Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

Abstract

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

The laminin-keratin link shields the nucleus from mechanical deformation and signalling

“…4m). Upon consecutive cycles of bead oscillations, we recorded a gradual increase in the mechanical resistance of the cytoskeleton, as quantified by the complex shear modulus G* 66 (Fig. 4n).…”

“…The complex shear modulus G* was measured for each oscillation cycle using the Micro-rheology routine of the LightACE, the control software of the optical tweezers instrument. For the computation of G*, the force was determined by means of the calibration-free "momentum method" 66 and the particle position was obtained using the measured stiffness of the trap.…”

Active microrheology with a single, time-shared laser trap

“…Thus, the accuracy of our method strictly depends on the ability to generate two identical traps with the same stiffness, k (pN/V), from a single laser source, as well as the bandwidth of the measurement of the relative voltage change (see Supplementary Text). This is straightforward in a set-up modulated with fast-scanning acusto-optic deflectors (AODs) [28, 29] and equipped with direct light momentum force detection [23]. In such configuration (Fig.…”

Volumetric imaging of fast cellular dynamics with deep learning enhanced bioluminescence microscopy

“…To generate bioluminescent Zebrafish embryos, 100 pg of lyn::OeNL mRNA was injected in 1-cell stage embryos to visualize the plasma membrane. Approximately two hours post-fertilization (2 hpf) at the 64-cell stage, embryos were dechorionated manually using a pair of forceps 74 and incubated in 400 μM fluorofurimazine (FFz, Promega) for 2 h. At sphere embryonic stage (4 hpf), embryos were mounted in a 35 mm No. 1 glass bottom μ -Dish (Cellvis) using 1% low melting point agarose (Promega) in Danieauś solution (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO 4 , 0.6 mM Ca(NO 3 ) 2 , and 5 mM HEPES).…”