Biology subjects

Männik, J.

Publications and source records attributed to Männik, J..

5 recordsLinked to original sources

bioRxiv2025

The role of active mRNA-ribosome dynamics and closing constriction in daughter chromosome separation in Escherichia coli

The mechanisms by which two sister chromosomes separate and partition into daughter cells in bacteria remain poorly understood. A recent theoretical model has proposed that out-of-equilibrium central dogma reactions involving mRNA and ribosomes play a significant role in this process. Here we test this idea in the Escherichia coli model system using high-throughput fluorescence microscopy in microfluidic devices. We compare our experimental observations with predictions from a reaction-diffusion model that includes central dogma-related reactions and excluded volume interactions between ribosomal subunits, polysomes, and chromosomal DNA. Our results show that the non-equilibrium reactions of ribosomes cause them to aggregate at the midcell, and this process facilitates the separation of the two daughter chromosomes. However, the observed effects are weaker in live cells than our one-dimensional reaction-diffusion model predicts. Rather than relying solely on active mRNA-ribosome dynamics, our data suggest that the closing division septum via steric interactions and potentially entropic forces between two DNA strands coupled to cell elongation act as additional mechanisms to ensure faithful partitioning of the nucleoids to two daughter cells. SignificanceThe mitotic spindle separates chromosomes in eukaryotic cells, but bacteria lack this structure. It remains unclear how bacterial chromosomes partition before cell division. It has been hypothesized that non-equilibrium dynamics of polysomes, that is, mRNA-ribosome complexes, actively drive the separation of bacterial chromosomes. Using quantitative microscopy combined with computational modeling, we show that polysome dynamics facilitates the separation of daughter chromosomes in Escherichia coli, but this process does not constitute the sole mechanism. Our findings suggest that the closing division septum via steric interactions and potentially entropic forces between the two DNA strands act as additional mechanisms.

biophysics↗

bioRxiv2024

Determining the rate-limiting processes for cell division in Escherichia coli

A critical cell cycle checkpoint for most bacteria is the onset of constriction when the septal peptidoglycan synthesis starts. According to the current understanding, the arrival of FtsN to midcell triggers this checkpoint in Escherichia coli. Recent structural and in vitro data suggests that recruitment of FtsN to the Z-ring leads to a conformational switch in actin-like FtsA, which links FtsZ protofilaments to the cell membrane and acts as a hub for the late divisome proteins. Here, we investigate this putative pathway using in vivo measurements and stochastic cell cycle modeling. Quantitatively upregulating protein concentrations and determining the resulting division timings shows that FtsN and FtsA numbers are not rate-limiting for the division in E. coli. However, at higher overexpression levels, they affect divisions: FtsN by accelerating and FtsA by inhibiting them. At the same time, we find that the numbers of FtsZ in the cell are rate-limiting for cell divisions in addition to other processes. Altogether, these findings suggest that instead of FtsN, FtsZ protofilaments drive the conformational switch of FtsA and lead to the onset of constriction. Our data is also suggestive that FtsA minirings are not present at any significant numbers in wild-type cells.

cell biology↗

bioRxiv2021

To bin or not to bin: analyzing single-cell growth data

Collection of high-throughput data has become prevalent in biology. Large datasets allow the use of statistical constructs such as binning and linear regression to quantify relationships between variables and hypothesize underlying biological mechanisms based on it. We discuss several such examples in relation to single-cell data and cellular growth. In particular, we show instances where what appears to be ordinary use of these statistical methods leads to incorrect conclusions such as growth being non-exponential as opposed to exponential and vice versa. We propose that the data analysis and its interpretation should be done in the context of a generative model, if possible. In this way, the statistical methods can be validated either analytically or against synthetic data generated via the use of the model, leading to a consistent method for inferring biological mechanisms from data. On applying the validated methods of data analysis to infer cellular growth on our experimental data, we find the growth of length in E. coli to be non-exponential. Our analysis shows that in the later stages of the cell cycle the growth rate is faster than exponential.

microbiology↗

bioRxiv2019

The effects of polydisperse crowders on the compaction of the Escherichia coli nucleoid

DNA binding proteins, supercoiling, macromolecular crowders, and transient DNA attachments to the cell membrane have all been implicated in the organization of the bacterial chromosome. However, it is unclear what role these factors play in compacting the bacterial DNA into a distinct organelle-like entity, the nucleoid. By analyzing the effects of osmotic shock and mechanical squeezing on Escherichia coli, we show that macromolecular crowders play a dominant role in the compaction of the DNA into the nucleoid. We find that a 30% increase in the crowder concentration from physiological levels leads to a 3-fold decrease in the nucleoids volume. The compaction is anisotropic, being higher along the long axes of the cell at low crowding levels. At higher crowding levels the compression becomes isotropic, implying that E. coli nucleoids lack a well-defined backbone. We furthermore show that the compressibility of the nucleoid is not significantly affected by cell growth rates and by prior treatment with rifampicin. The latter results point out that in addition to poly-ribosomes, soluble cytoplasmic proteins have a significant contribution in determining the size of the nucleoid.

biophysics↗

bioRxiv2018

Regulation of FtsZ levels in Escherichia coli in slow growth conditions

A key regulator of cell division in most walled bacteria is the FtsZ protein that assembles into protofilaments attached to the membrane at midcell. These dynamic protofilament assemblies, known as the Z-ring, act as a scaffold for more than two dozen proteins involved in synthesis of septal cell envelopes. What triggers the formation of the Z-ring during the cell cycle is poorly understood. In Escherichia coli model organism, the common view is that FtsZ concentration is constant throughout its doubling time and therefore regulation of assembly should be controlled by some yet to be identified protein-protein interactions. Here we show using quantitative analysis of newly developed fluorescent reporter that FtsZ concentration varies in a cell-cycle dependent manner in slow growth conditions and that upregulation of FtsZ synthesis correlates with the formation of the Z-ring. About 4-fold upregulation of FtsZ synthesis in the first half of the cell cycle is followed by its rapid degradation by ClpXP protease in the last 10% of the cell cycle. The initiation of rapid degradation coincides with dissociation of FtsZ from the septum. Altogether, our data indicate that the Z-ring formation in slow growth conditions in E. coli is controlled by a regulatory sequence where upregulation of an essential cell cycle factor is followed by its degradation.\n\nSignificanceFtsZ is the key regulator for bacterial cell division. It initiates division by forming a dynamic ring-like structure, the Z-ring, at the mid-cell. Here we show that, contrarily to the current paradigm, FtsZ concentration in Escherichia coli model organism varies throughout cell cycle in slow growth conditions. Faster FtsZ synthesis in the first half of the cell cycle is followed by its rapid degradation by ClpXP protease in the end of the cell cycle. Upregulation of FtsZ synthesis correlates with the formation of the Z-ring. Our data demonstrates that in slow growth E. coli cell division progresses according to paradigm where upregulation of essential cell cycle factor is followed by its degradation.

cell biology↗