Cell spheroid viscoelasticity is deformation-dependent.

Tissue surface tension influences cell sorting and tissue fusion. Earlier mechanical studies suggest that multicellular spheroids actively reinforce their surface tension with applied force. Here we study this open question through high-throughput microfluidic micropipette aspiration measurements on cell spheroids to identify the role of force duration and spheroid deformability. In particular, we aspirate spheroid protrusions of mice fibroblast NIH3T3 and human embryonic HEK293T homogeneous cell spheroids into micron-sized capillaries for different pressures and monitor their viscoelastic creep behavior. We find that larger spheroid deformations lead to faster cellular retraction once the pressure is released, regardless of the applied force. Additionally, less deformable NIH3T3 cell spheroids with an increased expression level of alpha-smooth muscle actin, a cytoskeletal protein upregulating cellular contractility, also demonstrate slower cellular retraction after pressure release for smaller spheroid deformations. Moreover, HEK293T cell spheroids only display cellular retraction at larger pressures with larger spheroid deformations, despite an additional increase in viscosity at these larger pressures. These new insights demonstrate that spheroid viscoelasticity is deformation-dependent and challenge whether surface tension truly reinforces at larger aspiration pressures.

Alternative splicing controls teneurin-3 compact dimer formation for neuronal recognition.

Neuronal network formation is facilitated by recognition between synaptic cell adhesion molecules at the cell surface. Alternative splicing of cell adhesion molecules provides additional specificity in forming neuronal connections. For the teneurin family of cell adhesion molecules, alternative splicing of the EGF-repeats and NHL domain controls synaptic protein-protein interactions. Here we present cryo-EM structures of the compact dimeric ectodomain of two teneurin-3 isoforms that harbour the splice insert in the EGF-repeats. This dimer is stabilised by an EGF8-ABD contact between subunits. Cryo-EM reconstructions of all four splice variants, together with SAXS and negative stain EM, reveal compacted dimers for each, with variant-specific dimeric arrangements. This results in specific trans-cellular interactions, as tested in cell clustering and stripe assays. The compact conformations provide a structural basis for teneurin homo- and heterophilic interactions. Altogether, our findings demonstrate how alternative splicing results in rearrangements of the dimeric subunits, influencing neuronal recognition and likely circuit wiring.

MukBEF-dependent chromosomal organization in widened Escherichia coli.

The bacterial chromosome is spatially organized through protein-mediated compaction, supercoiling, and cell-boundary confinement. Structural Maintenance of Chromosomes (SMC) complexes are a major class of chromosome-organizing proteins present throughout all domains of life. Here, we study the role of the Escherichia coli SMC complex MukBEF in chromosome architecture and segregation. Using quantitative live-cell imaging of shape-manipulated cells, we show that MukBEF is crucial to preserve the toroidal topology of the Escherichia coli chromosome and that it is non-uniformly distributed along the chromosome: it prefers locations toward the origin and away from the terminus of replication, and it is unevenly distributed over the origin of replication along the two chromosome arms. Using an ATP hydrolysis-deficient MukB mutant, we confirm that MukBEF translocation along the chromosome is ATP-dependent, in contrast to its loading onto DNA. MukBEF and MatP are furthermore found to be essential for sister chromosome decatenation. We propose a model that explains how MukBEF, MatP, and their interacting partners organize the chromosome and contribute to sister segregation. The combination of bacterial cell-shape modification and quantitative fluorescence microscopy paves way to investigating chromosome-organization factors in vivo.

Structural insights into the contactin 1 - neurofascin 155 adhesion complex.

Cell-surface expressed contactin 1 and neurofascin 155 control wiring of the nervous system and interact across cells to form and maintain paranodal myelin-axon junctions. The molecular mechanism of contactin 1 - neurofascin 155 adhesion complex formation is unresolved. Crystallographic structures of complexed and individual contactin 1 and neurofascin 155 binding regions presented here, provide a rich picture of how competing and complementary interfaces, post-translational glycosylation, splice differences and structural plasticity enable formation of diverse adhesion sites. Structural, biophysical, and cell-clustering analysis reveal how conserved Ig1-2 interfaces form competing heterophilic contactin 1 - neurofascin 155 and homophilic neurofascin 155 complexes whereas contactin 1 forms low-affinity clusters through interfaces on Ig3-6. The structures explain how the heterophilic Ig1-Ig4 horseshoe's in the contactin 1 - neurofascin 155 complex define the 7.4 nm paranodal spacing and how the remaining six domains enable bridging of distinct intercellular distances.

Putting on molecular weight: Enabling cryo-EM structure determination of sub-100-kDa proteins.

Significant advances in the past decade have enabled high-resolution structure determination of a vast variety of proteins by cryogenic electron microscopy single particle analysis. Despite improved sample preparation, next-generation imaging hardware, and advanced single particle analysis algorithms, small proteins remain elusive for reconstruction due to low signal-to-noise and lack of distinctive structural features. Multiple efforts have therefore been directed at the development of size-increase techniques for small proteins. Here we review the latest methods for increasing effective molecular weight of proteins <100 â€‹kDa through target protein binding or target protein fusion - specifically by using nanobody-based assemblies, fusion tags, and symmetric scaffolds. Finally, we summarize these state-of-the-art techniques into a decision-tree to facilitate the design of tailored future approaches, and thus for further exploration of ever-smaller proteins that make up the largest part of the human genome.

Mechanisms for Chromosome Segregation in Bacteria.

The process of DNA segregation, the redistribution of newly replicated genomic material to daughter cells, is a crucial step in the life cycle of all living systems. Here, we review DNA segregation in bacteria which evolved a variety of mechanisms for partitioning newly replicated DNA. Bacterial species such as Caulobacter crescentus and Bacillus subtilis contain pushing and pulling mechanisms that exert forces and directionality to mediate the moving of newly synthesized chromosomes to the bacterial poles. Other bacteria such as Escherichia coli lack such active segregation systems, yet exhibit a spontaneous de-mixing of chromosomes due to entropic forces as DNA is being replicated under the confinement of the cell wall. Furthermore, we present a synopsis of the main players that contribute to prokaryotic genome segregation. We finish with emphasizing the importance of bottom-up approaches for the investigation of the various factors that contribute to genome segregation.

Direct observation of independently moving replisomes in Escherichia coli.

The replication and transfer of genomic material from a cell to its progeny are vital processes in all living systems. Here we visualize the process of chromosome replication in widened E. coli cells. Monitoring the replication of single chromosomes yields clear examples of replication bubbles that reveal that the two replisomes move independently from the origin to the terminus of replication along each of the two arms of the circular chromosome, providing direct support for the so-called train-track model, and against a factory model for replisomes. The origin of replication duplicates near midcell, initially splitting to random directions and subsequently towards the poles. The probability of successful segregation of chromosomes significantly decreases with increasing cell width, indicating that chromosome confinement by the cell boundary is an important driver of DNA segregation. Our findings resolve long standing questions in bacterial chromosome organization.