QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 26 October 2004
doi: 10.1242/jcs.01501

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nagy, A. Articles by Kellermayer, M. S. Z. Search for Related Content PubMed PubMed Citation Articles by Nagy, A. Articles by Kellermayer, M. S. Z.

Differential actin binding along the PEVK domain of skeletal muscle titin

Attila Nagy¹, Paola Cacciafesta^1,*, László Grama¹, András Kengyel¹, András Málnási-Csizmadia² and Miklós S. Z. Kellermayer^1,

¹ Department of Biophysics, University of Pécs, Faculty of Medicine, Szigeti út 12. Pécs 7624, Hungary
² Department of Biochemistry, Eötvös University, Pázmány Péter sétány 1/c., Budapest 1117, Hungary

View larger version (18K): [in a new window]   Fig. 1. PEVK segments and fragments in skeletal muscle titin. (A) Motif layout of skeletal muscle titin PEVK domain (Greaser, 2001). White rectangles indicate PPAK motifs, and gray ones, polyE motifs. The boundaries of the PEVK segments and fragments expressed and studied in this work are indicated by segments with arrows on both ends. The pure polyE fragment was generated by tandem duplication of the region marked with a line with diamonds at both ends. (B) SDS-PAGE of the expressed and purified PEVK segments and fragments. (a) molecular weight standards, (b) PEVKI, (c) PEVKII, (d) PEVKIII, (e) PPAK, (f) polyE-rich and (g) pure polyE.

View larger version (43K): [in a new window]   Fig. 2. Interaction of PEVK segments with actin. (A) Effect of PEVK segments on in vitro acto-HMM motility. Velocity as a function of PEVKI, -II and -III segment concentration. Inset graph shows velocity as a function of log[PEVK segment]. The in vitro motility assay was carried out at a KCl concentration of 25 mM, HMM concentration of 40 µg/ml and ATP concentration of 1 mM. (B) Solid-state surface binding of F-actin by PEVK segments as a function of ionic strength (adjusted by varying KCl concentration). The total filament length per field of view at the initial concentration of 25 mM KCl, were 115 µm, 145 µm and 195 µm for the PEVKI, -II and -III segments, respectively. (C) Actin crosslinking assay with various PEVK fragments (i) PEVKI, (ii) PEVKII and (iii) PEVKIII. Bar, 20 µm.

View larger version (65K): [in a new window]   Fig. 3. Effect of calcium and Ca2+ regulatory proteins on the actin binding of PEVKII. (A) Solid-state surface binding of F-actin by PEVKII as a function of free Ca2+ concentration (pCa). (B) SDS-PAGE of isolated native thin filaments. Left lane, thin filament preparation; right lane, molecular weight standards. Tm, tropomyosin; TnT, troponin T; TnI, troponin I; TnC, troponin C. (C) Surface binding of native thin filaments by PEVKII. Only short filaments are observed, in accordance with the size of the native thin filaments (1 µm). (i) Negative control (BSA on the surface). (ii) Positive control (HMM-coated surface; pCa 3). (iii) PEVKII-coated surface; pCa 9. (iv) PEVKII-coated surface; pCa 3. Bar, 10 µm.

View larger version (31K): [in a new window]   Fig. 4. Interaction of various PEVK fragments with actin. (A) In vitro acto-HMM velocity as a function of PPAK and pure polyE fragment concentration. The assay was carried out in the presence of 140 mM KCl. The data were normalized to the velocity in the absence of PEVK fragment (concentration=0 µg/ml), which was 3.2 µm/s for the PPAK fragment and 3.0 µm/s for the pure polyE fragment. (B) Solid-state surface binding of F-actin by PPAK and pure polyE fragments as a function of KCl concentration. The total filament length per field of view at the initial concentration of 25 mM KCl were 400 µm and 1140 µm for the PPAK and pure polyE fragments, respectively. (C) F-actin crosslinking assay in the presence of various PEVK fragments. (i) Actin filaments in the presence of PPAK. (ii) Crosslinked actin filaments in the presence of pure polyE fragment. Bar 20 µm. (iii) Summary of the actin-crosslinking assay.

View larger version (38K): [in a new window]   Fig. 5. PEVK fragment–F-actin co-sedimentation assay. (A) SDS-PAGE of polyE-rich fragment-actin binding assay. S, supernatant; P, pellet. The total polyE concentrations for the S-P lane pairs in this assay were: 0.036, 0.048, 0.060, 0.072, 0.084 and 0.096 mg/ml from left to right of gel. (B) Bound PEVK fragment concentration as a function of total PEVK fragment concentration. Inset shows a magnified view of the low-concentration range.

View larger version (46K): [in a new window]   Fig. 6. Binding of IAF-labeled PEVKII to purified rabbit psoas myofibrils. (A) Confocal microscopic images of a myofibril incubated with a fluorescently labeled PEVKII segment. Phase-contrast (upper panel) and the corresponding fluorescence (lower panel) microscopic images are shown. Bar, 2 µm. (B) Grayscale intensity along the myofibril axis for both the phase contrast and fluorescence microscopic images.

View larger version (36K): [in a new window]   Fig. 7. Model of in situ PEVK-actin interaction in the sarcomere. Schematic arrangement of myofilaments in an extended and contracted sarcomere. Top panel, highly extended sarcomere. Middle panel, shortening sarcomere in which the PEVK domain is contracted. Bottom panel, shortening sarcomere in which the tip of the thick filament crossed the site of PEVK binding to the thin filament. The shaded area indicates the dimensions of the space occupied by the PEVK domain that defines the apparent concentration of the actin-binding sites in PEVK. As the dimensions of this space vary along the thin filament axis, the actin concentration is constant regardless of the size of the space. Arrows indicate the direction of movement during the contraction of a rather thick filament.