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.

Abstract-Magnetic particles were given to macrophage-like cultured cells J774A.1 to perform cytomagnetometry in which the magnetic field from the cells was measured and the mechanical parameters of the cytoplasm and the energy E r of the intracellular movement were estimated. The response of the cells to ATP-synthesis inhibitor MIA measured by cytomagnetometry was intriguing; the energy E r became larger contrary to our expectation. We have been searching for the reason for this phenomenon by the immunofluorescent microscopy and the use of other drugs and cell types. The results have yielded some clue to the question, which we discuss in the present paper. I. INTRODUCTIONCytomagnetometry has been used to investigate mechanical properties of the cytoplasm for nearly 20 years. It was first conjectured by Nemoto[1] that the relaxation phenomenon observed in the magnetic field from the human lung which inhaled magnetic dust was due to the intracellular movements of the alveolar macrophages in the lung which randomized the direction of the magnetic moments of the particles within the cells which had been magnetized by an external magnetic field immediately before the relaxation measurement. The energy responsible for the relaxation process, which we designate as E r was also estimated by the method of secondary magnetization of the lung. Later, macrophages in vitro were used and it was shown that the relaxation process was indeed due to the intracellular movements within cells [2][3][4]. Since then several researchers have used this technique for studying some aspects of the biomechanics of cells [5][6][7][8][9][10][11]. Of recent interest is the study by Wang et al. [8] on the transduction of mechanical stimuli on the outer surface of the cell to the inner cytoskeletal structure.In [1] the measurement method of E r was proposed based on the simplest model in which a magnetic particle goes under rotational Brownian motion which is impeded by apparent viscosity of the cytoplasm. This model seemed to be enough for estimating E r although other experimental results showed that phagosomes containing the particles experience elasticity as well as viscosity and the randomizing force.Models have been proposed for cytomagnetometry. The first model in [1] consisted of E r and apparent viscosity η. Viscoelastic components were added to the model and the behavior of the model was examined in detail in [10]. Experimental data were treated with the viscoelastic model but in a somewhat simpler way in [11]. A different model using viscoelasticity was proposed but not discussed here.In the present paper, we present our recent result with J774A.1 cells which showed intriguing behavior of E r when MIA was added. MIA is known to inhibit the glycolysis pathway in the ATP synthesis in cells. In the present paper, we present the results of the cytomagnetometric measurements and the microscopic observations of the cytoskeleton to discuss the strange behavior of E r . II. MODEL 1) Energy E r Responsible for Relaxation:Cytomagnetometry is p...