Bestselling Series. Harry Potter. Popular Features. New Releases. Description Actin is one of the most widespread proteins in eukaryotic cells. This book and its companion "Molecular Interactions of Actin.
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Actin Structure and Actin-Binding Proteins" provide an authoritative and opinionated view of the structure and function of this essential protein. Each section includes an historical perspective and a detailed commentary on actin protein chemistry, molecular and cell biology of actin. While some chapters review the body of knowledge of the subject, others contain new experimental data.
This book will appeal to research scientists seeking contemporary overviews of actin-myosin interaction and actin-based regulation.
Actin-Myosin Interaction: Structure, Function and Drug Discovery
Contributors include senior scientists as well as the new breed of younger scientists. Other books in this series. Add to basket. Drosophila Eye Development Kevin Moses. Pulsed contractions of an actin-myosin network drive apical constriction Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis.
Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and non-muscle myosin-II myosin belt underlying adherens junctions. However, it is unclear whether other force-generating mechanisms can drive this process. This study shows, with the use of real-time imaging and quantitative image analysis of Drosophila gastrulation, that the apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighbouring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained.
In contrast to the purse-string model, constriction pulses are powered by actin-myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inwards. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin-myosin network contractions, whereas expression of twist stabilizes the constricted state of the cell apex.
These results suggest a new model for apical constriction in which a cortical actin-myosin cytoskeleton functions as a developmentally controlled subcellular ratchet to reduce apical area incrementally Martin, During Drosophila gastrulation, apical constriction of ventral cells facilitates the formation of a ventral furrow and the subsequent internalization of the presumptive mesoderm.
Although myosin is known to localize to the apical cortex of constricting ventral furrow cells, it is not known how myosin produces force to drive constriction. Understanding this mechanism requires a quantitative analysis of cell and cytoskeletal dynamics. Methods were developed to reveal and quantify apical cell shape with Spider-GFP, a green fluorescent protein GFP -tagged membrane-associated protein that outlines individual cells.
Although the average apical area steadily decreased at a rate of about 5 microm 2 min -1 , individual cells showed transient pulses of rapid constriction that exceeded microm 2 min During the initial 2 min of constriction, weak constriction pulses were often interrupted by periods of cell stretching.
Tropomyosin‐Mediated Regulation of Cytoplasmic Myosins
However, at 2 min, constriction pulses increased in magnitude and cell shape seemed to be stabilized between pulses, leading to net constriction. Overall, cells underwent an average of 3. Constriction pulses were mostly asynchronous between adjacent cells. As a consequence, cell apices between constrictions seemed to be pulled by their constricting neighbours. Thus, apical constriction occurs by means of pulses of rapid constriction interrupted by pauses during which cells must stabilize their constricted state before reinitiating constriction Martin, To determine how myosin might generate force during pulsed constrictions, myosin and cell dynamics were simultaneously imaged by using myosin regulatory light chain spaghetti squash , or squ fused to mCherry Myosin-mCherry and Spider-GFP.
Discrete myosin spots and fibres present on the apical cortex formed a network that extended across the tissue. These myosin structures were dynamic, with apical myosin spots repeatedly increasing in intensity and moving together at about 40 nm s -1 to form larger and more intense myosin structures at the medial apical cortex. This process, which is referred to as myosin coalescence, resulted in bursts of myosin accumulation that were correlated with constriction pulses.
The peak rate of myosin coalescence preceded the peak constriction rate by s, suggesting that myosin coalescence causes apical constriction. Between myosin coalescence events, myosin structures, including fibres, remained present on the cortex, possibly maintaining cortical tension between constriction pulses. Contrary to the purse-string model, no significant myosin accumulation was seen at cell-cell junctions. To confirm that constriction involved medial myosin coalescence and not contraction of a circumferential purse-string, constriction rate was correlated with myosin intensity at either the medial or junctional regions of the cell.
Desmin: molecular interactions and putative functions of the muscle intermediate filament protein
Apical constriction was correlated more significantly with medial myosin, suggesting that, in contrast to the purse-string model, constriction is driven by contractions at the medial apical cortex Martin, Myosin coalescence resembled contraction of a cortical actin-myosin network. Therefore, to determine whether apical constriction is driven by pulsed contractions of the actin-myosin network, the organization of the cortical actin cytoskeleton was examined. In fibroblasts and keratocytes, actin network contraction bundles actin filaments into fibre-like structures.
Consistent with this expectation was the identification of an actin filament meshwork underlying the apical cortex in which prominent actin-myosin fibres spanning the apical cortex appeared specifically in constricting cells. An actin-myosin network contraction model would predict that myosin coalescence results from myosin spots exerting traction on each other through the cortical actin network.
To test whether myosin coalescence requires an intact actin network, the actin network was disrupted with cytochalasin D CytoD. Disruption of the actin network with CytoD resulted in apical myosin spots that localized together with actin structures and appeared specifically in ventral cells. Myosin spots in CytoD-injected embryos showed more rapid movement than those in control-injected embryos, suggesting that apical myosin spots in untreated embryos are constrained by the cortical actin network.
Although myosin movement was uninhibited in CytoD-treated embryos, myosin spots failed to coalesce and cells failed to constrict. Because myosin coalescence requires an intact actin network, it is proposed that pulses of myosin coalescence represent contractions of the actin-myosin network Martin, Because actin-myosin contractions occurred at the medial apical cortex, it was unclear how the actin-myosin network was coupled to adherens junctions. Before apical constriction, adherens junctions are present about 4 microm below the apical cortex.
As apical constriction initiated, these subapical adherens junctions gradually disappeared and adherens junctions simultaneously appeared apically at the same level as myosin. This apical redistribution of adherens junctions occurred at specific sites along cell edges midway between vertices. As apical constriction initiated, these sites bent inwards.
This bending depended on the presence of an intact actin network, which is consistent with contraction of the actin-myosin network generating force to pull junctions. Indeed, myosin spots undergoing coalescence were observed to lead adherens junctions as they transiently bent inwards. Thus, pulsed contraction of the actin-myosin network at the medial cortex seems to pull the cell surface inwards at discrete adherens junction sites, resulting in apical constriction Martin, The transcription factors Twist and Snail regulate the apical constriction of ventral furrow cells.
Snail is a transcriptional repressor whose target or targets are currently unknown, whereas Twist enhances snail expression and activates the expression of fog and t48 , which are thought to activate the Rho1 GTPase and promote myosin contractility. To examine the mechanism of pulsed apical constriction further, how Twist and Snail regulate myosin dynamics was tested. In contrast to wild-type ventral cells, in which myosin was concentrated on the apical cortex, twist and snail mutants accumulated myosin predominantly at cell junctions, similarly to lateral cells.
These ventral cells failed to constrict productively, which supported the cortical actin-myosin network contraction model, rather than the purse-string model, for apical constriction. Although myosin coalescence was inhibited in snail mutants, it still occurred in twist mutants, as did pulsed constrictions. This difference was also observed when Snail or Twist activity was knocked down by RNA-mediated interference.
However, the magnitude of constriction pulses in twistRNAi embryos was greater than that of twist mutant embryos, suggesting that the low level of Twist activity present in twistRNAi embryos enhances contraction efficiency by activating the expression of snail or other transcriptional targets. Myosin coalescence was inhibited in snail twist double mutants, demonstrating that the pulsed constrictions in twist mutants required snail expression. Thus, the expression of snail , not twist , initiates the actin-myosin network contractions that power constriction pulses Martin, It was therefore asked why the pulsed contractions that were observed in twistRNAi embryos failed to constrict cells.
Using Spider-GFP to visualize cell outlines, it was found that although constriction pulses were inhibited in snailRNAi embryos, constriction pulses still occurred in twistRNAi embryos. However, the constricted state of cells in twistRNAi embryos was not stabilized between pulses, resulting in fluctuations in apical area with little net constriction. This stabilization defect was not due to lower snail activity, because these fluctuations continued when snail expression was driven independently of twist by using the P[ sna ] transgene.
Although the frequency and magnitude of constriction pulses in such embryos were similar to those in control embryos, stretching events were significantly higher in twistRNAi ; P[ sna ] embryos, suggesting a defect in maintaining cortical tension. This defect might result from a failure to establish a dense actin meshwork, because both twist mutants and twistRNAi embryos had a more loosely arranged apical meshwork of actin spots and fibres than constricting wild-type cells did.
Thus, a 'ratchet' model is proposed for apical constriction, in which phases of actin-myosin network contraction and stabilization are repeated to constrict the cell apex incrementally. In contrast to the purse-string model, it was found that apical constriction is correlated with pulses of actin-myosin network contraction that occur on the apical cortex.
Pulsed cortical contractions could allow dynamic rearrangements of the actin network to optimize force generation as cells change shape.