Positive feedback interactions between microtubule and actin dynamics during cell motility

The migration of tissue cells requires interplay between the microtubule and actin cytoskeletal systems. Recent reports suggest that interactions of microtubules with actin dynamics creates a polarization of microtubule assembly behavior in cells, such that microtubule growth occurs at the leading edge and microtubule shortening occurs at the cell body and rear. Microtubule growth and shortening may activate Rac1 and RhoA signaling, respectively, to control actin dynamics. Thus, an actin-dependent gradient in microtubule dynamic-instability parameters in cells may feed back through the activation of specific signalling pathways to perpetuate the polarized actin-assembly dynamics required for cell motility.     

Actomyosin dynamics modulate microtubule deformation and growth during T-cell activation

Activation of T-cells leads to the formation of immune synapses (ISs) with antigen-presenting cells. This requires T-cell polarization and coordination between the actomyosin and microtubule cytoskeletons. The interactions between these two cytoskeletal components during T-cell activation are not well understood. Here, we elucidate the interactions between microtubules and actin at the IS with high-resolution fluorescence microscopy. We show that microtubule growth dynamics in the peripheral actin-rich region is distinct from that in the central actin-free region. We further demonstrate that these differences arise from differential involvement of Arp2/3- and formin-nucleated actin structures. Formin inhibition results in a moderate decrease in microtubule growth rates, which is amplified in the presence of integrin engagement. In contrast, Arp2/3 inhibition leads to an increase in microtubule growth rates. We find that microtubule filaments are more deformed and exhibit greater shape fluctuations in the periphery of the IS than at the center. Using small molecule inhibitors, we show that actin dynamics and actomyosin contractility play key roles in defining microtubule deformations and shape fluctuations. Our results indicate a mechanical coupling between the actomyosin and microtubule systems during T-cell activation, whereby different actin structures influence microtubule dynamics in distinct ways.     

Fluctuation Analysis of Centrosomes Reveals a Cortical Function of Kinesin-1

The actin and microtubule networks form the dynamic cytoskeleton. Network dynamics is driven by molecular motors applying force onto the networks and the interactions between the networks. Here we assay the dynamics of centrosomes in the scale of seconds as a proxy for the movement of microtubule asters. With this assay we want to detect the role of specific motors and of network interaction. During interphase of syncytial embryos of Drosophila, cortical actin and the microtubule network depend on each other. Centrosomes induce cortical actin to form caps, whereas F-actin anchors microtubules to the cortex. In addition, lateral interactions between microtubule asters are assumed to be important for regular spatial organization of the syncytial embryo. The functional interaction between the microtubule asters and cortical actin has been largely analyzed in a static manner, so far. We recorded the movement of centrosomes at 1 Hz and analyzed their fluctuations for two processes—pair separation and individual movement. We found that F-actin is required for directional movements during initial centrosome pair separation, because separation proceeds in a diffusive manner in latrunculin-injected embryos. For assaying individual movement, we established a fluctuation parameter as the deviation from temporally and spatially slowly varying drift movements. By analysis of mutant and drug-injected embryos, we found that the fluctuations were suppressed by both cortical actin and microtubules. Surprisingly, the microtubule motor Kinesin-1 also suppressed fluctuations to a similar degree as F-actin. Kinesin-1 may mediate linkage of the microtubule (+)-ends to the actin cortex. Consistent with this model is our finding that Kinesin-1-GFP accumulates at the cortical actin caps.     

Interaction of rat brain microtubule proteins and 6S tubulin with rabbit skeletal muscle actomysin.

The interaction between actomyosin from rabbit skeletal muscle and microtubule proteins or 6S tubulin from rat brain was investigated with respect to the change in ATPase activity and physicochemical properties. Myosin bound to both microtubule proteins and 6S tubulin at low ionic strength. In the aggregates the molar ratio of microtubule proteins or 6S tubulin to myosin was 0.5–1.5 or 1.5–2.5. The superprecipitation of actomyosin was inhibited by 6S tubulin. The degree of superprecipitation inhibition was dependent on the mixing order of myosin, actin, 6S tubulin, and ATP. When myosin was preincubated first with 6S tubulin, the inhibition was most marked. The actin activation of myosin Mg-ATPase was inhibited by both microtubule proteins and 6S tubulin with stronger effects by the latter. The preincubation of myosin with 6S tubulin prior to the addition of actin induced not only greater inhibition of ATPase but also the binding of a larger quantity of 6S tubulin to myosin than the preincubation of myosin with actin. The similar results were obtained with microtubule proteins.     

Motor proteins at the microtubule plus-end

The plus-end of the microtubule has a central role in the interactions that occur between the microtubule and actin cytoskeletons. The recent identification of a family of proteins that congregate at the plus-end is enabling an increased mechanistic understanding of how this cross talk is accomplished. These proteins, termed plus-end tracking proteins because they appear to associate with the plus-end as it grows, have already been shown to regulate microtubule dynamics and to facilitate the formation of connections between the plus-end and the actin-rich cortex. Several motor proteins, including an actin-based motor, microtubule-based motors that move towards either end of the microtubule and microtubule motors that depolymerize microtubule ends, can now be added to the list of plus-end tracking proteins. Here, we discuss how the presence of these motors at the plus-end seems to drive several fundamental cellular processes involving force generation at the interface between microtubule ends and the cortex, vesicle translocation following search and capture, microtubule disassembly and the delivery of signals to the cortex that govern actin assembly and cell polarity.     

Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1

The tumor suppressor protein adenomatous polyposis coli (APC) regulates cell protrusion and cell migration, processes that require the coordinated regulation of actin and microtubule dynamics. APC localizes in vivo to microtubule plus ends and actin-rich cortical protrusions, and has well-documented direct effects on microtubule dynamics. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal “basic” domain of APC (APC-B) potently nucleates the formation of actin filaments in vitro and stimulates actin assembly in cells. Nucleation is achieved by a mechanism involving APC-B dimerization and recruitment of multiple actin monomers. Further, APC-B nucleation activity is synergistic with its in vivo binding partner, the formin mDia1. Together, APC-B and mDia1 overcome a dual cellular barrier to actin assembly imposed by profilin and capping protein. These observations define a new function for APC and support an emerging view of collaboration between distinct actin assembly–promoting factors with complementary activities.     

Interaction between Microtubules and the Drosophila Formin Cappuccino and Its Effect on Actin Assembly

Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.     

Unique actin and microtubule arrays co-ordinate the differentiation of microspores to mature pollen in Nicotiana tabacum

The complex cellular events that occur during development of the male gametophyte of higher plants suggest a role for the cytoskeleton. This investigation has revealed that unique microtubule arrays mediate events that occur during microspore development; both actin and microtubule arrays have important roles during the asymmetrical microspore mitosis and unique actin arrays mediate events that occur during early pollen development. Migration of the nucleus to the generative pole during cellular polarization of the microspore is mediated by a microtubule cage that encloses the nucleus. Nuclear position at the generative pole is maintained by an actin net that tethers it to the pole prior to the asymmetrical mitosis. During entry into mitosis, the microtubule cage becomes modified and transforms into the asymmetrical mitotic spindle. Actin is localized within the region of the mitotic spindle and in the phragmoplast. Following mitosis, actin networks enclose first the generative cell and then the vegetative nucleus. These actin networks function during migration of the generative cell and vegetative nucleus toward the centre of the pollen grain. Mature pollen contains a dense cortical actin meshwork and a disc-shaped microtubule array enclosing the generative cell. The functional importance of the unique actin and microtubule arrays is verified by their targeted disruption with specific cytoskeletal inhibitors, which disrupt normal development and cellular morphology. In summary, these data provide evidence that the co-ordinated reorganization of unique actin and microtubule arrays is an essential determinant of microspore and pollen development.     

The effect of the depolymerization and disintegration of the microtubular system on the cytoskeleton of untransformed and transformed cells]

How important are the changes of microtubule control for the realization of actin cortex changes during neoplastic transformation? To answer this question we studied the actin cytoskeleton and intermediate filaments condition after colcemid destruction or taxol disintegration of microtubule system in non-transformed cells BALB/c 3T3 and in the same cells transformed by Ha-ras gene. We have come to a conclusion that the differences between non-transformed and transformed cells in the actin cytoskeleton organization remain the same after specific inhibitor action on the microtubules; after the microtubules are destroyed the differences between the two cell types appear in the intermediate filament organization; there are reasons to assume that changes in the actin cortex structure may play the central role in morphological transformation expression.     

The Ability to Induce Microtubule Acetylation Is a General Feature of Formin Proteins

Cytoplasmic microtubules exist as distinct dynamic and stable populations within the cell. Stable microtubules direct and maintain cell polarity and it is thought that their stabilization is dependent on coordinative organization between the microtubule network and the actin cytoskeleton. A growing body of work suggests that some members of the formin family of actin remodeling proteins also regulate microtubule organization and stability. For example, we showed previously that expression of the novel formin INF1 is sufficient to induce microtubule stabilization and tubulin acetylation, but not tubulin detyrosination. An important issue with respect to the relationship between formins and microtubules is the determination of which formin domains mediate microtubule stabilization. INF1 has a distinct microtubule-binding domain at its C-terminus and the endogenous INF1 protein is associated with the microtubule network. Surprisingly, the INF1 microtubule-binding domain is not essential for INF1-induced microtubule acetylation. We show here that expression of the isolated FH1 + FH2 functional unit of INF1 is sufficient to induce microtubule acetylation independent of the INF1 microtubule-binding domain. It is not yet clear whether or not microtubule stabilization is a general property of all mammalian formins; therefore we expressed constitutively active derivatives of thirteen of the fifteen mammalian formin proteins in HeLa and NIH3T3 cells and measured their effects on stress fiber formation, MT organization and MT acetylation. We found that expression of the FH1 + FH2 unit of the majority of mammalian formins is sufficient to induce microtubule acetylation. Our results suggest that the regulation of microtubule acetylation is likely a general formin activity and that the FH2 should be thought of as a dual-function domain capable of regulating both actin and microtubule networks.     

MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain

BACKGROUND: MAP2 and tau are abundant microtubule-associated proteins (MAPs) in neurons. The development of neuronal dendrites and axons requires a dynamic interaction between microtubules and actin filaments. MAPs represent good candidates to mediate such interactions. Although MAP2c and tau have similar, well-characterized microtubule binding activities, their actin interaction is poorly understood. RESULTS: Here, we show by using a cosedimentation assay that MAP2c binds F-actin. Upon actin binding, MAP2c organizes F-actin into closely packed actin bundles. Moreover, we show by using a deletion approach that MAP2c's microtubule binding domain (MTBD) is both necessary and sufficient for both F-actin binding and bundling activities. Surprisingly, even though the MAP2 and tau MTBDs share high sequence homology and possess similar microtubule binding activities, tau is unable to bind or bundle F-actin. Furthermore, experiments with chimeric proteins demonstrate that the actin binding activity fully correlates with the ability to promote neurite initiation in neuroblastoma cells. CONCLUSIONS: These results provide the first demonstration that the MAP2c and tau MTBD domains exhibit distinct properties, diverging in actin binding and neurite initiation activities. These results implicate a novel actin function for MAP2c in neuronal morphogenesis and furthermore suggest that actin interactions could contribute to functional differences between MAP2 and tau in neurons.     

Microtubules are a target for self-incompatibility signaling in Papaver pollen

Perception and integration of signals into responses is of crucial importance to cells. Both the actin and microtubule cytoskeleton are known to play a role in mediating diverse stimulus responses. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization. SI in Papaver rhoeas triggers a Ca(2+)-dependent signaling network to trigger programmed cell death (PCD), providing a neat way to inhibit and destroy incompatible pollen. We previously established that SI stimulates F-actin depolymerization and that altering actin dynamics can push pollen tubes into PCD. Very little is known about the role of microtubules in pollen tubes. Here, we investigated whether the pollen tube microtubule cytoskeleton is a target for the SI signals. We show that SI triggers very rapid apparent depolymerization of cortical microtubules, which, unlike actin, does not reorganize later. Actin depolymerization can trigger microtubule depolymerization but not vice versa. Moreover, although disruption of microtubule dynamics alone does not trigger PCD, alleviation of SI-induced PCD by taxol implicates a role for microtubule depolymerization in mediating PCD. Together, our data provide good evidence that SI signals target the microtubule cytoskeleton and suggest that signal integration between microfilaments and microtubules is required for triggering of PCD.     

New twists in actin–microtubule interactions

Actin filaments and microtubules are cytoskeletal polymers that participate in many vital cell functions including division, morphogenesis, phagocytosis, and motility. Despite the persistent dogma that actin filament and microtubule networks are distinct in localization, structure, and function, a growing body of evidence shows that these elements are choreographed through intricate mechanisms sensitive to either polymer. Many proteins and cellular signals that mediate actin–microtubule interactions have already been identified. However, the impact of these regulators is typically assessed with actin filament or microtubule polymers alone, independent of the other system. Further, unconventional modes and regulators coordinating actin–microtubule interactions are still being discovered. Here we examine several methods of actin–microtubule crosstalk with an emphasis on the molecular links between both polymer systems and their higher-order interactions.     

Focal loss of actin bundles causes microtubule redistribution and growth cone turning

It is commonly believed that growth cone turning during pathfinding is initiated by reorganization of actin filaments in response to guidance cues, which then affects microtubule structure to complete the turning process. However, a major unanswered question is how changes in actin cytoskeleton are induced by guidance cues and how these changes are then translated into microtubule rearrangement. Here, we report that local and specific disruption of actin bundles from the growth cone peripheral domain induced repulsive growth cone turning. Meanwhile, dynamic microtubules within the peripheral domain were oriented into areas where actin bundles remained and were lost from areas where actin bundles disappeared. This resulted in directional microtubule extension leading to axon bending and growth cone turning. In addition, this local actin bundle loss coincided with localized growth cone collapse, as well as asymmetrical lamellipodial protrusion. Our results provide direct evidence, for the first time, that regional actin bundle reorganization can steer the growth cone by coordinating actin reorganization with microtubule dynamics. This suggests that actin bundles can be potential targets of signaling pathways downstream of guidance cues, providing a mechanism for coupling changes in leading edge actin with microtubules at the central domain during turning.     

Filopodial actin bundles are not necessary for microtubule advance into the peripheral domain of Aplysia neuronal growth cones

Filopodial actin bundles guide microtubule assembly in the growth cone peripheral (P) domain and retrograde actin-network flow simultaneously transports microtubules rearward. Therefore, microtubule-end position is determined by the sum of microtubule assembly and retrograde transport rates. However, how filopodia actually affect microtubule assembly dynamics is unknown. To address this issue we quantitatively assessed microtubule and actin dynamics before and after selective removal of filopodia. Filopodium removal had surprisingly little effect on retrograde actin-flow rates or underlying network structures, but resulted in an approximate doubling of peripheral microtubule density and deeper penetration of microtubules into the P domain. The latter stemmed from less efficient coupling of microtubules to remaining actin networks and not from a change in microtubule polymer dynamics. Loss of filopodia also resulted in increased lateral microtubule movements and a more randomized microtubule distribution in the P domain. In summary, filopodia do not seem to be formally required for microtubule advance; however, their presence ensures radial distribution of microtubules in the P domain and facilitates microtubule transport by retrograde flow. The resulting dynamic steady state has interesting implications for rapid microtubule-positioning responses in the P domain.     

Interaction of the betaIV-tubulin isotype with actin stress fibers in cultured rat kidney mesangial cells.

Microtubules and actin filaments are two of the major components of the cytoskeleton. There is accumulating evidence for interaction between the two networks. Both the alpha- and beta-subunits of tubulin exist as numerous isotypes, some of which have been highly conserved in evolution. In an effort to better understand the functional significance of tubulin isotypes, we used a double immunofluorescence labeling technique to investigate the interactions between the tubulin beta-isotypes and the actin stress fiber network in cultured rat kidney mesangial cells, smooth-muscle-like cells from the renal glomerulus. Removal of the soluble cytoplasmic and nucleoplasmic proteins by detergent extraction caused the microtubule network to disappear while the stress fiber network was still present. In these extracted cells, the betaI- and betaII-tubulin isotypes were no longer present in the cytoplasm while the betaIV-isotype co-localized with actin stress fibers. Co-localization between betaIV-tubulin and actin stress fibers was also observed when the microtubule network was disrupted by the anti-tubulin drug colchicine and also by microinjection of the betaIV-tubulin antibody. Our results suggest that the betaIV isotype of tubulin may be involved in interactions between microtubules and actin.     

Dynein binds to beta-catenin and may tether microtubules at adherens junctions

Interactions between microtubule and actin networks are thought to be crucial for mechanical and signalling events at the cell cortex. Cytoplasmic dynein has been proposed to mediate many of these interactions. Here, we report that dynein is localized to the cortex at adherens junctions in cultured epithelial cells and that this localization is sensitive to drugs that disrupt the actin cytoskeleton. Dynein is recruited to developing contacts between cells, where it localizes with the junctional proteins beta-catenin and E-cadherin. Microtubules project towards these early contacts and we hypothesize that dynein captures and tethers microtubules at these sites. Dynein immunoprecipitates with beta-catenin, and biochemical analysis shows that dynein binds directly to beta-catenin. Overexpression of beta-catenin disrupts the cellular localization of dynein and also dramatically perturbs the organization of the cellular microtubule array. In cells overexpressing beta-catenin, the centrosome becomes disorganized and microtubules no longer appear to be anchored at the cortex. These results identify a novel role for cytoplasmic dynein in capturing and tethering microtubules at adherens junctions, thus mediating cross-talk between actin and microtubule networks at the cell cortex.     

Taxol reduces cytosolic E-cadherin and beta-catenin levels in nasopharyngeal carcinoma cell line TW-039: cross-talk between the microtubule- and actin-based cytoskeletons

Taxol affects microtubule dynamics by promoting microtubule assembly. To obtain a better insight into possible cross-talk between the microtubule- and actin-based cytoskeletons, we studied the short-term effects of Taxol treatment on the expression of actin and the E-cadherin/catenin complex in the nasopharyngeal carcinoma cell line TW-039 using immunofluorescence, immunoprecipitation, and immunoblotting methods. Morphologic changes in actin filaments, including ventral actin clumps and perijunctional actin blebs, were seen at Taxol concentrations > or =1 microM. Levels of detergent-soluble E-cadherin fell to 53% or 58% compared to controls in cells treated, respectively, with 1 or 5 microM Taxol, while levels of detergent-soluble beta-catenin fell to 76% or 74%. Levels of the detergent-soluble pool of alpha- and gamma-catenin and the detergent-insoluble pool of the E-cadherin/catenin complex were unchanged by Taxol treatment and no significant difference was seen in the levels of adenomatous polyposis coli or glycogen synthase-3beta or tyrosine phosphorylation patterns. These results suggest that modulation of microtubule dynamics by Taxol may have effects on the expression of actin and the cytosolic E-cadherin and beta-catenin in nasopharyngeal carcinoma cells through pathways not involving the phosphorylation of beta-catenin.     

Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte

Mutants in the actin nucleators Cappuccino and Spire disrupt the polarized microtubule network in the Drosophila oocyte that defines the anterior-posterior axis, suggesting that microtubule organization depends on actin. Here, we show that Cappuccino and Spire organize an isotropic mesh of actin filaments in the oocyte cytoplasm. capu and spire mutants lack this mesh, whereas overexpressed truncated Cappuccino stabilizes the mesh in the presence of Latrunculin A and partially rescues spire mutants. Spire overexpression cannot rescue capu mutants, but prevents actin mesh disassembly at stage 10B and blocks late cytoplasmic streaming. We also show that the actin mesh regulates microtubules indirectly, by inhibiting kinesin-dependent cytoplasmic flows. Thus, the Capu pathway controls alternative states of the oocyte cytoplasm: when active, it assembles an actin mesh that suppresses kinesin motility to maintain a polarized microtubule cytoskeleton. When inactive, unrestrained kinesin movement generates flows that wash microtubules to the cortex.     

Profilin connects actin assembly with microtubule dynamics

Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.