Figueroa-Cuilan, W. M. | Search bioRxiv

bioRxiv2026

Diel remodeling and cellular integration of the nitroplast

Nitrogen-fixing eukaryotes were not believed to exist in nature until the recent discovery of a N2-fixing organelle, or nitroplast, in the marine microalga Braarudosphaera bigelowii. This nitroplast (formerly known as UCYN-A2) has long been recognized as key cyanobacterial contributor to global oceanic N2 fixation. However, how this novel organelle is integrated and regulated within the architecture of a eukaryotic cell remains unclear. Here, we combine multiscale volumetric imaging with cryo-electron tomography to resolve the native architecture, cellular integration, and diel remodeling of the nitroplast in cultured and environmental cells. We find that the nitroplast occupies up to 10% of the cell volume and exhibits close interfaces with multiple host organelles through membrane contact sites, while integration of this metabolically demanding compartment does not disrupt global scaling of host organelles. Interestingly, the chloroplast-to-nitroplast volume ratio is conserved across distinct life stages. Cryo-electron tomography reveals that the nitroplast retains a reinforced four-layer cyanobacterial envelope and is additionally surrounded by two host-derived layers that remodel across the day-night cycle. During daytime N2 fixation, these host-derived barriers become locally discontinuous and the organelle interface becomes enriched with two distinct vesicle populations. Our findings suggest that dynamic control of organelle accessibility through transient membrane gating represents a fundamental strategy by which eukaryotic cells could domesticate new endosymbiotic functions during early organellogenesis.

cell biology↗

bioRxiv2022

Quantitative analysis of morphogenesis and growth dynamics in an obligate intracellular bacterium

Obligate intracellular bacteria of the order Rickettsiales include numerous arthropod-borne human pathogens. However, our understanding of the basic biology of Rickettsia species is limited by technical challenges imposed by their obligate intracellular lifestyle. To overcome this roadblock, we developed quantitative methods to assess the cell wall composition, intracellular growth, and morphology of Rickettsia parkeri, a human pathogen in the Spotted Fever Group of the Rickettsia genus. Analysis of the cell wall composition of R. parkeri revealed unique features including a high M3 monomer fraction and absence of LD-crosslinks. Using a novel fluorescence microscopy approach, we quantified the cell morphology of R. parkeri in live host cells and found that bacterial morphology is maintained stably during exponential growth in two different epithelial cell lines. To assess population growth kinetics in a high-throughput and high-resolution manner, we developed an imaging-based growth assay and applied this to determine the growth rate of up to 24 infected cultures at a time. We also sought to gain insight into the cell cycle regulation of R. parkeri. To this end, we developed methods to quantify the fraction of the population preparing to divide as well as those undergoing active constriction. These approaches permitted a quantitative analysis of cell cycle status across a population of R. parkeri. Finally, as a proof of concept, we applied the above tools to quantitatively determine how MreB, a bacterial actin homolog, contributes to the growth and morphogenesis of R. parkeri. Inhibition of MreB with the small molecule MP265 led to cell rounding and slowed growth, suggesting that MreB is required for the growth and shape maintenance of R. parkeri. Collectively, we developed a toolkit of high-throughput, quantitative tools to understand intracellular growth and morphogenesis of R. parkeri that is translatable to other obligate intracellular bacteria. AUTHOR SUMMARYThe obligate intracellular lifestyle of members of the bacterial order Rickettsiales, which includes important human pathogens, has hindered our progress in understanding their biology. Here we developed and applied high-throughput, quantitative tools to analyze essential features of rickettsial cell biology such as morphology and growth in living host cells. By applying these tools in a proof of concept, we showed that the bacterial actin homolog, MreB is required for the regulation of rod shape and intracytoplasmic growth.

microbiology↗