The dendritic cell cytoskeleton is critical for the formation of the immunological synapse

The binding of a T cell to an APC results in T cell actin cytoskeletal rearrangement leading to the formation of an immunological synapse. The APC cytoskeleton has been thought to play a passive role in this process. In this study, we demonstrate that dendritic cells (DC), unlike other APC, actively polarize their actin cytoskeleton during interaction with T cells. DC cytoskeletal rearrangement was critical for both the clustering and the activation of resting T cells. This study provides compelling evidence that the APC cytoskeleton plays an active role in the immunological synapse and may explain the unique ability of DC to activate resting T cells.     

Keratin 18 attenuates estrogen receptor α-mediated signaling by sequestering LRP16 in cytoplasm

Background

As a key player in suppression of colon tumorigenesis, Adenomatous Polyposis Coli (APC) has been widely studied to determine its cellular functions. However, inconsistencies of commercially available APC antibodies have limited the exploration of APC function. APC is implicated in spindle formation by direct interactions with tubulin and microtubule-binding protein EB1. APC also interacts with the actin cytoskeleton to regulate cell polarity. Until now, interaction of APC with the third cytoskeletal element, intermediate filaments, has remained unexamined.

Results

We generated an APC antibody (APC-M2 pAb) raised against the 15 amino acid repeat region, and verified its reliability in applications including immunoprecipitation, immunoblotting, and immunofluorescence in cultured cells and tissue. Utilizing this APC-M2 pAb, we immunoprecipitated endogenous APC and its binding proteins from colon epithelial cells expressing wild-type APC. Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), we identified 42 proteins in complex with APC, including β-catenin and intermediate filament (IF) proteins lamin B1 and keratin 81. Association of lamin B1 with APC in cultured cells and human colonic tissue was verified by co-immunoprecipitation and colocalization. APC also colocalized with keratins and remained associated with IF proteins throughout a sequential extraction procedure.

Conclusion

We introduce a versatile APC antibody that is useful for cell/tissue immunostaining, immunoblotting and immunoprecipitation. We also present evidence for interactions between APC and IFs, independent of actin filaments and microtubules. Our results suggest that APC associates with all three major components of the cytoskeleton, thus expanding potential roles for APC in the regulation of cytoskeletal integrity.     

E-Cadherin and the Cytoskeletal Network in Colorectal Cancer Development and Metastasis

Abnormalities in the expression and functional activity of cell adhesion molecules are implicated in the development and progression of the majority of colorectal cancers (CRC). Cell–cell adhesion molecule E-cadherin regulates cell polarity, differentiation, proliferation and migration through its intimate association to the actin cytoskeletal network. During colorectal carcinogenesis changes in intercellular adhesion and dynamic rearrangements in the actin cytoskeleton result in altered signalling and migration with loss of contact inhibition. The adenomatous polyposis coli (APC) protein, besides its established role in the β catenin/Wnt signalling pathway, can coordinate microtubule and actin organization during cell migration. The actin-bundling protein Fascin promotes cell motility and is overexpressed in CRC. Based on recent molecular and pathological studies, this review focusses on the role of these molecules sharing the common feature of being associated with the cytoskeletal network during colorectal carcinogenesis and metastasis. The potential use of these molecules as prognostic markers and/or therapeutic targets will also be discussed.     

E-cadherin and the cytoskeletal network in colorectal cancer development and metastasis

Abnormalities in the expression and functional activity of cell adhesion molecules are implicated in the development and progression of the majority of colorectal cancers (CRC). Cell-cell adhesion molecule E-cadherin regulates cell polarity, differentiation, proliferation and migration through its intimate association to the actin cytoskeletal network. During colorectal carcinogenesis changes in intercellular adhesion and dynamic rearrangements in the actin cytoskeleton result in altered signalling and migration with loss of contact inhibition. The adenomatous polyposis coli (APC) protein, besides its established role in the β catenin/Wnt signalling pathway, can coordinate microtubule and actin organization during cell migration. The actin-bundling protein Fascin promotes cell motility and is overexpressed in CRC. Based on recent molecular and pathological studies, this review focusses on the role of these molecules sharing the common feature of being associated with the cytoskeletal network during colorectal carcinogenesis and metastasis. The potential use of these molecules as prognostic markers and/or therapeutic targets will also be discussed.     

Adenomatous polyposis coli regulates axon arborization and cytoskeleton organization via its N-terminus

Conditional deletion of APC leads to marked disruption of cortical development and to excessive axonal branching of cortical neurons. However, little is known about the cell biological basis of this neuronal morphological regulation. Here we show that APC deficient cortical neuronal growth cones exhibit marked disruption of both microtubule and actin cytoskeleton. Functional analysis of the different APC domains revealed that axonal branches do not result from stabilized β-catenin, and that the C-terminus of APC containing microtubule regulatory domains only partially rescues the branching phenotype. Surprisingly, the N-terminus of APC containing the oligomerization domain and the armadillo repeats completely rescues the branching and cytoskeletal abnormalities. Our data indicate that APC is required for appropriate axon morphological development and that the N-terminus of APC is important for regulation of the neuronal cytoskeleton.     

APC蛋白的结构特征及其与细胞骨架的关系

编码APC蛋白(adenomatous polyposis coli,APC)基因的缺失突变会导致家族性和散发性的结肠癌,APC蛋白除了能直接参与Wnt信号途径调节β—catenin的浓度之外,最近的研究表明APC蛋白能够与细胞骨架的主要成分微管和微丝直接或间接结合,通过调节微管的解聚和聚合,间接调节染色体的分离,作为潜在的细胞骨架调节分子将细胞骨架与重要的细胞信号转导通路紧密联系在一起。     

The many faces of the tumor suppressor gene APC

Inactivation of the tumor suppressor adenomatous polyposis coli (APC) protein is a critical early step in the development of familial and sporadic colon cancer. Close examination of the function of APC has shown that it is a multifunctional protein involved in a wide variety of processes, including regulation of cell proliferation, cell migration, cell adhesion, cytoskeletal reorganization, and chromosomal stability. Tantalizing clues to the different functions of APC have been provided by the identification of proteins interacting with several discrete motifs within APC. Each of these putative functions could link APC inactivation with tumorigenesis. Here, we will summarize recent findings regarding the diverse role of APC. We will emphasize the interaction of APC with different binding partners, the role of these complex interactions for normal functioning of the cell, and how disruption of these interactions may play a role in tumor development. The rapid progress made recently shows the many faces of APC, leading to a constant reappreciation of this multitasking tumor suppressor protein.     

E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton

beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E- cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression.     

thoA介导的细胞骨架在肿瘤发生发展中的作用

RhoA是Ras超家族中具有GTP酶活性的一种小G蛋白分子。RhoA在肿瘤组织的高表达与肿瘤的恶性程度密切相关。另外,RhoA的酶活性通过信号通路参与和调节微丝（microfilament,MF）和微管（microtubule,MT）细胞骨架的重排。新近研究表明,活性RhoA调控细胞骨架改变,进而诱导细胞癌变及肿瘤细胞增殖、入侵、转移、屏障功能和凋亡等多种生命活动。因此,研究RhoA介导的细胞骨架在肿瘤发生发展中的作用具有重要意义。该文结合作者的最新研究成果,对RhoA及其分子机制作一综述。     

Structure-function relationships in smooth muscle: The missing links

Smooth muscle cells have developed a contractile machinery that allows them to exert tension on the surrounding extracellular matrix over their entire length. This has been achieved by coupling obliquely organized contractile filaments to a more-or-less longitudinal framework of cytoskeletal elements. Earlier structural data suggested that the cytoskeleton was composed primarily of intermediate filaments and played only a passive role. More recent findings highlight the segregation of actin isotypes and of actin-associated proteins between the contractile and cytoskeletal domains and raise the possibility that the cytoskeleton performs a more active function. Current efforts focus on defining the relative contributions of myosin cross-bridge cycling and actin-associated protein interactions to the maintenance of tension in smooth muscle tissue.     

Cofilin-mediated F-actin severing is regulated by the Rap GTPase and controls the cytoskeletal dynamics that drive lymphocyte spreading and BCR microcluster formation

When lymphocytes encounter APCs bearing cognate Ag, they spread across the surface of the APC to scan for additional Ags. This is followed by membrane contraction and the formation of Ag receptor microclusters that initiate the signaling reactions that lead to lymphocyte activation. Breakdown of the submembrane cytoskeleton is likely to be required for the cytoskeleton reorganization that drives cell spreading and for removing physical barriers that limit Ag receptor mobility. In this report, we show that Ag receptor signaling via the Rap GTPases promotes the dephosphorylation and activation of the actin-severing protein cofilin and that this results in increased severing of cellular actin filaments. Moreover, we show that this cofilin-mediated actin severing is critical for the changes in actin dynamics that drive B and T cell spreading, for the formation of BCR microclusters, and for the increased mobility of BCR microclusters within the plasma membrane after BCR engagement. Finally, using a model APC, we show that activation of this Rap-cofilin signaling module controls the amount of Ag that is gathered into BCR microclusters and that this is directly related to the magnitude of the resulting BCR signaling that is initiated during B cell-APC interactions. Thus, Rap-dependent activation of cofilin is critical for the early cytoskeletal changes and BCR reorganization that are involved in APC-dependent lymphocyte activation.     

Cytoskeleton and mitochondrial morphology and function

It has been well established that the cytoskeleton is an essential modulator of cell morphology and motility, intracytoplasmic transport and mitosis, however cytoskeletal linkage to the organelles has not been unequivocally demonstrated. Indeed, cytoskeleton appears to be essential in determining and modulating gene phenotype as a function of cellular environment. According to recent studies, the organization of the cytoskeleton network together with associated protein(s) could be essential in regulating mitochondrial function and particularly the permeability of the mitochondrial outer membrane to ADP. The aim of this chapter is to summarize the main properties of the cytoskeletal environment of mitochondria and the possible role(s) of this network in mitochondrial function in myocytes.     

Dynamic association of L-selectin with the lymphocyte cytoskeletal matrix

L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial venules (HEV). This study examines the biochemical basis and regulation of interactions between L-selectin, an integral transmembrane protein, and the lymphocyte cytoskeleton. Using a detergent-based extraction procedure, constitutive associations between L-selectin and the insoluble cytoskeletal matrix could not be detected. However, engagement of the L-selectin lectin domain by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-derived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytoskeleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosine kinase activity (staurosporine, calphostin C, or genistein), although L-selectin-mediated adhesion of human PBL was markedly suppressed by these agents. Exposure of human PBL or murine pre-B transfectants expressing full-length human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enhanced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimulation and did not support adhesion to HEV. These studies, when taken together with the previously demonstrated interaction between the L-selectin cytoplasmic domain and the cytoskeletal linker protein alpha-actinin, strongly implicate the actin-based cytoskeleton in dynamically controlling L-selectin adhesion.     

Stimulus-induced selective association of actin-associated proteins (alpha-actinin) and protein kinase C isoforms with the cytoskeleton of human neutrophils.

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein alpha-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal alpha-actinin and actin. Increased association of PKCbetaI and betaII with the cytoskeletal fraction of stimulated cells was also observed, with phorbol ester being more effective than chemotactic peptide. A fraction of phosphatase 2A was constitutively associated with the cytoskeleton independent of cell activation. None of the stimuli promoted association of vinculin or myosin II with the cytoskeleton. Phosphatase inhibitors okadaic acid and calyculin A prevented increases in cytoskeletal actin, alpha-actinin, and PKCbetaII induced by phorbol ester, suggesting the requirement for phosphatase activity in these events. Increases in cytoskeletal alpha-actinin and PKCbetaII showed differing sensitivity to agents that prevent actin polymerization (cytochalasin D, latrunculin A). Latrunculin A (1 microM) completely blocked PMA-induced increases in cytoskeletal alpha-actinin but reduced cytoskeletal recruitment of PKCbetaII only by 16%. Higher concentrations of latrunculin A (4 microM), which almost abolished the cytoskeletal actin pool, reduced cytoskeletal PKCbetaII by 43%. In conclusion, a selective enrichment of cytoskeletal and signaling proteins in the cytoskeleton of human neutrophils is induced by specific stimuli.     

Adenomatous Polyposis Coli (APC) in cell migration

Adenomatous Polyposis Coli (APC) protein is mostly known as a tumor suppressor that regulates Wnt signaling, but is also an important cytoskeletal protein. Mutations in the APC gene are linked to colorectal cancer and various neurological disorders and intellectual disabilities. Cytoskeletal functions of APC appear to have significant contributions to both types of these disorders. As a cytoskeletal protein, APC can regulate both actin and microtubule cytoskeletons, which together form the main machinery for cell migration. As APC is a multifunctional protein with numerous interaction partners, the complete picture of how APC regulates cell motility is still unavailable. However, some molecular mechanisms begin to emerge. Here, we review available information about roles of APC in cell migration and propose a model explaining how microtubules, using APC as an intermediate, can initiate leading edge protrusion in response to external signals by stimulating Arp2/3 complex-dependent nucleation of branched actin filament networks via a series of intermediate events.     

Zinc induces specific association of PKC with membrane cytoskeleton

Putative binding sites for zinc are present in the regulatory domain of protein kinase C but a distinct role for zinc has not yet been proposed. Here we show that micromolar concentrations of zinc chloride cause pure rat brain protein kinase C to localize in a detergent-insoluble, cytoskeletal fraction of red cell membranes and to bind to isolated cytoskeleton in the presence of phosphatidylserine. Attachment of protein kinase C to cytoskeleton was accompanied by enhanced expression of binding sites for 3H-phorbol ester, a regulatory ligand of protein kinase C. The active factor in the cytoskeleton was labile to protease suggesting that protein kinase C binds to a cytoskeletal protein.     

Age-dependent defects in TCR-triggered cytoskeletal rearrangement in CD4+ T cells

Previous research has shown that many of the CD4 T cells from older mice do not form functional immune synapses after conjugation with peptide-pulsed APC. We now show that the defect lies at a very early stage in the cytoskeletal reorganization that precedes movement of protein kinases and their substrates to the TCR/APC interface. Antagonist peptides presented to T cells from young mice induce migration of talin (but not paxillin, vinculin, or F-actin) to the APC contact zone, but CD4 T cells from older donors typically fail to show the talin polarization response. A spreading assay in which contact with anti-CD3-coated slides induces CD4 T cells to assume a conical shape and develop lammelopodia also shows a decline with age in the proportion of T cells that can initiate cytoskeletal changes in response to this simplified stimulus. Finally, the transition from detergent-soluble to cytoskeletal forms of the p16, p21, and p23 isoforms of CD3zeta in response to CD3/CD4/CD28 cross-linking is much stronger in young than in old T cells. Thus, defects in cytoskeletal reorganization triggered by initial contact between TCR and peptide-bearing APC precede, and presumably contribute to, defective activation of protein kinase-mediated signals in the first few minutes of the activation cascade in T cells from aged mice.     

Vinculin is proteolyzed by calpain during platelet aggregation: 95 kDa cleavage fragment associates with the platelet cytoskeleton

The focal adhesion protein vinculin contributes to cell attachment and spreading through strengthening of mechanical interactions between cell cytoskeletal proteins and surface membrane glycoproteins. To investigate whether vinculin proteolysis plays a role in the influence vinculin exerts on the cytoskeleton, we studied the fate of vinculin in activated and aggregating platelets by Western blot analysis of the platelet lysate and the cytoskeletal fractions of differentially activated platelets. Vinculin was proteolyzed into at least three fragments (the major one being approximately 95 kDa) within 5 min of platelet activation with thrombin or calcium ionophore. The 95 kDa vinculin fragment shifted cellular compartments from the membrane skeletal fraction to the cortical cytoskeletal fraction of lysed platelets in a platelet aggregation-dependent manner. Vinculin cleavage was inhibited by calpeptin and E64d, indicating that the enzyme responsible for vinculin proteolysis is calpain. These calpain inhibitors also inhibited the translocation of full-length vinculin to the cytoskeleton. We conclude that cleavage of vinculin and association of vinculin cleavage fragment(s) with the platelet cytoskeleton is an activation response that may be important in the cytoskeletal remodeling of aggregating platelets.     

Characterization of a 60S complex of the adenomatous polyposis coli tumor suppressor protein

The tumor suppressor protein adenomatous polyposis coli (APC) is a multifunctional protein with a well characterized role in the Wnt signal transduction pathway and roles in cytoskeletal regulation and cell polarity. The soluble pool of APC protein in colon epithelial tumor cells exists in two distinct complexes fractionating at approximately 20S and approximately 60S in size. The 20S complex contains components of the beta-catenin destruction complex and probably functions in the Wnt pathway. In this study, we characterized the molecular nature of the 60S APC- containing complex by examining known potential binding partners of APC. 60S APC did not contain EB1 or diaphanous, proteins that have been reported to interact with APC and are implicated in microtubule plus end stabilization. Nor did the two other microtubule associated proteins, MAP4 or KAP3, which is thought to link APC to kinesin motor proteins, associate with the 60S complex. Minor fractions of alpha-tubulin, gamma-tubulin and IQGAP1, a Rac1 and CDC42 effector that interacts with APC, specifically associated with APC in the 60S fraction. We propose that 60S APC is a discrete high molecular weight complex with a novel function in cytoskeletal regulation in epithelial cells apart from its well established role in targeting catenin destruction or its proposed role in microtubule plus end stabilization.