Microtubule binding proteins CLIP-170, EB1, and p150Glued form distinct plus-end complexes

Microtubule plus-end proteins CLIP-170 and EB1 dynamically track the tips of growing microtubules in vivo. Here we examine the association of these proteins with microtubules in vitro. CLIP-170 binds tubulin dimers and co-assembles into growing microtubules. EB1 binds tubulin dimers more weakly, so no co-assembly is observed. However, EB1 binds to CLIP-170, and forms a co-complex with CLIP-170 and tubulin that is recruited to growing microtubule plus ends. The interaction between CLIP-170 and EB1 is competitively inhibited by the related CAP-Gly protein p150Glued, which also localizes to microtubule plus ends in vivo. Based on these observations, we propose a model in which the formation of distinct plus-end complexes may differentially affect microtubule dynamics in vivo.     

CLIP-170/tubulin-curved oligomers coassemble at microtubule ends and promote rescues

BACKGROUND: CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. RESULTS: We used the N-terminal part of CLIP-170 (named H2), which contains the microtubule binding domains, to investigate how it modulates in vitro microtubule dynamics and structure. We found that H2 primarily promoted rescues (transitions from shrinkage to growth) of microtubules nucleated from pure tubulin and isolated centrosomes, and stimulated microtubule nucleation. Electron cryomicroscopy revealed that H2 induced the formation of tubulin rings in solution and curved oligomers at the extremities of microtubules in assembly conditions. CONCLUSIONS: These results suggest that CLIP-170 targets specifically at microtubule plus ends by copolymerizing with tubulin and modulates microtubule nucleation, polymerization, and rescues by the same basic mechanism with tubulin oligomers as intermediates.     

Codon optimization reveals critical factors for high level expression of two rare codon genes in Escherichia coli: RNA stability and secondary structure but not tRNA abundance

Platelet-derived growth factor (PDGF)-BB elicits a migratory response including reorganization of the actin cytoskeleton in different cell types. Here we have investigated the effects of PDGF-BB stimulation on beta(1) integrin containing focal adhesions in human diploid fibroblasts adhered to collagen type I. Stimulation with PDGF-BB dissociated focal adhesions and relocated beta(1) integrins from focal adhesions to the periphery of the cells. These changes were rapid and transient in character. Relocation of beta(1) integrins was prevented by inhibitors of phosphoinositide-3-kinase and protein kinase C. PDGF-BB stimulated fibroblasts exhibited an increased diffusion coefficient of cell surface beta(1) integrins as determined by fluorescence recovery of photobleaching. The cell surface expression of beta(1) integrins was not changed after stimulation with PDGF-BB. Our data suggest that PDGF-BB increases the dynamic properties of cell-surface beta(1) integrins, which most likely are important for the migratory response elicited by PDGF-BB.     

Regulation of cell adhesion to collagen via β1 integrins is dependent on interactions of filamin A with vimentin and protein kinase C epsilon

Cell adhesion and spreading on collagen, which are essential processes for development and wound healing in mammals, are mediated by β1 integrins and the actin and intermediate filament cytoskeletons. The mechanisms by which these separate cytoskeletal systems interact to regulate β1 integrins and cell spreading are poorly defined. We previously reported that the actin cross-linking protein filamin A binds the intermediate filament protein vimentin and that these two proteins co-regulate cell spreading. Here we used deletional mutants of filamin A to define filamin A-vimentin interactions and the subsequent phosphorylation and re-distribution of vimentin during cell spreading on collagen. Imaging of fixed and live cell preparations showed that phosphorylated vimentin is translocated to the cell membrane during spreading. Knockdown of filamin A inhibited cell spreading and the phosphorylation and re-distribution of vimentin. Knockdown of filamin A and/or vimentin reduced the cell surface expression and activation of β1 integrins, as indicated by immunoblotting of plasma membrane-associated proteins and shear force assays. In vitro pull-down assays using filamin A mutants showed that both vimentin and protein kinase C? bind to repeats 1-8 of filamin A. Reconstitution of filamin-A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation, and the cell surface expression of β1 integrins. We conclude that the binding of filamin A to vimentin and protein kinase Cε is an essential regulatory step for the trafficking and activation of β1 integrins and cell spreading on collagen.     

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to alpha-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.     

Cytoplasmic linker protein-170 enhances spreading and phagocytosis in activated macrophages by stabilizing microtubules

Activation of macrophages causes increased cell spreading, increased secretion of cytokines and matrix metalloproteinases, and enhanced phagocytosis. The intracellular mechanisms driving the up-regulation of these activities have not been completely clarified. We observe that classical activation of murine resident peritoneal or RAW 264.7 macrophages with a combination of IFN-gamma and LPS induces an increase in stabilized cytoplasmic microtubules (MTs), measured with an anti-acetylated alpha-tubulin Ab. We examined the mechanism of this MT stabilization and find that macrophage activation causes redistribution of the MT plus-end tracking protein, cytoplasmic linker protein-170 (CLIP-170). CLIP-170 is localized at the distal plus-ends of MTs in resting macrophages, but accumulates along the length of MTs in IFN-gamma/LPS-activated cells. A direct involvement of CLIP-170 in MT stabilization has not been thoroughly established. In this study, we show that expression of a mutant CLIP-170 chimeric protein (dominant-negative CLIP-170-GFP), lacking the MT-binding domain, prevents MT stabilization in activated RAW 264.7 macrophages. Furthermore, we find enhanced CLIP-170 association with MTs and MT stabilization by treating resting macrophages with okadaic acid, implicating the protein phosphatase 2A in CLIP-170 binding and MT stabilization in RAW 264.7 cells. Finally, we observed enhanced cell spreading and phagocytosis in both IFN-gamma/LPS-activated and okadaic acid-treated resting RAW 264.7 cells, which are markedly reduced in activated cells expressing dominant-negative CLIP-170-GFP. These results identify CLIP-170 as a key regulator of MT stabilization and establish a prominent role for stabilized MTs in cell spreading and phagocytosis in activated macrophages.     

Smooth muscle actin determines mechanical force-induced p38 activation

The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As alpha-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.65 pn/mum(2)) generated by magnetic fields were applied to collagen-coated magnetite beads bound to Rat-2 cells. Immunoblotting showed that p38alpha was the predominant p38 isoform. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the alpha2beta1 integrin. SMA was present almost entirely as filaments. Swinholide depolymerized SMA filaments and blocked force-induced p38 phosphorylation and force-induced increases of SMA. Knockdown of SMA (70% reduction) using RNA interference did not affect beta-actin but inhibited force-induced p38 phosphorylation by 50%. Inhibition of Rho kinase blocked SMA filament assembly, force-induced increases of SMA, and force-induced p38 activation. Force application increased SMA content and enhanced the association of phosphorylated p38 with SMA filaments. Blockade of p38 phosphorylation by SB203586 abrogated force-induced increases of SMA. In cells transfected with SMA promoter-beta-galactosidase fusion constructs, co-transfection with constitutively active p38 or MKK6 increased SMA promoter activity by 2.5-3-fold. Dominant negative p38 blocked force-induced activation of the SMA promoter. In SMA negative cells, there was no force-induced p38 phosphorylation. We conclude that force-induced p38 phosphorylation is dependent on an SMA filament-dependent pathway that uses a feed-forward amplification loop to synergize force-induced SMA expression with p38 activation.     

Force regulates smooth muscle actin in cardiac fibroblasts

Chronic ventricular pressure overload can regulate expression of alpha-smooth muscle actin (SMA) in cardiac fibroblasts, but it is unclear if force alone or the concomitant activity of angiotensin II is the principal regulatory factor. To test if SMA mRNA and protein in rat cardiac fibroblasts are regulated directly by force, we first induced SMA expression in cultured cells and then applied magnetically generated perpendicular forces through focal adhesions using collagen-coated magnetite beads. Continuous static forces (0.65 pN/micrometer(2)) selectively reduced SMA but not beta-actin mRNA and protein content within 4 h (to 55 +/- 9% of controls); SMA returned to baseline by 8 h. There was no change in SMA content after force application with either plasma or the cellular fibronectin IIIA domain, BSA, or poly-L-lysine beads. The early loss of SMA was apparently due to selective leakage into the cell culture medium. Treatment with angiotensin II (10 nM) abrogated the force-induced reduction of SMA and increased the levels of this protein. The stress kinase p38 was phosphorylated by force, whereas extracellular signal-regulated kinase 1/2 and c-Jun NH(2)-terminal kinase were unaffected. The p38 kinase inhibitor SB-203580 relieved the force-induced SMA reduction. We conclude that force-induced inhibition of SMA is mediated through the p38 kinase pathway, and this pathway antagonizes angiotensin II regulation of SMA.     

Mechanical properties of individual focal adhesions probed with a magnetic microneedle

A permanent magnetic microneedle was developed to apply tensional forces to integrin receptors via ligand-coated magnetic microbeads while optically analyzing the mechanical properties of individual focal adhesions. Force application (130 pN for 3 s) through activated beta1 integrins produced less bead displacement than when unligated integrins were stressed. This strengthening response differed markedly on a bead-by-bead basis, correlated directly with local focal adhesion assembly, and was similar when analyzed at 4 degrees C, indicating that it was due to passive material properties of the cell. Viscoelastic analysis clarified that recruitment of focal adhesion proteins increased the local elastic stiffness of the adhesion complex without changing its viscous behavior. These data indicate that individual focal adhesions exhibit distinct mechanical properties that depend upon local focal adhesion assembly, and that these local variations in micromechanics can be detected and analyzed within living cells using the permanent magnetic microneedle technique.     

Purification and analysis of authentic CLIP-170 and recombinant fragments.

We have purified authentic CLIP-170 (cytoplasmic linker protein of 170 kDa) and fragments comprising functional domains of the protein to characterize the structural basis of the function of CLIP-170. Analysis of authentic CLIP-170 and the recombinant fragments by electron microscopy after glycerol spraying/low angle rotary metal shadowing reveals CLIP-170 as a thin, 135-nm-long molecule with two kinks in its central rod domain, which are approximately equally spaced from the two ends of the protein. The central domain consisting of heptad repeats, which is alpha-helical in nature and forms a 2-stranded coiled-coil, mediates dimerization of CLIP-170. The rod domain harbors two kinks, each spaced approximately 37 nm from the corresponding end of the molecule, thus providing mechanical flexibility to the highly elongated molecule. The N-terminal domain of CLIP-170 binds to microtubules in vitro with a stoichiometry of one dimeric head domain per four tubulin heterodimers. Authentic CLIP-170 binds to microtubules with lower stoichiometry, indicating that the rod and tail domains affect microtubule binding of CLIP-170. These results document that CLIP-170 is a highly elongated polar molecule with the microtubule-binding domain and the organelle-interacting domains at opposite ends of the homodimer, thus providing a structural basis for the function of CLIP-170 as a microtubule-organelle linker protein.     

Assembly and mechanosensory function of focal adhesions: experiments and models

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.     

Interactions between CLIP-170, tubulin, and microtubules: implications for the mechanism of Clip-170 plus-end tracking behavior

CLIP-170 belongs to a group of proteins (+TIPs) with the enigmatic ability to dynamically track growing microtubule plus-ends. CLIP-170 regulates microtubule dynamics in vivo and has been implicated in cargo-microtubule interactions in vivo and in vitro. Though plus-end tracking likely has intimate connections to +TIP function, little is known about the mechanism(s) by which this dynamic localization is achieved. Using a combination of biochemistry and live cell imaging, we provide evidence that CLIP-170 tracks microtubule plus-ends by a preassociation, copolymerization, and regulated release mechanism. As part of this analysis, we find that CLIP-170 has a stronger affinity for tubulin dimer than for polymer, and that CLIP-170 can distinguish between GTP- and GDP-like polymer. This work extends the previous analysis of CLIP-170 behavior in vivo and complements the existing fluorescence microscope characterization of CLIP-170 interactions with microtubules in vitro. In particular, these data explain observations that CLIP-170 localizes to newly polymerized microtubules in vitro but cannot track microtubule plus-ends in vitro. These observations have implications for the functions of CLIP-170 in regulating microtubule dynamics.     

CLIP-170 facilitates the formation of kinetochore-microtubule attachments

CLIP-170 is a microtubule 'plus end tracking' protein involved in several microtubule-dependent processes in interphase. At the onset of mitosis, CLIP-170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have suggested an essential role for CLIP-170 during mitosis, the molecular function of CLIP-170 in mitosis has not yet been revealed. Here, we used a combination of high-resolution microscopy and RNAi-mediated depletion to study the function of CLIP-170 in mitosis. We found that CLIP-170 dynamically localizes to the outer most part of unattached kinetochores and to the ends of growing microtubules. In addition, we provide evidence that a pool of CLIP-170 is transported along kinetochore-microtubules by the dynein/dynactin complex. Interference with CLIP-170 expression results in defective chromosome congression and diminished kinetochore-microtubule attachments, but does not detectibly affect microtubule dynamics or kinetochore-microtubule stability. Taken together, our results indicate that CLIP-170 facilitates the formation of kinetochore-microtubule attachments, possibly through direct capture of microtubules at the kinetochore.     

The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis

Microtubule dynamics are modulated by regulatory proteins that bind to their plus ends (+TIPs [plus end tracking proteins]), such as cytoplasmic linker protein 170 (CLIP-170) or end-binding protein 1 (EB1). We investigated the role of +TIPs during phagocytosis in macrophages. Using RNA interference and dominant-negative approaches, we show that CLIP-170 is specifically required for efficient phagocytosis triggered by αMβ2 integrin/complement receptor activation. This property is not observed for EB1 and EB3. Accordingly, whereas CLIP-170 is dynamically enriched at the site of phagocytosis, EB1 is not. Furthermore, we observe that CLIP-170 controls the recruitment of the formin mDia1, an actin-nucleating protein, at the onset of phagocytosis and thereby controls actin polymerization events that are essential for phagocytosis. CLIP-170 directly interacts with the formin homology 2 domain of mDia1. The interaction between CLIP-170 and mDia1 is negatively regulated during αMβ2-mediated phagocytosis. Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of αMβ2 integrins.     

Model of coupled transient changes of Rac, Rho, adhesions and stress fibers alignment in endothelial cells responding to shear stress

Interactions of cell adhesions, Rho GTPases and actin in the endothelial cells' response to external forces are complex and not fully understood, but a qualitative understanding of the mechanosensory response begins to emerge. Here, we formulate a mathematical model of the coupled dynamics of cell adhesions, small GTPases Rac and Rho and actin stress fibers guiding a directional reorganization of the actin cytoskeleton. The model is based on the assumptions that the interconnected cytoskeleton transfers the shear force to the adhesion sites, which in turn transduce the force into a chemical signal that activates integrins at the basal surface of the cell. Subsequently, activated and ligated integrins signal and transiently de-activate Rho, causing the disassembly of actin stress fibers and inhibiting the maturation of focal complexes into focal contacts. Focal complexes and ligated integrins activate Rac, which in turn enhances focal complex assembly. When Rho activity recovers, stress fibers re-assemble and promote the maturation of focal complexes into focal contacts. Merging stress fibers self-align, while the elevated level of Rac activity at the downstream edge of the cell is translated into an alignment of the cells and the newly forming stress fibers in the flow direction. Numerical solutions of the model equations predict transient changes in Rac and Rho that compare well with published experimental results. We report quantitative data on early alignment of the stress fibers and its dependence on cell shape that agrees with the model.     

CLIP-170 links endocytic vesicles to microtubules.

Binding of endocytic carrier vesicles to microtubules depends on the microtubule-binding protein CLIP-170 in vitro. In vivo, CLIP-170 colocalizes with a subset of transferrin receptor-positive endocytic structures and, more extensively, with endosomal tubules induced by brefeldin A. The structure of CLIP-170 has been analyzed by cloning its cDNA. The predicted non-helical C- and N-terminal domains of the homodimeric protein are connected by a long coiled-coil domain. We have identified a novel motif present in a tandem repeat in the N-terminal domain of CLIP-170 that is involved in binding to microtubules. This motif is also found in the Drosophila Glued and yeast BIK1 proteins. These features, together with its very elongated structure, suggest that CLIP-170 belongs to a novel class of proteins, cytoplasmic linker proteins (CLIPs), mediating interactions of organelles with microtubules.     

Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1

Microtubule plus end binding proteins (+TIPs) localize to the dynamic plus ends of microtubules, where they stimulate microtubule growth and recruit signaling molecules. Three main +TIP classes have been identified (XMAP215, EB1, and CLIP-170), but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. Here, we report crystal structures of the tubulin binding domains of XMAP215 (yeast Stu2p and Drosophila Msps), EB1 (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces. Functional studies, however, reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and, in most cases, plus end tracking in living cells. We propose that +TIPs, although diverse in structure, share a common property of multimerizing tubulin, thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end.     

The microtubule cytoskeleton participates in control of beta2 integrin avidity

Leukocyte avidity is regulated by cytoskeletal constraints, which keep beta(2) integrins in an inactive mode. Releasing these constraints results in increased lateral mobility and clustering of integrins, effectively activating adhesion. At least part of the constraint on beta(2) integrins is due to actin; whether other cytoskeletal components are involved has not previously been investigated. Microtubules are a candidate for control of integrin rearrangement, because they modulate focal adhesions, which are sites of interaction between integrins and the cytoskeleton. Here we report that both depolymerization of microtubules by colchicine or nocodazole and stabilization of microtubules by taxol increased the lateral mobility of beta(2) integrins, activating adhesion. Increased integrin mobility was accompanied by an increase in tyrosine phosphorylation of paxillin, a biochemical event associated with activation of beta(2) integrins. Further, C3 exoenzyme, an inhibitor of Rho, blocked induction of integrin mobility by nocodazole, but not by taxol, suggesting that there are multiple microtubule-dependent pathways to integrin rearrangement, only some of which require Rho activity. Taken together, our data suggest that a dynamic microtubule system is required to regulate integrin-cytoskeleton interactions. Furthermore, these data demonstrate that microtubules participate in control of integrin rearrangement, one of the earliest steps in activation of integrin-mediated adhesion.