Magnetic Phagosome Motion in J774A.1 Macrophages: Influence of Cytoskeletal Drugs

Abstract

The role of the different cytoskeletal structures like microfilaments (MF), microtubuli (MT), and intermediate filaments (IF) in phagosome motion is unclear. These cytoskeletal units play an important role in macrophage function (migration, phagocytosis, phagosome transport). We investigated ferromagnetic phagosome motions by cell magnetometry. J774A.1 macrophages were incubated with 1.3-microm spherical magnetite particles for 24 h, after which more than 90% of the particles had been phagocytized. Phagosome motions can be caused either by the cell itself (relaxation) or by applying magnetic twisting forces, yielding cell stiffness and viscoelastic properties of the cytoskeleton. Apparent viscosity of the cytoplasm was non-Newtonian and showed a shear-rate-dependent power law behavior. Elastically stored energy does not force the magnetic phagosomes back to their initial orientation: 57% of the twisting shear was not recoverable. Cytoskeletal drugs, like Cytochalasin D (CyD, 2 - 4 microM), Colchicine (CoL, 10 microM), or Acrylamide (AcL, 40 mM) were added in order to disturb the different cytoskeletal structures. AcL disintegrates IF, but affected neither stochastic (relaxation) nor directed phagosome motions. CyD disrupts MF, resulting in a retarded stochastic phagosome motion (relative decay 0.53 +/- 0.01 after 5 min versus 0.34 +/- 0.01 in control), whereas phagosome twisting shows only a small response with a 9% increase of stiffness and a small reduction of recoverable strain. CoL depolymerizes the MT, inducing a moderately accelerated relaxation (relative decay 0.28 +/- 0.01 after 5 min) and a 10% increase of cell stiffness, where the pure viscous shear is increased and the viscoelastic recoil is inhibited by 40%. Combining the two drugs conserves both effects. After disintegrating either MF or MT, phagosome motion and cytoskeletal stiffness reflect the behavior of either MT or MF, respectively. The results verify that the dominant phagosome transport mechanism is MF-associated. MT depolymerization by CoL induces an activation of the F-actin synthesis, which may induce an accelerated relaxation and an increase of stiffness. Cell mechanical properties are not modulated by MF depolymerization, whereas MT depolymerization causes a loss of viscous resistance and a loss of cell elasticity. The mean energy for stochastic phagosome transport is 5*10(-18) Joules and corresponds to a force of 7 pN on a single 1.3-microm phagosome.

Effects of zinc oxide nanoparticles on Kupffer cell phagosomal motility, bacterial clearance, and liver function

“…In previous magnetometric studies, magnetic relaxation was found to be the result of cytoskeletal machinery and ATP. 43,44 Principal components of the cytoskeleton, the actin microfilaments are highly susceptible to oxidative damage and resulting deformations, such as cross-linking, may be enhanced by the presence of ZnO ENPs or dissolved Zn ions. Our data also explored whether IV exposure to ZnO ENPs may impair Kupffer cell phagosomal motion and activity, and in turn may increase the risk of infection following bacteremia.…”

Overexpression of myosin IB in living Entamoeba histolytica enhances cytoplasm viscosity and reduces phagocytosis

“…In addition, the cytoplasm and the cell cortex have been previously described in vivo as viscoelastic gels (Stossel, 1993;Bausch et al, 1999;Moller et al, 2000;Feneberg et al, 2001) but the role of myosin molecules in the gel mechanical properties still remains unclear. Notably, nothing has been described concerning a role for unconventional myosins.…”

385 Ultrafine particles cause cytoskeletal dysfunctions in macrophages: Role of intracellular calcium

“…Cytotoxic reactions can involve the energy metabolism of the cell, intracellular calcium transients [13], the dynamics of cytoskeletal filaments and the transport mechanisms of phagosomes [41,42]. A previous study has shown that destroying microfilaments by cytochalasin D resulted in a retarded relaxation and an increased stiffness [37,53]. Colchicine, which disrupts…”

“…We have developed protocols to study cytoskeleton associated functions in macrophages in vivo and in vitro, such as phagosome transport, mechanical integrity (viscoelastic properties, stiffness) and phagocytosis, using ferromagnetic microparticles [36][37][38]. In in vitro studies the magnetic microparticles (1.8 µm diameter) are incubated for 24 hours together with cultivated macrophages, after which more than 95% are ingested.…”