Hydrodynamic drag analysis of individual Drosophila centrosomes in physiological conditions. Two approaches were used: (A) Stokes law and (B) thermal-noise analysis. The plots further depict centrosome-neighborhood hydrodynamic size effects due the range of displacements used in each method (see the text for details). (C) Force versus velocity data for a centrosome in BRB80 buffer (blue dots) and a 2.10-μm-diameter polystyrene bead in DI water (red dots). Black dotted lines: Linear regressions. Inferred diameters for the centrosome and the 2.10-μm bead in this plot are 1.31 ± 0.01 and 2.10 ± 0.01 μm, respectively. (D) Power spectral density distribution of force fluctuations for a centrosome in BRB80 buffer (blue line) and a 2.10-μm-diameter polystyrene bead in DI water (red line). The units are volts squared per hertz (V²/Hz) since we measure a voltage proportional to the fluctuating force. Only fluctuations in the horizontal direction were measured. Black curves: Fittings to the power spectral density function described in the Materials and Methods section. At both low and high frequencies, electronic noise may add to the thermal fluctuations, causing deviations from the expected spectrum. The corner frequencies for the centrosome and the 2.10-μm bead in this plot are 693 ± 13 Hz and 1795 ± 16 Hz, respectively, and the corresponding diameters are 0.87 ± 0.01 and 2.14 ± 0.01 μm. These measurements were taken by the dual-beam laser tweezers, with each diode-laser running at 200 mW and wavelength 835 nm.

Electric force over single centrosomes at different pH values. (A) Experimental configuration: A single centrosome held in the optical trap is displaced from the center of the common lasers' focal spot (red halo; note that the light path is perpendicular to the plane of the plot) due to the electric field, E, between the two gold-plated tungsten electrodes (yellow rods). The circuit configuration is such that a positive force (F_electric) toward the anode implies a negatively charged specimen. (B) Centrosomes (left column) and streptavidin-coated, 2.10-μm-diameter beads (right column) were subjected to constant-voltage pulses of ∼1 s. Note that force strengths depend not only on the pH, but also on both the ionic strength and mobility of the buffering salt species for a given applied voltage. We used pulses of 3.5 V, except in DI water (3.9 V and 4.3 V for the experiments with centrosomes and beads, respectively), and except for the experiments with beads in TAE (3.1 V). Black lines: Low-pass filtered data to 1 Hz.

Hydrodynamic size of Drosophila centrosome in physiological conditions. Top row: Size distributions in BRB80 measured by (A) Stokes law (n = 97) and (B) thermal-noise analysis (n = 75). Bottom row: Size distributions in BRB80 with 5 mM CaCl₂ and no EGTA, measured by (C) Stokes law (n = 78) and (D) thermal-noise analysis (n = 91). Black lines in the histograms are Lorentzian fits. Size heterogeneity in the centrosome population may reflect either the progression of the nuclear cycles or the loss or gain of PCM throughout the centrosome purification procedure (26).

Effect of the pH on the centrosome size. Data points at each pH represent centrosome size distribution-mean values (measured by the thermal-noise method), and error bars are their corresponding full width at half-maximum. Note that different buffers have been used to cover the plotted range of pH (see Materials and Methods for details). The black lines are two independent linear regressions to the size data points corresponding to the two different electric behaviors of the centrosome with pH (q > 0 and q < 0). The red background indicates a negative-charge pH region (q < 0), and the blue background indicates a positive-charge pH region (q > 0). The vertical shaded bar delimits the isoelectric region. The horizontal dashed line marks the Drosophila centrosome size determined by EM (26).