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Visualization of the Polarity of Isolated Titin Molecules: A Single Globular Head on a Long Thin Rod As the M Band Anchoring Domain?

Rüdiger Nave, Dieter O. Fürst and Klaus Weber
The Journal of Cell Biology
Vol. 109, No. 5 (Nov., 1989), pp. 2177-2187
Stable URL: http://www.jstor.org/stable/1613717
Page Count: 11
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Visualization of the Polarity of Isolated Titin Molecules: A Single Globular Head on a Long Thin Rod As the M Band Anchoring Domain?
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Abstract

TII, the extractable form of titin, was purified from myofibrils and separated by high resolution gel permeation chromatography into two fractions ( TII A and TII B). Novel specimen orientation methods used before metal shadowing and EM result in striking pictures of the two forms. Molecules layered on mica become uniformly oriented when subjected to centrifugation. TII B comprises a very homogeneous fraction. All molecules reveal a single globular head at one end on a long and very thin rod of uniform diameter. The lengths of the rods have a very narrow distribution (900 ± 50 nm). TII A molecules seem lateral oligomers of TII B, attached to each other via the head regions. While dimers are the predominant species, trimers and some higher oligomers can also be discerned. Mild proteolysis destroys the heads and converts TII A and TII B into TII B-like rods. Similar molecules also result from titin purified from myofibrils by certain established purification schemes. Headless titin molecules show in gel electrophoresis only the TII band, while head bearing molecules give rise to two additional polypeptides at 165 and 190 kD. Immunoelectron microscopy of myofibrils identifies both titin-associated proteins as M band constituents. We speculate that in the polar images of TII the globular head region corresponds to the M band end of the titin molecules. This hypothesis is supported by immunoelectron micrographs of TII B molecules using titin antibodies of known epitope location in the half sarcomere. This proposal complements our previous immunoelectron microscopic data on myofibrils. They showed that epitopes present only on the nonextractable TI species locate to the Z line and its immediately adjacent region (Fürst, D. O., M. Osborn, R. Nave, and K. Weber. 1988. J. Cell Biol. 106:1563-1572). Thus, the two distinct ends of the titin molecule attach to Z and M band material respectively.

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