Cytoskeletal-assisted dynamics of the mitochondrial reticulum in living cells

Abstract

Subcellular organelle dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins, and lead to complex multiexponential relaxations that occur over a wide range of spatial and temporal scales. Here we report spatio-temporal measurements of the fluctuations of the mitochondrial reticulum in osteosarcoma cells by using Fourier imaging correlation spectroscopy, over time and distance scales of 10(-2) to 10(3) s and 0.5-2.5 microm. We show that the method allows a more complete description of mitochondrial dynamics, through the time- and length-scale-dependent collective diffusion coefficient D(k,tau), than available by other means. Addition of either nocodazole to disrupt microtubules or cytochalasin D to disassemble microfilaments simplifies the intermediate scattering function. When both drugs are used, the reticulum morphology of mitochondria is retained even though the cytoskeletal elements have been de-polymerized. The dynamics of the organelle are then primarily diffusive and can be modeled as a collection of friction points interconnected by elastic springs. This study quantitatively characterizes organelle dynamics in terms of collective cytoskeletal interactions in living cells.

Fig 1.

(A) Schematic of the FICS experimental geometry. Fluorescently labeled filaments are represented as N interconnected disks and the excitation grating as gray-scale bars. The filament cross-sectional diameter is ≈0.5 μm, whereas the grating fringe spacing, d_G, is adjusted between ≈0.56 and 2.6 μm. Signal fluctuations occur when regions of the reticulum separated by the distance d_G move relative to one another. (B) At any instant, a static particle configuration is uniquely described by a sum of vectors in the complex plane whose superposition is Ĉ = (^½ ∑ A_n exp[ik_G⋅r_n(t)] ≡ |Ĉ|exp[iα] with amplitude |Ĉ| and phase α.

Fig 2.

(A) Schematic of the actively stabilized FICS apparatus. An interference fringe pattern is produced in the sample plane of a fluorescence microscope by two coincident laser beams. One beam passes through an electro-optics phase modulator (Conoptics, Danbury, CT). A frequency generator (Keithley) is used to modulate the phase of the excitation grating from 0 to 2π at the angular frequency ω_G (=50,000 rad/s). Both the modulated fluorescence and transmitted excitation signal are collected with a fused-silica oil-immersion objective (Leica, Plan Fluotar, ×100, numerical aperture = 1.3). A dichroic beam-splitter (Chroma Technology, Brattleboro, VT) reflects the excitation (532 nm) and transmits the emission (583 nm; transmission efficiency 93%). The emission is passed through a long-pass interference filter (Omega, cutoff wavelength 570 nm, transmission efficiency 80% at 583 nm) and an excitation barrier filter. The filtered signal is imaged onto a thermoelectrically cooled photomultiplier tube (PMT, Hamamatsu, R1527) operating in current mode. The PMT output is detected by using a digital dual-phase lock-in amplifier (Stanford Research Systems, SR830) that is referenced to the RF-generator used to drive the phase-modulator. A computer, which controls an analog-to-digital data acquisition board (National Instruments), records separately the average background fluorescence intensity, κI₀Ĉ(0), the complex components of the demodulated signal, ReĈ and ImĈ, and the RPE. The image of the excitation grating is passed through a Ronche-ruling, and tightly focused onto a small-area avalanche photo-diode (APD, Pacific Silicon Sensor, Westlake Village, CA). The APD output is measured by using a phase-sensitive detector referenced to the RF-generator. A type I servo is used to generate a feedback signal (delivered to a Piezo-mounted mirror) to minimize the RPE between the excitation grating and the reference waveform. (B) Typical time course for the RPE with feedback engaged (red, rms phase-error = 3.9 nm) or disengaged (black, rms phase-error = 288 nm).

Fig 3.

Intermediate scattering functions, f(k_G,τ), for mitochondria in live osteosarcoma cells under different cytoskeletal conditions. The time-axis is plotted in reduced time units, τ₀ = [k_G²D₀]⁻¹ where D₀ = 1.8 × 10⁻⁴ μm²⋅s⁻¹. The Insets show typical specimens on which FICS experiments were conducted. (A) Control physiological conditions. (B) Depolymerization of microtubules using Nocodazole. (C) Depolymerization of microfilaments using Cytochalasin D. (D) Depolymerization of both microtubules and microfilaments. In all panels, the dotted curves show the expected decay of a purely diffusive system with f(k_G,τ) = exp[−k_G²D₀τ]. The symbols (× ≡ k_G = 7.8 μm⁻¹, ⋄ ≡ k_G = 3.9 μm⁻¹) indicate the Rouse fit with N = 100, b = 5.4 μm, and R_g = b(N/6)^1/2 = 22 μm.