␣B-crystallin, a major component of the vertebrate lens, is a chaperone belonging to the family of small heat shock proteins. These proteins form oligomers that bind to partially unfolded substrates and prevent denaturation. ␣B-crystallin in cardiac muscle binds to myofibrils under conditions of ischemia, and previous work has shown that the protein binds to titin in the I-band of cardiac fibers (Golenhofen, N., Arbeiter, A., Koob, R., and Drenckhahn, D. (2002) J. Mol. Cell. Cardiol. 34, 309 -319). This part of titin extends as muscles are stretched and is made up of immunoglobulin-like modules and two extensible regions (N2B and PEVK) that have no well defined secondary structure. We have followed the position of ␣B-crystallin in stretched cardiac fibers relative to a known part of the titin sequence. ␣B-crystallin bound to a discrete region of the I-band that moved away from the Z-disc as sarcomeres were extended. In the physiological range of sarcomere lengths, ␣B-crystallin bound in the position of the N2B region of titin, but not to PEVK. In overstretched myofibrils, it was also in the Ig region between N2B and the Z-disc. Binding between ␣B-crystallin and N2B was confirmed using recombinant titin fragments. The Ig domains in an eight-domain fragment were stabilized by ␣B-crystallin; atomic force microscopy showed that higher stretching forces were needed to unfold the domains in the presence of the chaperone. Reversible association with ␣B-crystallin would protect I-band titin from stress liable to cause domain unfolding until conditions are favorable for refolding to the native state.␣B-crystallin is one of several crystallins in the vertebrate lens. An important function of the protein is to act as a chaperone preventing partially unfolded proteins from forming aggregates that would make the lens opaque (1, 2). ␣B-crystallin is a member of the family of small heat shock proteins (sHSPs)1 . These are chaperones in multisubunit complexes that bind to proteins in the early stages of denaturation, holding them in a folding-competent state. ␣B-crystallin is also found in tissues other than the lens, including cardiac and skeletal muscle (3). The ␣B-crystallin content of cardiac muscle is 3-5% of the total soluble protein (4), and it protects the fibers from the effects of ischemia, preventing extensive structural damage (5-7). ␣B-crystallin moves from general distribution in the cytosol to myofibrils following ischemia (8, 9). Recent immunoelectron microscopy of rat heart has shown that ␣B-crystallin is in a narrow region of the I-band rather than in the Z-disc, as was previously thought, and is also associated with desmin filaments connecting neighboring myofibrils (10). When actin was extracted from pig heart myofibrils, ␣B-crystallin remained, suggesting that the protein was associated with titin in the I-band rather than with actin; and this was supported by co-purification of ␣B-crystallin with titin (10).Titin is a large (3 MDa) protein in striated muscle that spans half the sarcomere, forming an extens...

Article:Burkart, C., Qiu, F., Brendel, S. et al. ReuseUnless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version -refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher's website. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. Please cite this article as: Burkart, C . ,Q i u ,F . ,B r e n d e l ,S . ,B e n e s ,V . ,H aåg, P., Labeit, S., This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (700) extend from the Z -disc to the ends of the thick filaments, though, Sls(700) is only in the myofibril core. These shorter isoforms would contribute to the high stiffness of IFM. Other muscles in the thorax and legs have longer Sls isoforms with varying amounts of PEVK sequence; all span the I-band to the ends of the thick filaments. In muscles with longer Ibands, the proportion of PEVK sequence would determine the extensibility of the sarcomere. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Flightin is a multiply phosphorylated, 20-kD myofibrillar protein found in Drosophila indirect flight muscles (IFM). Previous work suggests that flightin plays an essential, as yet undefined, role in normal sarcomere structure and contractile activity. Here we show that flightin is associated with thick filaments where it is likely to interact with the myosin rod. We have created a null mutation for flightin, fln0, that results in loss of flight ability but has no effect on fecundity or viability. Electron microscopy comparing pupa and adult fln0 IFM shows that sarcomeres, and thick and thin filaments in pupal IFM, are 25–30% longer than in wild type. fln0 fibers are abnormally wavy, but sarcomere and myotendon structure in pupa are otherwise normal. Within the first 5 h of adult life and beginning of contractile activity, IFM fibers become disrupted as thick filaments and sarcomeres are variably shortened, and myofibrils are ruptured at the myotendon junction. Unusual empty pockets and granular material interrupt the filament lattice of adult fln0 sarcomeres. Site-specific cleavage of myosin heavy chain occurs during this period. That myosin is cleaved in the absence of flightin is consistent with the immunolocalization of flightin on the thick filament and biochemical and genetic evidence suggesting it is associated with the myosin rod. Our results indicate that flightin is required for the establishment of normal thick filament length during late pupal development and thick filament stability in adult after initiation of contractile activity.

Thick and thin filaments in asynchronous flight muscle overlap nearly completely and thick filaments are attached to the Z‐disc by connecting filaments. We have raised antibodies against a fraction of Lethocerus flight muscle myofibrils containing Z‐discs and associated filaments and also against a low ionic strength extract of myofibrils. Monoclonal antibodies were obtained to proteins of 800 kd (p800), 700 kd (p700), 400 kd (p400) and alpha‐actinin. The positions of the proteins in Lethocerus flight and leg myofibrils were determined by immunofluorescence and electron microscopy. p800 is in connecting filaments of flight myofibrils and in A‐bands of leg myofibrils. p700 is in Z‐discs of flight myofibrils and an immunologically related protein, p500, is in leg muscle Z‐discs. p400 is in M‐lines of both flight and leg myofibrils. Preliminary DNA sequencing shows that p800 is related to vertebrate titin and nematode twitchin. Molecules of p800 could extend from the Z‐disc a short way along thick filaments, forming a mechanical link between the two structures. All three high molecular weight proteins probably stabilize the structure of the myofibril.