The carboxy-terminus of the formin FMNL1É£ bundles actin to potentiate adenocarcinoma migration.

The formin family of proteins contributes to spatiotemporal control of actin cytoskeletal rearrangements during motile cell activities. The FMNL subfamily exhibits multiple mechanisms of linear actin filament formation and organization. Here we report novel actin-modifying functions of FMNL1 in breast adenocarcinoma migration models. FMNL1 is required for efficient cell migration and its three isoforms exhibit distinct localization. Suppression of FMNL1 protein expression results in a significant impairment of cell adhesion, migration, and invasion. Overexpression of FMNL1É£, but not FMNL1ß or FMNL1α, enhances cell adhesion independent of the FH2 domain and FMNL1É£ rescues migration in cells depleted of all three endogenous isoforms. While FMNL1É£ inhibits actin assembly in vitro, it facilitates bundling of filamentous actin independent of the FH2 domain. The unique interactions of FMNL1É£ with filamentous actin provide a new understanding of formin domain functions and its effect on motility of diverse cell types suggest a broader role than previously realized.

Non-canonical activity of the podosomal formin FMNL1γ supports immune cell migration.

Having previously located the formin FMNL1 in macrophage podosomes, we developed an in vivo model to assess the role of FMNL1 in the migration activities of primary macrophages. Deletion of FMNL1 in mice was genetically lethal; however, targeted deletion in macrophages was achieved by employing macrophage-specific Cre. Unchallenged FMNL1-deficient mice exhibited an unexpected reduction in tissue-resident macrophages despite normal blood monocyte numbers. Upon immune stimulus, the absence of FMNL1 resulted in reduced macrophage recruitment in vivo, decreased migration in two-dimensional in vitro culture and a decrease in the number of macrophages exhibiting podosomes. Of the three described isoforms of FMNL1 - α, ß and γ - only FMNL1γ rescued macrophage migration when expressed exogenously in depleted macrophages. Surprisingly, mutation of residues in the FH2 domain of FMNL1γ that disrupt barbed-end actin binding did not limit rescue of macrophage migration and podosome numbers. These observations suggest that FMNL1 contributes to macrophage migration activity by stabilizing the lifespan of podosomes without interaction of fast-growing actin termini.

Reliable and inexpensive expression of large, tagged, exogenous proteins in murine bone marrow-derived macrophages using a second generation lentiviral system.

Over the past two decades, researchers have struggled to efficiently express foreign DNA in primary macrophages, impeding research progress. The applications of lipofection, electroporation, microinjection, and viral-mediated transfer typically result in disruptions in macrophage differentiation and function, low expression levels of exogenous proteins, limited efficiency and high cell mortality. In this report, after extensive optimization, we present a method of expressing large tagged proteins at high efficiency, consistency, and low cost using lentiviral infection. This method utilizes laboratory-propagated second generation plasmids to produce efficient virus that can be stored for later use. The expression of proteins up to 150 kDa in size is achieved in 30-70% of cells while maintaining normal macrophage differentiation and morphology as determined by fluorescence microscopy and Western blot analysis. This manuscript delineates the reagents and methods used to produce lentivirus to express exogenous DNA in murine bone marrow-derived macrophages sufficient for single cell microscopy as well as functional assays requiring large numbers of murine bone marrow-derived macrophages.

Human Macrophages Utilize the Podosome Formin FMNL1 for Adhesion and Migration.

Macrophages play a crucial role in detecting, regulating, and resolving immune crises, requiring migration through complex extracellular matrices. Unwarranted macrophage inflammatory activity potentiates kidney disease, rheumatoid arthritis, and transplant rejection. Proper remodeling of the actin cytoskeleton, especially at adhesion structures, is essential to the translocation of macrophages. Macrophages form actin-rich adhesions termed "podosomes", giving them the capacity to make contacts with the substratum for traction through interstitial tissues. Macrophages express multiple formins, including FMNL1, Dia1, and Fhod1, with potential to impact actin remodeling involved in migration. Formins are a family of proteins that are best known for modifying the actin cytoskeleton via nucleation, elongation, bundling, and/or severing actin filaments. In this study we demonstrate that the formin FMNL1 is a key regulator of podosomes and is required for normal macrophage migration. Additionally, this is the first study to demonstrate defects in primary human cell migration resulting from specific formin silencing. Pharmacologic inhibition of all formin activity results in a significant decrease in podosome formation and normal macrophage migration. Furthermore, targeted suppression of FMNL1 results in decreases in macrophage migration similar to inhibition of all expressed macrophage formins. These novel findings suggest FMNL1 as a possible chemotherapeutic target to hinder macrophage migration, which could offer an innovative method for limiting unnecessary macrophage-mediated inflammation. We hypothesize that formins are required in podosome actin dynamics to support macrophage migration.

The multiplicity of human formins: Expression patterns in cells and tissues.

Formins are actin-binding proteins conserved across species from plants to humans. The formin family is defined by their common formin homology (FH2) domains. The 15 distinct human formins are involved in a broad range of cellular functions, including cell adhesion, cytokinesis, cell polarity, and cell morphogenesis. Their commonality is actin polymerization activity inherent to FH2 domains. Although still requiring much study, biochemical activity of formins has been carefully described. In contrast, much less is known of their activities in complex living systems. With the diversity of the formin family and the actin structures that they affect, an extensive future of study beckons. In this study, we report the expression level of all 15 formins in 22 different human cell and tissue types using quantitative real-time PCR. Identification of major themes in formin expression and documentation of expression profiles should facilitate the cellular study of formins.

Calcium Integrin Binding Protein Associates with Integrins αVß3 and αIIbß3 Independent of ß3 Activation Motifs.

The Calcium Integrin Binding protein (CIB) has been identified as interacting specifically with the cytoplasmic tail of the integrin αIIb domain to induce receptor activation and integrin αIIbß3 mediated cell adhesion to extracellular proteins. In K562 cells stably expressing mutated integrin αVß3, or chimeric αVß3 carrying αIIb cytoplasmic tail, we report that the interaction of CIB with ß3 integrins is not αIIbß3 specific but binds αIIb as well as αV cytoplasmic tail domains. A double mutation of two proline residues to alanine residues in the αIIb cytoplasmic domain, previously shown to disturb its conformation, inhibits chimeric αV/αIIbß3-CIB interaction. This demonstrates that αIIb cytoplasmic domain loop-like conformation is required for interaction with CIB. Moreover, mutations of ß3 cytoplasmic domain residues Tyr-747 and/or Tyr-759 to phenylalanine residues (Y747F, Y759F, and Y747,759F) as well as residues Ser-752 to proline or alanine (S752P and S752A), do not affect the αIIbß3 or αVß3 interaction with CIB. Since tyrosine residues Tyr-747 and/or Tyr-759 are the sites of tyrosine phosphorylation of ß3 subunit, these results suggest that the ß3 integrin-CIB interaction occurs through a ß3-phosphorylation independent mechanism. Likewise, ablation of conformation-dependent affinity change in ß3 Ser752Pro mutation had no affect on CIB-ß3 interaction. In summary, our results demonstrate that the αIIb-subunit integrin and CIB interaction is non-exclusive and requires the loop-like αIIb-cytoplasmic domain conformation. An interaction of CIB with αV-containing integrins provides an additional role for this molecule in keeping with its expression outside of platelets.

The formin FRL1 (FMNL1) is an essential component of macrophage podosomes.

Podosomes are highly dynamic actin-rich adhesion structures in cells of myeloid lineage and some transformed cells. Unlike transformed mesenchymal cell types, podosomes are the sole adhesion structure in macrophage and thus mediate all contact with adhesion substrate, including movement through complex tissues for immune surveillance. The existence of podosomes in inflammatory macrophages and transformed cell types suggest an important role in tissue invasion. The proteome, assembly, and maintenance of podosomes are emerging, but remain incompletely defined. Previously, we reported a formin homology sequence and actin assembly activity in association with macrophage beta-3 integrin. In this study we demonstrate by quantitative reverse transcriptase polymerase chain reaction and Western blotting that the formin FRL1 is specifically upregulated during monocyte differentiation to macrophages. We show that the formin FRL1 localizes to the actin-rich cores of primary macrophage podosomes. FRL1 co-precipitates with beta-3 integrin and both fixed and live cell fluorescence microscopy show that endogenous and overexpressed FRL1 selectively localize to macrophage podosomes. Targeted disruption of FRL1 by siRNA results in reduced cell adhesion and disruption of podosome dynamics. Our data suggest that FRL1 is responsible for modifying actin at the macrophage podosome and may be involved in actin cytoskeleton dynamics during adhesion and migration within tissues.

Macrophages of multiple sclerosis patients display deficient SHP-1 expression and enhanced inflammatory phenotype.

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of proinflammatory cytokine signaling, TLR signaling, and inflammatory gene expression. Furthermore, mice genetically lacking SHP-1 (me/me) display a profound susceptibility to inflammatory CNS demyelination relative to wild-type mice. In particular, SHP-1 deficiency may act predominantly in inflammatory macrophages to increase CNS demyelination as SHP-1-deficient macrophages display coexpression of inflammatory effector molecules and increased demyelinating activity in me/me mice. Recently, we reported that PBMCs of multiple sclerosis (MS) patients have a deficiency in SHP-1 expression relative to normal control subjects indicating that SHP-1 deficiency may play a similar role in MS as to that seen in mice. Therefore, it became essential to examine the specific expression and function of SHP-1 in macrophages from MS patients. Herein, we document that macrophages of MS patients have deficient SHP-1 protein and mRNA expression relative to those of normal control subjects. To examine functional consequences of the lower SHP-1, the activation of STAT6, STAT1, and NF-kappaB was quantified and macrophages of MS patients showed increased activation of these transcription factors. In accordance with this observation, several STAT6-, STAT1-, and NF-kappaB-responsive genes that mediate inflammatory demyelination were increased in macrophages of MS patients following cytokine and TLR agonist stimulation. Supporting a direct role of SHP-1 deficiency in altered macrophage function, experimental depletion of SHP-1 in normal subject macrophages resulted in an increased STAT/NF-kappaB activation and increased inflammatory gene expression to levels seen in macrophages of MS patients. In conclusion, macrophages of MS patients display a deficiency of SHP-1 expression, heightened activation of STAT6, STAT1, and NF-kappaB and a corresponding inflammatory profile that may be important in controlling macrophage-mediated demyelination in MS.

A Pyk2-Vav1 complex is recruited to beta3-adhesion sites to initiate Rho activation.

Integrin alphavbeta3-mediated adhesion of haemopoietic cells to vitronectin results in beta3 tyrosine phosphorylation and Rho activation which is necessary for adhesion. Previously, we have shown that the RhoGEF (Rho guanine-nucleotide-exchange factor) Vav1 could associate indirectly with alphavbeta3 during leucocyte adhesion to vitronectin. In the present study, we have identified the non-receptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) as the adaptor protein that links Vav1 with alphavbeta3. The association of Pyk2 and Vav1 with beta3 relies on the presence of Tyr747 in beta3, the primary site of beta3 phosphorylation. However, association of Pyk2 with Vav1 is independent of beta3 tyrosine phosphorylation. Formation of a Pyk2-Vav1 complex occurs upon cell adhesion and Pro717 of Pyk2 plays a key role in Pyk2 interaction with Vav1. Utilizing purified recombinant proteins, we confirmed the direct interaction between Pyk2 and Vav1 In vitro. Cells transfected with GFP (green fluorescent protein)-Pyk2-P717A demonstrated severely suppressed cytoskeletal reorganization, impaired Vav1 recruitment, decreased Rho GTPase activation and loss of cell adhesion. Using siRNA (small interfering RNA) to specifically reduce Pyk2 levels in cells resulted in disrupted association between Vav1 and beta3 and impaired cell adhesion. These results indicate that Pyk2 is a critical signalling molecule downstream of beta3 integrin tyrosine phosphorylation and mediates Vav1 recruitment to accomplish actin reorganization necessary for adhesion.

Purified integrin adhesion complexes exhibit actin-polymerization activity.

BACKGROUND: Cell adhesion and motility are accomplished through a functional linkage of the extracellular matrix with the actin cytoskeleton via adhesion complexes composed of integrin receptors and associated proteins. To determine whether this linkage is attained actively or passively, we isolated integrin complexes from nonadherent hematopoietic cells and determined their influence on the polymerization of actin. RESULTS: We observed that alpha(V)beta3 complexes are capable of dramatically accelerating the rate of actin assembly, resulting in actin fibers tethered at their growing ends by clustered integrins. The ability to enhance actin polymerization was dependent upon Arg-Gly-Asp-ligand-induced beta3 tyrosine phosphorylation, agonist-induced cellular activation, sequestration of Diaphanous formins, and clustering of the receptor. CONCLUSIONS: These results suggest that adhesion complexes actively promote actin assembly from their cytosolic face in order to establish a mechanical linkage with the extracellular matrix.

Tyrosine phosphorylation of beta3 integrin provides a binding site for Pyk2.

Integrins expressed on leukocytes possess the ability to maintain themselves in a non-adhesive state, thus preventing unwarranted adhesion and uncontrolled inflammation. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha(V)beta(3). Firm adhesion to the extracellular matrix and directed cellular motility requires the reorganization of the actin cytoskeleton. The ability of beta(3) to recruit signaling and scaffolding molecules to propagate alpha(V)beta(3) -mediated signals is regulated in part by the phosphorylation of the beta(3) cytoplasmic tail. The identities of integrin-associated signaling molecules within alpha(V)beta(3) podosomes and in particular the proximal binding partners of the beta(3) cytoplasmic tail are not completely known. Here we show that alpha(V)beta(3) ligation induces Pyk2-Tyr-402 phosphorylation and its association with the beta(3) cytoplasmic tail in a beta(3)-Tyr-747 phosphorylation-dependent manner. Pyk2 binding to the beta(3) cytoplasmic tail is direct and dependent upon Pyk2-Tyr-402 and beta(3) -Tyr-747 phosphorylations. These data identify Pyk2 as a phosphorylated beta(3) binding partner, providing a potential structural and signaling platform to achieve alpha(V)beta(3) -mediated remodeling of the actin cytoskeleton.

Beta3 tyrosine phosphorylation and alphavbeta3-mediated adhesion are required for Vav1 association and Rho activation in leukocytes.

Integrin alpha(v)beta(3)-mediated adhesion of hematopoietic cells to vitronectin results in activation of the Rho GTPases. Mutation of beta(3) tyrosine residue 747, previously shown to disrupt cell adhesion, results in sustained activation of Cdc42 and diminished Rac and Rho activity. We investigated the role of the hematopoietically restricted guanine nucleotide exchange factor Vav1 in alpha(v)beta(3)-mediated adhesion. We find that Vav1, a guanine nucleotide exchange factor for Rac and Rho, associates with alpha(v)beta(3) upon cell adhesion to vitronectin and that this association requires beta(3) tyrosine phosphorylation. Expression of exogenous Vav1 demonstrates that Y160F, but not wild type or the Vav1Y174F mutant, inhibits Rac and Rho activation during alpha(v)beta(3)-mediated cell adhesion to vitronectin. Cells expressing Vav1Y160F exhibit a sustained Cdc42 activation similar to nonphosphorylatable beta(3) mutants. In addition, cytoskeletal reorganization and cell adhesion are severely suppressed in Vav1Y160F-transfected cells, and Vav1Y160F fails to associate with beta(3) integrins. Furthermore, Vav1 itself is selectively phosphorylated upon tyrosine 160 after alpha(v)beta(3)-mediated adhesion, and the association between Vav1 and beta(3) occurs in specific response to adhesion to substrate. These studies describe a phosphorylation-dependent association between beta(3) integrin and Vav1 which is essential for cell progression to a Rho-dominant phenotype during cell adhesion.

Integrating an integrin: a direct route to actin.

Integrins were so named for their ability to link the extracellular and intracellular skeletons. Now almost 20 years into integrin research, numerous questions remain as to how this interaction is accomplished and how it is modified to achieve a desired phenotype. As the cell adhesion and actin assembly fields are merging in combined approaches, novel actin assembly mechanisms are being uncovered. Some of the earliest identified cytoplasmic linker molecules, believed to mediate integrin-actin binding, are once again the subject of scrutiny as potential dynamic mediators of cell anchorage. It seems plausible that each unique cellular morphology occurs as the result of activation of distinct actin assembly systems that are either stabilized by unique bundling and linker proteins or modified for progression to a new phenotype. While this research initiative is likely to continue rapidly in a forward fashion, it remains to be clarified how integrins assemble the most stable and basic cytoskeletal phenotype, the adherent cell with prominent stress fibers. Recent investigations point towards a shift in the current model of anchoring at the cell periphery by providing both mechanisms and evidence for de novo actin assembly orchestrated by the adhesion site. Lacking a complete pathway from integrin ligation to an integrated extracellular-intracellular skeleton in any single system, this review proposes a simple model of integrin-mediated stress fiber integration by drawing from work in multiple systems.

Beta 3 integrin phosphorylation is essential for Arp3 organization into leukocyte alpha V beta 3-vitronectin adhesion contacts.

Integrins play a pivotal role in self-regulated hematopoietic adhesion and migration. Leukocyte alpha(V)beta(3) integrin-mediated adhesion to vitronectin requires protein kinase C activation and phosphorylation on tyrosine 747 of the beta(3) cytoplasmic tail. We have previously shown that beta(3) phosphorylation is required for Rho activation. In this study, an antibody specific to phosphorylated beta(3) tyrosine 747 was used to localize phosphorylated alpha(V)beta(3) in vitronectin adhesive structures. Early adhesion contacts containing phosphorylated beta(3) preceded actin stress fiber formation. beta(3) phosphorylation decreased progressively throughout the course of adhesion coincident with the appearance of actin stress fibers. Time-dependent increases in colocalization of beta(3) with tyrosine 402 phosphorylated Pyk2 in similar adhesive structures was observed, providing evidence for downstream signaling complex formation. Surprisingly, Arp3 organized into similar adhesion contacts in cells expressing wild-type beta(3) but not in those expressing a nonphosphorylatable mutant of beta(3), suggesting that beta(3) phosphorylation is required for sequestration of Arp3 to adhesion complexes. Suppression of actin stress fiber formation by an inhibitor to Rho kinase disrupted Arp3 organization while prolonging beta(3) phosphorylation throughout the adhesion time course. These data confirm a requirement for beta(3) phosphorylation in alpha(V)beta(3)-mediated adhesion to vitronectin and suggest that beta(3) phosphorylation permits signaling complex assembly at the adhesion site necessary for actin stress fiber formation in leukocytes.

Integrin alphaIIb-subunit cytoplasmic domain mutations demonstrate a requirement for tyrosine phosphorylation of beta3-subunits in actin cytoskeletal organization.

Using truncated or mutated alphaIIb integrin cytoplasmic domains fused to the alphaV extracellular domain and expressed with the beta3 integrin subunit, we demonstrate that the double mutation of proline residues 998 and 999 to alanine (PP998/999AA), previously shown to disturb the C-terminal conformation of the alphaIIb integrin cytoplasmic domain, prevents tyrosine phosphorylation of beta3 integrin induced by Arg-Gly-Asp peptide ligation. This mutation also inhibits integrin mediated actin assembly and cell adhesion to vitronectin. In contrast, progressive truncation of the alphaIIb-subunit cytoplasmic domain did not reproduce these effects. Interestingly, the PP998/999AA mutations of alphaIIb did not affect beta3 tyrosine phosphorylation, cell adhesion, or actin polymerization induced by manganese. Exogenous addition of manganese was sufficient to rescue beta3 phosphorylation, cell adhesion, and actin assembly in cells expressing the PP998/999AA mutation when presented with a vitronectin substrate. Further, induction of the high affinity conformation of this mutant beta3 integrin by incubation with either Arg-Gly-Asp peptide or exogenous manganese was equivalent. These results suggest that the extracellular structure of beta3 integrins in the high affinity conformation is not directly related to the structure of the cytoplasmic face of the integrin. Moreover, the requirement for beta3 phosphorylation is demonstrated without mutation of the beta3 subunit. In support of our previous hypothesis of a role for beta3 phosphorylation in adhesion, these studies demonstrate a strong correlation between beta3 tyrosine phosphorylation and assembly of a cytoskeleton competent to support firm cell adhesion.

Heparin modulates integrin-mediated cellular adhesion: specificity of interactions with alpha and beta integrin subunits.

Heparin is known to influence the growth, proliferation, and migration of vascular cells, but the precise mechanisms are unknown. We previously demonstrated that unfractionated heparin (UH) binds to the platelet integrin alpha(IIb)beta(3), and enhances ligand binding. To help define the specificity and site(s) of heparin-integrin interactions, we employed the erythroleukemic K562 cell line, transfected to express specific integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)). By comparing K562 cells expressing a common alpha subunit (Kalpha(v)beta(3), Kalpha(v)beta(5)) with cells expressing a common beta subunit (Kalpha(v)beta(3), Kalpha(IIb)beta(3)), we observed that heparin differentially modulated integrin-mediated adhesion to vitronectin. UH at 0.5-7.5 microg/ml consistently enhanced the adhesion of beta(3) expressing cells (Kalpha(v)beta(3),Kalpha(IIb)beta(3)). In contrast, UH at 0.5-7.5 microg/ml inhibited Kalpha(v)beta(5) adhesion. Experiments using integrin-blocking antibodies, appropriate control ligands, and nontransfected native K562 cells revealed that heparin's actions were mediated by the specific integrins under study. Preincubation of heparin with Kalpha(v)beta(3) cells enhanced adhesion, while preincubation of heparin with the adhesive substrate (vitronectin) had minimal effect. There was a structural specificity to heparin's effect, in that a low molecular weight heparin and chondroitin sulfate showed significantly less enhancement of adhesion. These findings suggest that heparin's modulation of integrin-ligand interactions occurs through its action on the integrin. The inhibitory or stimulatory effects of heparin depend on the beta subunit type, and the potency is dictated by structural characteristics of the glycosaminoglycan.

Ligand-dependent activation of integrin alpha vbeta 3.

The ability of leukocytes to self-regulate adhesion during transendothelial and extravascular migration is fundamental to the performance of immune surveillance in complex extracellular matrices. Leukocyte adhesion is regulated through the modulation of integrin receptors such as alpha(v)beta(3). In this study, we examined the activation of alpha(v)beta(3) resulting from attachment to vitronectin or fibronectin. In K562 cells stably expressing transfected alpha(v)beta(3), adhesion to vitronectin required tyrosine phosphorylation of the beta(3) subunit and activation of phosphoinositide 3-kinase and protein kinase C. In contrast, adhesion to fibronectin proceeded without beta(3)-tyrosine phosphorylation or the activities of phosphoinositide 3-kinase or protein kinase C. Firm adhesion to both ligands and actin stress fiber formation required both Syk and Rho activity, suggesting that each ligand employs unique signaling pathways to achieve an active integrin complex, likely merging at a common RhoGEF such as Vav. Distinct signaling by a single integrin species interacting with different ligands permits initiation of additional cellular processes specific to the current task and provides an explanation for what has been described as promiscuous ligand specificity among integrins.

Kinetic regulation of beta 3 integrin tyrosine phosphorylation.

Tyrosine phosphorylation of beta(3) integrins is a permissive stage in the activation of alpha(IIb)beta(3) and alpha(v)beta(3) in platelets and leukocytes, respectively. In this study we demonstrated direct phosphorylation of beta(3) integrins as a result of interaction with soluble monomeric ligand, and we characterized the differential kinetics of beta(3) phosphorylation as a consequence of alpha subunit pairing. We found that beta(3) phosphorylation is initiated by RGD peptide binding in a dose-dependent and saturable fashion with alpha(IIb)beta(3) becoming phosphorylated and dephosphorylated more rapidly than alpha(v)beta(3). Site mapping of phosphate incorporation reveals significant phosphorylation at Tyr-747 in both beta(3) integrin species with incorporation at Tyr-759 found at significant levels only in alpha(IIb)beta(3). Mutation of cytoplasmic beta(3) tyrosine residues in a transfection model prevents cell adhesion via these integrins. These data demonstrate that recognition of ligand is sufficient to induce beta(3) tyrosine phosphorylation and suggests that this event is regulated by the alpha subunit pairing of beta(3).