RESUMO
Directed migration of smooth muscle cells (SMCs) from the media to the intima and their subsequent proliferation are key events in atherosclerosis as these cells contribute to the bulk and stability of atheromatous plaques. We showed previously that two cytoskeleton-associated proteins, RHAMM and ARPC5, play important roles in rear polarization of the microtubule organizing centre (MTOC), directed migration, and in maintaining cell division fidelity. These proteins were analyzed to predict additional potential interacting partners using the bioinformatics programs BLAST, ClustalW, and PPI Spider. We identified spectrin alpha, a protein with a known role in actin polymerization as part of the pathway. We show that in migrating SMCs spectrin alpha localizes at the nodes of the actin net, and it partially colocalizes with RHAMM in the perinuclear region. In dividing SMCs spectrin alpha is present at spindle poles and midbody. Moreover, we show that spectrin alpha and RHAMM interact in a complex. Using siRNA to knockdown spectrin disrupted SMC migration, MTOC polarization, and the assembly of a polygonal actin net dorsolateral of the nucleus. Spectrin alpha knockdown also disrupted the organization of the bipolar spindle, chromosome division, and cytokinesis during cell division. The identification of interacting partners such as spectrin alpha and the decoding of pathways involved in polarity regulation during the migration of smooth muscle cells in atherosclerosis is important for identifying atherosclerosis biomarkers and developing therapeutic agents to block atherosclerotic plaque formation.
Assuntos
Divisão Celular , Movimento Celular , Centro Organizador dos Microtúbulos/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima/metabolismo , Placa Aterosclerótica/metabolismo , Espectrina/metabolismo , Fuso Acromático/metabolismo , Animais , Células Cultivadas , Centro Organizador dos Microtúbulos/patologia , Miócitos de Músculo Liso/patologia , Neointima/patologia , Placa Aterosclerótica/patologia , Ratos , Fuso Acromático/patologiaRESUMO
Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.
Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Segregação de Cromossomos , Histona Desmetilases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Ligação ProteicaRESUMO
The rapid proliferation of smooth muscle cells (SMCs) contributes to atherosclerotic plaque formation and neointimal thickening in other occlusive vascular diseases. In cancer cells, rapid cell proliferation is often accompanied by DNA damage, division aberrations, elevated cell apoptosis, or accumulation of abnormal cells. However, little is known about division fidelity in vascular disorders. We have analyzed the cell division fidelity during the rapid SMC proliferation that occurs after balloon injury of the rat carotid artery using en face confocal microscopy of the full thickness of the vessel wall. SMCs newly migrated to the neointima had increased division defects and increased apoptosis compared with SMCs in the subjacent media, despite comparable mitosis rates. Protein kinase Cα and the receptor for hyaluronic acid-mediated motility (RHAMM) regulate division fidelity in cultured neointimal SMCs. The centrosomal targeting sequence of RHAMM was required for localization to the mitotic spindle and spindle organization. Dynein and RHAMM colocalized in the spindle area and were part of a complex. Dynein inhibition caused spindle defects similar to RHAMM or protein kinase C inhibition. Our study uncovered abnormalities in rapidly proliferating SMCs after arterial injury that could contribute to the growth of atherosclerotic plaques and reduce plaque stability by triggering apoptosis, and it described a mechanism by which RHAMM and dynein coordinate division fidelity in neointimal SMCs.
Assuntos
Aterosclerose/patologia , Artérias Carótidas/patologia , Divisão Celular , Progressão da Doença , Animais , Apoptose , Artérias Carótidas/metabolismo , Divisão do Núcleo Celular , Proliferação de Células , Centrossomo/metabolismo , Segregação de Cromossomos , Citocinese , Dineínas/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Receptores de Hialuronatos/química , Receptores de Hialuronatos/metabolismo , Masculino , Índice Mitótico , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Neointima/metabolismo , Neointima/patologia , Ligação Proteica , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Fuso Acromático/metabolismo , Túnica Média/metabolismo , Túnica Média/patologiaRESUMO
Cadherins aggregate and stabilize cell-cell junctions through interactions with adjacent cells. In addition, N-cadherin and E-cadherin concentrate at free edges or at the lamellipodia of migrating cells and are found within large vesicles called macropinosomes, which develop from membrane ruffles. The binding properties of cadherins have not previously been associated with the localization of cadherins at membrane ruffles; however, we report that the dorsal, ventral and lateral membrane contacts that occur as a result of the overlap of membrane ruffles aggregate N-cadherin, and that both N-cadherin and E-cadherin promote macropinosome closure and fluid-phase uptake in macropinosomes. These data reveal a previously unsuspected function for cadherin-mediated cell-cell adhesion molecules in the closure of cell-autonomous membrane contacts at membrane ruffles, resulting in macropinocytosis.
Assuntos
Caderinas/fisiologia , Comunicação Celular/fisiologia , Pinocitose/fisiologia , Animais , Becaplermina , Caderinas/metabolismo , Adesão Celular/fisiologia , Comunicação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dextranos/metabolismo , Humanos , Camundongos , Músculo Liso/citologia , Músculo Liso/metabolismo , Pinocitose/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismoRESUMO
Directed migration of smooth muscle cells (SMCs) from the media to the intima in arteries occurs during atherosclerotic plaque formation and during restenosis after angioplasty or stent application. The polarized orientation of the microtubule-organizing center (MTOC) is a key determinant of this process, and we therefore investigated factors that regulate MTOC polarity in vascular SMCs. SMCs migrating in vivo from the medial to the intimal layer of the rat carotid artery following balloon catheter injury were rear polarized, with the MTOC located posterior of the nucleus. In tissue culture, migrating neointimal cells maintained rear polarization, whereas medial cells were front polarized. Using phosphoproteomic screening and mass spectrometry, we identified ARPC5 and RHAMM as protein kinase C (PKC)-phosphorylated proteins associated with rear polarization of the MTOC in neointimal SMCs. RNA silencing of ARPC5 and RHAMM, PKC inhibition, and transfection with a mutated nonphosphorylatable ARPC5 showed that these proteins regulate rear polarization by organizing the actin and microtubule cytoskeletons in neointimal SMCs. Both ARPC5 and RHAMM, in addition to PKC, were required for migration of neointimal SMCs.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Polaridade Celular , Proteínas da Matriz Extracelular/metabolismo , Receptores de Hialuronatos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Miócitos de Músculo Liso/patologia , Proteína Quinase C-alfa/metabolismo , Túnica Íntima/patologia , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Animais , Artérias/metabolismo , Artérias/patologia , Contagem de Células , Movimento Celular , Núcleo Celular/metabolismo , Proteínas da Matriz Extracelular/antagonistas & inibidores , Inativação Gênica , Masculino , Modelos Biológicos , Mutação/genética , Fosforilação , Proteína Quinase C-alfa/antagonistas & inibidores , Pseudópodes/metabolismo , Ratos , Ratos Sprague-Dawley , Túnica Íntima/metabolismo , Túnica Média/metabolismo , Túnica Média/patologiaRESUMO
Migration of smooth muscle cells from the arterial media to the intima is central to several vascular pathologies including restenosis. This study demonstrates that, like directional migration of other cells, smooth muscle migration is accompanied by a dramatic, polarized reorganization of the cell cytoskeleton that is accompanied by activation of the Rho GTPase Cdc42 and inactivation of glycogen synthase kinase-3beta. We also show, for the first time, that signals generated at the posterior-lateral aspects of wound edge cells by the cell-cell adhesion molecule N-cadherin are required for polarization and rapid migration of vascular smooth muscle. Importantly, when a cohort of migrating smooth muscle cells encounter CHO cells or the A10 smooth muscle cell line, neither of which expresses N-cadherin, polarity is only slightly suppressed. However, when smooth muscle cells encounter stably transfected, N-cadherin-expressing A10 cells or (N-cadherin-expressing) vascular endothelium, they rapidly lose their polarized phenotype. The latter finding indicates that endothelial signaling to innermost smooth muscle cells via N-cadherin may be critical to normal vessel wall stability. We infer that asymmetrical distribution of N-cadherin is necessary for the establishment of cell polarity during migration and that N-cadherin ligation is highly effective in abrogating polarized migration. Finally, we showed that endothelial cell polarity does not depend on N-cadherin; therefore, this molecule may be an attractive target for therapies to prevent restenosis without suppressing endothelial repair and risking late thrombosis.
Assuntos
Caderinas/metabolismo , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Endotélio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Células CHO , Caderinas/genética , Adesão Celular/fisiologia , Linhagem Celular , Células Cultivadas , Cricetinae , Cricetulus , Endotélio Vascular/citologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Músculo Liso Vascular/citologia , Ratos , Suínos , Transfecção , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Originally characterized as regulators of cytokinesis, septins were later implicated in other cellular processes. Recent studies show that septins have a broader role in microtubule-dependent processes, such as karyokinesis, exocytosis, and maintenance of cell shape. Many members of the septin family have been shown to colocalize or interact with the microtubule cytoskeleton, suggesting that these might be general properties of septins. Septins could play an important role in regulating microtubule dynamics by interacting with microtubule-associated proteins (MAPs) that modulate microtubule stability. Being able to associate with both microtubules and actin, septins can play an important role as adaptors between the two cytoskeletons and as regulators of processes in which both actin and microtubules are involved. As septins are associated with various neurodegenerative diseases and cancer, a better understanding of the biology of septins and their interactions with microtubules is important in order to develop possible therapeutic strategies for these diseases.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Animais , HumanosRESUMO
During early development in Drosophila, pseudocleavage furrows in the syncytial embryo prevent contact between neighboring spindles, thereby ensuring proper chromosome segregation. Here we demonstrate that the GTPase Ran regulates pseudocleavage furrow organization. Ran can exert control on pseudocleavage furrows independently of its role in regulating the microtubule cytoskeleton. Disruption of the Ran pathway prevented pseudocleavage furrow formation and restricted the depth and duration of furrow ingression of those pseudocleavage furrows that did form. We found that Ran was required for the localization of the septin Peanut to the pseudocleavage furrow, but not anillin or actin. Biochemical assays revealed that the direct binding of the nuclear transport receptors importin alpha and beta to anillin prevented the binding of Peanut to anillin. Furthermore, RanGTP reversed the inhibitory action of importin alpha and beta. On expression of a mutant form of anillin that lacked an importin alpha and beta binding site, inhibition of Ran no longer restricted the depth and duration of furrow ingression in those pseudocleavage furrows that formed. These data suggest that anillin and Peanut are involved in pseudocleavage furrow ingression in syncytial embryos and that this process is regulated by Ran.
Assuntos
Proteínas Contráteis/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas dos Microfilamentos/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Bovinos , Proteínas Contráteis/química , Citocinese , Drosophila melanogaster , Cinética , Microtúbulos/metabolismo , Mitose , Modelos Biológicos , Transgenes , Tubulina (Proteína)/metabolismoRESUMO
The Ran pathway has been shown to have a role in spindle assembly. However, the extent of the role of the Ran pathway in mitosis in vivo is unclear. We report that perturbation of the Ran pathway disrupted multiple steps of mitosis in syncytial Drosophila embryos and uncovered new mitotic processes that are regulated by Ran. During the onset of mitosis, the Ran pathway is required for the production, organization, and targeting of centrosomally nucleated microtubules to chromosomes. However, the role of Ran is not restricted to microtubule organization, because Ran is also required for the alignment of chromosomes at the metaphase plate. In addition, the Ran pathway is required for postmetaphase events, including chromosome segregation and the assembly of the microtubule midbody. The Ran pathway mediates these mitotic events, in part, by facilitating the correct targeting of the kinase Aurora A and the kinesins KLP61F and KLP3A to spindles.
Assuntos
Segregação de Cromossomos , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Metáfase , Fuso Acromático/metabolismo , Proteína ran de Ligação ao GTP/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Aurora Quinases , Centrossomo/metabolismo , Cromossomos/metabolismo , Proteínas de Drosophila/análise , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Cinesinas/análise , Cinesinas/metabolismo , Metáfase/genética , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/enzimologia , Proteína ran de Ligação ao GTP/antagonistas & inibidoresRESUMO
We showed previously that in crane-fly spermatocytes myosin is required for tubulin flux [Silverman-Gavrila and Forer, 2000a: J Cell Sci 113:597-609], and for normal anaphase chromosome movement and contractile ring contraction [Silverman-Gavrila and Forer, 2001: Cell Motil Cytoskeleton 50:180-197]. Neither the identity nor the distribution of myosin(s) were known. In the present work, we used immunofluorescence and confocal microscopy to study myosin during meiosis-I of crane-fly spermatocytes compared to tubulin, actin, and skeletor, a spindle matrix protein, in order to further understand how myosin might function during cell division. Antibodies to myosin II regulatory light chain and myosin II heavy chain gave similar staining patterns, both dependent on stage: myosin is associated with nuclei, asters, centrosomes, chromosomes, spindle microtubules, midbody microtubules, and contractile rings. Myosin and actin colocalization along kinetochore fibers from prometaphase to anaphase are consistent with suggestions that acto-myosin forces in these stages propel kinetochore fibres poleward and trigger tubulin flux in kinetochore fibres, contributing in this way to poleward chromosome movement. Myosin and actin colocalization at the cell equator in cytokinesis, similar to studies in other cells [e.g., Fujiwara and Pollard, 1978: J Cell Biol 77:182-195], supports a role of actin-myosin interactions in contractile ring function. Myosin and skeletor colocalization in prometaphase spindles is consistent with a role of these proteins in spindle formation. After microtubules or actin were disrupted, myosin remained in spindles and contractile rings, suggesting that the presence of myosin in these structures does not require the continued presence of microtubules or actin. BDM (2,3 butanedione, 2 monoxime) treatment that inhibits chromosome movement and cytokinesis also altered myosin distributions in anaphase spindles and contractile rings, consistent with the physiological effects, suggesting also that myosin needs to be active in order to be properly distributed.