Figure 9.

The RAm1-P1 fluorescent conjugate is a cellular client-based thermo-labile sensor of proteostasis network capacity in E. coli. (a) P1 selectively binds to and reacts with RA in E. coli affording the RAm1-P1 conjugate. Thus, only cells transformed with RA exhibit conjugate fluorescence in the confocal image. (b) The pre-formed RAm1-P1 conjugate formed granular aggregate structures as observed by confocal fluorescence imaging (white arrows) after heating at 45 °C for 10 min, serving as a sensor of proteostasis network capacity insufficiency. (c) Transcriptional reprogramming of E. coli by over-expressing the σ³²-I54N heat shock response transcription factor enhances the proteostasis network capacity of the cytosol, protecting the preformed RAm1-P1 conjugate from aggregating upon application of thermal stress. This is reflected by the lack of granular structures in rightmost confocal image in comparison to the image on the left where aggregation is observed because cytosolic proteostasis network capacity was not preemptively enhanced. Sample preparation and imaging details are described in the Supporting Information, Experimental Section. NT = non-transformed. Images were taken using a Zeiss LSM710 confocal microscope.