TY - JOUR
AU - Atomi,, Yoriko
AB - Abstract αB-crystallin is highly expressed in the heart and slow skeletal muscle; however, the roles of αB-crystallin in the muscle are obscure. Previously, we showed that αB-crystallin localizes at the sarcomere Z-bands, corresponding to the focal adhesions of cultured cells. In myoblast cells, αB-crystallin completely colocalizes with microtubules and maintains cell shape and adhesion. In this study, we show that in beating cardiomyocytes α-tubulin and αB-crystallin colocalize at the I- and Z-bands of the myocardium, where it may function as a molecular chaperone for tubulin/microtubules. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the striated patterns of GFP-αB-crystallin fluorescence recovered quickly at 37°C. FRAP mobility assay also showed αB-crystallin to be associated with nocodazole-treated free tubulin dimers but not with taxol-treated microtubules. The interaction of αB-crystallin and free tubulin was further confirmed by immunoprecipitation and microtubule sedimentation assay in the presence of 1–100 μM calcium, which destabilizes microtubules. Förster resonance energy transfer analysis showed that αB-crystallin and tubulin were at 1–10 nm apart from each other in the presence of colchicine. These results suggested that αB-crystallin may play an essential role in microtubule dynamics by maintaining free tubulin in striated muscles, such as the soleus or cardiac muscles. αB-crystallin, dynamics (cytoskeleton), FRAP, microtubules, small heat shock protein αB-crystallin is a member of the family of small heat shock proteins (sHSPs) that function as a molecular chaperone and maintain protein homeostasis by preventing substrate aggregation (1). We have previously shown that αB-crystallin is expressed at high levels in the striated muscle, such as the beating heart and the slow soleus anti-gravitational muscle that is stretched periodically. These tissues respond to mechanical and physical stress and also maintain high oxidative metabolism (2). Slow anti-gravitational striated muscles are well developed in humans and have evolved according to the demands of endurance activities performed with a standing posture, such as walking and running, necessitating the support of body weight under gravity (3). There are two types of skeletal muscles. αB-crystallin is expressed at higher levels in slow-twitch soleus muscles compared to fast-twitch muscles (4–6). The expression pattern of αB-crystallin is muscle fibre type dependent and decreases based on the myosin isotype in the order of Type I > Type IId > Type IIA > Type IIB (7). We screened for marker proteins to elucidate the mechanisms of slow muscle atrophy and found that αB-crystallin expression specifically and rapidly decreased in a hindlimb suspension model (4, 5, 8). Interestingly, αB-crystallin levels did not decrease in the stretched muscles, even with suspension (3, 4), suggesting that αB-crystallin expression responded to mechanical stress (3). Although the cytoskeletal structure is required to transduce mechanical stress, only desmin intermediate filaments and other muscle-specific cytoskeleton proteins, such as titin and nebulin (9), are reported. In fact, the causal gene of ‘desmin’-related myopathy has been identified as the R120G mutant of αB-crystallin (10). However, microtubules localize along intermediate filaments in many cell types, particularly those of mesenchymal cells. Therefore, the cytoskeletal protein tubulin, which comprises microtubules, hypothesized to contribute to the responsiveness to mechanical stress, particularly endurance type stress, constantly occurring in slow skeletal muscle. αB-crystallin modulates the assembly of the intermediate filament vimentin (11), stabilizes actin filaments in a phosphorylation-dependent manner (12), directly associates with the microtubule-associated protein (MAP) in microtubules (13) and affects microtubule assembly in lens epithelial cells (14). We have previously reported strong and interesting correlations between tubulin and αB-crystallin expression among various striated muscles with respect to physiological and metabolic characteristics (2). Although we did not determine the colocalization of these two proteins in striated muscles, we suggested that there is functional interaction between the two and that this interaction was important for both myoblast and myotube differentiation (8, 13). We hypothesized that the microtubule network is essential for transducing mechanical stress induced by ordinary contraction that occurs in mature striated muscle in living and moving multicellular organisms, even after differentiation. These prominent properties of microtubules may be related to the microtubule’s intrinsic dynamic instability, in which αB-crystallin may play a key role. Because, it works as a chaperone for the free form of ‘tubulin dimer’ of microtubules (15, 16). Previous studies using immunostaining and immunoelectron microscopy have revealed that αB-crystallin and HSP27 partly localize to Z-bands and I-bands in skeletal muscles or cardiomyocytes (5, 17). However, in these earlier studies, the proteins examined were in a fixed state, which does not allow to study the molecular dynamics that occur within cells and precludes understanding of dynamic states in continuously contracting slow muscles and cardiomyocytes. The constant autonomous beating of cardiomyocytes makes this cell type a good model to observe in vivo responses to a variety of mechanical stimuli under different conditions. In this study, we transfected rat neonatal cardiomyocytes with GFP-αB-crystallin and used fluorescent recovery after photobleaching (FRAP) to investigate how αB-crystallin interacts with the cytoskeleton and microtubules in living cardiomyocytes. We showed that αB-crystallin localized at Z- and I-bands in living cardiomyocytes during physiological muscle contraction (without added stress). Our results suggested that αB-crystallin dynamically interacted with Z- and I-band proteins, e.g. tubulin/microtubules, in the normal state. Additionally, constitutive expression of αB-crystallin together with its chaperone activity and interaction with tubulin may be important for the rapid responses to mild mechanical stress conditions, such as physiological heart beating. Materials and Methods Mice This study was approved by the Ethical Committee for Animal Experiments at Tokyo University of Agriculture and Technology. Preparation of mouse myocardial (ventricular) and soleus fibres B6 female mice were anaesthetized with isoflurane, and sacrificed. Heart and soleus muscles were removed, washed with microtubule stabilizing buffer (100 mM PIPES, 1 mM MgCl2, 1 mM EGTA, 2 M glycerol), warmed at 37°C and then fixed with microtubule stabilizing buffer containing 10% formaldehyde (Sigma-Aldrich, Co., St. Louis, MO, USA) for 3 h. Using tweezers, the heart was dissociated along the running of myocardial fibres, and the soleus muscle was split under a microscope into single or few muscle fibres. During this process, the tissue was maintained in phosphate-buffered saline (PBS). Culture of neonatal rat cardiomyocytes Primary cultures of neonatal rat cardiomyocytes were prepared according to Matoba et al. (18). Hearts were removed from 1- to 2-day-old Wistar rats after decapitation and placed in PBS. The hearts were washed with PBS, and the aorta and atria were removed. The ventricles were minced into 3-mm3 fragments that were then enzymatically digested four times for 8 min each with 7.5 ml PBS containing 0.2% collagenase (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The cells were collected by centrifugation at 300 g and incubated in 100-mm culture dishes for 1 h at 37°C in a CO2 incubator. Non-adherent cardiomyocytes were harvested and seeded into 35-mm glass base dishes (AGC Techno Glass Co., Ltd., Shizuoka, Japan) (5 × 105 cells/dish). The cardiomyocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and a penicillin–streptomycin–neomycin antibiotic mixture. The culture medium was then exchanged with MEM supplemented with 10% calf serum and 1 mM BrdU to inhibit proliferation of non-myocytes. The cells were then transfected with vectors expressing green fluorescent protein (GFP)-αB-crystallin, enhanced green fluorescent protein (EGFP)-C1 or EGFP-α-tubulin using Lipofectamine 2000 according to the manufacturer’s protocol. GFP-αB-crystallin, N-terminal fusion of human αB-crystallin to EGFP, was prepared as reported previously (19). pEGFP-C1, pEGFP-α-tubulin, pEYFP (yellow fluorescent protein)-α-tubulin vectors were purchased from Clontech (Mountain View, CA, USA). Two or three days after transfection, the cells were treated with heat shock and/or then observed. pCFP (cyan fluorescent protein)-αB-crystallin was prepared by subcloning the α-tubulin cDNA into CFP vector. Immunostaining The myocardial and soleus fibres were blocked with PBS containing 1% BSA, 0.04% saponin, 0.05% NaN3 at room temperature for 2 h, and then incubated overnight using a primary antibody (DM1A) diluted 200-fold. Washing was performed three times for 20 min using PBS (containing 0.04% saponin, 0.05% NaN3) and then allowed to stand at room temperature for 2 h with an anti-mouse secondary antibody. Thereafter, it was washed again and incubated overnight with a primary antibody (αB-crystallin). After washing, it was allowed to stand at room temperature for 2 h with an anti-rabbit secondary antibody. Hoechst 33342 (1:10,000) was used once for nuclear staining at the time of washing. Myocytes were briefly washed with 37°C PBS and fixed with either FME (4% formaldehyde (neutralized), 2 mM MgCl2, 2 mM EGTA) or FME containing 0.03% Triton X-100. The fixed cells were washed several times with PBS and kept in PBS containing 1% BSA and 0.02% sodium azide. Cells were immunohistochemically stained with primary antibodies, followed by staining the secondary antibodies. Antibodies The following antibodies were used in this study: rabbit anti-αB-crystallin antibodies (isolated in our laboratory; 1:5,000 dilution for western blotting, 1:100 dilution for immunostaining), as described previously (15); mouse monoclonal anti-α-tubulin antibodies (clone DM1A; Sigma T6199; 1:50 dilution for immunostaining, 1:3,000 dilution for western blotting); mouse monoclonal anti-α-sarcomeric actin antibodies (A2172; 1:30 dilution; Sigma-Aldrich); and mouse monoclonal anti-desmin antibodies (D1033; 1:30 dilution; Sigma-Aldrich); mouse monoclonal anti-GFP antibodies (632375; 1:3,000 dilution for western blotting, 1:50 dilution for immunostaining; Clontech); rhodamine-conjugated anti-rabbit IgG (AP192R; 1:50 dilution; Chemicon); FITC-conjugated goat anti-mouse IgG (55514; 1:50 dilution; Cappel); anti-mouse Alexa 546 (A11030; 1:50 dilution; Life Technologies); anti-rabbit Alexa 488 (A11034; 1:50 dilution; Life Technologies); and horseradish peroxidase (HRP)-conjugated anti-mouse antibodies (NA931; 1:3,000 dilution; GE); Goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 647 (A32728; 1:200; Invitrogen); goat anti-rabbit IgG (H + L) secondary antibody, Alexa Fluor 555 (A-21429; 1:300; Invitrogen); and Hoechst 33342 (H3570; 1:10,000; Invitrogen). Microscopy Live-cell and fixed-cell sample imaging were performed with Carl Zeiss LSM510 confocal microscope or a Nikon A1RMP confocal microscope. For live cell experiments, the microscope was equipped with a temperature-controlled chamber and CO2 supply. Live cell experiments were performed at 37 or 44.5°C and 5% CO2. Fluorescence recovery after photobleaching Primary cultured rat neonatal cardiomyocytes transfected with pEGFP-αB-crystallin, pEGFP-α-tubulin or pEGFP-C1 were incubated in culture medium at 37°C with either the microtubule-stabilizing drug taxol (10 μM, 15–60 min) or the destabilizing drug nocodazole (10 μM, 15–60 min). The frame including the region of interest (ROI) was imaged before bleaching. The striated region of the beating cardiomyocytes was targeted for photobleaching and monitoring of fluorescence recovery. The ROI was 50 pixel × 50 pixel. The left corner area (10 pixel × 10 pixel) of the ROI was photobleached with 10 scans (total bleach time 50 ms) of the ROI with 2.2 mW laser power (488 nm). Imaging of the area was resumed immediately after photobleaching and continued every 40 ms for 10 s. Data correction The ROIs of the bleached region RB(t) and total cell RT(t) were measured in each trial, where t=0 is the time at breaching. Based on the previous work (20), the background and photofading corrections were operated by Ct=RBt-BRTt-B,(1) where B is ROI of the background. To show the recovery rate after bleaching, normalized fluorescence intensity Nt was calculated by Nt=Ct-C(0)CPre-C(0)×100,(2) where CPre is an average value of the four points in Ct before bleaching. By the normalization, Nt is about 100% before bleaching and Nt is 0% at bleaching. Curve fitting To evaluate the property of the recovery, we conducted curve fitting for the average of Nt over trials in each condition. For the curve fitting, we used Ft=A1-e-τt t≥0 ,(3) where A represents the rate of the mobile fractions after the convergence and τ is the constant parameter. The ROI of the bleached region is entirely recovered if A = 100%, otherwise some fractions (100-A %) remained in the bleached region. T1/2 represents half-life. The curve fitting was performed based on the custom-made MATLAB program. Some data were filtered before the curve fitting as shown in Supplementary Data. Immunoprecipitation Neonatal rat cardiomyocytes were lysed with lysis buffer (80 mM PIPES [pH 6.8], 1 mM MgCl2, 1 mM EGTA, 1% NP-40, protease inhibitor cocktail [Calbiochem]) and left on ice for 10 min. The lysate was centrifuged at 4°C at 15,000 × g for 15 min. Immunoprecipitation was performed by PierceTM Co-Immunoprecipitation Kit according to manufacturer’s instruction. Briefly, the αB-crystallin antibody was first immobilized for 2 h using AminoLink Plus coupling resin. The resin was then washed and incubated with lysate for 1 h at 37°C. A negative control to assess non-specific binding received the same treatment as the co-immunoprecipitation samples, including the normal rabbit IgG. After incubation, the resin was again washed, and protein was eluted using elution buffer. The co-precipitated proteins were boiled in SDS-PAGE sample buffer for 5 min, and SDS-PAGE and immunoblotting were carried out using monoclonal anti-α-tubulin antibodies (T6199; Sigma) as the primary antibody and HRP-conjugated anti-mouse antibodies as the secondary antibody (NA931; GE). Immunoblotting was detected with ECL reagents (RPN2232; GE). Co-precipitation of microtubule fraction from porcine heart The isolation of microtubules from porcine heart lysate was carried out as previously described (21). Briefly, porcine heart (Shibaura Zoki, Tokyo) were homogenized in 1.5 volume of PME buffer at 4°C, followed by centrifugation at 30,000 × g for 15 min and 180,000 × g for 90 min at 4°C to remove the highly expressed muscle proteins (myosin and actin). Ca2+ concentrations (1–100 μM) in the solution are adjusted by calcium activating buffer (22, 23) containing calcium methanesulphonate and potassium methanesulphonate. Taxol (20 μM) and GTP (1 mM) were also added and incubated for 15 min at 37°C for promoting microtubule assembly. The solution was overlaid onto 5% sucrose in PME containing 20 μM taxol and centrifuged at 30,000 × g for 25 min at 37°C to pellet microtubules. Fractions were analysed by immunoblotting. Förster resonance energy transfer Primary cultured rat neonatal cardiomyocytes transfected with CFP-αB-crystallin and YFP-tubulin were incubated at 37°C in culture medium with 0.1 ng/ml colchicine for 2 h. The dish was transferred into a stage chamber at 37°C. Galvano laser with a wavelength of 457.0 nm was applied, and observation was performed with a 40× water immersion lens (resolution: 6.0, channels: 32, binning: 1, wavelength: 465.3–657.3). The spectrum profile was used to examine the intensity of light with a wavelength of 535 nm. Data were collected using Nikon A1RMP confocal microscope. FRET ratio was calculated by intensity of YFP divided by intensity of CFP in 1 μm x 1 μm square area with or without vacuole. The intensity of background was subtracted. n = 10. Statistical analysis Data are expressed as means ± standard deviation for a given number of observations. Comparisons between two normally distributed groups were made using an unpaired Student’s t-test. Results Colocalization of striated αB-crystallin and tubulin in the surface layer of mouse formalin-fixed soleus and myocardium Previously, we have found that αB-crystallin is localized in Z-bands of isolated myofibrils of rat soleus muscle by both immunofluorescence and immunoelectron microscopy (5). In this study, we performed double immunostaining and found that there is partial overlap in the localization of αB-crystallin and tubulin in the fixed mouse soleus muscle and myocardium fibres. In both muscles, αB-crystallin was localized in the I-band and Z-band, and tubulin was confirmed to partially colocalize with αB-crystallin near the sarcolemma of both striated muscles (Fig. 1). Fig. 1 Open in new tabDownload slide Immunohistochemistry of αB-crystallin and tubulin in soleus skeletal muscle (A) and myocardium (ventricle) (B). Muscle fibres treated from both muscles of normal bred mice were fixed with formalin, and immunostained with anti-αB-crystallin antibody (left) and anti-α-tubulin antibody (middle). DNA is shown by arrowhead in merged panel. αB-crystallin in both soleus and ventricular muscles was localized in the I-band and Z-band, and tubulin was colocalized with αB-crystallin near the sarcolemma (arrow). Bars, (A): 20 μm; (B): 10 μm. Inset: High magnification image (Bar is (A) and (B) 10 μm). Fig. 1 Open in new tabDownload slide Immunohistochemistry of αB-crystallin and tubulin in soleus skeletal muscle (A) and myocardium (ventricle) (B). Muscle fibres treated from both muscles of normal bred mice were fixed with formalin, and immunostained with anti-αB-crystallin antibody (left) and anti-α-tubulin antibody (middle). DNA is shown by arrowhead in merged panel. αB-crystallin in both soleus and ventricular muscles was localized in the I-band and Z-band, and tubulin was colocalized with αB-crystallin near the sarcolemma (arrow). Bars, (A): 20 μm; (B): 10 μm. Inset: High magnification image (Bar is (A) and (B) 10 μm). Localization of GFP-αB-crystallin in living rat neonatal cardiomyocytes To investigate the dynamic localization of αB-crystallin in living cardiomyocytes, GFP-αB-crystallin vector was transfected into primary rat neonatal cardiomyocytes. Expression of the GFP-αB-crystallin fusion protein in the transfected cells was confirmed by SDS-PAGE and western blotting (Fig. 2A and B). To confirm whether the transfected GFP-αB-crystallin has the same localization as endogenous αB-crystallin, the transfected cells were fixed and immunostained with anti-αB-crystallin antibody (Fig. 2C). In fixed cells, GFP-αB-crystallin colocalized with endogenous αB-crystallin wherein cytoplasmic and membranous staining that had a slightly striated pattern was observed. GFP-αB-crystallin was not localized in the nucleus, although the GFP tag alone did. Fig. 2 Open in new tabDownload slide GFP-αB-crystallin expression in cardiomyocytes. (A) Proteins in cardiomyocyte homogenates (10 μg) were separated by SDS-PAGE. Lane 1: untransfected lysate; Lane 2: GFP-C1-transfected cardiomyocyte lysate; and Lane 3: GFP-αB-crystallin-transfected cardiomyocyte lysate. (B) Immunoblot analysis of cardiomyocyte lysates. Lanes 1 and 2 have 4 and 20 ng of recombinant αB-crystallin, respectively. Lane 3: untransfected lysate; Lanes 4, 6: GFP-C1-transfected cardiomyocyte lysate; Lanes 5, 7: GFP-αB-crystallin-transfected cardiomyocyte lysate; Lanes 1–5: anti-αB-crystallin antibody; Lanes 6, 7: anti-GFP-antibody. (C) GFP-αB-crystallin colocalizes with endogenous αB-crystallin in fixed cardiomyocytes. Cardiomyocytes transfected with pEGFP-αB-crystallin or pEGFP were fixed and immunostained with anti-αB-crystallin antibody. GFP-αB-crystallin colocalized with endogenous αB-crystallin while GFP localized in the nucleus. Bar = 10 μm. Fig. 2 Open in new tabDownload slide GFP-αB-crystallin expression in cardiomyocytes. (A) Proteins in cardiomyocyte homogenates (10 μg) were separated by SDS-PAGE. Lane 1: untransfected lysate; Lane 2: GFP-C1-transfected cardiomyocyte lysate; and Lane 3: GFP-αB-crystallin-transfected cardiomyocyte lysate. (B) Immunoblot analysis of cardiomyocyte lysates. Lanes 1 and 2 have 4 and 20 ng of recombinant αB-crystallin, respectively. Lane 3: untransfected lysate; Lanes 4, 6: GFP-C1-transfected cardiomyocyte lysate; Lanes 5, 7: GFP-αB-crystallin-transfected cardiomyocyte lysate; Lanes 1–5: anti-αB-crystallin antibody; Lanes 6, 7: anti-GFP-antibody. (C) GFP-αB-crystallin colocalizes with endogenous αB-crystallin in fixed cardiomyocytes. Cardiomyocytes transfected with pEGFP-αB-crystallin or pEGFP were fixed and immunostained with anti-αB-crystallin antibody. GFP-αB-crystallin colocalized with endogenous αB-crystallin while GFP localized in the nucleus. Bar = 10 μm. Expression of GFP-αB-crystallin was also examined in living and beating cardiomyocytes (Fig. 3A). In the majority of living cells, GFP-αB-crystallin had a diffuse cytoplasmic distribution and a striated pattern. In contrast, control GFP that localized in the nucleus and cytoplasm did not show a striated pattern. To further characterize GFP-αB-crystallin localization, cardiomyocytes transfected with GFP-αB-crystallin were exposed to severe heat shock treatment (Fig. 3C). Heat shock was used as a severe stress condition to compare with the physiological condition. Based on the method described by van de Klundert et al. (17), living cardiomyocytes transfected with GFP-αB-crystallin were incubated at 44.5°C for 1 h and observed by microscopy. In living cells exposed to heat stress, αB-crystallin localization showed a more clear striated pattern relative to that of non-stressed cells. Together, these results show that GFP-αB-crystallin behaves similarly to endogenous αB-crystallin during heat shock. Fig. 3 Open in new tabDownload slide GFP-αB-crystallin shows a striated fluorescence pattern in living cardiomyocytes. GFP-αB-crystallin was transfected into cardiomyocytes and the living cells were observed with a microscope. (A, B) No-stress; (C, D) Heat stress at 44.5°C for 1 h. A striated pattern of GFP-αB-crystallin was observed while GFP had a diffuse fluorescence pattern with nuclear localization. Bar =10 μm. Fig. 3 Open in new tabDownload slide GFP-αB-crystallin shows a striated fluorescence pattern in living cardiomyocytes. GFP-αB-crystallin was transfected into cardiomyocytes and the living cells were observed with a microscope. (A, B) No-stress; (C, D) Heat stress at 44.5°C for 1 h. A striated pattern of GFP-αB-crystallin was observed while GFP had a diffuse fluorescence pattern with nuclear localization. Bar =10 μm. To investigate the relation of αB-crystallin with the cytoskeleton, cardiomyocytes were fixed and immunostained with antibodies against α-tubulin, desmin, or α-sarcomeric actin (Fig. 4) after heat shock at 44.5°C. αB-crystallin showed a staining pattern that was similar to desmin and colocalized with tubulin but not actin. The striated GFP-αB-crystallin staining pattern mimicked the Z- and I-band structures but not the A-band. Fig. 4 Open in new tabDownload slide αB-crystallin immunohistochemistry in cardiomyocytes after heat shock. Heat-shocked cardiomyocytes were fixed with formalin and immunostained with anti-αB-crystallin antibody together with anti-α-tubulin (A), anti-desmin (B) or anti α-actin (C) antibodies. αB-crystallin localized in the I-band. Inset: magnified images. Bar =10 μm. Fig. 4 Open in new tabDownload slide αB-crystallin immunohistochemistry in cardiomyocytes after heat shock. Heat-shocked cardiomyocytes were fixed with formalin and immunostained with anti-αB-crystallin antibody together with anti-α-tubulin (A), anti-desmin (B) or anti α-actin (C) antibodies. αB-crystallin localized in the I-band. Inset: magnified images. Bar =10 μm. FRAP analysis showed that αB-crystallin was dynamic under physiological conditions in rat neonatal cardiomyocytes Our observations suggested that αB-crystallin localized in the I- and Z-bands during contraction of living cardiomyocytes, both physiologically (37°C) and during heat shock (44.5°C). To investigate the dynamic properties of αB-crystallin, we performed FRAP assays under physiological conditions (Fig. 5), which provide information about the intrinsic mobility of αB-crystallin molecules within cardiomyocytes as a function of time. The region showing GFP-αB-crystallin-striated fluorescence patterns was photobleached (area within the white box, Fig. 5). Images were taken before and immediately after photobleaching. Scanning and imaging times were 124 and 39 ms, respectively. As living cardiomyocytes are beating, the fluorescence intensity of the ROI fluctuated during beating. The striated fluorescence pattern of GFP-αB-crystallin in the photobleached area rapidly recovered within 1 s after bleaching (T1/2 = 0.0971); however, in the presence of heat stress, it did not recover. This result showed that GFP-αB-crystallin in the striated region was highly dynamic in the physiological state but not under heat shock conditions. Fig. 5 Open in new tabDownload slide FRAP analysis of GFP-αB mobility. (A) Cardiomyocytes transiently expressing GFP alone or GFP-αB-crystallin were imaged with a Zeiss LSM510 microscope. A single whole cell is shown in each leftmost panel, and enlarged time-lapse views of the area indicated by the white square with pseudo-colours are shown to the right. GFP fluorescence was photobleached in the red-boxed area in the ‘prebleach’ image. Images were obtained before and immediately after photobleaching. Scanning times were 124 and 39 ms for photobleaching and imaging, respectively. Bar =10 μm. (B) The fluorescence intensity in each bleached area shown in (A) was plotted as a function of time. Points show normalized FRAP data for all trials in each condition. Oscillation line and error bars show average and standard deviation for normalized FRAP data at each time, respectively. Solid line shows fitting curve for averaged data. A and τ are parameters of fitting curve defined in equation (3). Fig. 5 Open in new tabDownload slide FRAP analysis of GFP-αB mobility. (A) Cardiomyocytes transiently expressing GFP alone or GFP-αB-crystallin were imaged with a Zeiss LSM510 microscope. A single whole cell is shown in each leftmost panel, and enlarged time-lapse views of the area indicated by the white square with pseudo-colours are shown to the right. GFP fluorescence was photobleached in the red-boxed area in the ‘prebleach’ image. Images were obtained before and immediately after photobleaching. Scanning times were 124 and 39 ms for photobleaching and imaging, respectively. Bar =10 μm. (B) The fluorescence intensity in each bleached area shown in (A) was plotted as a function of time. Points show normalized FRAP data for all trials in each condition. Oscillation line and error bars show average and standard deviation for normalized FRAP data at each time, respectively. Solid line shows fitting curve for averaged data. A and τ are parameters of fitting curve defined in equation (3). Interaction of αB-crystallin with tubulin in rat neonatal cardiomyocyte lysates and tubulin/microtubules purified from porcine heart tissue To examine the binding of αB-crystallin to tubulin in cardiomyocytes, we performed immunoprecipitation (Fig. 6A). The cardiomyocyte lysates were incubated with an antibody to αB-crystallin, and the resulting immunoprecipitates were analysed for the presence of α-tubulin by immunoblotting. The results demonstrated that the antibody to αB-crystallin, but not the control IgG, immunoprecipitated tubulin, confirming the interaction between αB-crystallin and tubulin in cardiomyocytes. The faint but recognizable bands in the blot of αB-crystallin at control lane may arise from the interaction of the control IgG with the immnoglobulin-like alpha-crystallin domain (24) present in all sHSPs including αB-crystallin. The beating of cardiomyocytes is regulated by the oscillation of intracellular calcium concentration ([Ca2+]i), and [Ca2+]i is maintained at several 100 nM at rest but increases to about several micro M at cardiac contraction (25, 26). Calcium induces disassembly of microtubules (27) and the structure of the disassembled free tubulin dimer is unstable (28, 29). We analysed the interaction of αB-crystallin with tubulin in the presence of calcium. Porcine heart tissue extract was adjusted to various calcium concentrations close to physiological concentrations, and then the amount of co-precipitated αB-crystallin with taxol-induced assembled tubulin was examined (Fig. 6B). The amount of tubulin and co-precipitated αB-crystallin increased depending on the calcium concentration. These results showed that calcium concentration oscillation, such as during cardiac contraction, affects the interaction between αB-crystallin and tubulin. This result was consistent with our previous findings that αB-crystallin contributes to microtubules resistance to disassembly by enhanced association with either tubulin dimer directly or microtubules through MAPs in the presence of calcium (13). Fig. 6 Open in new tabDownload slide Interaction of αB-crystallin with tubulin in cardiomyocytes. (A) Co-immunoprecipitation (CO-IP) assay to detect the interaction between αB-crystallin and tubulin in rat neonatal cardiomyocytes. αB-crystallin antibody and control antibody (normal rabbit IgG) were used for CO-IP, and αB-crystallin and tubulin were visualized following western blotting with anti αB-crystallin and α-tubulin antibodies, respectively. (B) Effect of calcium concentration on co-precipitation of αB-crystallin by taxol-dependent tubulin precipitation. MT in porcine heart lysate were precipitated with 20 μM taxol and various Ca2+ concentration (0, 1, 10, 100 μM), then αB-crystallin co-precipitated with MT was analysed by western blotting. αB-crystallin and tubulin are positive controls purified from pig lens and pig brain, respectively. The lysate is from porcine heart. Fig. 6 Open in new tabDownload slide Interaction of αB-crystallin with tubulin in cardiomyocytes. (A) Co-immunoprecipitation (CO-IP) assay to detect the interaction between αB-crystallin and tubulin in rat neonatal cardiomyocytes. αB-crystallin antibody and control antibody (normal rabbit IgG) were used for CO-IP, and αB-crystallin and tubulin were visualized following western blotting with anti αB-crystallin and α-tubulin antibodies, respectively. (B) Effect of calcium concentration on co-precipitation of αB-crystallin by taxol-dependent tubulin precipitation. MT in porcine heart lysate were precipitated with 20 μM taxol and various Ca2+ concentration (0, 1, 10, 100 μM), then αB-crystallin co-precipitated with MT was analysed by western blotting. αB-crystallin and tubulin are positive controls purified from pig lens and pig brain, respectively. The lysate is from porcine heart. Mobility of αB-crystallin after microtubule destabilization and stabilization by FRAP analysis in rat neonatal cardiomyocytes αB-crystallin interacts with tubulin and functions as a molecular chaperone in vitro. To investigate the interaction between αB-crystallin and microtubules in living cardiomyocytes, we used microscopy imaging and FRAP to analyse both microtubule and αB-crystallin dynamics in the presence of taxol (microtubule stabilizing drug) or nocodazole (microtubule destabilizing drug) (Fig. 7). Fig. 7 Open in new tabDownload slide Effect of tubulin stabilization/destabilization drugs on GFP-tubulin and GFP-αB-crystallin mobility. (A) Effects of tubulin stabilization/destabilization drugs on GFP-tubulin mobility. Upper images show fluorescent microscopic images of GFP-tubulin-transfected cardiomyocytes. Arrow: microtubules, arrowhead: distribution of free tubulin. (B, C) The average fluorescence intensity of GFP-tubulin (B) and GFP-αB-crystallin (C) in each bleached area was plotted as a function of time (FRAP analysis). Left column: untreated cells (control), centre: cells treated with nocodazole, right: cells treated with taxol (n = 5). Fig. 7 Open in new tabDownload slide Effect of tubulin stabilization/destabilization drugs on GFP-tubulin and GFP-αB-crystallin mobility. (A) Effects of tubulin stabilization/destabilization drugs on GFP-tubulin mobility. Upper images show fluorescent microscopic images of GFP-tubulin-transfected cardiomyocytes. Arrow: microtubules, arrowhead: distribution of free tubulin. (B, C) The average fluorescence intensity of GFP-tubulin (B) and GFP-αB-crystallin (C) in each bleached area was plotted as a function of time (FRAP analysis). Left column: untreated cells (control), centre: cells treated with nocodazole, right: cells treated with taxol (n = 5). In untreated control cardiomyocytes, microscopy imaging showed that many microtubules (arrow) extended from the microtubule organizing centre to the peripheral regions, and free tubulin dimers (arrowhead) were also observed (Fig. 7A, left). Following treatment of cardiomyocytes with 10 μM nocodazole, most cytosolic microtubules disappeared, and only free tubulin remained (arrowhead) (Fig. 7A, middle). In contrast, taxol (10 μM) treatment resulted in the reduction of free cytosolic tubulin in favour of microtubules (arrow) (Fig. 7A, right). We next performed FRAP analysis of GFP-α-tubulin and GFP-αB-crystallin under each condition to analyse microtubule dynamics (Fig. 7B and C). In control cardiomyocytes, mobile fraction of GFP-α-tubulin and GFP-αB-crystallin represented about 0.517 and 0.753 of cells in the ROI, respectively. Two FRAP patterns were observed for GFP-αB-crystallin in the normal state. One population showed only about 40% recovery, and the other was almost 100% recovery. It may depend on the ROI and/or on the presence of fast-mobility unfolded tubulin-bound αB-crystallin in local calcium concentration at the time of observation. In the presence of nocodazole, mobile fraction (A), constant parameter (⁠ τ ⁠) and half-life (T1/2) of GFP-α-tubulin and GFP-αB-crystallin were A = 0.802, τ = 10.621, T1/2 = 0.065 and A = 0.869, τ = 10.397, T1/2 = 0.0664, respectively (Fig. 7B). In addition, in nocodazole treatment, although the oscillation pattern was observed in the FRAP for GFP-αB-crystallin by cardiac contraction, no difference in the FRAP pattern was observed among ROIs. The amount of free mobile αB-crystallin also increased due to the increase in the free tubulin population by addition of nocodazole. These results suggested that αB-crystallin recognized nocodazole-bound tubulin homogenously (versus two population present in GFP-αB-crystallin FRAP analysis probably because of temporal calcium oscillation). Although the change in the free mobile αB-crystallin evaluated by FRAP analysis upon nocodazole treatment seemed to be subtle, different dynamic properties relative to control were observed, as shown in Fig. 7C. Moreover, in the presence of taxol, the mobile fraction of GFP-α-tubulin was 0.313, yet mobile fraction of GFP-αB-crystallin was 0.918. The GFP-αB-crystallin dynamics were consistent with those of GFP-α-tubulin in nocodazole-treated but not in taxol-treated living cardiomyocytes. These results implied that αB-crystallin interacted with free dimer tubulin. Because the striated pattern of GFP-α-tubulin fluorescence was not observed in cardiomyocyte Z-bands in this case, photobleaching was performed in areas where microtubules were clearly formed. Fluorescence of cells transfected with a vector expressing the GFP tag alone was not affected by either nocodazole or taxol, suggesting that almost all of the GFP tag molecules were free (data not shown). Colocalization of αB-crystallin and tubulin in the presence of colchicine in Förster resonance energy transfer analysis in rat neonatal cardiomyocytes To get an information whether αB-crystallin interacts with tubulin in living cells, we performed an assay to detect tubulin-αB-crystallin proximity by Förster resonance energy transfer in cardiomyocyte (30). Here, the CFP coupled to the N-terminus of αB-crystallin served as the FRET donor, while the YFP-tubulin served as the FRET acceptor. In this assay, the yellow fluorescence intensity will increase when acceptor YFP-tubulin present in the vicinity (1–10 nm) of CFP-αB-crystallin by energy transfer (Fig. 8). In the presence of microtubule destabilizer colchicine, FRET was observed in an autophagosome-like structure (Fig. 8D) formed by entrapment of endoplasmic reticulum (ER) and tubulin as detected in colchicine myopathy (31). Intensity of YFP divided by intensity of CFP in area with and without vacuole is 0.7397 ± 0.08163 and 0.5648 ± 0.09289, respectively. The ratio in the area with vacuole is significantly higher compared with the area without vacuole (p < 0.0005, n=10) This may reflect that the interaction between tubulin and αB-crystallin is very quick and temporal. Fig. 8 Open in new tabDownload slide Colocalization of αB-crystallin and tubulin in the vacuole of cardiomyocytes. (A) Snapshots at wavelength range from 465.3 to 656.3 nm (Left). Snapshots at wavelength range from 477.3 to 482.1 nm ( CFP, right upper) and from 531.4 to 536.2 nm ( YFP, right lower). The image is a cell in medium with 0.1 ng/ml colchicine for 2 h. The dots are vacuoles. Bar =10 μm. (B) Model of FRET (32). A is αB-crystallin. B is tubulin. When those are closer than 10 nm, FRET occurs. Fig. 8 Open in new tabDownload slide Colocalization of αB-crystallin and tubulin in the vacuole of cardiomyocytes. (A) Snapshots at wavelength range from 465.3 to 656.3 nm (Left). Snapshots at wavelength range from 477.3 to 482.1 nm ( CFP, right upper) and from 531.4 to 536.2 nm ( YFP, right lower). The image is a cell in medium with 0.1 ng/ml colchicine for 2 h. The dots are vacuoles. Bar =10 μm. (B) Model of FRET (32). A is αB-crystallin. B is tubulin. When those are closer than 10 nm, FRET occurs. Discussion Numerous studies have suggested that sHSPs are expressed in various conditions and contribute to the maintenance of organisms (33). However, due to the structure of the sHSPs, they exist as extremely unstable oligomers, and it has been proposed that the unstable form itself is important for maintaining the denatured protein in a state that facilitates refolding (34). The details of chaperone function are still unknown. Furthermore, sHSPs are more conducive in extending healthy life expectancy (35), but the reason is still unknown. In this study, we hypothesized that ‘pulsation’ itself is a mechanical stress that induces αB-crystallin in beating cardiomyocytes. In the body, the heart keeps beating, and an active human does not only walk in a standing position but also performs activities in a standing position every day. The movement of these tissues and of the body causes mechanical mild stress on various cells, such as skeletal muscle cells, cardiomyocytes, chondrocytes, osteocytes and fibroblasts. αB-crystallin is constitutively expressed in tissues where such mild mechanical stress is loaded. That is, assembly and disassembly between microtubules and tubulin must occur continuously in these cells. Mild mechanical stress may have a ‘hormesis effect’, which is named to explain ‘good effects brought by mild stress’, relating to oxidative stress, physical exercise (36) and mitochondrial hormesis (37). This phenomena may be intrinsically a cause induced by ‘dynamic instability’ of tubulin/microtubule system, which is one of house-keeping cell system structure relating to reactive oxygen species and microtubule close to sarcolemma in heart (38). Although dynamic property of heart and skeletal muscle tissue has hardly been characterized, the width of sarcomere, observed by an endoscope with a diameter of 350 μm, fluctuates during muscle contraction in humans and mice (39). Muscle contraction occurs constantly during daily activities, especially in antigravity muscles that are the basis of all activities. The colocalization of αB-crystallin and tubulin was partially observed in the cell membrane of formalin-fixed soleus muscle and myocardial fibres (Fig. 1), which may reflect sarcomere fluctuation associated with muscle contraction in vivo. Although studies on microtubules in muscle are very limited, dystrophin is one of the MAPs (40), and microtubules increase in myocardial pathology (41). The results of this study show that the essence of the dynamic instability of the microtubule system is to maintain constitutive expression of αB-crystallin, a molecular chaperone responsive to mechanical stress, in tissues that undergo constitutive stress even after differentiation. We have shown that αB-crystallin maintains cell shape and adhesion in myoblast cells both in non-stressed and stretch-stress conditions, confirming that αB-crystallin is a resilience chaperone (42, 43). αB-crystallin interacts with tubulin/microtubules in myoblasts and skeletal muscle (2, 8, 13, 15, 16, 44, 44). Consistent with this, we also confirmed the interaction of αB-crystallin with tubulin using immunoprecipitation, co-precipitation with tubulin induced by taxol treatment and FRAP analysis in cardiomyocytes. In skeletal muscles and cardiomyocytes, muscle contraction involves calcium release from the sarcoplasmic reticulum that results in local microtubules disassembly (38). Our results indicated that calcium increases the interaction of taxol-induced tubulin/microtubule precipitation and αB-crystallin. αB-crystallin may protect free tubulin dimers that are increased by calcium treatment. Actually, microtubule disassembly induced by the addition of Ca2+ recovers more quickly in the presence of αB-crystallin in vitro (13). In this experiment, when taxol is added to cells, free tubulin dimers disappear from the cytosol because most tubulin dimers assemble to form microtubules. In the paper showing the results of experiments using C6 glioma cells by Kato et al. (44), the levels of both mRNA and protein of αB-crystallin markedly decreased when cultured C6 glioma cells were treated with microtubule-stabilizing drug taxol. By adding taxol, the interaction between free tubulin and αB-crystallin in cytoplasm may reduce by decreasing the amount of αB-crystallin and free tubulin dimer. On the other hand, in this in vitro experiment (fig. 6B), both free tubulin and αB-crystallin are present in the supernatant after homogenization of porcine myocardial tissue. αB-crystallin may recognize and bind to calcium-dependent conformational changed tubulin dimer, although the mechanism is unknown. After that, αB-crystallin and tubulin seem to co-precipitate by the addition of taxol. Although αB-crystallin may regulate microtubule assembly/disassembly by binding to MAPs, maintenance of free tubulin dimers is also considered to contribute to the maintenance of microtubules. Moreover, during constant contractile stress that occurs during heartbeats, Z-band proteins are expected to undergo structure alterations (46, 47). Recently, lattice defects in microtubule have also been observed by the activity of microtubule-severing enzymes and physical interactions in the crowded cellular environment (48). In the myocardium, because microtubules receive a mild mechanical load with every beat, αB-crystallin may interact with free tubulin dimers near microtubules to maintain microtubules, which are skeletal structures supporting tension. Thus, an important role of αB-crystallin may be the protection of unstable microtubules in continuously beating cardiomyocytes by maintaining physiological levels of native tubulin. When cardiomyocytes were treated with microtubule stabilizing or destabilizing agents (taxol or nocodazole, respectively), the dynamics of tubulin and αB-crystallin changed. An explanation for this result is as follows. Nocodazole binding to tubulin may induce a conformational change in the tubulin dimer similar to colchicine, which competes with nocodazole for binding to tubulin sites that are located at the α-/β-tubulin interface (49). Furthermore, nocodazole binds at a location that prevents curved tubulin from adopting a straight structure (50). We have reported that αB-crystallin interacts with free tubulin dimers and protects tubulin from stress-induced denaturation (15, 16), and temperature-induced structural transitions of tubulin can also occur (51). In fact, tubulin does not bind to αB-crystallin at 4°C, but it does bind at 37°C (15). Additionally, nocodazole induces the synthesis and accumulation of αB-crystallin (43). Based on this chaperone hypothesis, the increase in αB-crystallin activity that occurs following nocodazole treatment of cardiomyocytes may be due to αB-crystallin recognition of nocodazole-bound free tubulin dimers, as was observed in myocytes cultured at 37°C, and these dimers may have different conformations from those found at 4°C. Because the main function of αB-crystallin is to preserve folding intermediates (52), αB-crystallin may recognize conformational changes in tubulin induced by nocodazole binding, and the dynamic properties of αB-crystallin shown here by FRAP analysis may be consistent with those of tubulin following nocodazole treatment. Although the change in the free mobile αB-crystallin evaluated by FRAP analysis following nocodazole treatment seemed to be subtle, dynamic properties different from control were observed (Fig. 7C). After the addition of taxol, αB-crystallin may not recognize fully assembled and stabilized microtubules that are also less dynamic. Instead, αB-crystallin may recognize tubulin and MAPs when microtubule turnover occurs (13). This possibility is supported by the finding that αB-crystallin binds to extracted heat-stable MAPs in a concentration-dependent manner (unpublished data). Additionally, taxol treatment decreased the GFP-tubulin dynamics but increased GFP-αB-crystallin dynamics in cardiac myocytes. This result may be due to the absence of αB-crystallin binding to taxol-stabilized microtubules. In the present study, oscillations in the pattern of FRAP were observed in synchronization with myocardial cell beating (Supplementary Fig. S1). Beating frequency was higher upon microtubule destabilization by nocodazole treatment than upon microtubule stabilization by taxol treatment, and this result is consistent with other studies (53, 54). Upon nocodazole treatment, the beating effect in FRAP pattern of GFP-tubulin was not observed. Because GFP-tubulin is diffusely distributed within the cells by nocodazole, the influence of the movement of ROI by beating may be small. As the three types of cytoskeletons interact with each other, destroying one of them results in severe effects. In the future, we would like to investigate the changes in the kinetic pattern of αB-crystallin after microtubule injury. Arany et al. (55) have reported that microtubule destabilizers (e.g. colchicine) and stabilizers (e.g. taxol) induce expression of the transcriptional coactivator PGC1α in C2C12 myotubes. Interestingly, PGC1α is induced by continuous mild heat stress (39°C) during differentiation (fusion) of both human and C2C12 confluent-cultured myoblasts and promotes the expression of Type I slow myosin compared with fast Type II myosin (56). Tissues that highly and constitutively express heat shock proteins are adaptable and show high plasticity, particularly when they remain dynamic and maintain proteostasis. It was found that αB-crystallin and YFP-tubulin were located 1–10 nm apart from each other in an autophagosome-like vesicle structure in the presence of colchicine, a microtubule assembly inhibitor. αB-crystallin and tubulin were often observed to colocalize just below the muscle cell membrane (Fig. 1) suggesting that αB-crystallin functions as a molecular chaperone that connects the microtubule network with the cellular inner membrane system such as the muscle sarcolemma, Golgi apparatus, endoplasmic reticulum and mitochondria. These inner membrane organelles and microtubules develop in the surface of the slow muscle fibers (57, 58), supporting higher oxidative metabolism and proteostasis and must be supported by αB-crystallin. It has been suggested αB-crystallin functions in recognizing the degeneration of drug-bound tubulin and targets it for degradation. αB-crystallin is a molecular chaperone that also contributes to the proteolytic system as part of the Skp, Cullin, F-box containing complex as E3-ligaze of cyclin D1 (59). During severe heat stress, αB-crystallin clearly localized not only Z-bands but also M-bands. Both the Z-band and the M-band form a mechanical link with the extracellular environment via integrins, and also serve as protein degradation sites, as can be seen from the M-band localization of MURF (60), which connects microtubules to the ubiquitin proteasome system. Therefore, there may be more substrates for αB-crystallin besides tubulin/microtubules. Pioneering discoveries such in skeletal muscle research such as MyoD master gene for differentiation, satellite cells as stem cells and the Ca2+ signalling molecule has pushing forward the tide of developmental biology and molecular biology. Research on PGC1α and longevity gene Sirtuins, which induce slow muscle, is at the forefront of aging research but does not simultaneously explain the morphology and function of proteins that guarantee activity in a structure-dependent manner. This is related to the fact that physical activity for health is divided into metabolic syndrome and locomotive syndrome, both of which have not led to the study of molecular chaperones. Proteins are read from genes, but the actual form of the protein conforms to the space in which the ion concentration in the body/cell differs depending on the protein. A review of skeletal muscle and sarcomere structure and proteolysis (61, 62) has also been published in relation to molecular chaperones, however there is no description of tubulin/microtubule. This is the first study to show that cells themselves are composed of proteins that change their structure extremely dynamically and momentarily and that their dynamics are a great basis for plasticity. Conclusion We showed by FRAP analysis that αB-crystallin interacts rapidly with Z-bands in beating cardiomyocytes. FRET analysis also confirmed colocalization of αB-crystallin and tubulin in the presence of colchicine, a microtubule assembly inhibitor. In contrast, during severe heat stress, the dynamic mobility of αB-crystallin dramatically decreased. Thus, αB-crystallin may interact with the cytoskeletal proteins (both assembled and free forms) localized around cardiomyocyte Z- and I-bands where proteins are constantly receiving mechanical stimuli and are subjects of mechanical stress. Cytoskeletal maintenance is absolutely necessary for muscle contraction (46), and the chaperone function of αB-crystallin that involves interactions with all three cytoskeleton protein families is likely essential for maintaining the dynamic structure that is required for proper contractile function in slow skeletal muscle and heart tissue, in which αB-crystallin is highly and constitutively expressed. Supplementary Data Supplementary Data are available at JB Online. Acknowledgements We are grateful to Dr Maki Yamaguchi and Prof. Shigeru Takemori at Jikei University for providing the calcium activating buffer. We would like to thank Dr Tokuko Haraguchi at National Institute of Information and Communications Technology and Dr Takeshi Shimi (current affiliation: Tokyo Tech World Research Hub Initiative) for supports of FRAP experiments. Funding Y.A. received Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (#10480004; #11167218; #1230800; #13022213; #21650171; #23650416; #26560323), research grant from Japan Space Utilization Promotion Center (FY1999-2001) and Research grant from Japan Space Forum (FY1997-2000). This work was partly supported by a Sasagawa Scientific Research Grant from the Japan Science Society to E.O.-F. Author Contributions E.O.-F. performed the experiments and wrote the first draft of the manuscript. S.H. and A.A. performed the experiments and wrote a part of the draft of the manuscript. S.F. performed the FRAP data analysis. M.S. constructed a vector, supervised a part of microscopy analysis and edited the manuscript. Y.A. was responsible for conceptualization and methodology and supervised the work, and edited entire manuscript. Conflict of Interest The authors declare no conflicts of interest associated with this manuscript. References 1 Horwitz J. 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Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Dynamic localization of αB-crystallin at the microtubule cytoskeleton network in beating heart cells
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvaa025
DA - 2020-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/dynamic-localization-of-b-crystallin-at-the-microtubule-cytoskeleton-yVh0U4KaqO
SP - 125
EP - 137
VL - 168
IS - 2
DP - DeepDyve
ER -