Kinesin motility is driven by subdomain dynamics

The microtubule (MT)-associated motor protein kinesin utilizes its conserved ATPase head to achieve diverse motility characteristics. Despite considerable knowledge about how its ATPase activity and MT binding are coupled to the motility cycle, the atomic mechanism of the core events remain to be found. To obtain insights into the mechanism, we performed 38.5 microseconds of all-atom molecular dynamics simulations of kinesin-MT complexes in different nucleotide states. Local subdomain dynamics were found to be essential for nucleotide processing. Catalytic water molecules are dynamically organized by the switch domains of the nucleotide binding pocket while ATP is torsionally strained. Hydrolysis products are 'pulled' by switch-I, and a new ATP is 'captured' by a concerted motion of the α0/L5/switch-I trio. The dynamic and wet kinesin-MT interface is tuned for rapid interactions while maintaining specificity. The proposed mechanism provides the flexibility necessary for walking in the crowded cellular environment.

Cytoskeletal Mechanics Regulating Amoeboid Cell Locomotion

Migrating cells exert traction forces when moving. Amoeboid cell migration is a common type of cell migration that appears in many physiological and pathological processes and is performed by a wide variety of cell types. Understanding the coupling of the biochemistry and mechanics underlying the process of migration has the potential to guide the development of pharmacological treatment or genetic manipulations to treat a wide range of diseases. The measurement of the spatiotemporal evolution of the traction forces that produce the movement is an important aspect for the characterization of the locomotion mechanics. There are several methods to calculate the traction forces exerted by the cells. Currently the most commonly used ones are traction force microscopy methods based on the measurement of the deformation induced by the cells on elastic substrate on which they are moving. Amoeboid cells migrate by implementing a motility cycle based on the sequential repetition of four phases. In this paper, we review the role that specific cytoskeletal components play in the regulation of the cell migration mechanics. We investigate the role of specific cytoskeletal components regarding the ability of the cells to perform the motility cycle effectively and the generation of traction forces. The actin nucleation in the leading edge of the cell, carried by the ARP2/3 complex activated through the SCAR/WAVE complex, has shown to be fundamental to the execution of the cyclic movement and to the generation of the traction forces. The protein PIR121, a member of the SCAR/WAVE complex, is essential to the proper regulation of the periodic movement and the protein SCAR, also included in the SCAR/WAVE complex, is necessary for the generation of the traction forces during migration. The protein Myosin II, an important F-actin cross-linker and motor protein, is essential to cytoskeletal contractility and to the generation and proper organization of the traction forces during migration.

The SCAR/WAVE complex is necessary for proper regulation of traction stresses during amoeboid motility

Cell migration requires a tightly regulated, spatiotemporal coordination of underlying biochemical pathways. Crucial to cell migration is SCAR/WAVE–mediated dendritic F-actin polymerization at the cell's leading edge. Our goal is to understand the role the SCAR/WAVE complex plays in the mechanics of amoeboid migration. To this aim, we measured and compared the traction stresses exerted by Dictyostelium cells lacking the SCAR/WAVE complex proteins PIR121 (pirA−) and SCAR (scrA−) with those of wild-type cells while they were migrating on flat, elastic substrates. We found that, compared to wild type, both mutant strains exert traction stresses of different strengths that correlate with their F-actin levels. In agreement with previous studies, we found that wild-type cells migrate by repeating a motility cycle in which the cell length and strain energy exerted by the cells on their substrate vary periodically. Our analysis also revealed that scrA− cells display an altered motility cycle with a longer period and a lower migration velocity, whereas pirA− cells migrate in a random manner without implementing a periodic cycle. We present detailed characterization of the traction-stress phenotypes of the various cell lines, providing new insights into the role of F-actin polymerization in regulating cell–substratum interactions and stresses required for motility.

Myosin II Is Essential for the Spatiotemporal Organization of Traction Forces during Cell Motility

Amoeboid motility requires spatiotemporal coordination of biochemical pathways regulating force generation and consists of the quasi-periodic repetition of a motility cycle driven by actin polymerization and actomyosin contraction. Using new analytical tools and statistical methods, we provide, for the first time, a statistically significant quantification of the spatial distribution of the traction forces generated at each phase of the cycle (protrusion, contraction, retraction, and relaxation). We show that cells are constantly under tensional stress and that wild-type cells develop two opposing “pole” forces pulling the front and back toward the center whose strength is modulated up and down periodically in each cycle. We demonstrate that nonmuscular myosin II complex (MyoII) cross-linking and motor functions have different roles in controlling the spatiotemporal distribution of traction forces, the changes in cell shape, and the duration of all the phases. We show that the time required to complete each phase is dramatically increased in cells with altered MyoII motor function, demonstrating that it is required not only for contraction but also for protrusion. Concomitant loss of MyoII actin cross-linking leads to a force redistribution throughout the cell perimeter pulling inward toward the center. However, it does not reduce significantly the magnitude of the traction forces, uncovering a non–MyoII-mediated mechanism for the contractility of the cell.

Mechanisms behind changes in gastric acid and bicarbonate outputs during the human interdigestive motility cycle

Human gastric interdigestive acid and bicarbonate outputs vary cyclically in association with the migrating motor complex (MMC). These phenomena were studied in 26 healthy volunteers by constant-flow gastric perfusion, with continuous recording of pH and Pco2 in mixed gastric effluent and concomitant open-tip manometry of gastroduodenal motility. Stable acid and bicarbonate outputs were registered during less than 50% of the MMC cycle. Acid secretion started to increase 71 +/- 3% into the cycle, with maximum output during antral phase III. Bicarbonate output increased biphasically 1) 40 +/- 5% into the cycle, coinciding with reflux of bile, and 2) at the end of duodenal phase III when the aspirate was devoid of bile. The bicarbonate peak associated with phase III was abolished by atropine (0.01 mg/kg iv, n = 8) and by pyloric occlusion (n = 9) but remained unchanged after omeprazole (n = 10). The acid peak was abolished by both atropine and omeprazole. It is concluded that the MMC-related changes in acid and alkaline outputs represent two different and independent phenomena. Acid secretion cyclicity is due to periodical variations in cholinergic stimulation of the parietal cells. In contrast, the phase III-associated increase in bicarbonate output is due to duodenogastric reflux.

Effect of neural blockades, gastrointestinal regulatory peptides, and diversion of gastroduodenal contents on periodic pancreatic secretion in the preruminant calf

The role of nerves, gastrointestinal peptides, and gastroduodenal contents in the regulation of pancreatic periodic function were studied in preruminant calves. Nine male, Friesian calves were surgically fitted with pancreatic and duodenal catheters, abomasal and duodenal cannulae, and duodenal electrodes. Pancreatic secretion oscillated in phase with the duodenal migrating myoelectric complex. Pancreatic secretion and duodenal motility were abolished by intravenous atropine (5 μg∙kg−1∙min−1). The frequency of pancreatic and duodenal cycles was similarly increased by motilin and decreased by pituitary adenylate cyclase activating polypeptide-27; secretin lengthened duodenal but not pancreatic cycles, resulting in loss of synchronization; cholecystokinin-8 and secretin increased pancreatic secretion (all infusions at 120 pmol∙kg−1∙h−1); intraduodenal lidocaine (2%) or diversion of gastroduodenal contents reduced pancreatic secretion without altering periodicity. In conclusion, generation of pancreatic as well as of duodenal periodicity in the calf depends upon cholinergic neural efferent input. Secretin, cholecystokinin-8, pituitary adenylate cyclase activating polypeptide, duodenal contents, and mucosal afferent receptors seem to have relatively minor regulatory roles but can modulate the level of pancreatic secretion. The importance of enteric neural influence from the duodenum and the role of motilin in the regulation of pancreatic periodicity and its synchronization with the duodenal motility cycle remain to be determined.Key words: pancreatic juice, duodenal migrating myoelectric complex, secretin, motilin, pituitary adenylate cyclase activating polypeptide, atropine, lidocaine, calf.

Relationship between interdigestive duodenal motility and fluid transport in humans

In 22 healthy volunteers distal duodenal fluid absorption was related to the interdigestive motility cycle. Fluid absorption was measured with a triple-lumen perfusion technique, and motility was registered with a low-compliance pneumohydraulic system. Pancreatic and biliary secretions were estimated by measurement of bilirubin and amylase release into the duodenal segment. Duodenal fluid absorption rate changed during the interdigestive motility cycle; the highest absorption rate was registered during phase I (low-motor activity) and absorption rate then decreased in parallel with increasing motor activity during phase II (r = -0.69, P less than 0.001). In late phase II a net fluid secretion was frequently registered, together with an increased release of bilirubin into the duodenal lumen. This pattern was seen during perfusion with both glucose-containing (30 mM) and glucose-free solutions. The results show that duodenal fluid absorption rate changes markedly during the interdigestive motility cycle. This effect may be a hydrodnamic phenomenon or may be due to activation of a neural secretory mechanism during phase II.