Overlapping and essential roles for molecular and mechanical mechanisms in mycobacterial cell division

Mechanisms to control cell division are essential for cell proliferation and survival. Bacterial cell growth and division require the coordinated activity of peptidoglycan synthases and hydrolytic enzymes to maintain mechanical integrity of the cell wall. Recent studies suggest that cell separation is governed by mechanical forces. How mechanical forces interact with molecular mechanisms to control bacterial cell division in space and time is poorly understood. Here we use a combination of atomic force microscope imaging, nanomechanical mapping and nanomanipulation to show that enzymatic activity and mechanical forces serve overlapping and essential roles in mycobacterial cell division. We find that mechanical stress gradually accumulates in the cell wall, concentrated at the future division site, culminating in rapid (millisecond) cleavage of nascent sibling cells. Inhibiting cell wall hydrolysis delays cleavage; conversely, locally increasing cell wall stress causes instantaneous and premature cleavage. Cells deficient in peptidoglycan hydrolytic activity fail to locally decrease their cell wall strength and undergo natural cleavage, instead forming chains of non-growing cells. Cleavage of these cells can be mechanically induced by local application of stress with an atomic force microscope. These findings establish a direct link between actively controlled molecular mechanisms and passively controlled mechanical forces in bacterial cell division.

Mechanical and Functional Study of Cell Physiology at the Nanoscale

Pascal Damian Odermatt

By live-cell imaging of biological samples dynamic cellular processes can be resolved. Fluorescence microscopy (FM) and atomic force microscopy (AFM) are both capable of imaging live cells. By combining these techniques structural as well as functional information of the same biological samples are measured. While the dynamics of specific proteins can be imaged by FM, the AFM provides the structural context. To study how intracellular processes affect the mechanics, structure and kinetics of cells at the nanoscale we combined a high-speed AFM (HS-AFM) and an inverted optical microscope (OM) into one instrument. With this system cell physiological processes at different time scales are imaged. Mammalian cell migration happening at minute time scales is studied by correlated super-resolution microscopy and HS-AFM. Bacterial cell separation happening within 10s of milliseconds is characterized as well as bacterial growth over multiple generations. Additionally to imaging, the capability of the AFM to quantitatively measure mechanical properties of tissues is deployed in combination with FM on mouse corneal tissues. Different to most other rod-shaped bacteria where division happens through constriction of the cell wall at midcell, mycobacteria keep the shape of their outer cell envelope unchanged during septation. The septal wall is formed by peptidoglycan branching off from the outer cell envelop eventually splitting the cell into two sister compartments. Nanomechanical measurements show that from the initiation of septation the stiffness at the septum increases significantly. High-temporal resolution imaging of the following bacterial separation process indicated that the sibling cells separate within tens of milliseconds. The connection of the outer cell envelop at the former septum is broken apart suggesting a mechanical break. The timing of the cell separation is regulated by the tensile stress at the septum and the ultimate tensile strength of the cell wall material. The positions where these separations occur are closely associated with morphological features on the undulating cell surface. These features are formed at the poles and migrate towards midcell as the cell grows. Interestingly, sibling cell separation is restricted to the center-most wave-trough making these features the first characteristic associated with division site placement. Experiments with mutant strains show that even asymmetric divisions occur within wave-troughs emphasizing their role in division site placement. Chronic inflammation induces an increase in the stiffness of the corneal tissue and is associated with dramatic changes in the composition of the tissue. Stem cells migrating along this tissue layer of increased stiffness experience increased activation of mechanotransduction pathways. This eventually leads to induction of a skin-like rather than corneal-like character of these regenerating stem cells. It demonstrates that the stiffness of the tissue and thus pathways involved in mechanotransduction decisively influence the stem cell fate.

EPFL2016

Time-lapse high-resolution microscopy to study the morphogenesis of microorganisms

Mélanie Thérèse Marie Hannebelle

Mycobacterium tuberculosis, the etiological agent for the tuberculosis disease, is a bacterial pathogen thought to infect about a quarter of the global human population. It is the first cause of death among infectious diseases, and is most prevalent in low-income populations. Much remains unknown about how these bacteria grow, divide, and manage to persist in the host even during month-long antibiotic treatments. Bacteria are typically at the edge of the spatial resolution that conventional optical microscopy techniques can offer, and so new tools are required to study bacterial substructures or surface morphologies with nanometer resolution. We developed a platform for automated optical microscopy and atomic force microscopy (AFM) in order to study the morphogenesis of mycobacteria. What is the growth dynamics of mycobacteria? Opposite models coexist in the literature for the pole growth dynamics of mycobacteria: the unipolar model and the bipolar model. We show that the growth dynamics of mycobacteria follows a new end take off NETO model. There is a surprising similarity between the NETO growth dynamics of mycobacteria and of fission yeast, which hints at shared mechanisms for pole growth in evolutionarily distant pole growing organisms. How do pole-growing organisms maintain their shape through insertion of new cell wall material at the tip? To explore further pole growth morphogenesis, we developed a method for imaging with AFM the pole of a growing rod-shaped microorganism. We observed the formation of a stiff fibril mesh on the pole of fission yeast cells, and showed that this mesh is stretched as the cell grows and insert new cell wall material at the tip. How do mycobacteria divide into two sibling cells? Another essential aspect of morphogenesis of rod-shaped cell is the division of a mother cell into two sibling cells. Using AFM, we measured the relative contributions of mechanical forces and molecular mechanisms driving the division process in mycobacteria. We showed that there is a concentration of mechanical stress at the future division site, using a combination of COMSOL finite element modeling and AFM measurements while controlling the cell turgor pressure. The accumulation of mechanical stress, in combination of enzymatic activity, ultimately leads to mechanical fracture and separation of the sibling bacteria. Conventional optical microscopy techniques tend to have low resolution and high speed, while conventional AFMs have comparatively high resolution and low speed. We designed and built a 3D-printed structured illumination (SIM) module, the openSIM, as an add-on for fluorescence microscopes. It provides structured illumination to obtain SIM images with higher resolution. With the aim of improving the imaging speed of AFM, we implemented photothermal actuation in a tip-scanning AFM head, and quantified the design constraints for high-speed photothermal off-resonance tapping imaging. AFM is most often used to scan and acquire high-resolution images of a sample. Its nanometer precision make it a unique tool for other applications. We combined an AFM head with a volcano-shaped probe for electrogram measurements, and demonstrated simultaneous recording of extracellular field potentials and contraction of the cell. This new tool opens the way for future studies exploring the intriguing links between mechanobiology and electrophysiology, with single-cell resolution.

EPFL2020

Gradient of Rigidity in the Lamellipodia of Migrating Cells Revealed by Atomic Force Microscopy

Giovanni Dietler, Sandor Kasas, Jean-Jacques Meister, Alexandre Yersin

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity pro. le resembled closely the actin density pro. le, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.