The structure of the complex of CCM1/KRIT1 and HEG1 defines a new mode of membrane protein anchorage important for cell–cell junctions and cardiovascular development.
The products of genes that cause cerebral cavernous malformations (CCM1/KRIT1, CCM2, and CCM3) physically interact. CCM1/KRIT1 links this complex to endothelial cell (EC) junctions and maintains junctional integrity in part by inhibiting RhoA. Heart of glass (HEG1), a transmembrane protein, associates with KRIT1. In this paper, we show that the KRIT1 band 4.1, ezrin, radixin, and moesin (FERM) domain bound the HEG1 C terminus (Kd = 1.2 µM) and solved the structure of this assembly. The KRIT1 F1 and F3 subdomain interface formed a hydrophobic groove that binds HEG1(Tyr1,380-Phe1,381), thus defining a new mode of FERM domain–membrane protein interaction. This structure enabled design of KRIT1(L717,721A), which exhibited a >100-fold reduction in HEG1 affinity. Although well folded and expressed, KRIT1(L717,721A) failed to target to EC junctions or complement the effects of KRIT1 depletion on zebrafish cardiovascular development or Rho kinase activation in EC. These data establish that this novel FERM–membrane protein interaction anchors CCM1/KRIT1 at EC junctions to support cardiovascular development.
The cyclical protrusion and retraction of the leading edge is a hallmark of many migrating cells involved in processes such as development, inflammation, and tumorigenesis. The molecular identity of signaling mechanisms that control these cycles has remained unknown. Here, we used live cell imaging of biosensors to monitor spontaneous morphodynamic and signaling activities, and employed correlative image analysis to examine the role of cAMP-activated Protein Kinase A (PKA) in protrusion regulation. PKA activity at the leading edge is closely synchronized with rapid protrusion and with the activity of RhoA. Ensuing PKA phosphorylation of RhoA and the resulting increased interaction between RhoA and RhoGDI establishes a negative feedback that controls the cycling of RhoA activity at the leading edge. Thus, cooperation between PKA, RhoA, and a RhoGDI forms a pacemaker that governs the morphodynamic behavior of migrating cells.
Cell migration; cAMP activated Protein Kinase A; Rho GTPase; cell protrusion; RhoGDI
To determine talin1's role in osteoclasts, we mated TLN1fl/fl mice with those expressing cathepsin K-Cre (CtsK-TLN1) to delete the gene in mature osteoclasts or with lysozyme M-Cre (LysM-TLN1) mice to delete TLN1 in all osteoclast lineage cells. Absence of TLN1 impairs macrophage colony-stimulating factor (M-CSF)-stimulated inside-out integrin activation and cytoskeleton organization in mature osteoclasts. Talin1-deficient precursors normally express osteoclast differentiation markers when exposed to M-CSF and receptor activator of nuclear factor κB (RANK) ligand but attach to substrate and migrate poorly, arresting their development into mature resorptive cells. In keeping with inhibited resorption, CtsK-TLN1 mice exhibit an ∼5-fold increase in bone mass. Osteoclast-specific deletion of Rap1 (CtsK-Rap1), which promotes talin/β integrin recognition, yields similar osteopetrotic mice. The fact that the osteopetrosis of CtsK-TLN1 and CtsK-Rap1 mice is substantially more severe than that of those lacking αvβ3 is likely due to added failed activation of β1 integrins. In keeping with osteoclast dysfunction, mice in whom talin is deleted late in the course of osteoclastogenesis are substantially protected from ovariectomy-induced osteoporosis and the periarticular osteolysis attending inflammatory arthritis. Thus, talin1 and Rap1 are critical for resorptive function, and their selective inhibition in mature osteoclasts retards pathological bone loss.
Talin binding to the integrin β tail alters the β transmembrane domain’s topology, resulting in integrin activation.
Talin binding to integrin β tails increases ligand binding affinity (activation). Changes in β transmembrane domain (TMD) topology that disrupt α–β TMD interactions are proposed to mediate integrin activation. In this paper, we used membrane-embedded integrin β3 TMDs bearing environmentally sensitive fluorophores at inner or outer membrane water interfaces to monitor talin-induced β3 TMD motion in model membranes. Talin binding to the β3 cytoplasmic domain increased amino acid side chain embedding at the inner and outer borders of the β3 TMD, indicating altered topology of the β3 TMD. Talin’s capacity to effect this change depended on its ability to bind to both the integrin β tail and the membrane. Introduction of a flexible hinge at the midpoint of the β3 TMD decoupled the talin-induced change in intracellular TMD topology from the extracellular side and blocked talin-induced activation of integrin αIIbβ3. Thus, we show that talin binding to the integrin β TMD alters the topology of the TMD, resulting in integrin activation.
Talin-mediated integrin activation drives integrin-based adhesions. A simple binary switch—vinculin competitively displacing RIAM from talin—is found to play a central role in the maturation and evolving functions of integrin-based adhesions.
Talin-mediated integrin activation drives integrin-based adhesions. Here we examine the roles of two proteins that induce talin–integrin interactions—vinculin and Rap1-GTP-interacting adaptor molecule (RIAM)—in the formation and maturation of integrin-based adhesions. RIAM-containing adhesions are primarily in the lamellipodium; RIAM is subsequently reduced in mature focal adhesions due to direct competition with vinculin for talin-binding sites. We show that vinculin binding to talin induces Rap1-independent association of talin with integrins and resulting integrin activation, in sharp contrast to Rap1-dependent RIAM-induced activation. Vinculin stabilizes adhesions, increasing their ability to transmit force, whereas RIAM played a critical role in lamellipodial protrusion. Thus displacement of RIAM by vinculin acts as a molecular switch that mediates the transition of integrin-based adhesions from drivers of lamellipodial protrusion to stable, force-bearing adhesions. Consequently changes in the abundance of two multiprotein modules within maturing adhesions, one regulated by Rap1 and one by tension, result in the temporal evolution of adhesion functions.
The PAR complex targets Tiam1 to adhesions, where it interacts with talin to promote adhesion-induced Rac1 activation, cell spreading, and migration.
Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration.
The depletion of ARF6 in epithelial cysts causes a striking inversion of glandular orientation. This requires temporal Rac1 inactivation and is accompanied in basement membrane cultures by improperly assembled laminins. In collagen I, these inverted cysts promote integrin-linked fibril linearization reminiscent of matrix remodeling in disease.
Epithelial cysts comprise the structural units of the glandular epithelium. Although glandular inversion in epithelial tumors is thought to be a potential mechanism for the establishment of metastatic disease, little is known about the morphogenic cues and signaling pathways that govern glandular polarity and organization. Using organotypic cultures of Madin-Darby canine kidney cells in reconstituted basement membrane, we show that cellular depletion of the small GTP-binding protein ARF6 promotes the formation of inverted cysts, wherein the apical cell membrane faces the cyst exterior, and the basal domain faces the central lumen, while individual cell polarity is maintained. These cysts are also defective in interactions with laminin at the cyst–matrix interface. This inversion of glandular orientation is accompanied by Rac1 inactivation during early cystogenesis, and temporal activation of Rac1 is sufficient to recover the normal cyst phenotype. In an unnatural collagen I microenvironment, ARF6-depleted, inverted epithelial cysts exhibit some loss of cell polarity, a marked increase in Rho activation and Rac1 inactivation, and striking rearrangement of the surrounding collagen I matrix. These studies demonstrate the importance of ARF6 as a critical determinant of glandular orientation and the matrix environment in dictating structural organization of epithelial cysts.
Background and Purpose
Cerebral cavernous malformations (CCMs) are characterized by grossly dilated capillaries, associated with vascular leak and hemorrhage, and occur in sporadic or inherited (autosomal dominant) forms with mutations in one of three gene loci (CCM 1, 2 or 3). We previously reported that the CCM1 protein (KRIT1) localizes to endothelial cell-cell junctions and loss of KRIT1 leads to junctional instability associated with activation of RhoA and its effector Rho kinase (ROCK). Although ROCK inhibition has been proposed as potential therapy for CCM, there has been no demonstration of a therapeutic effect on CCM lesion genesis in vivo.
Our recently generated a model of CCM1 disease (Ccm1+/−Msh2−/−) was treated with ROCK inhibitor fasudil (100 mg/kg/day administered in drinking water from weaning to 5 months of age), or placebo, and blindly assessed CCM lesion burden by systematic survey of animals’ brains. For comparison, we also assessed therapeutic effect in previously described Ccm2+/−Trp53−/− mice, treated with the same dose and duration of fasudil and placebo.
Fasudil treated Ccm1+/−Msh2−/− mice had a significantly decreased prevalence of CCM lesions compared to placebo controls. Lesions in treated animals were smaller and less likely associated with hemorrhage, inflammation and endothelial proliferation, and exhibited decreased expression of ROCK activation biomarkers. A therapeutic effect was also documented in Ccm2+/−Trp53−/− mice.
This represents the first report of therapeutic benefit of pharmacological therapy in development and progression of CCMs, and indicates that ROCK activation is a critical step in CCM lesion genesis and maturation.
Cerebral cavernous malformation; cavernous angioma; ROCK; Fasudil; therapy
Endothelial cell–cell junctions regulate vascular permeability, vasculogenesis, and angiogenesis. Familial cerebral cavernous malformations (CCMs) in humans result from mutations of CCM2 (malcavernin, OSM, MGC4607), PDCD10 (CCM3), or KRIT1 (CCM1), a Rap1 effector which stabilizes endothelial cell–cell junctions. Homozygous loss of KRIT1 or CCM2 produces lethal vascular phenotypes in mice and zebrafish. We report that the physical interaction of KRIT1 and CCM2 proteins is required for endothelial cell–cell junctional localization, and lack of either protein destabilizes barrier function by sustaining activity of RhoA and its effector Rho kinase (ROCK). Protein haploinsufficient Krit1+/− or Ccm2+/− mouse endothelial cells manifested increased monolayer permeability in vitro, and both Krit1+/− and Ccm2+/− mice exhibited increased vascular leak in vivo, reversible by fasudil, a ROCK inhibitor. Furthermore, we show that ROCK hyperactivity occurs in sporadic and familial human CCM endothelium as judged by increased phosphorylation of myosin light chain. These data establish that KRIT1–CCM2 interaction regulates vascular barrier function by suppressing Rho/ROCK signaling and that this pathway is dysregulated in human CCM endothelium, and they suggest that fasudil could ameliorate both CCM disease and vascular leak.
Changes in expression of PEA-15 contribute to diabetes, tumor invasion, and cellular senescence. PEA-15 increases activation of the ERK MAP kinase pathway; the present study shows that it does so by interfering with ERK1/2 phosphorylation of FRS2, terminator of downstream signaling from FGF receptors.
Changes in cellular expression of phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) are linked to insulin resistance, tumor cell invasion, and cellular senescence; these changes alter the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway. Here, we define the mechanism whereby increased PEA-15 expression promotes and sustains ERK1/2 activation. PEA-15 binding prevented ERK1/2 membrane recruitment and threonine phosphorylation of fibroblast receptor substrate 2α (FRS2α), a key link in fibroblast growth factor (FGF) receptor activation of ERK1/2. This reduced threonine phosphorylation led to increased FGF-induced tyrosine phosphorylation of FRS2α, thereby enhancing downstream signaling. Conversely, short hairpin RNA-mediated depletion of endogenous PEA-15 led to reduced FRS2α tyrosine phosphorylation. Thus, PEA-15 interrupts a negative feedback loop that terminates growth factor receptor signaling downstream of FRS2α. This is the dominant mechanism by which PEA-15 activates ERK1/2 because genetic deletion of FRS2α blocked the capacity of PEA-15 to activate the MAP kinase pathway. Thus, PEA-15 prevents ERK1/2 localization to the plasma membrane, thereby inhibiting ERK1/2-dependent threonine phosphorylation of FRS2α to promote activation of the ERK1/2 MAP kinase pathway.
CD98hc (CD98 heavy chain, 4F2 antigen, Slc3a2) was discovered as a lymphocyte activation antigen. Deletion of CD98hc in B cells leads to complete failure of B cell proliferation, plasma cell formation, and antibody secretion. Here we examined the role of T cell CD98 in cell-mediated immunity and autoimmune disease pathogenesis by specifically deleting it in murine T cells. Deletion of T cell CD98 prevented experimental autoimmune diabetes associated with dramatically reduced T cell clonal expansion. Nevertheless initial T cell homing to pancreatic islets was unimpaired. In sharp contrast to B cells, CD98-null T cells showed only modestly impaired antigen-driven proliferation and nearly normal homeostatic proliferation. Furthermore, these cells were activated by antigen leading to cytokine production (CD4) and efficient cytolytic killing of targets (CD8). The integrin binding domain of CD98 was necessary and sufficient for full clonal expansion, pointing to a role for adhesive signaling in T cell proliferation and autoimmune disease. When we expanded CD98-null T cells in vitro, they adoptively transferred diabetes, establishing that impaired clonal expansion was responsible for protection from disease. Thus the integrin binding domain of CD98 is required for antigen-driven T cell clonal expansion in the pathogenesis of an autoimmune disease and may represent a useful therapeutic target.
Side chains of Lys/Arg near transmembrane domain (TMD)1–3 membrane-water interfaces can “snorkel” placing their positive charge near negatively-charged phospholipid head groups4–6; however, snorkeling's functional effects are obscure. Integrin β TMDs exhibit such conserved basic amino acids; here we used nuclear magnetic resonance (NMR) spectroscopy7, 8 to show that integrin β3(Lys716) helps determine β3 TMD topography. The αIIbβ3 TMD structure suggests that precise β3 TMD crossing angles enable the assembly of outer and inner membrane “clasps” (OMC and IMC) that hold the αβ TMD together to limit transmembrane signalling9 . Mutation of β3(Lys716) caused dissociation of αIIbβ3 TMDs and integrin activation. To confirm that altered topography of β3(Lys716) mutants activated αIIbβ3, we utilized directed evolution of β3(K716A) to identify substitutions restoring default state. Introduction Pro(711) at the midpoint of β3 TMD (A711P) increased αIIbβ3 TMD association and inactivated integrin αIIbβ3(A711P,K716A). β3(Pro711) introduced a TMD kink of 30 ± 1° precisely at the OMC/IMC border, thereby decoupling the tilt between these segments. Thus, widely-occurring snorkeling residues in TMDs can help maintain TMD topography and membrane-embedding thereby regulating transmembrane signalling.
Neutrophil recruitment to sites of inflammation involves P-selectin dependent rolling. Quantitative dynamic footprinting is a useful tool to visualize the topography of the neutrophil footprint as it interacts with the substrate. However, elucidating the role of specific proteins in addition to topography requires simultaneous visualization of two fluorochromes.
To validate dual-color quantitative dynamic footprinting, mouse neutrophils were labeled with the membrane dyes DiO and DiI and perfused into microchannels coated with P-selectin-Fc. Footprints of rolling neutrophils were recorded as two separate images, one for each fluorochrome. To assess the localization of the cytoskeletal protein paxillin, we applied dual-color quantitative dynamic footprinting to DiO stained neutrophils of mice expressing an mCherry-paxillin fusion protein.
The footprint topographies obtained from DiO and DiI in the plasma membrane were identical. The z-coordinates of the microvilli tips obtained with the two fluorochromes in the footprint were also identical. Paxillin was found to be localized to some, but not all ridges in the neutrophil footprint.
Our data suggest that the spectral properties of the fluorochrome do not affect the results. Dual-color quantitative dynamic footprinting will be useful for simultaneous visualization of two fluorochromes in the footprint of rolling cells.
DqDF; P-selectin; footprint; TIRF
Tumor inflammation promotes angiogenesis, immunosuppression and tumor growth, but the mechanisms controlling inflammatory cell recruitment to tumors are not well understood. We found that a range of chemoattractants activating G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and Toll-like/IL-1 receptors (TLR/IL1Rs) unexpectedly initiate tumor inflammation by activating the PI3-kinase isoform p110γ in Gr1+CD11b+ myeloid cells. Whereas GPCRs activate p110γ in a Ras/p101 dependent manner, RTKs and TLR/IL1Rs directly activate p110γ in a Ras/p87-dependent manner. Once activated, p110γ promotes inside-out activation of a single integrin, α4β1, causing myeloid cell invasion into tumors. Pharmacological or genetic blockade of p110γ suppressed inflammation, growth and metastasis of implanted and spontaneous tumors, revealing an important therapeutic target in oncology.
In vitro studies indicate that binding of talin to the β3 integrin cytoplasmic domain (tail) results in integrin αIIbβ3 (GPIIb–IIIa) activation. Here we tested the importance of talin binding for integrin activation in vivo and its biological significance by generating mice harboring point mutations in the β3 tail. We introduced a β3(Y747A) substitution that disrupts the binding of talin, filamin, and other cytoplasmic proteins and a β3(L746A) substitution that selectively disrupts interactions only with talin. Platelets from animals homozygous for each mutation showed impaired agonist-induced fibrinogen binding and platelet aggregation, providing proof that inside-out signals that activate αIIbβ3 require binding of talin to the β3 tail. β3(L746A) mice were resistant to both pulmonary thromboembolism and to ferric chloride–induced thrombosis of the carotid artery. Pathological bleeding, measured by the presence of fecal blood and development of anemia, occurred in 53% of β3(Y747A) and virtually all β3-null animals examined. Remarkably, less than 5% of β3(L746A) animals exhibited this form of bleeding. These results establish that αIIbβ3 activation in vivo is dependent on the interaction of talin with the β3 integrin cytoplasmic domain. Furthermore, they suggest that modulation of β3 integrin–talin interactions may provide an attractive target for antithrombotics and result in a reduced risk of pathological bleeding.
The Ras family of small GTPases regulates cell proliferation, spreading, migration and apoptosis, and malignant transformation by binding to several protein effectors. One such GTPase, R-Ras, plays distinct roles in each of these processes, but to date, identified R-Ras effectors were shared with other Ras family members (e.g., H-Ras). We utilized a new database of Ras-interacting proteins to identify RLIP76 (RalBP1) as a novel R-Ras effector. RLIP76 binds directly to R-Ras in a GTP-dependent manner, but does not physically associate with the closely related paralogues H-Ras and Rap1A. RLIP76 is required for adhesion-induced Rac activation and the resulting cell spreading and migration, as well as for the ability of R-Ras to enhance these functions. RLIP76 regulates Rac activity through the adhesion-induced activation of Arf6 GTPase and activation of Arf6 bypasses the requirement for RLIP76 in Rac activation and cell spreading. Thus, we identify a novel R-Ras effector, RLIP76, which links R-Ras to adhesion-induced Rac activation through a GTPase cascade that mediates cell spreading and migration.
Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment.
Antagonists to α4 integrin show promise for several autoimmune and inflammatory diseases but may exhibit mechanism-based toxicities. We tested the capacity of blockade of α4 integrin signaling to perturb functions involved in inflammation, while limiting potential adverse effects. We generated and characterized mice bearing a Y991A mutation in α4 integrin [α4(Y991A) mice], which blocks paxillin binding and inhibits α4 integrin signals that support leukocyte migration. In contrast to the embryonic-lethal phenotype of α4 integrin–null mice, mice bearing the α4(Y991A) mutation were viable and fertile; however, they exhibited defective recruitment of mononuclear leukocytes into thioglycollate-induced peritonitis. α4 Integrins are essential for definitive hematopoiesis; however, the α4(Y991A) mice had intact lymphohematopoiesis and, with the exception of reduced Peyer’s patches, normal architecture and cellularity of secondary lymphoid tissues. We conclude that interference with α4 integrin signaling can selectively impair mononuclear leukocyte recruitment to sites of inflammation while sparing vital functions of α4 integrins in development and hematopoiesis.
Cell cycle progression is dependent on the nuclear localization and transcriptional effects of activated extracellular signal-regulated kinase (ERK)1 and ERK2 mitogen-activated protein (MAP) kinases (ERK1/2). Phosphoprotein enriched in astrocytes (PEA-15) binds ERK1/2 and inhibits their nuclear localization, thus blocking cell proliferation. Here, we report that phosphorylation of PEA-15 blocks its interaction with ERK1/2 in vitro and in vivo and that phosphorylation of both Ser104 and Ser116 is required for this effect. Using phosphomimetic and nonphosphorylatable mutants of PEA-15, we found that PEA-15 phosphorylation abrogates its capacity to block the nuclear localization and transcriptional activities of ERK1/2; this phosphorylation therefore enables the proliferation of cells that express high levels of PEA-15. Additionally, we report that PEA-15 phosphorylation can modulate nontranscriptional activities of ERK1/2, such as the modulation of the affinity of integrin adhesion receptors. Finally, we used a novel anti-phospho-specific PEA-15 antibody to establish that PEA-15 is phosphorylated in situ in normal mammary epithelium. These results define a novel posttranslational mechanism for controlling the subcellular localization of ERK1/2 and for specifying the output of MAP kinase signaling.
Cerebral cavernous malformations (CCMs) are vascular lesions of the central nervous system appearing as multicavernous, blood-filled capillaries, leading to headache, seizure and hemorrhagic stroke. CCM occurs either sporadically or as an autosomal dominant disorder caused by germline mutation of one of the three genes: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. Surgically resected human CCM lesions have provided molecular and immunohistochemical evidence for a two-hit (germline plus somatic) mutation mechanism. In contrast to the equivalent human genotype, mice heterozygous for a Ccm1- or Ccm2-null allele do not develop CCM lesions. Based on the two-hit hypothesis, we attempted to improve the penetrance of the model by crossing Ccm1 and Ccm2 heterozygotes into a mismatch repair-deficient Msh2−/− background. Ccm1+/−Msh2−/− mice exhibit CCM lesions with high penetrance as shown by magnetic resonance imaging and histology. Significantly, the CCM lesions range in size from early-stage, isolated caverns to large, multicavernous lesions. A subset of endothelial cells within the CCM lesions revealed somatic loss of CCM protein staining, supporting the two-hit mutation mechanism. The late-stage CCM lesions displayed many of the characteristics of human CCM lesions, including hemosiderin deposits, immune cell infiltration, increased endothelial cell proliferation and increased Rho-kinase activity. Some of these characteristics were also seen, but to a lesser extent, in early-stage lesions. Tight junctions were maintained between CCM lesion endothelial cells, but gaps were evident between endothelial cells and basement membrane was defective. In contrast, the Ccm2+/−Msh2−/− mice lacked cerebrovascular lesions. The CCM1 mouse model provides an in vivo tool to investigate CCM pathogenesis and new therapies.
In response to antigen stimulation, PKCθ translocates to the T cell plasma membrane, becoming highly focused at the immunological synapse (IS). cis-Acting sequences that regulate IS retention are not known. It is shown that a catalytically competent PKCθ kinase domain is essential for IS retention but not for membrane translocation.
Protein kinase Cθ (PKCθ) is a serine/threonine kinase that plays an essential role in antigen-regulated responses of T lymphocytes. Upon antigen stimulation, PKCθ is rapidly recruited to the immunological synapse (IS), the region of contact between the T cell and antigen-presenting cell. This behavior is unique among T cell PKC isoforms. To define domains of PKCθ required for retention at the IS, we generated deletion and point mutants of PKCθ. We used quantitative imaging analysis to assess IS retention of PKCθ mutants in antigen-stimulated T cell clones. Deletion of the kinase domain or site-directed mutation of a subset of known PKCθ phosphorylation sites abrogated or significantly reduced IS retention, respectively. IS retention did not correlate with phosphorylation of specific PKCθ residues but rather with kinase function. Thus PKCθ catalytic competence is essential for stable IS retention.
Rap1 stabilizes cell–cell junctions by directly binding to KRIT1, displacing it from microtubules and enabling localization at the junctions.
Activation of Rap1 small GTPases stabilizes cell–cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1–Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ∼40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell–cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell–cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.
Substrate rigidity profoundly impacts cellular behaviors such as migration, gene expression, and cell fate. Total Internal Reflection Fluorescence (TIRF) microscopy enables selective visualization of the dynamics of substrate adhesions, vesicle trafficking, and biochemical signaling at the cell-substrate interface. Here we apply high-refractive-index silicone gels to perform TIRF microscopy on substrates with a wide range of physiological elastic moduli and simultaneously measure traction forces exerted by cells on the substrate.
Using both in vivo and in vitro protein kinase C (PKC) θ mutant models, we found that PKCθ, the PKC isoform predominantly expressed in skeletal muscle, is required for myoblast fusion and myofiber growth, by regulating focal adhesion kinase activity and, in turn, the expression of the pro-fusion genes caveolin-3 and β1D-integrin.
Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKCθ, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKCθ is strongly up-regulated following freeze injury–induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKCθ knockout and muscle-specific PKCθ dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKCθ mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKCθ mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKCθ in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKCθ-null myoblasts. We thus propose that PKCθ signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.
Shroom2 regulates proper endothelial morphogenesis through a direct interaction with Rho kinase and the subsequent assembly of a cortical actomyosin network. Loss of Shroom2 results in decreased cell contractility and cytoskeletal organization and increased cell migration and angiogenesis.
The intrinsic contractile, migratory, and adhesive properties of endothelial cells are central determinants in the formation of vascular networks seen in vertebrate organisms. Because Shroom2 (Shrm2) is expressed within the endothelium, is localized to cortical actin and cell–cell adhesions, and contains a conserved Rho kinase (Rock) binding domain, we hypothesized that Shrm2 may participate in the regulation of endothelial cell behavior during vascular morphogenesis. Consistent with this hypothesis, depletion of Shrm2 results in elevated branching and sprouting angiogenic behavior of endothelial cells. This is recapitulated in human umbilical vein endothelial cells and in a vasculogenesis assay in which differentiated embryonic stem cells depleted for Shrm2 form a more highly branched endothelial network. Further analyses indicate that the altered behavior observed following Shrm2 depletion is due to aberrant cell contractility, as evidenced by decreased stress fiber organization and collagen contraction with an increase in cellular migration. Because Shrm2 directly interacts with Rock, and Shrm2 knockdown results in the loss of Rock and activated myosin II from sites of cell–cell adhesion, we conclude that Shrm2 facilitates the formation of a contractile network within endothelial cells, the loss of which leads to an increase in endothelial sprouting, migration, and angiogenesis.