Biology subjects

Heinrich, D.

Publications and source records attributed to Heinrich, D..

6 recordsLinked to original sources

bioRxiv2022

Insights in the complex DegU, DegS, Spo0A regulation system of Paenibacillus polymyxa by CRISPR-Cas9-based targeted point mutations

Despite being unicellular organisms, bacteria undergo complex regulation mechanisms which coordinate different physiological traits. Among others, DegU, DegS, and Spo0A are the pleiotropic proteins which govern various cellular responses and behaviors. However, the functions and regulatory networks between these three proteins are rarely described in the highly interesting bacterium Paenibacillus polymyxa. In this study, we investigate the roles of DegU, DegS, and Spo0A by introduction of targeted point mutations facilitated by a CRISPR-Cas9-based system. In total, five different mutant strains were generated: the single mutants DegU Q218*, DegS L99F, Spo0A A257V, the double mutant DegU Q218* DegS L99F, and the triple mutant DegU Q218* DegS L99F Spo0A A257V. Characterization of the wild type and the engineered strains revealed differences in swarming behavior, genetic competence, sporulation, and viscosity formation of the culture broth. In particular, the double mutant DegU Q218* DegS L99F showed significant increase in regard to the genetic competence as well as a stable exopolysaccharides formation. Furthermore, we highlight similarities and differences of the roles of DegU, DegS, and Spo0A between P. polymyxa and related species. Finally, this study provides novel insights in the complex regulatory system of P. polymyxa DSM 365. ImportanceTo date, only limited knowledge is available on how complex cellular behaviors are regulated in P. polymyxa. In this study, we investigate three regulatory proteins which play a role in governing different physiological traits. Precise targeted point mutations are introduced to their respective genes by employing a highly efficient CRISPR-Cas9-based system. Characterization of the strains revealed some similarities, but also differences, with the model bacterium Bacillus subtilis in regard to the regulation of cellular behaviors. Furthermore, we identified several strains which have superior performance in comparison to the wild type strain. Overall, our study provides novel insights which will be of importance in understanding how multiple cellular processes are regulated in Paenibacillus species.

molecular biology↗

bioRxiv2021

Effect of G4C2-Repeat Expansions on the Motion of Lysosomes Inside Neurites.

The G4C2 hexanucleotide repeat expansion in the c9orf72 locus is one among a plethora of mutations associated with amyotrophic lateral sclerosis. It accounts for the majority of disease cases. The exact processes underlying the pathology of this mutation remain elusive, yet recent evidence suggests a mechanism that disrupts axonal trafficking. Here, we used a neuronal cell line with and without the G4C2 repeats, and implemented time-resolved local mean squared displacement analysis to characterize the motion of lysosomes inside neurites. Neurites were either aligned along chemically patterned lines, or oriented randomly on the substrate. We confirmed that in the presence of the G4C2 repeats, lysosome motion was affected. Lysosomes had a smaller reach exhibited lower velocity, especially inside aligned neurites. At the same time they became more active with increasing length of the G4C2 repeats when the neurites were randomly oriented. The duration of diffusive and super-diffusive lysosome transport remained unaffected for both neurite geometries and for all lengths of the repeats, but the displacement and velocity was decreased on varying the repeat number and neurite geometry. Lastly, the ratio of anterograde/retrograde/neutral trajectories was affected disparately for the two neurite geometries. Our observations support the hypothesis that impaired axonal trafficking emerges in the presence of the G4C2 hexanucleotide repeat expansion.

biophysics↗

bioRxiv2021

Impact of neurite alignment on organelle motion.

Intracellular transport is pivotal for cell growth and survival. Malfunctions in this process have been associated with devastating neurodegenerative diseases, posing a need for deeper understanding of the involved mechanisms. Here, we used an experimental methodology that lead neurites of differentiated PC12 cells in either of two configurations: an one-dimensional, where the neurites align along lines, or a two-dimensional configuration, where the neurites adopt a random orientation and shape on a fiat substrate. We subsequently monitored the motion of functional organelles, the lysosomes, inside the neurites. Implementing a time-resolved analysis of the mean-squared displacement, we quantitatively characterized distinct motion modes of the lysosomes. Our results indicate that neurite alignment gives rise to faster diffusive and super-diffusive lysosomal motion in comparison to the situation where the neurites are randomly oriented. After inducing lysosome swelling through an osmotic challenge by sucrose, we confirmed the predicted slowdown in diffusive mobility. Surprisingly we found that the swelling-induced mobility change affected each of the (sub-/super-) diffusive motion modes differently and depended on the alignment configuration of the neurites. Our findings imply that intracellular transport is significantly and robustly dependent on cell morphology, which might be in part controlled by the extracellular matrix.

biophysics↗

bioRxiv2019

Chemotaxis and topotaxis add vectorially for amoeboid cell migration

Cells encounter a wide variety of physical and chemical cues when navigating their native environments. However, their response to multiple simultaneous cues is not yet clear. In particular, the influence of topography, in the presence of a chemotactic gradient, on their migratory behavior is understudied. Here, we investigate the effects of topographical guidance on highly motile amoeboid cell migration (topotaxis) generated by asymmetrically placed micropillars. The micropillar field allows for an additional, natural chemotactic gradient in two different directions, thereby revealing the relevance of topotaxis in the presence of cell migration directed by chemical gradients (chemotaxis). Interestingly, we found that the topotactic drift generated by the pillar field is conserved during chemotaxis. We show that the drifts generated by both these cues add up linearly. A coarse-grained analysis as a function of pillar spacing subsequently revealed that the strength and direction of the topotactic drift is determined by (i) the pore size, (ii) space between pores, and (iii) the effective diffusion constant of the cells. Finally, we argue that topotaxis must be conserved during chemotaxis, as it is an emergent property of both the asymmetric properties of the pillar field and the inherent stochasticity of (biased) amoeboid migration.

biophysics↗

bioRxiv2019

Metabolic cost calculations of gait using musculoskeletal energy models, a comparison study

This paper compares predictions of metabolic energy expenditure in gait using seven metabolic energy expenditure models to assess their correlation with experimental data. Ground reaction forces, marker data, and pulmonary gas exchange data were recorded for six walking trials at combinations of two speeds, 0.8 m/s and 1.3 m/s, and three inclines, -8% (downhill), level, and 8% (uphill). The metabolic cost, calculated with the metabolic energy models was compared to the metabolic cost from the pulmonary gas exchange rates. A repeated measures correlation showed that all models correlated well with experimental data, with correlations of at least 0.9. The model by Bhargava et al. [7] and the model by Lichtwark and Wilson [21] had the highest correlation, 0.96. The model by Margaria [23] predicted the increase in metabolic cost following a change in dynamics best in absolute terms.

physiology↗

bioRxiv2016

Reversible metamorphosis in a bacterium

The cell wall is a shape-defining structure that envelopes almost all bacteria. One of its main functions is to serve as a protection barrier to environmental stresses. Bacteria can be forced in a cell wall-deficient state under highly specialized conditions, which are invariably aimed at interrupting cell wall synthesis. Therefore, the relevance of such cells has remained obscure. Here we show that many filamentous actinomycetes have a natural ability to generate a new, cell wall-deficient cell type in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the canonical mycelial mode-of-growth. Remarkably, prolonged exposure of S-cells to hyperosmotic stress yielded variants that are able to proliferate indefinitely without their cell wall. This is the first report that demonstrates the formation of wall-deficient cells as a natural adaptation strategy and their potential transition into stable wall-less forms solely caused by prolonged exposure to osmotic stress. Given that actinomycetes are potent antibiotic producers, our work also provides important insights into how biosynthetic gene clusters and resistance determinants may disseminate into the environment.

microbiology↗