3D immuno-confocal image reconstruction of fibroblast cytoskeleton and nucleus architecture

Computational models of cellular structures generally rely on simplifying approximations and assumptions that limit biological accuracy. This study presents a comprehensive image processing pipeline for creating unified three-dimensional (3D) reconstructions of the cell cytoskeletal networks and nuclei. Confocal image stacks of these cellular structures were reconstructed to 3D isosurfaces (Imaris), then tessellations were simplified to reduce the number of elements in initial meshes by applying quadric edge collapse decimation with preserved topology boundaries (MeshLab). Geometries were remeshed to ensure uniformity (Instant Meshes) and the resulting 3D meshes exported (ABAQUS) for downstream application. The protocol has been applied successfully to fibroblast cytoskeletal reorganisation in the scleral connective tissue of the eye, under mechanical load that mimics internal eye pressure. While the method herein is specifically employed to reconstruct immunofluorescent confocal imaging data, it is also more widely applicable to other biological imaging modalities where accurate 3D cell structures are required.     

In vitro cleavage of eIF4GI but not eIF4GII by HIV-1 protease and its effects on translation in the rabbit reticulocyte lysate system

The vertebrate muscle Z-band organizes and tethers antiparallel actin filaments in adjacent sarcomeres and hence propagates the tension generated by the actomyosin interaction during muscular contraction. The axial width of the Z-band varies with fibre and muscle type: fast twitch muscles have narrow (approximately 30-50 nm) Z-bands, while slow-twitch and cardiac muscles have wide (approximately 100-140 nm) Z-bands. In electron micrographs of longitudinal sections of fast fibres like those found in fish body white muscle, the Z-band appears as a characteristic zigzag layer of density connecting the mutually offset actin filament arrays in adjacent sarcomeres. Wide Z-bands in slow fibres such as the one studied here (bovine neck muscle) show a stack of three or four zigzag layers. The variable Z-band width incorporating variable numbers of zigzag layers presumably relates to the different mechanical properties of the respective muscles. Three-dimensional reconstructions of Z-bands reveal that individual zigzag layers are often composed of more than one set of protein bridges, called Z-links, probably alpha-actinin, between oppositely oriented actin filaments. Fast muscle Z-bands comprise two or three layers of Z-links. Here we have applied Fourier reconstruction methods to obtain clear three-dimensional density maps of the Z-bands in beef muscle. The bovine slow muscle investigated here reveals a Z-band comprising six sets of Z-links, which, due to their shape and the way their projected densities overlap, appear in longitudinal sections as either three or four zigzag layers, depending on the lattice view. There has been great interest recently in the suggestion that Z-band variability with fibre type may be due to differences in the repetitive region (tandem Z-repeats) in the Z-band part of titin (also called connectin). We discuss this in the context of our results and present a systematic classification of Z-band types according to the numbers of Z-links and titin Z-repeats.     

Phosphorylation of titin modulates passive stiffness of cardiac muscle in a titin isoform-dependent manner

We investigated the effect of protein kinase A (PKA) on passive force in skinned cardiac tissues that express different isoforms of titin, i.e., stiff (N2B) and more compliant (N2BA) titins, at different levels. We used rat ventricular (RV), bovine left ventricular (BLV), and bovine left atrial (BLA) muscles (passive force: RV > BLV > BLA, with the ratio of N2B to N2BA titin, approximately 90:10, approximately 40:60, and approximately 10:90%, respectively) and found that N2B and N2BA isoforms can both be phosphorylated by PKA. Under the relaxed condition, sarcomere length was increased and then held constant for 30 min and the peak passive force, stress-relaxation, and steady-state passive force were determined. Following PKA treatment, passive force was significantly decreased in all muscle types with the effect greatest in RV, lowest in BLA, and intermediate in BLV. Fitting the stress-relaxation data to the sum of three exponential decay functions revealed that PKA blunts the magnitude of stress-relaxation and accelerates its time constants. To investigate whether or not PKA-induced decreases in passive force result from possible alteration of titin-thin filament interaction (e.g., via troponin I phosphorylation), we conducted the same experiments using RV preparations that had been treated with gelsolin to extract thin filaments. PKA decreased passive force in gelsolin-treated RV preparations with a magnitude similar to that observed in control preparations. PKA was also found to decrease restoring force in skinned ventricular myocytes of the rat that had been shortened to below the slack length. Finally, we investigated the effect of the beta-adrenergic receptor agonist isoprenaline on diastolic force in intact rat ventricular trabeculae. We found that isoprenaline phosphorylated titin and that it reduced diastolic force to a degree similar to that found in skinned RV preparations. Taken together, these results suggest that during beta-adrenergic stimulation, PKA increases ventricular compliance in a titin isoform-dependent manner.     

Zasp/Cypher internal ZM-motif containing fragments are sufficient to co-localize with alpha-actinin--analysis of patient mutations

Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) has an important role in maintaining Z-disc stability in striated and cardiac muscle. ZASP/Cypher interacts through its PDZ domain with the major Z-disc actin cross-linker, alpha-actinin. ZASP/Cypher also has a conserved sequence called the ZM-motif, and it is found in two alternatively spliced exons 4 and 6. We have shown earlier that the ZM-motif containing internal regions of two related proteins ALP and CLP36 interact with alpha-actinin rod region, and that the ZM-motif is important in targeting ALP to the alpha-actinin containing structures in cell. Here, we show that the ZASP/Cypher internal fragments containing either ZM exon 4 or 6 co-localized with alpha-actinin in cultured myoblasts and nonmuscle cells. Fragments of 130 residues around the ZM-consensus were sufficient for localization, which is similar to our previous results of ALP. Moreover, ZASP/Cypher protein interacted directly with the alpha-actinin rod and competed with ALP in binding to the rod. During the inhibition of stress fiber assembly ZASP/Cypher and alpha-actinin co-localization could be partially disturbed, suggesting that ZASP/Cypher is bound to alpha-actinin mainly when alpha-actinin is localizing in stress fibers. Many point mutations found in cardiomyopathy patients are located in the internal region of ZASP/Cypher. However, we found no evidence that human patient mutations in the internal domain would affect the ZASP/Cypher co-localization with alpha-actinin, or that the mutations would destabilize the ZASP/Cypher protein.     

Spatial distribution of the transverse stiffness of relaxed and activated rat soleus muscle fibers

The transverse stiffness of glycerinated and demembranated fibers from the soleus muscle of Wistar rats in different functional states was measured by atomic force microscopy. It was demonstrated that the transverse stiffness of relaxed fibers near the Z disk is approximately twofold higher as compared with the M-line region. However, the stiffness of glycerinated fibers in the Z-disk and M-line regions is considerably lower than that of demembranated fibers. The values of mechanical parameters of activated fibers are significantly higher as compared with the relaxed fibers. However, the stiffness of activated glycerinated fibers near the Z disk approximately doubled as compared with the relaxed state, whereas the stiffness of the Z-disk region in demembranated fibers increased more than fourfold. The stiffness of both glycerinated and demembranized fibers near the M-line increased approximately threefold.     

In-vivo determination of 3D muscle architecture of human muscle using free hand ultrasound

Muscle architecture is an important parameter affecting the muscle function. Most of the previous studies on in-vivo muscle architecture have used in 2D ultrasound. The importance of the third dimension has not been much explored due to lack of appropriate methods. DT-MRI has been used to study muscle architecture in 3D, however, due to long scan times of about 15 min DT-MRI has not been suitable to study active muscle contractions. The purpose of this study was to develop and validate methods to determine in-vivo muscle fascicle orientations in 3D using ultrasound. We have used 2D ultrasound and a 3D position tracker system to find the 3D fascicle orientation in 3D space. 2D orientations were obtained by using automated methods developed in our previous studies and we have extended these in the current study to obtain the 3D muscle fascicle orientation in 3D space. The methods were validated using the physical phantom and we found that the mean error in the measurement was less than 0.5° in each of the three co-ordinate planes. These methods can be achieved with short scan times (less than 2 min for the gastrocnemii) and will thus enable future studies to quantify 3D muscle architecture during sub-maximal voluntary contractions.     

Effects of Corticosterone on Connectin Content and Protein Breakdown in Rat Skeletal Muscle

We examined the effects of a glucocorticoid, corticosterone, on calpain activity, connectin content and protein breakdown in rat muscle. The results indicated that calpain activity was increased by corticosterone and thus breakdown of connectin was stimulated followed by increased breakdown of skeletal muscle protein.     

Functional architecture of the SR calcium store in uterine smooth muscle

Sarcoplasmic reticulum (SR) is abundant in uterine smooth muscle cells. The functional role of this organelle in the regulation of uterine myocytes is not fully understood. The data available in the literature suggest that SR plays a dual role: as a source of calcium and as a calcium sink shaping calcium transients produced by membrane depolarisation and uterotonic agonists. Advances in digital imaging techniques including confocal microscopy of isolated living cells, and the development of methods for direct measurement of intraluminal calcium, has triggered a substantial increase in the number of publications elucidating the role of intracellular stores in calcium signalling. In this paper we review the literature and our own work on the SR calcium store in uterine smooth muscle cells.     

Alpha spectrin is essential for morphogenesis and body wall muscle formation in Caenorhabditis elegans

A common feature of multicellular animals is the ubiquitous presence of the spectrin cytoskeleton. Although discovered over 30 yr ago, the function of spectrin in non-erythrocytes has remained elusive. We have found that the spc-1 gene encodes the only alpha spectrin gene in the Caenorhabditis elegans genome. During embryogenesis, alpha spectrin localizes to the cell membrane in most if not all cells, starting at the first cell stage. Interestingly, this localization is dependent on beta spectrin but not beta(Heavy) spectrin. Furthermore, analysis of spc-1 mutants indicates that beta spectrin requires alpha spectrin to be stably recruited to the cell membrane. Animals lacking functional alpha spectrin fail to complete embryonic elongation and die just after hatching. These mutant animals have defects in the organization of the hypodermal apical actin cytoskeleton that is required for elongation. In addition, we find that the process of elongation is required for the proper differentiation of the body wall muscle. Specifically, when compared with myofilaments in wild-type animals the myofilaments of the body wall muscle in mutant animals are abnormally oriented relative to the longitudinal axis of the embryo, and the body wall muscle cells do not undergo normal cell shape changes.     

Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells

Very long, elastic connectin/titin molecules position the myosin filaments at the center of a sarcomere by linking them to the Z line. The behavior of the connectin filaments during sarcomere formation in differentiating chicken skeletal muscle cells was observed under a fluorescent microscope using the antibodies to the N terminal (located in the Z line), C terminal (M line), and C zone (myosin filament) regions of connectin and was compared to the incorporation of -actinin and myosin into forming sarcomeres. In early stages of differentiating muscle cells, the N terminal region of connectin was incorporated into a stress fiber-like structure (SFLS) together with -actinin to form dots, whereas the C terminal region was diffusely distributed in the cytoplasm. When both the C and N terminal regions formed striations in young myofibrils, the epitope to the C zone of A-band region, that is the center between the A-I junction and the M-line, initially was diffuse in appearance and later formed definite striations. It appears that it took some time for the N and C terminal regions of connectin to form a regular organization in a sarcomere. Thus the two ends of the connectin filaments were first fixed followed by the specific binding of the middle portion onto the myosin filament during sarcomere formation.     

Stimulus-Induced Selective Association of Actin-Associated Proteins (α-Actinin) and Protein Kinase C Isoforms with the Cytoskeleton of Human Neutrophils

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein α-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal α-actinin and actin. Increased association of PKCβI and βII with the cytoskeletal fraction of stimulated cells was also observed, with phorbol ester being more effective than chemotactic peptide. A fraction of phosphatase 2A was constitutively associated with the cytoskeleton independent of cell activation. None of the stimuli promoted association of vinculin or myosin II with the cytoskeleton. Phosphatase inhibitors okadaic acid and calyculin A prevented increases in cytoskeletal actin, α-actinin, and PKCβII induced by phorbol ester, suggesting the requirement for phosphatase activity in these events. Increases in cytoskeletal α-actinin and PKCβII showed differing sensitivity to agents that prevent actin polymerization (cytochalasin D, latrunculin A). Latrunculin A (1 μM) completely blocked PMA-induced increases in cytoskeletal α-actinin but reduced cytoskeletal recruitment of PKCβII only by 16%. Higher concentrations of latrunculin A (4 μM), which almost abolished the cytoskeletal actin pool, reduced cytoskeletal PKCβII by 43%. In conclusion, a selective enrichment of cytoskeletal and signaling proteins in the cytoskeleton of human neutrophils is induced by specific stimuli.     

The D3-creatine dilution method non-invasively measures muscle mass in mice

Developing accurate methods to quantify age-related muscle loss (sarcopenia) could greatly accelerate development of therapies to treat muscle loss in the elderly, as current methods are inaccurate or expensive. The current gold standard method for quantifying sarcopenia is dual-energy X-ray absorptiometry (DXA) but does not measure muscle directly—it is a composite measure quantifying “lean mass” (muscle) excluding fat and bone. In humans, DXA overestimates muscle mass, which has led to erroneous conclusions about the importance of skeletal muscle in human health and disease. In animal models, DXA is a popular method for measuring lean mass. However, instrumentation is expensive and is potentially limited by anesthesia concerns. Recently, the D₃-creatine (D₃Cr) dilution method for quantifying muscle mass was developed in humans and rats. This method is faster, cheaper, and more accurate than DXA. Here, we demonstrate that the D₃Cr method is a specific assay for muscle mass in mice, and we test associations with DXA and body weight. We evaluated the D₃Cr method compared to DXA-determined lean body mass (LBM) in aged mice and reported that DXA consistently overestimates muscle mass with age. Overall, we provide evidence that the D₃Cr dilution method directly measures muscle mass in mice. Combined with its ease of use, accessibility, and non-invasive nature, the method may prove to more quickly advance development of preclinical therapies targeting sarcopenia.     

Age-related changes in human skeletal muscle microstructure and architecture assessed by diffusion-tensor magnetic resonance imaging and their association with muscle strength

Diffusion-tensor magnetic resonance imaging (DT-MRI) offers objective measures of muscle characteristics, providing insights into age-related changes. We used DT-MRI to probe skeletal muscle microstructure and architecture in a large healthy-aging cohort, with the aim of characterizing age-related differences and comparing these to muscle strength. We recruited 94 participants (43 female; median age = 56, range = 22–89 years) and measured microstructure parameters—fractional anisotropy (FA) and mean diffusivity (MD)—in 12 thigh muscles, and architecture parameters—pennation angle, fascicle length, fiber curvature, and physiological cross-sectional area (PCSA)—in the rectus femoris (RF) and biceps femoris longus (BFL). Knee extension and flexion torques were also measured for comparison to architecture measures. FA and MD were associated with age (β = 0.33, p = 0.001, R² = 0.10; and β = −0.36, p < 0.001, R² = 0.12), and FA was negatively associated with Type I fiber proportions from the literature (β = −0.70, p = 0.024, and R² = 0.43). Pennation angle, fiber curvature, fascicle length, and PCSA were associated with age in the RF (β = −0.22, 0.26, −0.23, and −0.31, respectively; p < 0.05), while in the BFL only curvature and fascicle length were associated with age (β = 0.36, and −0.40, respectively; p < 0.001). In the RF, pennation angle and PCSA were associated with strength (β = 0.29, and 0.46, respectively; p < 0.01); in the BFL, only PCSA was associated with strength (β = 0.43; p < 0.001). Our results show skeletal muscle architectural changes with aging and intermuscular differences in the microstructure. DT-MRI may prove useful for elucidating muscle changes in the early stages of sarcopenia and monitoring interventions aimed at preventing age-associated microstructural changes in muscle that lead to functional impairment.     

胰岛素影响自发性高血压大鼠血管平滑肌细胞增殖及肌动蛋白分布的丝裂原激酶的机制探讨

血管平滑肌细胞增殖的同时伴有细胞内肌动蛋白的改变,这种改变受PKC-MAPK信号转导途径调控,但目前机制尚不清楚。为探讨胰岛素对PKC-MAPK信号转导途径参与调控血管平滑肌细胞增殖及细胞内肌动蛋白分布的影响,本研究用PKC抑制剂预处理SHR在鼠体外培养的血管平滑肌细胞,观察预处理的血管平滑肌细胞经胰岛素刺激后细胞内DNA的合成、MAPK的活性、表达及细胞内肌动蛋白的分布。发现,胰岛素刺激后可使血管平滑肌细胞增殖,同时伴有[^3H]TdR掺入增加、MAPK活性及表达与对照组比较明显升高。这些作用可被PKC抑制剂阻断。胰岛素在刺激血管平滑肌细胞增殖的同时也使细胞内肌动蛋白重新分布,这一效应也可被PKC抑制剂阻断。 上述结果提示,胰岛素使血管平滑肌细胞增殖的效应可能与MAPK信号转导途径有关。