Differential mobility and self-association of Arc/Arg3.1 in the cytoplasm and nucleus of living cells

ABSTRACTArc, also known as Arg3.1, is an activity-dependent immediate-early gene product that plays essential roles in memory consolidation. A pool of Arc is located in the postsynaptic cytoplasm, where it promotes AMPA receptor endocytosis and cytoskeletal remodeling. However, Arc is also found in the nucleus, a major portion being associated with promyelocytic leukemia nuclear bodies (PML-NBs). Nuclear Arc has been implicated in epigenetic control of gene transcription associated with learning and memory. In this study, we use a battery of fluorescence nanoimaging approaches to characterize the behavior of Arc in living cells. Our results indicate that in the cytoplasm, Arc exists predominantly as monomers and dimers associated with slowly diffusing particles. In contrast, nuclear Arc is almost exclusively monomeric and displays a higher diffusivity than cytoplasmic Arc. We further show that Arc moves freely and rapidly between PML-NBs and the nucleoplasm, and that its movement within PML-NBs is relatively unobstructed.

Biased activation of β2-AR/Gi/GRK2 signal pathway attenuated β1-AR sustained activation induced by β1-adrenergic receptor autoantibody

Heart failure is the terminal stage of many cardiac diseases, in which β1-adrenoceptor (β1-AR) autoantibody (β1-AA) has a causative role. By continuously activating β1-AR, β1-AA can induce cytotoxicity, leading to cardiomyocyte apoptosis and heart dysfunction. However, the mechanism underlying the persistent activation of β1-AR by β1-AA is not fully understood. Receptor endocytosis has a critical role in terminating signals over time. β2-adrenoceptor (β2-AR) is involved in the regulation of β1-AR signaling. This research aimed to clarify the mechanism of the β1-AA-induced sustained activation of β1-AR and explore the role of the β2-AR/Gi-signaling pathway in this process. The beating frequency of neonatal rat cardiomyocytes, cyclic adenosine monophosphate content, and intracellular Ca2+ levels were examined to detect the activation of β1-AA. Total internal reflection fluorescence microscopy was used to detect the endocytosis of β1-AR. ICI118551 was used to assess β2-AR/Gi function in β1-AR sustained activation induced by β1-AA in vitro and in vivo. Monoclonal β1-AA derived from a mouse hybridoma could continuously activate β1-AR. β1-AA-restricted β1-AR endocytosis, which was reversed by overexpressing the endocytosis scaffold protein β-arrestin1/2, resulting in the cessation of β1-AR signaling. β2-AR could promote β1-AR endocytosis, as demonstrated by overexpressing/interfering with β2-AR in HL-1 cells, whereas β1-AA inhibited the binding of β2-AR to β1-AR, as determined by surface plasmon resonance. ICI118551 biasedly activated the β2-AR/Gi/G protein-coupled receptor kinase 2 (GRK2) pathway, leading to the arrest of limited endocytosis and continuous activation of β1-AR by β1-AA in vitro. In vivo, ICI118551 treatment attenuated myocardial fiber rupture and left ventricular dysfunction in β1-AA-positive mice. This study showed that β1-AA continuously activated β1-AR by inhibiting receptor endocytosis. Biased activation of the β2-AR/Gi/GRK2 signaling pathway could promote β1-AR endocytosis restricted by β1-AA, terminate signal transduction, and alleviate heart damage.

Death Receptors DR4 and DR5 Undergo Spontaneous and Ligand-Mediated Endocytosis and Recycling Regardless of the Sensitivity of Cancer Cells to TRAIL

Tumor necrosis factor-associated ligand inducing apoptosis (TRAIL) induces apoptosis through the death receptors (DRs) 4 and 5 expressed on the cell surface. Upon ligand stimulation, death receptors are rapidly internalized through clathrin-dependent and -independent mechanisms. However, there have been conflicting data on the role of death receptor endocytosis in apoptotic TRAIL signaling and possible cell type-specific differences in TRAIL signaling have been proposed. Here we have compared the kinetics of TRAIL-mediated internalization and subsequent recycling of DR4 and DR5 in resistant (HT-29 and A549) and sensitive (HCT116 and Jurkat) tumor cell lines of various origin. TRAIL stimulated the internalization of both receptors in a concentration-dependent manner with similar kinetics in sensitive and resistant cell lines without affecting the steady-state expression of DR4 and DR5 in cell lysates. Using the receptor-selective TRAIL variant DR5-B, we have shown that DR5 is internalized independently of DR4 receptor. After internalization and elimination of TRAIL from culture medium, the receptors slowly return to the plasma membrane. Within 4 h in resistant or 6 h in sensitive cells, the surface expression of receptors was completely restored. Recovery of receptors occurred both from newly synthesized molecules or from trans-Golgi network, as cycloheximide and brefeldin A inhibited this process. These agents also suppressed the expression of cell surface receptors in a time- and concentration-dependent manner, indicating that DRs undergo constitutive endocytosis. Inhibition of receptor endocytosis by sucrose led to sensitization of resistant cells to TRAIL and to an increase in its cytotoxic activity against sensitive cells. Our results confirm the universal nature of TRAIL-induced death receptor endocytosis, thus cell sensitivity to TRAIL can be associated with post-endocytic events.

Molecular Pathogenesis and Regulation of the miR-29-3p-Family: Involvement of ITGA6 and ITGB1 in Intra-Hepatic Cholangiocarcinoma

The aggressive nature of intrahepatic cholangiocarcinoma (ICC) renders it a particularly lethal solid tumor. Searching for therapeutic targets for ICC is an essential challenge in the development of an effective treatment strategy. Our previous studies showed that the miR-29-3p-family members (miR-29a-3p, miR-29b-3p and miR-29c-3p) are key tumor-suppressive microRNAs that control many oncogenic genes/pathways in several cancers. In this study, we searched for therapeutic targets for ICC using the miR-29-3p-family as a starting point. Our functional studies of cell proliferation, migration and invasion confirmed that the miR-29-3p-family act as tumor-suppressors in ICC cells. Moreover, in silico analysis revealed that “focal adhesion”, “ECM-receptor”, “endocytosis”, “PI3K-Akt signaling” and “Hippo signaling” were involved in oncogenic pathways in ICC cells. Our analysis focused on the genes for integrin-α6 (ITGA6) and integrin-β1 (ITGB1), which are involved in multiple pathways. Overexpression of ITGA6 and ITGB1 enhanced malignant transformation of ICC cells. Both ITGA6 and ITGB1 were directly regulated by the miR-29-3p-family in ICC cells. Interestingly, expression of ITGA6/ITGB1 was positively controlled by the transcription factor SP1, and SP1 was negatively controlled by the miR-29-3p-family. Downregulation of the miR-29-3p-family enhanced SP1-mediated ITGA6/ITGB1 expression in ICC cells. MicroRNA-based exploration is an attractive strategy for identifying therapeutic targets for ICC.

CD13 is a critical regulator of cell–cell fusion in osteoclastogenesis

The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell–cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion.

BRET-based effector membrane translocation assay monitors GPCR-promoted and endocytosis-mediated Gq activation at early endosomes

G protein–coupled receptors (GPCRs) are gatekeepers of cellular homeostasis and the targets of a large proportion of drugs. In addition to their signaling activity at the plasma membrane, it has been proposed that their actions may result from translocation and activation of G proteins at endomembranes—namely endosomes. This could have a significant impact on our understanding of how signals from GPCR-targeting drugs are propagated within the cell. However, little is known about the mechanisms that drive G protein movement and activation in subcellular compartments. Using bioluminescence resonance energy transfer (BRET)–based effector membrane translocation assays, we dissected the mechanisms underlying endosomal Gq trafficking and activity following activation of Gq-coupled receptors, including the angiotensin II type 1, bradykinin B2, oxytocin, thromboxane A2 alpha isoform, and muscarinic acetylcholine M3 receptors. Our data reveal that GPCR-promoted activation of Gq at the plasma membrane induces its translocation to endosomes independently of β-arrestin engagement and receptor endocytosis. In contrast, Gq activity at endosomes was found to rely on both receptor endocytosis-dependent and -independent mechanisms. In addition to shedding light on the molecular processes controlling subcellular Gq signaling, our study provides a set of tools that will be generally applicable to the study of G protein translocation and activation at endosomes and other subcellular organelles, as well as the contribution of signal propagation to drug action.