Fall 2020, 2019

Carboxysomes, prototypical bacterial microcompartments (BMCs) found in cyanobacteria, are large (~1 GDa) and essential protein complexes that enhance CO2 fixation. While carboxysome biogenesis has been elucidated, the activity dynamics, lifetime, and degradation of these structures have not been investigated, owing to the inability of tracking individual BMCs over time in vivo. We have developed a fluorescence-imaging platform to simultaneously measure carboxysome number, position, and activity over time in a growing cyanobacterial population, allowing individual carboxysomes to be clustered on the basis of activity and spatial dynamics. We have demonstrated both BMC degradation, characterized by abrupt activity loss followed by polar recruitment of the deactivated complex, and a subclass of ultraproductive carboxysomes. Together, our results reveal the BMC life cycle after biogenesis and describe the first method for measuring activity of single BMCs in vivo. Single cell and organelle measurements reveal activity dynamics and degradation of the carbon-fixing cyanobacterial carboxysome. Single cell and organelle measurements reveal activity dynamics and degradation of the carbon-fixing cyanobacterial carboxysome.

Photosynthetic organisms regulate their responses to many diverse stimuli in an effort to balance light harvesting with utilizable light energy for carbon fixation and growth (source–sink regulation). This balance is critical to prevent the formation of reactive oxygen species that can lead to cell death. However, investigating the molecular mechanisms that underlie the regulation of photosynthesis in cyanobacteria using ensemble-based measurements remains a challenge due to population heterogeneity. Here, to address this problem, we used long-term quantitative time-lapse fluorescence microscopy, transmission electron microscopy, mathematical modelling and genetic manipulation to visualize and analyse the growth and subcellular dynamics of individual wild-type and mutant cyanobacterial cells over multiple generations. We reveal that mechanical confinement of actively growing Synechococcus sp. PCC 7002 cells leads to the physical disassociation of phycobilisomes and energetic decoupling from the photosynthetic reaction centres. We suggest that the mechanical regulation of photosynthesis is a critical failsafe that prevents cell expansion when light and nutrients are plentiful, but when space is limiting. These results imply that cyanobacteria must convert a fraction of the available light energy into mechanical energy to overcome frictional forces in the environment, providing insight into the regulation of photosynthesis and how microorganisms navigate their physical environment.

Non-invasively focusing light into strongly scattering media, such as biological tissue, is highly desirable but challenging. Recently, ultrasonically guided wavefront-shaping technologies have been developed to address this limitation. So far, the focusing resolution of most implementations has been limited by acoustic diffraction. Here, we introduce nonlinear photoacoustically guided wavefront shaping (PAWS), which achieves optical diffraction-limited focusing in scattering media. We develop an efficient dual-pulse excitation approach to generate strong nonlinear photoacoustic signals based on the Grueneisen relaxation effect. These nonlinear photoacoustic signals are used as feedback to guide iterative wavefront optimization. As a result, light is effectively focused to a single optical speckle grain on the scale of 5-7 μm, which is ~10 times smaller than the acoustic focus, with an enhancement factor of ~6,000 in peak fluence. This technology has the potential to benefit many applications that require a highly confined strong optical focus in tissue.