Intracellular phagosome shell is rigid enough to transfer outside torque to the inner spherical particle

TL;DR

Novel roto-translational coupled dynamics is reported, and it is shown that this coupling manifests itself as sustained fluxes in phase space, a signature of broken detailed balance.

Abstract

Intracellular phagosomes have a lipid bilayer encapsulated fluidic shell outside the particle, on the outer side of which, molecular motors are attached. An optically trapped spherical birefringent particle phagosome provides an ideal platform to probe fluidity of the shell, as the inner particle is optically confined both in translation and in rotation. Using a recently reported method to calibrate the translation and pitch rotations - yielding a spatial resolution of about 2 nm and angular resolution of 0.1 degrees - we report novel roto-translational coupled dynamics. We also suggest a new technique where we explore the correlation between the translation and pitch rotation to study extent of activity. Given that a spherical birefringent particle phagosome is almost a sphere, the fact that it turns due to the activity of the motors is not obvious, even implying high rigidity of shell. Applying a minimal model for the roto-translational coupling, we further show that this coupling manifests itself as sustained fluxes in phase space, a signature of broken detailed balance.

Figures (14)

• Figure 1: Schematic for study of pitch rotation adn translation of phagosomes inside cell (a) The six degrees of freedom of an optically trapped sphere. (b) Schematic of experimental setup. (c) The sample chamber will cells attached to the top surface and are maintained within a layer of DMEM media. Phagocytosed beads are trapped with the focused beam. (d) The power spectral densities (PSDs) for X translation and pitch rotation of a phagocytosed bead in a cell fitted to eq. Obtained fitting parameter is used to calibrate the PSD. Panels (e) and (f) show oppositely directed motors pulling on a birefringent cargo. The darker shaded region indicate the plane of optic axis. Detachment of some motors and new attachments formed in (f) causes the cargo to move forward and rotate, shown by rotation of optic axis.
• Figure 2: Tug-of-war like motion demonstrated by phagosome. 4D trajectory of phagosome with (a) time shown in colour, (b) pitch angle shown in colour, (c) position of the bead inside cell captured with a bright field microscope. White arrows indicate the direction of to and fro motion (d) 2D trajectory in X-Y plane extracted from video tracking. (e) Time series for X and pitch for tracked with QPD with respective force and torque scale bars. (f) Corresponding MSD curves for X (blue open circle) and pitch (red open circle). (g) CCF of X and pitch rotation from experimental data (red curve) and fit to Eq.\ref{['eq:CCF']} (black curve). (h) Broken detailed balance is demonstrated by showing circulating current from the vector plot of the configuration current, see Eq.\ref{['eq:current']}. Using the parameters obtained by fit to the data from (g), we do a vector plot of the configuration current, see Eq.\ref{['eq:current']}, which is overlaid on the pseudo-color plot of the steady-state probability distribution, see Eq.\ref{['eq:probS']}. (i) Oppositely directed teams of motors pulling on a cargo along a cytoskeletal track causes the birefringent cargo to rotate. The optics axis shown as the black solid line indicates the orientation of the birefringent sphere. Right end directed team of motors pulls on it causing it to rotate in clockwise sense (1-2). The other team of motors take over to cause reversal in direction and rotation in anticlockwise direction (2-3 and 3-4).
• Figure 3: Trajectory of phagocytosed bead while encountering an obstacle (a) 4D trajectory, with the fourth dimension, pitch angle, shown as colour. The projections in XY and XZ plane are shown in grey. The sudden bump (marked with black arrow and within dashed circle in XY projection) indicate curving of trajectory around a possible obstacle which is accompanied by a large change in angle (in dark red). (b) Time series for simultaneous X (blue) and pitch (red) motion recorded with QPD with respective force and torque scale bars (c) MSD curve for X (blue open circle) and pitch (red open circle) with respective exponents shown as $\alpha_x$ and $\alpha_{pitch}$ indicating super diffusive and near diffusive behaviour respectively (d)bright field image of the cell showing position of the trapped phagocytosed bead enclosed in white dashed circle (e) CCF of pitch and X (in red) fitted to eq. (\ref{['eq:CCF']}) (in black line). (f) Using the fit to the data, we do a vector plot of the configuration current in phase space, see Eq.\ref{['eq:current']}, which is overlaid on the pseudo-color plot of the steady-state probability distribution, see Eq.\ref{['eq:probS']}. (g) cartoon showing a spherical cargo dragged over an obstacle by motors (1-3) with black dashed line showing the direction of optic axis.
• Figure 4: Change in plane of motion as point of attachment changes from horizontal to a vertical filament. (a) 4-dimensional trajectory of the PBB inside cell showing change in plane of trajectory from horizontal to vertical (shown within dashed ellipse) along with large change in pitch angle(in red) (b) X and pitch displacement recorded with QPD with respective force and torque scale bars (c) corresponding MSDs for pitch and X with exponents indicating near diffusive($\approx 1$) and super diffusive($>1$) behaviour respectively. (d) Position of the phagocytosed bead under examination (enclosed within white dashed circle) as seen under bright field. (e) CCF of pitch and X fitted to Eq. (\ref{['eq:CCF']}). (f) A vector plot of the configuration current, see Eq.\ref{['eq:current']}, which is overlaid on the pseudo-color plot of the steady-state probability distribution, see Eq.\ref{['eq:probS']}. This plot clearly shows circulating current, a signature of broken detailed balance in the system. (g) and (h) Change in pitch angle when point of contact of PBB shifts from a horizontally placed filament to a vertically placed filament
• Figure 5: Complete rotation of phagocytosed hexagonal NaYF$_4$ crystals under intracellular activity in absence of optical trap (A) Scanning Electron Microscopy(SEM) images of NaYF$_4$ crystals with hexagonal prism shape (b) The hexagonal faces are called the 'face on' sides and the four rectangular faces are called 'side on' sides (c) When a crystal in 'side on'(A in (a)) orientation undergoes a pitch rotation, it orients 'face on'(C in (a)) via an intermediate orientation(B) in (a). (d)-(f)Phagocytosed NaYF$_4$ crystal inside MCF7 cell observed under bright field microscopy. Time lapse of the particle within dashed orange rectangle arrow shows a 90 degree flipping from (d) to (f). The particular crystal is seen to change orientation from A to C via B indicating rotation by 90 degree