Actin-based cell motility: myofibril contraction - Essay Example

BIOL263-Cellular Biology Laboratory John Frankis Dr. Karl Fath 5/16/07 Actin-Based Cell Motility: Myofibril Contraction Introduction: Muscle contraction is a very important physiological behavior to human. All physical movement for example lifting arms, swallowing food, heart beating involved muscles contraction. The mechanism of muscle contraction is studied by a lot of researchers. However, the most widely accepted theory was the "filament sliding model" proposed by Huxley and Niedergerke (1954), and Huxley and Hanson (1954). A skeletal muscle is made up of subunits called the fasicles. Fasicles are bundles of elongated muscle fibers which extend for the length of the muscle. The muscle fiber is segmented into distinct sectional bands. Within each muscle cell are numerous myofibrils, which also extend for the length of the muscle cell. Sarcomeres are the basic contractile subunit of myofibrils (Remedios, 2007). Within the myofibrils subunit, there are thin and thick filaments. The thin filament is made up of actin while the thick filament is made up of myosin. These two filaments are the main constituent of the filament sliding model. During a muscle contraction, a crossbridge cycle was initiated. When the nervous system sends a synaptic signal to the muscle fiber through the release of acetylcholine into the synaptic clefts, it causes the sarcoplasmic reticulum to release calcium ion Ca2+. Ca2+ binds to the troponin (Stephen, 2000). This activated the myosin ATPase and ATP is hydrolyzed by to ADP and Pi. ATP initially dissociates actomyosin into actin and myosin; i.e. the thick filaments will be detached from the thin filaments. It is bind to the myosin head in the thick filaments (Barane and Barane, 2002). When ATP is hydrolyzed, myosin is attached to the actin through the shift of tropomyosin to the actin site. A crossbridge is formed.It is believed that the angle of crossbridge attachment is 90o. The actin-myosin interaction triggers the sequential release of Pi and ADP from the myosin head, resulting in the working stroke (Barane and Barane, 2002). It is thought that the energy stored in the myosin molecule brings about a conformational change in the crossbridge tilting the angle from 90o to 45o. This tilting pulls the actin filament about 10 nm toward the center of the sarcomere, while the energy stored in myosin is utilized (Barane and Barane, 2002). The repeat formation and breaking of the crossbridge results the sliding of both the filament and shortening of the sarcomere (Stephen, 2000). Thus, in this study, we aimed to observed the changes of the rabbit muscle during the contraction. Materials and Methods: Samples: Glycerinated rabbit (Sylvilagus floridanus) psoas muscle. Reagents: 100 mM KCl, 5.0 mM Pipes buffer, 4mM EGTA, 4 mM MgCl2, pH 7.0 0.1 M sodium pyrophosphate at pH 6.4. Equipments and apparatus: Clean microscope slides and cover glasses Pasteur pipettes Filter paper strips Bucket with ice A 2 cm segment of glycerinated rabbit (Sylvilagus floridanus) psoas muscle was soaked for 30 minutes in ice-cold standard salt solution: 100 mM KCl, 5.0 mM Pipes buffer, 4 mM EGTA, 4 mM MgCl2, pH 7.0. This washed out most of the glycerol and placed the myofibrils in the basic buffer. The fibers and some of the salt solution were moved to a small Petri dish set inside a larger Petri dish of ice. Then, they were all placed on the stage of a dissecting microscope. Under the microscope, by using dissecting needles, the fibers were shredded longitudinally until they were less than 0.2 mm in diameter. The shredded muscle fibers were kept cold during the class and fibers were taken out as needed for observations. A small drop containing muscle fibers was transferred by using a Pasteur pipette attached to a rubber bulb to the center of a microscope slide. The muscles contraction was observed under both the dissecting microscope and compound microscope. The slide was placed under a dissecting microscope. Then, the zoom was adjusted and focused to see the myofibril. A drop of the ATP solution was added to the myofibrils. The changes on the entire myofibril were observed. A small drop containing muscle fibers was transferred to the center of another microscope slide that had double-sticky tape spacers. A cover glass was applied to the preparation. The finest filaments were examined at low and high power and observations were recorded. A small drop of immersion oil was applied to the center of the field of view. The 100X objective was carefully swung into place. The film of oil was continuous between the cover slip and the objective lens. The fine focus knob was used to bring the myofibrils into sharp focus. Measurements were made for the sarcomeres length and width of various bands. Mixtures of solutions were prepared according to the following: A wet mount of another fibril was prepared. The fibril was located under the microscope and the attachment to the slide was tested by drawing standard salt solution applied using a Pipette-man through the chamber with a wick of filter paper and solution 1. After that, a drop of ATP was added. The band lengths and sarcomere lengths were measured and a drawing was prepared. Another wet mount of fibrils was made. This time the solutions were added in this order: standard salt solution, solution 2, ATP, solution 1, more ATP. Finally, a drop of solution 3 was drawn into the chamber. Observations were made on the protein myosin. Results: Table 1: Measurements of Sarcomere Length (Steps 1 and 3) Condition Sarcomere Length Sarcomere Length Sarcomere Length Sarcomere Length Average Sarcomere Length (Division) Average sarcomere length (m) Initial 0.30 od 0.30 od 0.30 od 0.30 od 0.30 od 3.0 m ATP solution 0.30 od 0.30 od 0.30 od 0.30 od 0.30 od 3.0 m Table 2: Measurements of Sarcomere length and widths of various bands (Step 4, 5, and 6) Solution Sarcomere Length A band (Bright) I band (Dark) 1 od 2 od 3 od Avg. od L m 1 od 2 od 3 od Avg. od L m 1 od 2 od 3 od Avg. od L m Initial(Relax) 0.30 0.30 0.30 0.30 3.0 0.10 0.10 0.10 0.10 1.0 0.20 0.20 0.20 0.20 2.0 Solution1 0.25 0.25 0.30 0.27 2.7 0.10 0.10 0.10 0.10 1.0 0.15 0.15 0.15 0.15 1.5 Solution2 0.20 0.20 0.20 0.20 2.0 0.10 0.10 0.10 0.10 1.0 0.10 0.10 0.10 0.10 1.0 Solution3 0.50 0.55 0.45 0.50 5.0 0.15 0.20 0.20 0.18 1.8 0.30 0.25 0.25 0.27 2.7 ATP 0.20 0.20 0.15 0.18 1.8 0.15 0.15 0.10 0.13 1.3 0.05 0.05 0.05 0.05 0.5 Step 6 0.15 0.15 0.10 0.13 1.3 0.10 0.10 0.05 0.08 0.8 0.05 0.05 0.05 0.05 0.5 Step 7 0.30 0.35 0.30 0.32 3.2 0.10 0.10 0.10 0.10 1.0 0.20 0.25 0.20 0.22 2.2 Table 3: Drawings of the Sarcomeres with Band I and Band A. Solution 1 ATP Solution 2 Step 6 Solution 3 Step 7 Figure 1: Observation of a normal myofibril with no added solutions. The average sarcomere length was 3.0 micrometers. Figure 1 showed the initial state of the rabbit muscle. The average length was about 3.0 micrometers. Band I width was 2.0 which Band A width was around 1.0 micrometer. Figure 2: Observation of myofibril with added profusion solution 1. The average sarcomere length was 2.7 micrometers. Figure 2 showed the state of muscle rabbit after calcium chloride was added to it. Band I shortened while Band A remained the same. This reduced the overall length of the sarcomere. This meant that Band I (which was the thin filament) was pulled against Band A (the thick filament). Figure 3: Observation of a myofibril with added perfusion solution 2. The average sarcomere length was 2.0 micrometers. Figure 3 exhibited the sarcomere length at 2.0 micrometers after solution with EGTA was added. EGTA is a chelating agent. The purpose to add in EGTA is to detach the thick filament from the thin filament and return the muscle to the equilibrium state despite the initial length. Figure 4: Observation of a myofibril with added perfusion solution 3. The average sarcomere was 5.0 micrometers. In the NaPPi solution, the length of the sarcomere increased to 5.0 micrometers. NAPPi solubilized myosin causing the Band A's length to increase to 1.8 micrometer. This resulted the increased of the whole sarcomere length. Figure 5: Observation of a myofibril with added profusion ATP. The average sarcomere was 1.8 micrometers. ATP provides the energy to muscle contraction. It can be seen that in figure 5, when ATP was added upon the muscle, the contraction of the muscle become more intact. This was proven by the shortening of sarcomere from 3.0 micrometer to 1.8 micrometer. Figure 6: Observation of a myofibril with added Standard Salt solution, solution 2, ATP, Solution 1, and more ATP. The average sarcomere length was 1.3 micrometers. In figure 6, the whole sarcomere length became 1.3 micrometers only when all the factors: ATP and calcium ions, were added upon. Increased amount of ATP would cause more contraction. An additional amount of ATP solution was added thus causing the muscle to shorten to 1.3 micrometer compared to adding ATP solution alone (1.8 micrometer). Figure 7: Observation of a myofibril solubilized added all the solution with added solution 3. The average sarcomere length was 3.2 micrometers. Meanwhile in figure 7, the sarcomere length was 3.2 micrometers when the NaPPi solution was added after all the solution was apply on the muscle. As in figure 6, the muscle shortened before NaPPi was added. This was due to the sufficient supply of calcium ions and ATP which provided energy for the contraction. However, when NaPPi was added, the sarcomere became longer. This meant that the whole muscle relaxed. NaPPi solubilized the myosin head making the whole muscle to expand. This compound was usually used in the food industry to soften the meat after rigor mortis. It increased the water holding capacity of the muscles, which meant that the sarcomere elongate due to increase water being hold between the thick and thin filament. The hydrolysis of the myosin too broke the crossbridge of between both the filament and bringing the muscle to a relaxed state. Discussion: The results of this laboratory experiment demonstrated that when a muscle was contracted, the length of the sarcomere shortened. Band I which contained the thin filaments in the muscles shortened the most while Band A's length remained quite constant during the muscle's contraction. ATP was the compound that promoted the contraction of the muscles. Addition amount of ATP contributed additional contraction to the muscle as in step 6; the actin was pulled against myosin very tightly. The degree of muscle contraction increased in the following sequence: solution 1, solution 2, solution 3 and step 6. In solution 1 (Table 2), the calcium ion triggered the contraction. The finding was similar to those of Heilbrunn and Wiercinsky, which showed that calcium ions were the only ions that caused muscle contraction. Some kinetic experiments have suggested that troponin reduces the affinity of actin for myosin in the presence of EGTA. Chiarandini and his colleagues found out that this was true when the EGTA was in an isotonic state with the muscles at 85mM. The muscle fibers were depolarized and were unable to contract even if they were hyperpolarized. For this study, the molarity of EGTA was 5mM. Thus, the result was differ from Hitchcock and Chiarandini findings. A more detailed study focus on influence of different molarity to the degree of the depolarization by EGTA to the muscles could be conducted. In step 6, it was observable that increased ATP would result in an increase of muscle contraction. Thus, the contraction was a continuous process. As for solution 3 and step 7, it can be seen that the width of band A increased. The NaPPi solution solubilized the myosin in the thick filament in band A, therefore, this exposed the thin filament, which could be observed as the widen band I. The experiment gave an overview of the effect of different solutions towards the muscles' contraction. More observations could be obtained by changing the molarity of the reagents. Conclusion To conclude, muscle contraction was influenced by the availability of the calcium ion and ATP. Phosphate solubilized myosin which relax the muscles and prevent it from contracting. However, EGTA at a lower dose could cause contraction which at a higher dose would inhibit such reaction. References Barane, M. and Barene, K. (2002) Regulation of muscle contraction. Obtained from http://www.uic.edu/classes/phyb/phyb516/regulationmusclecontru3.htm on 20th April, 2007. Barane, M. and Barene, K. (2002b) Contractile protein: Myosin. Obtained from http://www.uic.edu/classes/phyb/phyb516/myosinu3.htm#myo on 20th April, 2007. Barane, M. and Barene, K. (2002c) Contractile protein: Actin. Obtained from http://www.uic.edu/classes/phyb/phyb516/actinu3.htm#Actin on 20th April, 2007. Chiarandini, D.J., Sanchez , J. A. and Stefani, E. (1980) Effect of calcium withdrawal on mechanical threshold in skeletal muscle fibers of the frog. J Physiology, 303:153-163. Hitchcock, S.E. (1973) Regulation of Muscle Contraction: Effect of Calcium on the Affinity of Troponin for Actin and Tropomyosin. Biochemistry, 12(13):2509. Huxley, H.E., Brown, W. & Holmes, K.C. (1965) Constancy of axial spacing's in frog Sartorius muscle during contraction. Nature 206, 1358. Huxley, A.F. and Niedergerke, R. (1954). Structural changes in muscle during contraction. Interference microscopy of living muscle fibers. Nature, 173, 971-973. Huxley, H.E. and Hanson, J. (1954). Changes in the cross-striations of muscle during contraction and stretch and their structural interpretation. Nature, 173, 973-976. Remedios, C.R. (2007) Skeletal Muscle Structure. Obtained from http://www.anatomy.usyd.edu.au/mru/lectures/lecture1.pdf on 13th May, 2007. Richitson, G. (2007) Bio 301 Human Physiology : Muscles. Obtained from http://people.eku.edu/ritchisong/RITCHISO//301notes3.htm on 22nd April, 2007. Stephen, S.B. (2000) The mechanism of muscle contraction. Obtained from http://meat.tamu.edu/muscontract.html on 12th May, 2007. Read More