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Biosynthesis and Structural Composition of Clustered Membrane Proteins

M.M. Falk, P. Shen, U. Lauf, B.N.G. Giepmans, S.-C. Chen*

* Northwest Hospital, Seattle, WA

A number of oligomeric proteins, including receptors for acetylcholine, aquaporin-0, aquaporin-4, tight junction occludins and claudins, and gap junction channels aggregate into tight clusters, arrays, or strands in the plasma membrane. However, the signals that regulate their tight packing are not well understood. Previously, we studied the synthesis and assembly of gap junction proteins (connexins) into half (connexons), and complete gap junction channels. However, many questions related to the biosynthesis and turnover of gap junction channels remain: (1) How and in which oligomeric or clustered stage do the connexins traffic from the Golgi complex to the plasma membrane? (2) How and where are the connexons integrated into the membrane? (3) How do connexons in apposing membranes register and dock and how is channel formation initiated? (4) How and why do the channels cluster? (5) How are the channels regulated and which signaling molecules are involved? (6) How are the channels within the plaque turned over?

To address these questions in live cells, we tagged the gap junction proteins a₁(Cx43), ß₁(Cx32), and ß₂(Cx26) with the autofluorescent tracers green fluorescent protein and its cyan and yellow variants. We combined this reporter technology with high-resolution fluorescence deconvolution microscopy, dual-color live-cell and time-lapse imaging, and 3-dimensional volume reconstruction. Comprehensive analysis verified that the tagged connexins trafficked, assembled, and packed normally. As indicated by dye microinjection analyses, the resultant channels were fully functional even under conditions in which the channels were assembled solely from tagged connexins. This combined technology enabled us for the first time to quantitatively identify the distribution of connexins and gap junction channels within cells and to analyze the packing behavior of gap junction channels assembled from different connexin isotypes.

We found that the structural composition of gap junction plaques assembled from more than a single connexin isotype strictly depends on the subclass characteristics of the connexin isotypes (Fig. 1). Furthermore, the high-resolution images and 3-dimensional volume reconstructions revealed a structural organization of gap junctions never seen before in such detail in live cells and provided a realistic impression of entire gap junctions as they appear in the plasma membranes of adjoining cells (Fig. 2). Finally, our results enabled us to reinterpret earlier studies obtained with immunofluorescent techniques and freeze-fracture electron microscopy in fixed and cryo-preserved cells. The previously described technology also provides a sound system for studying the dynamics and underlying principles intrinsic to the biosynthesis, turnover, and degradation of clustered membrane proteins.

PUBLICATIONS

Falk, M.M. Biosynthesis and structural composition of gap junction intercellular membrane channels. Eur. J. Cell Biol., in press.

Falk, M.M. Cell-free expression for analyzing the membrane integration, oligomerization, and assembly characteristics of gap junction connexins. Methods 20:165, 2000.

Falk, M.M. Connexins/connexons: Cell-free expression. Methods Mol. Biol., in press.

Falk, M.M., Lauf, U. High-resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structure and function of gap junctions. Microsc. Res. Tech., in press.