth. It has no measurable effects on MT nucleation, polymerization, catastrophe, or bundling. A direct interaction with ase1p (PRC1/MAP65) targets cls1p to regions of antiparallel MT overlap. These findings show how a MT-stabilizing factor attached to specific sites on MTs can help to generate MT structures that have both dynamic and stable components.     

Localization and possible function of 20 kDa actin-modulating protein (actolinkin) in the sea urchin egg

We have previously described a novel actin-capping protein, a 20,000-molecular weight protein (20K protein)-actin complex (20K-A) isolated from sea urchin eggs. In the present study, the localization and possible function of this 20K protein were investigated. The 20K protein was localized in the sea urchin egg cortex. Its distribution in the cortex as revealed by immunofluorescence microscopy did not change during or after fertilization up to the first mitosis, but it was concentrated to some extent in the cleavage furrow region. Exogenously added actin polymerized on the cortex isolated from unfertilized egg; however, actin did not polymerize on the cortex extracted with 0.6 M KCl, that is, the cell membrane, which lost the 20K protein. The cell membrane preincubated with 20K-A restored the activity to grow actin filaments. When decorated with myosin subfragment 1, almost all the actin filaments showed the arrowhead configuration pointing away from the membrane, indicating that they were connected to the membrane at their barbed ends. These results strongly suggest that the 20K protein connects actin filaments to the plasma membrane of sea urchin eggs. Because of this property we call this protein "actolinkin".     

Crosslinkers and motors organize dynamic microtubules to form stable bipolar arrays in fission yeast

Microtubule (MT) nucleation not only occurs from centrosomes, but also in large part from dispersed nucleation sites. The subsequent sorting of short MTs into networks like the mitotic spindle requires molecular motors that laterally slide overlapping MTs and bundling proteins that statically connect MTs. How bundling proteins interfere with MT sliding is unclear. In bipolar MT bundles in fission yeast, we found that the bundler ase1p localized all along the length of antiparallel MTs, whereas the motor klp2p (kinesin-14) accumulated only at MT plus ends. Consequently, sliding forces could only overcome resistant bundling forces for short, newly nucleated MTs, which were transported to their correct position within bundles. Ase1p thus regulated sliding forces based on polarity and overlap length, and computer simulations showed these mechanisms to be sufficient to generate stable bipolar bundles. By combining motor and bundling proteins, cells can thus dynamically organize stable regions of overlap between cytoskeletal filaments.     

The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.