Covalent Labeling of Proteins with Fluorescent Compounds for Imaging Applications

The labeling of proteins with fluorescent compounds for microscopy has allowed a greater understanding of biological processes. The preparation of fluorescent proteins is the first step in development of their use in microscopy. Methods are described to label and characterize a protein as an example of the general approach for other proteins. Skeletal muscle alpha-actinin was labeled with either fluorescein-5-maleimide or 5-iodoaceamidofluorescein and the reaction characterized. The maleimide reaction was much more rapid and efficient than the iodoacetamide reaction giving a coupling efficiency of 65% under the given ration conditions. The fluorescein-5-maleimide alpha-actinin was functionally characterized and there was essentially no influence on the fluorescein label on the F-actin binding properties of alpha-actinin. The fluorescein alpha-actinin was also shown to specifically bind to the Z-line of isolated myofibrils. A general outline and discussion are presented on how to label and characterize proteins for use in microscopy

Measurement of Calcium Dissociation Rates from Troponin C in Rigor Skeletal Myofibrils

Ca2+ dissociation from the regulatory domain of troponin C may influence the rate of striated muscle relaxation. However, Ca2+ dissociation from troponin C has not been measured within the geometric and stoichiometric constraints of the muscle fiber. Here we report the rates of Ca2+ dissociation from the N-terminal regulatory and C-terminal structural domains of fluorescent troponin C constructs reconstituted into rabbit rigor psoas myofibrils using stopped-flow technology. Chicken skeletal troponin C fluorescently labeled at Cys 101, troponin CIAEDANS, reported Ca2+ dissociation exclusively from the structural domain of troponin C at ∼0.37, 0.06, and 0.07/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Ca2+ dissociation from the regulatory domain was observed utilizing fluorescently labeled troponin C containing the T54C and C101S mutations. Troponin CMIANST54C,C101S reported Ca2+ dissociation exclusively from the regulatory domain of troponin C at >1000, 8.8, and 15/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Interestingly, troponin CIAANST54C,C101S reported a biphasic fluorescence change upon Ca2+ dissociation from the N- and C-terminal domains of troponin C with rates that were similar to those reported by troponin CMIANST54C,C101S and troponin CIAEDANS at all levels of the troponin C systems. Furthermore, the rate of Ca2+ dissociation from troponin C in the myofibrils was similar to the rate of Ca2+ dissociation measured from the troponin C-troponin I complexes. Since the rate of Ca2+ dissociation from the regulatory domain of TnC in myofibrils is similar to the rate of skeletal muscle relaxation, Ca2+ dissociation from troponin C may influence relaxation

Influence of ADP on Cross-Bridge-Dependent Activation of Myofibrillar Thin Filaments

AbstractContraction of skeletal muscle is regulated by calcium at the level of the thin filament via troponin and tropomyosin. Studies have indicated that strong cross-bridge binding is also involved in activation of the thin filament. To further test this, myofibrils were incubated with a wide range of fluorescent myosin subfragment 1(fS1) at pCa 9 or pCa 4 with or without ADP. Sarcomere fluorescence intensity and the fluorescence intensity ratio (non-overlap region/overlap region) were measured to determine the amount and location of bound fS1 in the myofibril. There was lower sarcomere fluorescence intensity with ADP compared to without ADP for both calcium levels. Similar data were obtained from biochemical measures of bound fS1, validating the fluorescence microscopy measurements. The intensity ratio, which is related to activation of the thin filament, increased with increasing [fS1] with or without ADP. At pCa 9, the fluorescence intensity ratio was constant until 80–160 nM fS1 without ADP conditions, then it went up dramatically and finally attained saturation. The dramatic shift of the ratio demonstrated the cooperative character of strong cross-bridge binding, and this was not observed at high calcium. A similar pattern was observed with ADP in that the ratio was right-shifted with respect to total [fS1]. Saturation was obtained with both the fluorescence intensity and ratio data. Plots of intensity ratio as a function of normalized sarcomere intensity (bound fS1) showed little difference between with and without ADP. This suggests that the amount of strongly bound fS1, not fS1 state (with or without ADP) is related to activation of the thin filament

Smooth Muscle Adherens Junctions Associated Proteins are Stable at the Cell Periphery During Relaxation and Activation

This study was performed to determine the stability of the adherens junction (AJ)-associated proteins at the smooth muscle cell (SMC) plasma membrane during relaxing and activating conditions. Dog stomach, ileum, colon, and trachea tissues were stored in Ca2+-free PSS or regular PSS or were activated in 10 μM carbachol in PSS before rapid freezing. The tissues were subsequently sectioned and immunoreacted using antibodies for vinculin, talin, fibronectin, and caveolin to determine their cellular distribution in these tissues under these conditions. In all four tissues and under all three conditions, the distribution of these four proteins remained localized to the periphery of the cell. In transverse tissue sections, the AJ-associated proteins formed a distinct punctate pattern around the periphery of the SMCs at the plasma membrane. These domains alternated with the caveolae (as identified by the presence of caveolin). In longitudinal tissue sections, the AJ-associated proteins formed continuous tracks or staves, while the caveolae remained punctate in this dimension as well. Caveolin is not present in the tapered ends of the SMCs, where the AJ-associated proteins appear continuous around the periphery. Densitometry of the fluorophore distribution of these proteins showed no shift in their localization from the SMC periphery when the tissues were relaxed or when they were activated before freezing. These results suggest that under physiologically relaxing and activating conditions, AJ-associated proteins remain stably localized at the plasma membrane

Adherens Junction-Associated Protein Distribution Differs in Smooth Muscle Tissue and Acutely Isolated Cells

This study was designed to examine how smooth muscle (SM) cell (SMC) isolation affects the distribution of some adherens junction (AJ) complex-associated proteins. Immunofluorescence procedures for identifying protein distribution were used on gastrointestinal and tracheal SM tissues and freshly isolated SMCs from dogs and rabbits. As confirmed by force measurements, relaxation, Ca2+ depletion, and cholinergic activation of SM tissues do not cause significant redistribution of the AJ-associated proteins vinculin, talin, or fibronectin away from the plasma membrane. Unlike SMCs in tissue, freshly isolated SMCs show a variable peripheral/ cytoplasmic vinculin and talin distribution that is not altered by activation. Enzymatic treatment of SM tissues (as done for the first step of SMC isolation) results in loss of fibronectin immunoreactivity in SMCs still in the tissue but fails to cause redistribution of vinculin, talin, or caveolin away from the periphery. The loss of fibronectin immunofluorescence with enzymatic digestion correlates significantly with loss of tissue force production. These results confirm that the AJ-associated proteins vinculin and talin do not redistribute throughout SMCs in tissues when relaxed, when generating force, or after enzymatic digestion. In addition, in freshly isolated SMCs, the distribution of these proteins is significantly altered in ~50% of the SMCs. The cause of this redistribution is currently unknown, as is the impact on intracellular signaling and mechanics of these cells. Use of these two systems (SMCs in tissues vs. freshly isolated SMCs) provides an ideal situation for studying the role of the AJ in SMC signaling and mechanics