A method for spatially resolved local intracellular mechanochemical sensing and organelle manipulation

Abstract

Because both the chemical and mechanical properties of living cells play crucial functional roles, there is a strong need for biophysical methods to address these properties simultaneously. Here we present a novel (to our knowledge) approach to measure local intracellular micromechanical and chemical properties using a hybrid magnetic chemical biosensor. We coupled a fluorescent dye, which serves as a chemical sensor, to a magnetic particle that is used for measurement of the viscoelastic environment by studying the response of the particle to magnetic force pulses. As a demonstration of the potential of this approach, we applied the method to study the process of phagocytosis, wherein cytoskeletal reorganization occurs in parallel with acidification of the phagosome. During this process, we measured the shear modulus and viscosity of the phagosomal environment concurrently with the phagosomal pH. We found that it is possible to manipulate phagocytosis by stalling the centripetal movement of the phagosome using magnetic force. Our results suggest that preventing centripetal phagosomal transport delays the onset of acidification. To our knowledge, this is the first report of manipulation of intracellular phagosomal transport without interfering with the underlying motor proteins or cytoskeletal network through biochemical methods.

Figure 1

(a) Actual trajectory of a magnetic particle internalized by a cell over a duration of 62 s. A magnetic pulse was applied every 30 s for a duration of 1 s. The successive magnetic pulses are labeled with symbols. Passive microrheology is carried out over the nonlabeled part of the trajectory (when the force is switched off), and active microrheology is carried out in the labeled part of the track (when the force is switched on). The start and end of the particle trajectory are marked in the figure. (b) MSD of the particle trajectory where the particle carries out Brownian motion. The MSD data were fitted with Eq. 1 (solid curve) to determine the diffusion coefficient. The diffusion coefficient calculated from the fit was found to be 1.74 × 10⁻⁴μm²/s. Only the first 10% of data points (0.4 s) were used for the fit. (c) Displacement response (circles), normalized by force, of a bead inside the macrophage cytoplasm as a result of the application of a 900 pN force step (dashed line) and a fit (solid line) to the Voigt-Maxwell model (Eq. 2). The fast elastic response followed by a slower viscous response can be clearly seen in the curve. The dotted lines indicate the slope of the viscous part of the curve, which is a measure of the damping coefficient γ₀ and the y-intercept, which is a measure of the spring constant k. Inset: Voigt-Maxwell model for a viscoelastic body. The model consists, in series, of a viscoelastic body (a dashpot characterized by damping coefficient γ₁ in parallel with a spring of spring constant k) with a dashpot characterized by damping coefficient γ₀.

Figure 2

Evolution of shear modulus (solid squares) and viscosity (open squares) in the immediate vicinity of the phagosome measured with the active microrheology approach, and the pH of the phagosome (open circles) as a function of time during phagosome acidification. Viscosity measured with passive microrheology (solid) is also plotted. A clear difference in viscosity between the active and passive methods (of 2 orders of magnitude) is seen.

Figure 3

(a–d) Location of a particle internalized (dotted circle) by a RAW 264.7 macrophage. Time-lapse images show the location of the phagosome in the absence and presence of an external force. The two frames are 10 min apart. Scale bar: 5 μm. (a and b) Normal transport of a phagosome from the plasma membrane toward the perinuclear region. (c and d) In the presence of an external force, the phagosome stays at the plasma membrane for the entire duration of experiment. The white arrow shows the direction of applied force. The two frames are 10 min apart. (e) An example of two individual pH curves, one each for normal transport (squares) and hindered phagosomal transport (circles). The particles were internalized at t = 0 min (or t₀). Onset of acidification for the phagosome with hindered transport is delayed by 20 min, compared with 5 min for the phagosome with normal centripetal transport. (f) Distribution of the time of onset of acidification for the two conditions: control experiment with normal phagosomal transport (circles, n = 15) and hindered phagosomal transport due to magnetic force (squares, n = 12). The flat line in the curves indicates the averages for the two conditions.

Figure 4

Repeated acidification cycles of a phagosome with hindered transport.