اشترك بالحزمة الذهبية واحصل على وصول غير محدود شمرا أكاديميا
تسجيل مستخدم جديدDirect measurement of cell wall stress-stiffening and turgor pressure in live bacterial cells
273
0
0.0
(
0
)
اسأل ChatGPT حول البحث
ﻻ يوجد ملخص باللغة العربية
We study intact and bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. We find strong evidence of power-law stress-stiffening in the E. coli cell wall, with an exponent of 1.22 pm 0.12, such that the wall is significantly stiffer in intact cells (E = 23 pm 8 MPa and 49 pm 20 MPa in the axial and circumferential directions) than in unpressurized sacculi. These measurements also indicate that the turgor pressure in living cells E. coli is 29 pm 3 kPa.
قيم البحث
اقرأ أيضاً
Bacterial processes ranging from gene expression to motility and biofilm formation are constantly challenged by internal and external noise. While the importance of stochastic fluctuations has been appreciated for chemotaxis, it is currently believed
that deterministic long-range fluid dynamical effects govern cell-cell and cell-surface scattering - the elementary events that lead to swarming and collective swimming in active suspensions and to the formation of biofilms. Here, we report the first direct measurements of the bacterial flow field generated by individual swimming Escherichia coli both far from and near to a solid surface. These experiments allowed us to examine the relative importance of fluid dynamics and rotational diffusion for bacteria. For cell-cell interactions it is shown that thermal and intrinsic stochasticity drown the effects of long-range fluid dynamics, implying that physical interactions between bacteria are determined by steric collisions and near-field lubrication forces. This dominance of short-range forces closely links collective motion in bacterial suspensions to self-organization in driven granular systems, assemblages of biofilaments, and animal flocks. For the scattering of bacteria with surfaces, long-range fluid dynamical interactions are also shown to be negligible before collisions; however, once the bacterium swims along the surface within a few microns after an aligning collision, hydrodynamic effects can contribute to the experimentally observed, long residence times. As these results are based on purely mechanical properties, they apply to a wide range of microorganisms.
Cell migration and mechanics are tightly regulated by the integrated activities of the various cytoskeletal networks. In cancer cells, cytoskeletal modulations have been implicated in the loss of tissue integrity, and acquisition of an invasive pheno
type. In epithelial cancers, for example, increased expression of the cytoskeletal filament protein vimentin correlates with metastatic potential. Nonetheless, the exact mechanism whereby vimentin affects cell motility remains poorly understood. In this study, we measured the effects of vimentin expression on the mechano-elastic and migratory properties of the highly invasive breast carcinoma cell line MDA231. We demonstrate here that vimentin stiffens cells and enhances cell migration in dense cultures, but exerts little or no effect on the migration of sparsely plated cells. These results suggest that cell-cell interactions play a key role in regulating cell migration, and coordinating cell movement in dense cultures. Our findings pave the way towards understanding the relationship between cell migration and mechanics, in a biologically relevant context.
Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensi
ons and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Forster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.
Key to collective cell migration is the ability of cells to rearrange their position with respect to their neighbors. Recent theory and experiments demonstrated that cellular rearrangements are facilitated by cell shape, with cells having more elonga
ted shapes and greater perimeters more easily sliding past their neighbors within the cell layer. Though it is thought that cell perimeter is controlled primarily by cortical tension and adhesion at each cells periphery, experimental testing of this hypothesis has produced conflicting results. Here we studied collective cell migration in an epithelial monolayer by measuring forces, cell perimeters, and motion, and found all three to decrease with either increased cell density or inhibition of cell contraction. In contrast to previous understanding, the data suggest that cell shape and rearrangements are controlled not by cortical tension or adhesion at the cell periphery but rather by the stress fibers that produce tractions at the cell-substrate interface. This finding is confirmed by an experiment showing that increasing tractions reverses the effect of density on cell shape and rearrangements. Our study therefore reduces the focus on the cell periphery by establishing cell-substrate traction as a major physical factor controlling cell shape and motion in collective cell migration.
Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Due to these issues, long-term tracking of proteins in confined and crowded environments,
such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5-nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used GFPs with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.
سجل دخول لتتمكن من نشر تعليقات
التعليقات
جاري جلب التعليقات
سجل دخول لتتمكن من متابعة معايير البحث التي قمت باختيارها
