The authors note that that in the constructs EcadTSMod, EcadTSModΔcyto, and EcadTSModΔmTFP, the Förster resonance energy transfer (FRET) acceptor fluorophore was monomeric enhanced yellow fluorescent protein (mEYFP)-not Venus as was originally reported. The photophysical properties of mEYFP and Venus, including absorption spectra, emission spectra, and fluorescence quantum yields are highly similar, leading to negligible changes in the Förster radius with the FRET donor monomeric Teal Fluorescent Protein (mTFP) (1). This substitution thus does not affect the FRET data reported in the original manuscript or their interpretation. The protein mEYFP is based on EYFP (Clontech) with the addition of the mutation A206K to suppress dimerization (2). www.pnas.org/cgi
The sympathetic nervous system regulates cardiac function through the activation of adrenergic receptors (ARs). β1 and β2ARs are the primary sympathetic receptors in the heart and play different roles in regulating cardiac contractile function and remodeling in response to injury. In this study, we examine the targeting and trafficking of β1 and β2ARs at cardiac sympathetic synapses in vitro. Sympathetic neurons form functional synapses with neonatal cardiac myocytes in culture. The myocyte membrane develops into specialized zones that surround contacting axons and contain accumulations of the scaffold proteins SAP97 and AKAP79/150 but are deficient in caveolin-3. The β1ARs are enriched within these zones, whereas β2ARs are excluded from them after stimulation of neuronal activity. The results indicate that specialized signaling domains are organized in cardiac myocytes at sites of contact with sympathetic neurons and that these domains are likely to play a role in the subtype-specific regulation of cardiac function by β1 and β2ARs in vivo.
Previous research suggested that α 2A and α 2C adrenergic receptor (AR) subtypes have overlapping but unique physiological roles in neuronal signaling; however, the basis for these dissimilarities is not completely known. To better understand the observed functional differences between these autoreceptors, we investigated targeting and signaling of endogenously expressed α 2A and α 2C ARs in cultured sympathetic ganglion neurons (SGN). At Days 1 and 4, α 2A and α 2C ARs could be readily detected in SGN from wild-type mice. By Day 8, α 2A ARs were targeted to cell body, as well as axonal and dendritic sites, whereas α 2C ARs were primarily localized to an intracellular vesicular pool within the cell body and proximal dendritic projections. Expression of synaptic vesicle marker protein SV2 did not differ at Day 8 nor co-localize with either subtype. By Day 16, however, α 2C ARs had relocated to somatodendritic and axonal sites and, unlike α 2A ARs, co-localized with SV2 at synaptic contact sites. Consistent with a functional role for α 2 ARs, we also observed that dexmedetomidine stimulation of cultured SGN more efficiently inhibited depolarization-induced calcium entry into older, compared to younger, cultures. These results provide direct evidence of distinct developmental patterns of endogenous α 2A and α 2C AR targeting and function in a native cell system and that maturation of SGN in culture leads to alterations in neuronal properties required for proper targeting. More importantly, the colocalization at Day 16 of α 2C ARs at sites of synaptic contact may partially explain the differential modulation of neurotransmitter release and responsiveness to action potential frequency observed between α 2A and α 2C ARs in SGN.
The role of endocytic proteins and the molecular mechanisms underlying epithelial cell cohesion and tumor dissemination are not well understood. Here, we report that the endocytic F-BAR-containing CDC42-interacting protein 4 (CIP4) is required for ERBB2- and TGF-β1-induced cell scattering, breast cancer (BC) cell motility and invasion into 3D matrices, and conversion from ductal breast carcinoma in situ to invasive carcinoma in mouse xenograft models. CIP4 promotes the formation of an E-cadherin-CIP4-SRC complex that controls SRC activation, E-cadherin endocytosis, and localized phosphorylation of the myosin light chain kinase, thereby impinging on the actomyosin contractility required to generate tangential forces to break cell-cell junctions. CIP4 is upregulated in ERBB2-positive human BC, correlates with increased distant metastasis, and is an independent predictor of poor disease outcome in subsets of BC patients. Thus, it critically controls cell-cell cohesion and is required for the acquisition of an invasive phenotype in breast tumors.
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