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In JoVE (1)
- Espaço-Temporal Manipulação de Atividade GTPase Pequeno em nível subcelular e em escala de tempo de segundos em células vivas
Other Publications (5)
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Articles by Scot Kuo in JoVE
Espaço-Temporal Manipulação de Atividade GTPase Pequeno em nível subcelular e em escala de tempo de segundos em células vivas
Robert DeRose1, Christopher Pohlmeyer1, Nobuhiro Umeda1,2, Tasuku Ueno1,2, Tetsuo Nagano2, Scot Kuo1,3, Takanari Inoue1
1Department of Cell Biology, Center for Cell Dynamics, Johns Hopkins University, 2Graduate School of Pharmaceutical Sciences, University of Tokyo, 3Biomedical Engineering, Johns Hopkins University
Um método para espácio-temporal controlo da actividade GTPase pequena pela luz é descrito. Este método baseia-se em rapamicina induzida heterodimerização FKBP-FRB e foto-enjaulamento sistemas. Otimização de luz irradiação permite a ativação espaço-temporalmente controlada de pequenas GTPases no nível subcelular.
Other articles by Scot Kuo on PubMed
Current Biology : CB. Feb, 2003 | Pubmed ID: 12593799
The intracellular movement of the bacterial pathogen Listeria monocytogenes has helped identify key molecular constituents of actin-based motility (recent reviews ). However, biophysical as well as biochemical data are required to understand how these molecules generate the forces that extrude eukaryotic membranes. For molecular motors and for muscle, force-velocity curves have provided key biophysical data to distinguish between mechanistic theories. Here we manipulate and measure the viscoelastic properties of tissue extracts to provide the first force-velocity curve for Listeria monocytogenes. We find that the force-velocity relationship is highly curved, almost biphasic, suggesting a high cooperativity between biochemical catalysis and force generation. Using high-resolution motion tracking in low-noise extracts, we find long trajectories composed exclusively of molecular-sized steps. Robust statistics from these trajectories show a correlation between the duration of steps and macroscopic Listeria speed, but not between average step size and speed. Collectively, our data indicate how the molecular properties of the Listeria polymerization engine regulate speed, and that regulation occurs during molecular-scale pauses.
Dynacortin Contributes to Cortical Viscoelasticity and Helps Define the Shape Changes of Cytokinesis
The EMBO Journal. Apr, 2004 | Pubmed ID: 15014435
During cytokinesis, global and equatorial pathways deform the cell cortex in a stereotypical manner, which leads to daughter cell separation. Equatorial forces are largely generated by myosin-II and the actin crosslinker, cortexillin-I. In contrast, global mechanics are determined by the cortical cytoskeleton, including the actin crosslinker, dynacortin. We used direct morphometric characterization and laser-tracking microrheology to quantify cortical mechanical properties of wild-type and cortexillin-I and dynacortin mutant Dictyostelium cells. Both cortexillin-I and dynacortin influence cytokinesis and interphase cortical viscoelasticity as predicted from genetics and biochemical data using purified dynacortin proteins. Our studies suggest that the regulation of cytokinesis ultimately requires modulation of proteins that control the cortical mechanical properties that establish the force-balance that specifies the shapes of cytokinesis. The combination of genetic, biochemical, and biophysical observations suggests that the cell's cortical mechanical properties control how the cortex is remodeled during cytokinesis.
Proceedings of the National Academy of Sciences of the United States of America. Feb, 2006 | Pubmed ID: 16461463
Cell cortices rearrange dynamically to complete cytokinesis, crawlin response to chemoattractant, build tissues, and make neuronal connections. Highly enriched in the cell cortex, actin, myosin II, and actin crosslinkers facilitate cortical movements. Because cortical behavior is the consequence of nanoscale biochemical events, it is essential to probe the cortex at this level. Here, we use high-resolution laser-based particle tracking to examine how myosin II mechanochemistry and dynacortin-mediated actin crosslinking control cortex dynamics in Dictyostelium. Consistent with its low duty ratio, myosin II does not directly drive active bead motility. Instead, myosin II and dynacortin antagonistically regulate other active processes in the living cortex.
Interactions Between Myosin and Actin Crosslinkers Control Cytokinesis Contractility Dynamics and Mechanics
Current Biology : CB. Apr, 2008 | Pubmed ID: 18372178
Contractile networks are fundamental to many cellular functions, particularly cytokinesis and cell motility. Contractile networks depend on myosin-II mechanochemistry to generate sliding force on the actin polymers. However, to be contractile, the networks must also be crosslinked by crosslinking proteins, and to change the shape of the cell, the network must be linked to the plasma membrane. Discerning how this integrated network operates is essential for understanding cytokinesis contractility and shape control. Here, we analyzed the cytoskeletal network that drives furrow ingression in Dictyostelium.
Proceedings of the National Academy of Sciences of the United States of America. Jan, 2009 | Pubmed ID: 19104041
Proximity to membranes is required of actin networks for many key cell functions, including mechanics and motility. However, F-actin rigidity should hinder a filament's approach to surfaces. Using confocal microscopy, we monitor the distribution of fluorescent actin near nonadherent glass surfaces. Initially uniform, monomers polymerize to create a depletion zone where F-actin is absent at the surface but increases monotonically with distance from the surface. At its largest, depletion effects can extend >35 microm, comparable with the average, mass-weighted filament length. Increasing the rigidity of actin filaments with phalloidin increases the extent of depletion, whereas shortening filaments by using capping protein reduces it proportionally. In addition, depletion kinetics are faster with higher actin concentrations, consistent with faster polymerization and faster Brownian-ratchet-driven motion. Conversely, the extent of depletion decreases with actin concentration, suggesting that entropy is the thermodynamic driving force. Quantitatively, depletion kinetics and extent match existing actin kinetics, rigidity, and lengths. However, explaining depletion profiles and concentration dependence (power-law of -1) requires modifying the rigid rod model. Within cells, surface depletion should slow membrane-associated F-actin reactions another approximately 10-fold beyond hydrodynamically slowed diffusion of filaments (approximately 10-fold). In addition, surface depletion should cause membranes to bend spontaneously toward filaments. Such depletion principles underlie the thermodynamics of all surface-associated reactions with mechanical structures, ranging from DNA to filaments to networks. For various functions, cells must actively resist the thermodynamics of depletion.