A MEC-2/stomatin condensate liquid-to-solid phase transition controls neuronal mechanotransduction during touch sensing.

A growing body of work suggests that the material properties of biomolecular condensates ensuing from liquid-liquid phase separation change with time. How this aging process is controlled and whether the condensates with distinct material properties can have different biological functions is currently unknown. Using Caenorhabditis elegans as a model, we show that MEC-2/stomatin undergoes a rigidity phase transition from fluid-like to solid-like condensates that facilitate transport and mechanotransduction, respectively. This switch is triggered by the interaction between the SH3 domain of UNC-89 (titin/obscurin) and MEC-2. We suggest that this rigidity phase transition has a physiological role in frequency-dependent force transmission in mechanosensitive neurons during body wall touch. Our data demonstrate a function for the liquid and solid phases of MEC-2/stomatin condensates in facilitating transport or mechanotransduction, and a previously unidentified role for titin homologues in neurons.

Axonal plasticity in response to active forces generated through magnetic nano-pulling.

Mechanical force is crucial in guiding axon outgrowth before and after synapse formation. This process is referred to as "stretch growth." However, how neurons transduce mechanical input into signaling pathways remains poorly understood. Another open question is how stretch growth is coupled in time with the intercalated addition of new mass along the entire axon. Here, we demonstrate that active mechanical force generated by magnetic nano-pulling induces remodeling of the axonal cytoskeleton. Specifically, the increase in the axonal density of microtubules induced by nano-pulling leads to an accumulation of organelles and signaling vesicles, which, in turn, promotes local translation by increasing the probability of assembly of the "translation factories." Modulation of axonal transport and local translation sustains enhanced axon outgrowth and synapse maturation.

Exploring cell and tissue mechanics with optical tweezers.

Cellular and tissue biosystems emerge from the assembly of their constituent molecules and obtain a set of specific material properties. To measure these properties and understand how they influence cellular function is a central goal of mechanobiology. From a bottoms-up, physics or engineering point-of-view, such systems are a composition of basic mechanical elements. However, the sheer number and dynamic complexity of them, including active molecular machines and their emergent properties, makes it currently intractable to calculate how biosystems respond to forces. Because many diseases result from an aberrant mechanotransduction, it is thus essential to measure this response. Recent advances in the technology of optical tweezers have broadened their scope from single-molecule applications to measurements inside complex cellular environments, even within tissues and animals. Here, we summarize the basic optical trapping principles, implementations and calibration procedures that enable force measurements using optical tweezers directly inside cells of living animals, in combination with complementary techniques. We review their versatility to manipulate subcellular organelles and measure cellular frequency-dependent mechanics in the piconewton force range from microseconds to hours. As an outlook, we address future challenges to fully unlock the potential of optical tweezers for mechanobiology.

An asymmetric mechanical code ciphers curvature-dependent proprioceptor activity.

A repetitive gait cycle is an archetypical component within the behavioral repertoire of many animals including humans. It originates from mechanical feedback within proprioceptors to adjust the motor program during locomotion and thus leads to a periodic orbit in a low-dimensional space. Here, we investigate the mechanics, molecules, and neurons responsible for proprioception in Caenorhabditis elegans to gain insight into how mechanosensation shapes the orbital trajectory to a well-defined limit cycle. We used genome editing, force spectroscopy, and multiscale modeling and found that alternating tension and compression with the spectrin network of a single proprioceptor encodes body posture and informs TRP-4/NOMPC and TWK-16/TREK2 homologs of mechanosensitive ion channels during locomotion. In contrast to a widely accepted model of proprioceptive "stretch" reception, we found that proprioceptors activated locally under compressive stresses in-vivo and in-vitro and propose that this property leads to compartmentalized activity within long axons delimited by curvature-dependent mechanical stresses.

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers.

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.

Positioning Accuracy in Holographic Optical Traps.

Spatial light modulators (SLMs) have been widely used to achieve dynamic control of optical traps. Often, holographic optical tweezers have been presumed to provide nanometer or sub-nanometer positioning accuracy. It is known that some features concerning the digitalized structure of SLMs cause a loss in steering efficiency of the optical trap, but their effect on trap positioning accuracy has been scarcely analyzed. On the one hand, the SLM look-up-table, which we found to depend on laser power, produces positioning deviations when the trap is moved at the micron scale. On the other hand, phase quantization, which makes linear phase gratings become phase staircase profiles, leads to unexpected local errors in the steering angle. We have tracked optically trapped microspheres with sub-nanometer accuracy to study the effects on trap positioning, which can be as high as 2 nm in certain cases. We have also implemented a correction strategy that enabled the reduction of errors down to 0.3 nm.

The nucleus measures shape changes for cellular proprioception to control dynamic cell behavior.

The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within tissues remains largely unknown. Here, using a zebrafish model, we show that the nucleus, the biggest cellular organelle, functions as an elastic deformation gauge that enables cells to measure cell shape deformations. Inner nuclear membrane unfolding upon nucleus stretching provides physical information on cellular shape changes and adaptively activates a calcium-dependent mechanotransduction pathway, controlling actomyosin contractility and migration plasticity. Our data support that the nucleus establishes a functional module for cellular proprioception that enables cells to sense shape variations for adapting cellular behavior to their microenvironment.