Biology subjects

Ojkic, N.

Publications and source records attributed to Ojkic, N..

8 recordsLinked to original sources

bioRxiv2025

Deep-learning deconvolution and segmentation of fluorescent membranes for high-precision bacterial cell-size profiling

Evolutionary studies in bacteria have emphasized genetic and metabolic diversity, while cell-size variation has received less attention. Here we introduce MEDUSSA, a high-throughput method for precise bacterial cell-size profiling based on automatic segmentation of fluorescent membrane images, well suited to studying cell-size diversity. The approach uses deep-learning-based membrane deconvolution, segmentation models fine-tuned for fluorescent-membrane images, and error-corrected cell measurement to extract accurate sizes from individual bacterial cells regardless of shape, size, chaining, or clustering. Our method overcomes limitations of phase-contrast segmentation, yielding reliable single-cell dimensions. We applied MEDUSSA to six strains of Priestia megaterium and found over twofold differences in cell volume across strains, largely driven by differences in cell width. We identified a partially-functional PBP1 allele that underlies the reduced width of one strain. Together, these results demonstrate the power of comparative analyses in bacterial cell biology and expand the toolkit to investigate the evolution of bacterial cell size.

cell biology↗

bioRxiv2025

Dynein synergises with EB1 to facilitate cortex-microtubule encounter and proper spindle positioning in metaphase

To rotate the bulky mitotic spindle, astral microtubules are pulled by cortex-bound dynein. We investigate whether dynein regulates astral microtubules, independent of its classical microtubule-pulling role by combining super-resolution live-cell microscopy with optogenetic tools to disrupt and measure astral microtubule distributions and spindle movements. Localised photoinactivation of EB1 reveals that microtubule ends reaching distinct cortical regions differently influence spindle movements. Using a dynein inhibitor gradient, we separate dyneins role in spindle positioning (maintaining pole-to-cortex distance) from its role in directing spindle movements. EB1 photoinactivation of whole spindles uncovers a previously unrecognised synergistic role for dynein and EB1 in maintaining astral microtubules reaching the cortex. We demonstrate the EB1-dependent role for dynein in maintaining astral microtubule growth is independent of its classical cortical roles by analysing dynamic changes in spindle position, displacement rates and cortical dynein localisation and by depleting dyneins cortical platform, LGN. Our finding that Dynein synergises with EB1 to regulate astral microtubule, independent of cortical pulling, has implications for how spindle positioning is correctly established in cells. HIGHLIGHTSO_LISystematic quantification of spindle movements and microtubule lengths reveals dyneins dual role in microtubule growth and pulling. C_LIO_LIMicrotubules reaching distinct cortex regions differently regulate spindle movements. C_LIO_LIEB1 photoinactivation shows dynein promotes EB1-independent astral microtubule growth. C_LIO_LIReduced dynein causes shorter asters and asymmetric spindle positioning, without reducing spindle movements. C_LIO_LINoncortical dynein regulates cortex-microtubule encounter; cortical dynein controls spindle movement. C_LI

cell biology↗

bioRxiv2021

Antibiotic resistance via bacterial cell shape-shifting

Bacteria have evolved to develop multiple strategies for antibiotic resistance by effectively reducing intra-cellular antibiotic concentrations or antibiotic binding affinities, but the role of cell morphology on antibiotic resistance remains poorly characterized. By analyzing cell morphological data of different bacterial species under antibiotic stress, we find that bacterial cells robustly reduce surface-to-volume ratio in response to most types of antibiotics. Using quantitative modelling we show that by reducing surface-to-volume ratio, bacteria can effectively reduce intracellular antibiotic concentration by decreasing antibiotic influx. The model predicts that bacteria can increase surface-to-volume ratio to promote antibiotic dilution if efflux pump activity is reduced, in agreement with data on membrane-transport inhibitors. Using the particular example of ribosome-targeting antibiotics, we present a systems-level model for the regulation of cell shape under antibiotic stress, and discuss feedback mechanisms that bacteria can harness to increase their fitness in the presence of antibiotics.

microbiology↗

bioRxiv2021

Bacterial cell shape control by nutrient-dependent synthesis of cell division inhibitors

By analysing cell shape and size of the bacterium Bacillus subtilis under nutrient perturbations, protein depletion, and antibiotic treatments we find that cell geometry is extremely robust, reflected in a well-conserved scaling relation between surface area (S) and volume (V), S ~ V{gamma}, with{gamma} = 0.85. We develop a molecular model supported by single-cell simulations to predict that the surface-to-volume scaling exponent{gamma} is regulated by nutrient-dependent production of metabolic enzymes that act as cell division inhibitors in bacteria. Using theory that is supported by experimental data, we predict the modes of cell shape transformations in different bacterial species and propose a mechanism of cell shape adaptation to different nutrient perturbations. For organisms with high surface-to-volume scaling exponent{gamma} , such as B. subtilis, cell width is not sensitive to growth rate changes, whereas organisms with low{gamma} , such as A. baumannii, cell shape adapts readily to growth rate changes. SIGNIFICANCEHow bacteria regulate their size and shapes to optimise their growth fitness in different nutrient environments remains largely unknown. By analysing the surface area and volume of rod-shaped B. subtilis exposed to different nutrient conditions and antibiotics we find that cells preserve a power law scaling between surface area and volume. We show that the surface-to-volume scaling is extremely robust and is regulated by nutrient-dependent synthesis of cell division inhibitors. By analysing different bacterial types, we find that cells conserve the surface-to-volume scaling exponent that is typical for each species, implying distinct mechanisms for morphological adaptation in each organism.

biophysics↗

bioRxiv2020

Nutrient-dependent trade-offs between ribosomes and division protein synthesis control bacterial growth and cell shape

Cell size control emerges from a regulated balance between the rates of cell growth and division. In bacteria, simple quantitative laws connect cellular growth rate to ribosome abundance. However, it remains poorly understood how translation regulates bacterial cell size and shapes under growth perturbations. Here we develop a whole-cell model for growth dynamics in rod-shaped bacteria that links ribosomal abundance with cell geometry, division control, and the extracellular environment. Our study reveals that cell size maintenance under nutrient perturbations requires a balanced trade-off between ribosomes and division protein synthesis. Deviations from this trade-off relationship are predicted under translational perturbations, leading to distinct modes of cell morphological changes, in agreement with single-cell experimental data on Escherichia coli. Furthermore, by calibrating our model with experimental data, we predict how combinations of nutrient-, translational- and shape perturbations can be chosen to optimize bacterial growth fitness and antibiotic resistance.

systems biology↗

bioRxiv2019

A roadblock-and-kill model explains the action of the DNA-targeting antibiotic ciprofloxacin

Fluoroquinolones - antibiotics that cause DNA damage by inhibiting DNA topoisomerases - are clinically important, but their mechanism of action is not yet fully understood. In particular, the dynamical response of bacterial cells to fluoroquinolone exposure has hardly been investigated, although the SOS response, triggered by DNA damage, is often thought to play a key role. Here we investigate growth inhibition of the bacterium Escherichia coli by the fluoroquinolone ciprofloxacin at low doses (up to 5x the minimum inhibitory concentration). We measure the long-term and short-term (dynamic) response of the growth rate and DNA production rate to ciprofloxacin, at both population- and single-cell level. We show that despite the molecular complexity of DNA metabolism, a simple `roadblock-and-kill model focusing on replication fork blockage and DNA damage by ciprofloxacin-poisoned DNA topoisomerase II (gyrase) quantitatively reproduces long-term growth rates. The model also predicts dynamical changes in DNA production rate in wild type E. coli and in an SOS-deficient mutant, following a step-up of ciprofloxacin. Our work reveals new insights into the dynamics of fluoroquinolone action, with important implications for predicting the rate of resistance evolution. Most importantly, our model explains why the response is delayed: it takes many doubling times to fragment the DNA sufficiently to inhibit gene expression. Our model also challenges the view that the SOS response plays a central role: the dynamical response is controlled by the timescale of DNA replication and gyrase binding/unbinding to the DNA rather than by the SOS response. More generally, our work highlights the importance of including biophysical processes in biochemical-systems models to fully understand bacterial response to antibiotics.

systems biology↗

bioRxiv2019

Universal surface-to-volume scaling and aspect ratio homeostasis in rod-shaped bacteria

Rod-shaped bacterial cells can readily adapt their lengths and widths in response to environmental changes. While many recent studies have focused on the mechanisms underlying bacterial cell size control, it remains largely unknown how the coupling between cell length and width results in robust control of rod-like bacterial shapes. In this study we uncover a universal surface-to-volume scaling relation in Escherichia coli and other rod-shaped bacteria, resulting from the preservation of cell aspect ratio. To explain the mechanistic origin of aspect-ratio control, we propose a quantitative model for the coupling between bacterial cell elongation and the accumulation of an essential division protein, FtsZ. This model reveals a mechanism for why bacterial aspect ratio is independent of cell size and growth conditions, and predicts cell morphological changes in response to nutrient perturbations, antibiotics, MreB or FtsZ depletion, in quantitative agreement with experimental data.

biophysics↗

bioRxiv2016

Cell wall remodeling drives engulfment during Bacillus subtilis sporulation

When starved, the Gram-positive bacterium Bacillus subtilis forms durable spores for survival. Sporulation initiates with an asymmetric cell division, creating a large mother cell and a small forespore. Subsequently, the mother cell membrane engulfs the forespore in a phagocytosis-like process. However, the force generation mechanism for forward membrane movement remains unknown. Here, we show that membrane migration is driven by cell wall remodeling at the leading edge of the engulfing membrane, with peptidoglycan synthesis and degradation mediated by penicillin binding proteins in the forespore and a cell wall degradation protein complex in the mother cell. We propose a simple model for engulfment in which the junction between the septum and the lateral cell wall moves around the forespore by a mechanism resembling the template model. Hence, we establish a biophysical mechanism for the creation of a force for engulfment based on the coordination between cell wall synthesis and degradation.

biophysics↗