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Unformatted text preview: Microtubule Plus-End-Tracking Proteins Target Gap Junctions Directly from the Cell Interior to Adherens Junctions Robin M. Shaw 1,5 , Alex J. Fay 2,5 , Manojkumar A. Puthenveedu 3 , Mark von Zastrow 3 , Yuh- Nung Jan 4 , and Lily Y. Jan 4,* 1 Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, CA 94143, 2 Graduate Group in Biophysics, University of California, San Francisco, CA 94143, 3 Departments of Psychiatry and Cellular & Molecular Pharmacology, University of California, San Francisco, CA 94143, 4 Howard Hughes Medical Institute and Departments of Physiology and Biochemistry, University of California, San Francisco, CA 94143, USA SUMMARY Gap junctions are intercellular channels that connect the cytoplasms of adjacent cells. For gap junctions to properly control organ formation and electrical synchronization in the heart and the brain, connexin-based hemichannels must be correctly targeted to cell-cell borders. While it is generally accepted that gap junctions form via lateral diffusion of hemichannels following microtubule- mediated delivery to the plasma membrane, we provide evidence for direct targeting of hemichannels to cell-cell junctions through a pathway that is dependent on microtubules; through the adherens- junction proteins N-cadherin and β-catenin; through the microtubule plus-end-tracking protein (+TIP) EB1; and through its interacting protein p150(Glued). Based on live cell microscopy that includes fluorescence recovery after photobleaching (FRAP), total internal reflection fluorescence (TIRF), deconvolution, and siRNA knockdown, we propose that preferential tethering of microtubule plus ends at the adherens junction promotes delivery of connexin hemichannels directly to the cell- cell border. These findings support an unanticipated mechanism for protein delivery to points of cell- cell contact. INTRODUCTION Gap junctions, which are formed by the serial coupling of hemichannels of adjacent cells, allow direct sharing of ions and small cytoplasmic molecules. Each hemichannel is a hexamer of connexin, and the most common isoform is Connexin43 (Cx43). Gap junctions reside at cell- cell borders, where their density is of critical importance. In the heart, for example, gap junctions are concentrated at the intercalated disc (ID) that joins the ends of cardiomyocytes, and this is where they ensure propagation of action potentials (Gros and Jongsma, 1996;Shaw and Rudy, 1997). Altered Cx43 gap junction distribution following cardiac ischemia contributes to malignant ischemic arrhythmias (Kaprielian et al., 1998;Peters et al., 1997;Shaw and Rudy, 1997). Preventing or reversing this process offers a strategy to repair damaged heart (Abraham et al., 2005;Reinecke et al., 2004). Understanding the molecular mechanism of gap *Correspondence: firstname.lastname@example.org 5 These authors contributed equally to this work....
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This note was uploaded on 11/01/2010 for the course PRC 1234 taught by Professor All during the Spring '10 term at HKU.
- Spring '10
- The Hours