Quantitative Time-Lapse Microscopy for Assessing Extracellular Matrix Stiffness-Dependent Bacterial Dissemination
Trascrizione
Take a multi-well plate with adherent human endothelial cell monolayers on collagen-coated polyacrylamide hydrogels of varying stiffness.
Cells on stiffer hydrogels undergo significant cytoskeletal reorganization, increasing intracellular tension, compared to softer hydrogels.
Wash cells with media and add engineered Listeria monocytogenes — expressing a fluorescent protein under the actin assembly-inducing protein (ActA) promoter.
Centrifuge to facilitate cell-bacteria contact. Incubate to mediate bacterial endocytosis.
Wash with media. Treat with gentamicin to eliminate non-internalized bacteria. Incubate.
Internalized bacteria release pore-forming toxins, disrupting phagosomal membranes. Cytosolic bacteria upregulate ActA and fluorescent protein expression, labeling intracellular bacteria.
ActA proteins induce actin polymerization, forming an actin comet tail propelling the bacteria forward, causing internalization by neighboring cells, and promoting dissemination.
Post-incubation, use dye-containing media to stain cell nuclei. Replace media with media containing gentamicin.
Perform time-lapse microscopy.
Softer hydrogels promote rapid bacteria intercellular spread compared to stiffer hydrogels with increased intracellular tension, suggesting matrix stiffness-dependent bacterial dissemination.
