Mechanical Properties of Re-constituted Actin Networks at an Oil/Water Interface Determined by Microrheology
Ershov, D.S. ; Cohen Stuart, M.A. ; Gucht, J. van der - \ 2012
Soft Matter 8 (2012). - ISSN 1744-683X - p. 5896 - 5903.
particle-tracking microrheology - cytoskeletal protein networks - micropipette aspiration - myosin-filaments - molecular motors - polymer networks - living cells - cortex - microscopy - membranes
There have been various attempts to investigate the mechanical properties of the actin cortex in cells, but the factors that control them remain poorly understood. To make progress, we develop a reconstituted model of the actin cortex that mimics its structure. We attach actin filaments to lipids lining the surface of an oil droplet using biotin–streptavidin bonds. In this way we can form a thin actin network that can be visualized and studied by confocal microscopy. Our approach allows incorporation of different actin-binding and motor proteins into this 2D network and characterization of their effect on its mechanical properties in a quantitative way. To study the viscoelasticity of the network, we use passive particle tracking microrheology, which allows storage and loss moduli to be extracted from the mean square displacement of tracer particles. We show that adding cross-linkers to the cortex increases its elasticity by several orders of magnitude and addition of myosin in the presence of ATP results in a strong and rapid stiffening of the network. This approach opens up a variety of possibilities to study viscoelastic properties of the actin cortex in vitro, allowing incorporation of any protein of interest into the system.
Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array
Schuster, M. ; Kilaru, S. ; Fink, G. ; Collemare, J.A.R. ; Roger, Y. ; Steinberg, G. - \ 2011
Molecular Biology of the Cell 22 (2011)19. - ISSN 1059-1524 - p. 3645 - 3657.
fungus ustilago-maydis - tug-of-war - lipid-droplet transport - cytoplasmic dynein - molecular motors - intracellular-transport - vesicle transport - cargo transport - caenorhabditis-elegans - polarity orientation
The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to the growing cell tip (anterograde). Occasionally, EEs run up to 90 mu m in one direction. The underlying MT array consists of unipolar MTs at both cell ends and antipolar bundles in the middle region of the cell. Cytoplasmic MT-organizing centers, labeled with gamma-tubulin ring complex protein, are distributed along the antipolar MTs but are absent from the unipolar regions. Dynein colocalizes with EEs for 10-20 mu m after they have left the cell tip. Inactivation of temperature-sensitive dynein abolishes EE motility within the unipolar MT array, whereas long-range motility is not impaired. In contrast, kinesin-3 is continuously present, and its inactivation stops long-range EE motility. This indicates that both motors participate in EE motility, with dynein transporting the organelles through the unipolar MT array near the cell ends, and kinesin-3 taking over at the beginning of the medial antipolar MT array. The cooperation of both motors mediates EE movements over the length of the entire cell.
Hydrodynamic flow caused by active transport along cytoskeletal elements
Houtman, D. ; Pagonabarraga, I. ; Lowe, C.P. ; Esseling-Ozdoba, A. ; Emons, A.M.C. ; Eiser, E. - \ 2007
Europhysics Letters 78 (2007)1. - ISSN 0295-5075 - p. 1 - 5.
We develop a simple lattice model to describe the hydrodynamic influence of active mass transport along bio-filaments on freely diffusing mass in the cell. To quantify the overall mass transport we include Brownian motion, excluded volume interactions, active transport along the filaments, and hydrodynamic interactions. The model shows that the hydrodynamic forces induced by molecular motors attached to the filaments give rise to a non-negligible flux close to the filament. This additional flux appears to have two effects. Depending on the degree of filament occupation it can exert a sufficiently large influence on unbound motors and cargo to modify their transport and also regulate the flux of motors bound to the filament. We expect such a mechanism is important in situations found in plant cells, where directional transport spans the entire cell. In particular, it can explain the cytoplasmic streaming observed in plant cells.