Pulling, failing, and adaptive mechanotransduction of macrophage filopodia.

Macrophages use filopodia to withdraw particles toward the cell body for phagocytosis. This can require substantial forces, which the cell generates after bio-mechanical stimuli are transmitted to the filopodium. Adaptation mechanisms to mechanical stimuli are essential for cells, but can a cell iteratively improve filopodia pulling? If so, the underlying mechanic adaptation principles organized on the protein level are unclear. Here, we tackle this problem using optically trapped 1 µm beads, which we tracked interferometrically at 1 MHz during connection to the tips of dorsal filopodia of macrophages. We observe repetitive failures while the filopodium tries to pull the bead out of the optical trap. Analyses of mean bead motions and position fluctuations on the nano-meter and microsecond scale indicate mechanical ruptures caused by a force-dependent actin-membrane connection. We found that beads are retracted three times slower under any load between 5 and 40 pN relative to the no-load transport, which has the same speed as the actin retrograde flow obtained from fluorescent speckle tracking. From this duty ratio of pulling velocities, we estimated a continuous on/off binding with τoff = 2⋅τon, with measured off times τoff = 0.1-0.5 s. Remarkably, we see a gradual increase of filopodia pulling forces from 10 to 30 pN over time and after failures, which points toward an unknown adaptation mechanism. Additionally, we see that the attachment strength and friction between the bead and filopodium tip increases under load and over time. All observations are typical for catch-bond proteins such as integrin-talin complexes. We present a mechanistic picture of adaptive mechanotransduction, which formed by the help of mathematical models for repetitive tip ruptures and reconnections. The analytic mathematical model and the stochastic computer simulations, both based on catch-bond lifetimes, confirmed our measurements. Such catch-bond characteristics could also be important for other immune cells taking up counteracting pathogens.

100 Hz ROCS microscopy correlated with fluorescence reveals cellular dynamics on different spatiotemporal scales.

Fluorescence techniques dominate the field of live-cell microscopy, but bleaching and motion blur from too long integration times limit dynamic investigations of small objects. High contrast, label-free life-cell imaging of thousands of acquisitions at 160 nm resolution and 100 Hz is possible by Rotating Coherent Scattering (ROCS) microscopy, where intensity speckle patterns from all azimuthal illumination directions are added up within 10 ms. In combination with fluorescence, we demonstrate the performance of improved Total Internal Reflection (TIR)-ROCS with variable illumination including timescale decomposition and activity mapping at five different examples: millisecond reorganization of macrophage actin cortex structures, fast degranulation and pore opening in mast cells, nanotube dynamics between cardiomyocytes and fibroblasts, thermal noise driven binding behavior of virus-sized particles at cells, and, bacterial lectin dynamics at the cortex of lung cells. Using analysis methods we present here, we decipher how motion blur hides cellular structures and how slow structure motions cover decisive fast motions.

Miniature scanning light-sheet illumination implemented in a conventional microscope.

Living cells are highly dynamic systems responding to a large variety of biochemical and mechanical stimuli over minutes, which are well controlled by e.g. optical tweezers. However, live cell investigation through fluorescence microscopy is usually limited not only by the spatial and temporal imaging resolution but also by fluorophore bleaching. Therefore, we designed a miniature light-sheet illumination system that is implemented in a conventional inverted microscope equipped with optical tweezers and interferometric tracking to capture 3D images of living macrophages at reduced bleaching. The horizontal light-sheet is generated with a 0.12 mm small cantilevered mirror placed at 45° to the detection axis. The objective launched illumination beam is reflected by the micro-mirror and illuminates the sample perpendicular to the detection axis. Lateral and axial scanning of both Gaussian and Bessel beams, together with an electrically tunable lens for fast focusing, enables rapid 3D image capture without moving the sample or the objective lens. Using scanned Bessel beams and line-confocal detection, an average axial resolution of 0.8 µm together with a 10-15 fold improved image contrast is achieved.