Farooqui, M. | Search bioRxiv

bioRxiv2023

Liquid-liquid interface microchannels with tunable mechanical properties mimic tissue deformations during neutrophil interstitial migration in vivo

Leukocytes navigate through interstitial spaces resulting in deformation of both the motile leukocytes and surrounding cells. Creating an in vitro system that models the deformable cellular environment encountered in vivo has been challenging. Here, we engineer microchannels with a liquid-liquid interface that exerts confining pressures (200-3000 Pa) similar to cells in tissues, and, thus, is deformable by cell generated forces. Consequently, the balance between migratory cell-generated and interfacial pressures determines the degree of confinement. Pioneer cells that first contact the interfacial barrier require greater deformation forces to forge a path for migration, and as a result migrate slower than trailing cells. Critically, resistive pressures are tunable by controlling the curvature of the liquid interface, which regulates motility. By granting cells autonomy in determining their confinement, and tuning environmental resistance, interfacial deformations are made to match those of surrounding cells in vivo during interstitial neutrophil migration in a larval zebrafish model. We discover that, in this context, neutrophils employ a bleb-based mechanism of force generation to deform a barrier exerting cell-scale confining pressures. Significance StatementImmune cells sense physical forces provided by surrounding cellular tissues to regulate their motility. Here, we introduce the use of liquid-liquid interfaces to model forces exerted by surrounding cells during interstitial motility in vivo. Neutrophils interacting with the interface employ a bleb-based mechanism of force generation to induce interfacial deformation. This work furthers our understanding of the mechanisms employed by immune cells to traverse through deformable barriers akin to cells in the body, and introduces a pioneering technology enabling the study of cell interaction with soft materials.

bioengineering↗

bioRxiv2020

Auto-inhibitory regulation of DNA binding by the C-terminal tails of the mitochondrial transcription factors Mtf1 and TFB2M

The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of the Saccharomyces cerevisiae and human mitochondrial RNA polymerases, respectively. These transcription factors directly interact with the non-template strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it is expected that the DNA binding activity of the mitochondrial transcription factors would be regulated to prevent DNA binding at inappropriate times. However, there is little information on how mitochondrial DNA transcription is regulated. While studying the C-tail deletion mutants of Mtf1 and TFB2M, we stumbled upon a new finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations show that Mtf1 with an intact C-tail has no affinity for the DNA but the deletion of C-tail greatly increases the DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as absolute as in Mtf1. Analysis of available TFB2M structures show that the C-tail makes intramolecular interactions with the DNA binding groove in the free factor, which we propose masks the DNA binding activity. Further studies show that the RNA polymerase relieves autoinhibition by interacting with the C-tail and engaging it in complex formation. Thus, our biochemical and structural analysis identify previously unknown autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate the DNA binding activity and aid in specific assembly of the initiation complexes.

biochemistry↗