Increasing antibiotic resistance in bacteria is a critical issue that often leads to infections or other morbidities. Mechanical properties of the bacterial cell wall, such as thickness or elastic modulus, may contribute to the ability of a bacterial cell to resist antibiotics. Techniques like atomic force microscopy (AFM) are used to quantify bacterial cell mechanical properties and image cell structures at nanoscale resolutions. An additional benefit of AFM is the ability to probe samples submerged in liquids, meaning that live bacteria can be imaged or evaluated in environments that more accurately simulate in vivo conditions as compared to other methods like electron microscopy. However, because AFM measurements are highly sensitive to small perturbations in the deflection of the tip of a sensor probe brought into contact with the specimen, immobilization of bacteria prior to measurement is essential for accurate measurements. Traditional chemical fixatives crosslink the molecules within the bacterial cell wall, which prevent the bacteria from locomotion. While effective for imaging, chemical crosslinkers are known to affect the measured stiffness of eukaryotic cells and also may affect the measured stiffness of the bacterial cell wall. Alternative immobilization methods include Cell-Tak™, an adhesive derived from marine mussels that does not interact with the bacterial wall and filters with known pore sizes which entrap bacteria. Previous studies have examined the effect of these immobilization methods on successful imaging of bacteria but have not addressed differences in measured modulus. This study compares the effects of immobilization methods including chemical fixatives, mechanical entrapment in filters, and Cell-Tak™ on the stiffness of the bacterial cell wall as measured by force spectroscopy.
more »
« less
Mechanical expansion microscopy
This chapter describes two mechanical expansion microscopy methods with accompanying step-by-step protocols. The first method, mechanically resolved expansion microscopy, uses non-uniform expansion of partially digested samples to provide the imaging contrast that resolves local mechanical properties. Examining bacterial cell wall with this method, we are able to distinguish bacterial species in mixed populations based on their distinct cell wall rigidity and detect cell wall damage caused by various physiological and chemical perturbations. The second method is mechanically locked expansion microscopy, in which we use a mechanically stable gel network to prevent the original polyacrylate network from shrinking in ionic buffers. This method allows us to use anti-photobleaching buffers in expansion microscopy, enabling detection of novel ultra-structures under the optical diffraction limit through super-resolution single molecule localization microscopy on bacterial cells and whole-mount immunofluorescence imaging in thick animal tissues. We also discuss potential applications and assess future directions.
more »
« less
- Award ID(s):
- 1923534
- PAR ID:
- 10310706
- Editor(s):
- Guichard, P.; Hamel, V.
- Date Published:
- Journal Name:
- Methods in cell biology
- Volume:
- 161
- ISSN:
- 0091-679X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
The spongy mesophyll is a complex, porous tissue found in plant leaves that enables carbon capture and provides mechanical stability. Unlike many other biological tissues, which remain confluent throughout development, the spongy mesophyll must develop from an initially confluent tissue into a tortuous network of cells with a large proportion of intercellular airspace. How the airspace in the spongy mesophyll develops while the tissue remains mechanically stable is unknown. Here, we use computer simulations of deformable polygons to develop a purely mechanical model for the development of the spongy mesophyll tissue. By stipulating that cell wall growth and remodelling occurs only near void space, our computational model is able to recapitulate spongy mesophyll development observed inArabidopsis thalianaleaves. We find that robust generation of pore space in the spongy mesophyll requires a balance of cell growth, adhesion, stiffness and tissue pressure to ensure cell networks become porous yet maintain mechanical stability. The success of this mechanical model of morphogenesis suggests that simple physical principles can coordinate and drive the development of complex plant tissues like the spongy mesophyll.more » « less
-
Abstract Background and AimsEndosidins are a group of low-molecular-weight compounds, first identified by ‘chemical biology’ screening assays, that have been used to target specific components of the endomembrane system. In this study, we employed multiple microscopy-based screening techniques to elucidate the effects of endosidin 5 (ES5) on the Golgi apparatus and the secretion of extracellular matrix (ECM) components in Penium margaritaceum. These effects were compared with those caused by treatments with brefeldin A and concanamycin A. Penium margaritaceum’s extensive Golgi apparatus and endomembrane system make it an outstanding model organism for screening changes to the endomembrane system. Here we detail changes to the Golgi apparatus and secretion of ECM material caused by ES5. MethodsChanges to extracellular polymeric substance (EPS) secretion and cell wall expansion were screened using fluorescence microscopy. Confocal laser scanning microscopy and transmission electron microscopy were used to assess changes to the Golgi apparatus, the cell wall and the vesicular network. Electron tomography was also performed to detail the changes to the Golgi apparatus. Key ResultsWhile other endosidins were able to impact EPS secretion and cell wall expansion, only ES5 completely inhibited EPS secretion and cell wall expansion over 24 h. Short treatments of ES5 resulted in displacement of the Golgi bodies from their typical linear alignment. The number of cisternae decreased per Golgi stack and trans face cisternae in-curled to form distinct elongate circular profiles. Longer treatment resulted in a transformation of the Golgi body to an irregular aggregate of cisternae. These alterations could be reversed by removal of ES5 and returning cells to culture. ConclusionsES5 alters secretion of ECM material in Penium by affecting the Golgi apparatus and does so in a markedly different way from other endomembrane inhibitors such as brefeldin A and concanamycin A.more » « less
-
One of the central problems in animal and plant developmental biology is deciphering how chemical and mechanical signals interact within a tissue to produce organs of defined size, shape, and function. Cell walls in plants impose a unique constraint on cell expansion since cells are under turgor pressure and do not move relative to one another. Cell wall extensibility and constantly changing distribution of stress on the wall are mechanical properties that vary between individual cells and contribute to rates of expansion and orientation of cell division. How exactly cell wall mechanical properties influence cell behavior is still largely unknown. To address this problem, a novel, subcellular element computational model of growth of stem cells within the multilayered shoot apical meristem (SAM) of Arabidopsis thaliana is developed and calibrated using experimental data. Novel features of the model include separate, detailed descriptions of cell wall extensibility and mechanical stiffness, deformation of the middle lamella, and increase in cytoplasmic pressure generating internal turgor pressure. The model is used to test novel hypothesized mechanisms of formation of the shape and structure of the growing, multilayeredSAMbased onWUSconcentration of individual cells controlling cell growth rates and layer-dependent anisotropic mechanical properties of subcellular components of individual cells determining anisotropic cell expansion directions. Model simulations also provide a detailed prediction of distribution of stresses in the growing tissue which can be tested in future experiments.more » « less
-
Mechanical bonds arise between molecules that contain interlocked subunits, such as one macrocycle threaded through another. Within polymers, these linkages will confer distinctive mechanical properties and other emergent behaviors, but polymerizations that form mechanical bonds efficiently and use simple monomeric building blocks are rare. In this work, we introduce a solid-state polymerization in which one monomer infiltrates crystals of another to form a macrocycle and mechanical bond at each repeat unit of a two-dimensional (2D) polymer. This mechanically interlocked 2D polymer is formed as a layered solid that is readily exfoliated in common organic solvents, enabling spectroscopic characterization and atomic-resolution imaging using advanced electron microscopy techniques. The 2D mechanically interlocked polymer is easily prepared on multigram scales, which, along with its solution processibility, enables the facile fabrication of composite fibers with Ultem that exhibit enhanced stiffness and strength.more » « less