Quantitatve assay of dissociated tissue cell motility in vitro

Summary A simple method for quantitating the motile properties of a dissociated tissue cell preparation is presented. A cell population is allowed to grow over a glass cover slip coated with Scarlet Red-containing Formvar. At confluence, the preparation is cut with a razor to remove a portion of the cells and the underlying pigmented Formvar and then returned to culture conditions. Using the cut edge as the starting line, cell motility can be easily measured. In this assay irradiated and nonirradiated 3T3 cells show similar motility characteristics over a 3-day observation period supporting the interpretation that the observed movement is due primarily to active cell motility rather than cell growth. This work was supported in part by American Cancer Society Grant IN-31-5-5.     

The electrical resistivity of cytoplasm.

The apparent cytoplasmic resistivity of two different giant cells has been measured using an extension of a previously developed single microelectrode technique. Each cell is penetrated by a metal microelectrode whose complex impedance is measured as a function of frequency between 500 kHz and 5.7 MHz. By plotting the measured impedance data on the complex Z plane and extrapolating the data to infinite frequency, the substantial effects of electrode polarization can be overcome. For Aplysia giant neurons and muscle fibers of the giant barnacle, the extrapolated cytoplasmic specific resistivities are 40 and 74 omega-cm, respectively, at infinite frequency. The barnacle data are in excellent agreement with sarcoplasmic resistivity values derived from the measured cable properties of other marine organisms, and from high frequency conductivity cell measurements in intact barnacle muscle tissue. In the Aplysia neurons, the frequency-dependent part of the electrode impedance is larger when the electrode is in a cell than when it is in an electrolyte solution with the same specific resistivity as the aqueous cytoplasm; however, the phase angle of the frequency-dependent component of the electrode impedance is the same in both cases. This suggests that the high apparent values of cytoplasmic resistivity found using the single microelectrode technique at lower frequencies probably reflect an artifact caused by reduction of the effective surface area of the electrode by intracellular membranes, with a corresponding increase in its polarization impedance.     

Effects of pH and ATP on the cytoplasm of Amoeba proteus: relation between cytoplasm motility and actin polymerization

The effect of pH and ATP was studied on isolated cytoplasm of Amoeba proteus. These two parameters were shown to influence both the motility and the organization of actin filaments in the isolated cytoplasm. Furthermore, our results demonstrate that there is a relationship between the motility and the polymeric state of actin. When the isolated cytoplasm is non-motile, actin is highly polymerized into long filaments arranged parallel in bundles. When this cytoplasm is motile, however, actin can either be weakly polymerized, i.e. observed as few short filaments, or can be polymerized in long branched filaments forming a loose network.     

Cell motility and thermodynamic fluctuations tailoring quantum mechanics for biology

Cell motility underlying muscle contraction is an instance of thermodynamics tailoring quantum mechanics for biology. Thermodynamics is intrinsically multi-agential in admitting energy consumers in the form of energy-deficient thermodynamic fluctuations. The onset of sliding movement of an actin filament on myosin molecules in the presence of ATP molecules to be hydrolyzed demonstrates that thermodynamic fluctuations transform their nature so as to accommodate themselves to energy transduction subject to the first law of thermodynamics. The transition from transversal to longitudinal fluctuations of an actin filament with the increase of ATP concentration coincides with the change in the nature of energy consumers acting upon thermal energy in the light of the first law, eventually embodying a uniform sliding movement of an actin filament.     

The structure of cortical cytoplasm

Actin-rich cortical cytoplasm of phagocytic leucocytes forms pseudopodia and controls cell shape and movement by generating directional propulsive and contractile forces. Proteins purified from leucocytes form and deform an actin matrix. Actin-binding protein (ABP) cross-links actin filaments into a three-dimensional lattice with perpendicular branches. This structure, which can be visualized in the electron microscope, is consistent with physical properties of actin-ABP matrices. Gelsolin binds one end of actin filaments with high affinity in the presence of calcium; acumentin, another protein, constitutively binds the other end with low affinity. Together these proteins can control actin filament length and thereby regulate expansion (propulsion) or collapse of the actin network. The assembly state of the network also controls myosin-based contractile forces. A tug-of-war decides the direction of lattice movement, regions of lesser structure tending to move toward regions of greater structure.     

Actin-dependent,retrograde motility of surface-attached beads and aggregating pigment granules in dissociated teleost retinal pigment epithelial cells

Teleost retinal pigment epithelial (RPE) cells contain pigment granules within apical projections which undergo actin-dependent, bi-directional motility. Dissociated RPE cells in culture attach to the substrate and extend apical projections in a radial array from the central cell body. Pigment granules within projections can be triggered to aggregate or disperse by the presence or absence of 1 mM cAMP. Aminated, fluorescent latex beads attached to the dorsal surface of apical projections and moved in the retrograde direction, towards the cell body. Bead rates on RPE cells with aggregating or fully aggregated pigment granules were 2.2 +/- 0.5 and 2.6 +/- 0.2 microm/min (mean +/- SEM), respectively, similar to rates of aggregating (retrograde) pigment granule movement (2.0 +/- 0.4 microm/min). Bead rates were slightly slower on cells with fully dispersed or dispersing pigment granules (1.5 +/- 0.1 and 1.5 +/- 0.4 microm/min). Movements of surface-attached beads and aggregating pigment granules were closely correlated in the distal portions of apical projections, but were more independent of each other in proximal regions of the projections. The actin disrupting drug, cytochalasin D (CD), reversibly halted retrograde bead movements, suggesting that motility of surface-attached particles is actin-dependent. In contrast, the microtubule depolymerizing drug, nocodazole, had no effect on retrograde bead motility. The similar characteristics and actin-dependence of retrograde bead movements and aggregating pigment granules suggest a correlation between these two processes.     

Oogenesis of Tilapia mossambica. III. The cytoplasm of previtellogenic oocytes]

Using methods of light and electron microscopy and of autoradiography, the morphology of cytoplasm in previtellogenic oocytes of tilapia mossambique was studied. Similar to other bony fishes, mitochondria at the early previtellogenic oocytes are mostly located in the perinuclear cytoplasm to be later distributed over the whole volume of growing oocytes. The Golgi complex is poorly developed. In the peripheral regions of the late previtellogenic oocytes, stickform mitochondria, pinocytotic vesicles and microvilli are observed, along with the perioocyte space formation. In the cytoplasm of previtellogenic oocytes polyribosomes appear. No differences in 3H-leucine incorporation intensity was noticed in oocytes of different previtellogenic stages. The characteristic feature of tilapia mossambique previtellogenic oocytes, in comparison with other bony fishes, is the presence of fat droplets in their cytoplasm.