Double-membrane-spanning gap junction (GJ) channels cluster into two-dimensional arrays, termed plaques, to provide direct cell-to-cell communication. GJ plaques often contain circular, channel-free domains ( approximately 0.05-0.5 mum in diameter) identified >30 y ago and termed nonjunctional membrane (NM) domains. We show, by expressing the GJ protein connexin43 (Cx43) tagged with green fluorescent protein, or the novel photoconvertible fluorescent protein Dendra2, that NM domains appear to be remnants generated by the internalization of small GJ channel clusters that bud over time from central plaque areas. Channel clusters internalized within seconds forming endocytic double-membrane GJ vesicles ( approximately 0.18-0.27 mum in diameter) that were degraded by lysosomal pathways. Surprisingly, NM domains were not repopulated by surrounding channels and instead remained mobile, fused with each other, and were expelled at plaque edges. Quantification of internalized, photoconverted Cx43-Dendra2 vesicles indicated a GJ half-life of 2.6 h that falls within the estimated half-life of 1-5 h reported for GJs. Together with previous publications that revealed continuous accrual of newly synthesized channels along plaque edges and simultaneous removal of channels from plaque centers, our data suggest how the known dynamic channel replenishment of functional GJ plaques can be achieved. Our observations may have implications for the process of endocytic vesicle budding in general.

Direct cell-cell communication mediated by plasma membrane-spanning gap junction (GJ) channels is vital to all aspects of cellular life. Obviously, GJ intercellular communication (GJIC) requires precise regulation, and it is known that controlled biosynthesis and degradation, and channel opening and closing (gating) are exploited. We discovered that cells internalize GJs in response to various stimuli. Here, we report that GJ internalization is a clathrin-mediated endocytic process that utilizes the vesicle-coat protein clathrin, the adaptor proteins adaptor protein complex 2 and disabled 2, and the GTPase dynamin. To our knowledge, we are first to report that the endocytic clathrin machinery can internalize double-membrane vesicles into cells.

During the inflammatory response, activation of G-protein coupled receptors (GPCRs) by inflammatory mediators rapidly leads to inhibition of gap junction intercellular communication (GJIC); however, the steps that lead to this inhibition are not known. Combining high-resolution fluorescence microscopy and functional assays, we found that activation of the GPCRs PAR-1 and ET A/B by their natural inflammatory mediator agonists, thrombin and endothelin-1, resulted in rapid and acute internalization of gap junctions (GJs) that coincided with the inhibition of GJIC followed by increased vascular permeability. The endocytosis protein clathrin and the scaffold protein ZO-1 appeared to be involved in GJ internalization, and ZO-1 was partially displaced from GJs during the internalization process. These findings demonstrate that GJ internalization is an efficient mechanism for modulating GJIC in inflammatory response.

Background and Purpose. An important part of treatment planning in physical therapy is effective goal setting. The Guide to Physical Therapist Practice recommends that therapists should identify the patient's goals and objectives during the initial examination in order to maximize outcomes. The purpose of this study was to examine whether therapists seek to involve patients in goal setting and, if so, what methods they use. Therapists' attitudes toward participation and patient satisfaction with the examination were also examined. Subjects and Methods. Twenty-two physical therapists audiotaped the initial examination of 73 elderly patients (Xϭ76.4 years of age, SDϭ7.1, rangeϭ65-94). The audiotaped examinations were then scored using the Participation Method Assessment Instrument (PMAI) to determine the frequency of attempts made by therapists to involve patients in goal setting. Therapists and patients completed surveys following the examinations. Results. Therapists' use of participation methods during examinations ranged from a minimum of 1 to a maximum of 19 out of 21 possible items on the PMAI. The therapists stated that they believed that it is important to include patients in goal-setting activities and that outcomes will be improved if patients participate. Patients also indicated that participation is important to them. Discussion and Conclusion. In most cases, the therapists did not fully take advantage of the potential for patient participation in goal setting. Patient and therapist education is needed regarding methods for patient participation during initial goal-setting activities. [Baker SM, Marshak HH, Rice GT, Zimmerman GJ. Patient participation in physical therapy goal setting. Phys Ther. 2001;81:1118 -1126