Search Results (4)

The growth of pollen tubes, which depends on actin filaments, is pivotal for plant reproduction. Pharmacological experiments showed that while oryzalin and brefeldin A treatments had no significant effect on the lipid droplets (LDs) trafficking, while 2,3-butanedione monoxime (BDM), latrunculin B, SMIFH2, and cytochalasin D treatments slowed down LDs trafficking, in such a manner that only residual wobbling was observed, suggesting that trafficking of LDs in pollen tube is related to F-actin. While the trafficking of LDs in the wild-type pollen tubes and in myo11-2, myo11b1-1, myo11c1-1, and myo11c2-1 single mutants and myo11a1-1/myo11a2-1 double mutant were normal, their trafficking slowed down in a myosin-XI double knockout (myo11c1-1/myo11c2-1) mutant. These observations suggest that Myo11C1 and Myo11C2 motors are involved in LDs movement in pollen tubes, and they share functional redundancy. Hence, LDs movement in Arabidopsis pollen tubes relies on the actomyosin system. Full article

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca²⁺-sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin’s ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca²⁺. These changes in the behavior of tropomyosin and the troponin–tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca²⁺-sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca²⁺-dependent movement and the ability of the troponin–tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility. Full article

Background and Objectives: Muscle fatigue is characterised by (1) loss of force, (2) decreased maximal shortening velocity and (3) a greater resistance to stretch that could be due to reduced intracellular Ca²⁺ and increased Pi, which alter cross bridge kinetics. Materials and Methods: To investigate this, we used (1) 2,3-butanedione monoxime (BDM), believed to increase the proportion of attached but non-force-generating cross bridges; (2) Pi that increases the proportion of attached cross bridges, but with Pi still attached; and (3) reduced activating Ca²⁺. We used permeabilised rat soleus fibres, activated with pCa 4.5 at 15 °C. Results: The addition of 1 mM BDM or 15 mM Pi, or the lowering of the Ca²⁺ to pCa 5.5, all reduced the isometric force by around 50%. Stiffness decreased in proportion to isometric force when the fibres were activated at pCa 5.5, but was well maintained in the presence of Pi and BDM. Force enhancement after a stretch increased with the length of stretch and Pi, suggesting a role for titin. Maximum shortening velocity was reduced by about 50% in the presence of BDM and pCa 5.5, but was slightly increased by Pi. Neither decreasing Ca²⁺ nor increasing Pi alone mimicked the effects of fatigue on muscle contractile characteristics entirely. Only BDM elicited a decrease of force and slowing with maintained stiffness, similar to the situation in fatigued muscle. Conclusions: This suggests that in fatigue, there is an accumulation of attached but low-force cross bridges that cannot be the result of the combined action of reduced Ca²⁺ or increased Pi alone, but is probably due to a combination of factors that change during fatigue. Full article

At a resting sarcomere length of approximately 2.2 µm bony fish muscles put into rigor in the presence of BDM (2,3-butanedione monoxime) to reduce rigor tension generation show the normal arrangement of myosin head interactions with actin filaments as monitored by low-angle X-ray diffraction. However, if the muscles are put into rigor using the same protocol but stretched to 2.5 µm sarcomere length, a markedly different structure is observed. The X-ray diffraction pattern is not just a weaker version of the pattern at full overlap, as might be expected, but it is quite different. It is compatible with the actin-attached myosin heads being in a different conformation on actin, with the average centre of cross-bridge mass at a higher radius than in normal rigor and the myosin lever arms conforming less to the actin filament geometry, probably pointing back to their origins on their parent myosin filaments. The possible nature of this new rigor cross-bridge conformation is discussed in terms of other well-known states such as the weak binding state and the ‘roll and lock’ mechanism; we speculate that we may have trapped most myosin heads in an early attached strong actin-binding state in the cross-bridge cycle on actin. Full article