Assessing the growth kinetics and stoichiometry of Escherichia coli at the single-cell level.

Microfluidic cultivation and single-cell analysis are inherent parts of modern microbial biotechnology and microbiology. However, implementing biochemical engineering principles based on the kinetics and stoichiometry of growth in microscopic spaces remained unattained. We here present a novel integrated framework that utilizes distinct microfluidic cultivation technologies and single-cell analytics to make the fundamental math of process-oriented biochemical engineering applicable at the single-cell level. A combination of non-invasive optical cell mass determination with sub-pg sensitivity, microfluidic perfusion cultivations for establishing physiological steady-states, and picoliter batch reactors, enabled the quantification of all physiological parameters relevant to approximate a material balance in microfluidic reaction environments. We determined state variables (biomass concentration based on single-cell dry weight and mass density), biomass synthesis rates, and substrate affinities of cells grown in microfluidic environments. Based on this data, we mathematically derived the specific kinetics of substrate uptake and growth stoichiometry in glucose-grown Escherichia coli with single-cell resolution. This framework may initiate microscale material balancing beyond the averaged values obtained from populations as a basis for integrating heterogeneous kinetic and stoichiometric single-cell data into generalized bioprocess models and descriptions.

Mapping developmental maturation of inner hair cell ribbon synapses in the apical mouse cochlea.

Ribbon synapses of cochlear inner hair cells (IHCs) undergo molecular assembly and extensive functional and structural maturation before hearing onset. Here, we characterized the nanostructure of IHC synapses from late prenatal mouse embryo stages (embryonic days 14-18) into adulthood [postnatal day (P)48] using electron microscopy and tomography as well as optical nanoscopy of apical turn organs of Corti. We find that synaptic ribbon precursors arrive at presynaptic active zones (AZs) after afferent contacts have been established. These ribbon precursors contain the proteins RIBEYE and piccolino, tether synaptic vesicles and their delivery likely involves active, microtubule-based transport pathways. Synaptic contacts undergo a maturational transformation from multiple small to one single, large AZ. This maturation is characterized by the fusion of ribbon precursors with membrane-anchored ribbons that also appear to fuse with each other. Such fusion events are most frequently encountered around P12 and hence, coincide with hearing onset in mice. Thus, these events likely underlie the morphological and functional maturation of the AZ. Moreover, the postsynaptic densities appear to undergo a similar refinement alongside presynaptic maturation. Blockwise addition of ribbon material by fusion as found during AZ maturation might represent a general mechanism for modulating ribbon size.