Combining single-molecule and expansion microscopy in fission yeast to visualize protein structures at the nanostructural level.

In this work, we have developed an expansion microscopy (ExM) protocol that combines ExM with photoactivated localization microscopy (ExPALM) for yeast cell imaging, and report a robust protocol for single-molecule and expansion microscopy of fission yeast, abbreviated as SExY. Our optimized SExY protocol retains about 50% of the fluorescent protein signal, doubling the amount obtained compared to the original protein retention ExM (proExM) protocol. It allows for a fivefold, highly isotropic expansion of fission yeast cells, which we carefully controlled while optimizing protein yield.

Conformational Dynamics of the Most Efficient Carboxylase Contributes to Efficient CO Fixation.

Crotonyl-CoA carboxylase/reductase (Ccr) is one of the fastest CO fixing enzymes and has become part of efficient artificial CO-fixation pathways in vitro, paving the way for future applications. The underlying mechanism of its efficiency, however, is not yet completely understood. X-ray structures of different intermediates in the catalytic cycle reveal tetramers in a dimer of dimers configuration with two open and two closed active sites.

Raw Data to Results: A Hands-On Introduction and Overview of Computational Analysis for Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) is an advanced microscopy method that uses the blinking of fluorescent molecules to determine the position of these molecules with a resolution below the diffraction limit (∼5-40 nm). While SMLM imaging itself is becoming more popular, the computational analysis surrounding the technique is still a specialized area and often remains a "black box" for experimental researchers. Here, we provide an introduction to the required computational analysis of SMLM imaging, post-processing and typical data analysis.

Intersubunit Coupling Enables Fast CO-Fixation by Reductive Carboxylases.

Enoyl-CoA carboxylases/reductases (ECRs) are some of the most efficient CO-fixing enzymes described to date. However, the molecular mechanisms underlying the extraordinary catalytic activity of ECRs on the level of the protein assembly remain elusive. Here we used a combination of ambient-temperature X-ray free electron laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from .

Discovery of a New Chemoeffector for Chemoreceptor Tsr and Identification of a Molecular Mechanism of Repellent Sensing.

Motile bacteria use chemotaxis to search for nutrients and escape from harmful chemicals. While the sensing mechanisms for chemical attractants are well established, the molecular details of chemorepellent detection are poorly understood. Here, by using combined computational and experimental approaches to screen potential chemoeffectors for the chemoreceptor Tsr, we identified a specific chemorepellent, 1-aminocyclohexanecarboxylic acid (ACHC).

An overview of bioinformatics, genomics, and transcriptomics resources for bryophytes.

Bryophytes are useful models for the study of plant evolution, development, plant-fungal symbiosis, stress responses, and gametogenesis. Additionally, their dominant haploid gametophytic phase makes them great models for functional genomics research, allowing straightforward genome editing and gene knockout via CRISPR or homologous recombination. Until 2016, however, the only bryophyte genome sequence published was that of Physcomitrium patens.

Bacterial Small Membrane Proteins: the Swiss Army Knife of Regulators at the Lipid Bilayer.

Small membrane proteins represent a subset of recently discovered small proteins (≤100 amino acids), which are a ubiquitous class of emerging regulators underlying bacterial adaptation to environmental stressors. Until relatively recently, small open reading frames encoding these proteins were not designated genes in genome annotations. Therefore, our understanding of small protein biology was primarily limited to a few candidates associated with previously characterized larger partner proteins.

Actin-generated force applied during endocytosis measured by Sla2-based FRET tension sensors.

Mechanical forces are integral to many cellular processes, including clathrin-mediated endocytosis, a principal membrane trafficking route into the cell. During endocytosis, forces provided by endocytic proteins and the polymerizing actin cytoskeleton reshape the plasma membrane into a vesicle. Assessing force requirements of endocytic membrane remodeling is essential for understanding endocytosis.

Production and Characterization of Motile and Chemotactic Bacterial Minicells.

Minicells are nanosized membrane vesicles produced by bacteria. Minicells are chromosome-free but contain cellular biosynthetic and metabolic machinery, and they are robust due to the protection provided by the bacterial cell envelope, which makes them potentially highly attractive in biomedical applications. However, the applicability of minicells and other nanoparticle-based delivery systems is limited by their inefficient accumulation at the target.

Coregulation of gene expression by White collar 1 and phytochrome in Ustilago maydis.

Ustilago maydis encodes ten predicted light-sensing proteins. The biological functions of only a few of them are elucidated. Among the characterized ones are two DNA-photolyases and two rhodopsins that act as DNA-repair enzymes or green light-driven proton pumps, respectively.

Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis.

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP that constitutes the anchor to the plasma membrane.

The role of genetic manipulation and in situ modifications on production of bacterial nanocellulose: A review.

Natural polysaccharides are well-known biomaterials because of their availability and low-cost, with applications in diverse fields. Cellulose, a renowned polysaccharide, can be obtained from different sources including plants, algae, and bacteria, but recently much attention has been paid to the microorganisms due to their potential of producing renewable compounds. In this regard, bacterial nanocellulose (BNC) is a novel type of nanocellulose material that is commercially synthesized mainly by Komagataeibacter spp.

Mutual functional dependence of cyclase-associated protein 1 (CAP1) and cofilin1 in neuronal actin dynamics and growth cone function.

Neuron connectivity depends on growth cones that navigate axons through the developing brain. Growth cones protrude and retract actin-rich structures to sense guidance cues. These cues control local actin dynamics and steer growth cones towards attractants and away from repellents, thereby directing axon outgrowth.

HAG1 and SWI3A/B control of male germ line development in P. patens suggests conservation of epigenetic reproductive control across land plants.

Bryophytes as models to study the male germ line: loss-of-function mutants of epigenetic regulators HAG1 and SWI3a/b demonstrate conserved function in sexual reproduction. With the water-to-land transition, land plants evolved a peculiar haplodiplontic life cycle in which both the haploid gametophyte and the diploid sporophyte are multicellular. The switch between these phases was coined alternation of generations.

The iron-sulfur scaffold protein HCF101 unveils the complexity of organellar evolution in SAR, Haptista and Cryptista.

Background: Nbp35-like proteins (Nbp35, Cfd1, HCF101, Ind1, and AbpC) are P-loop NTPases that serve as components of iron-sulfur cluster (FeS) assembly machineries. In eukaryotes, Ind1 is present in mitochondria, and its function is associated with the assembly of FeS clusters in subunits of respiratory Complex I, Nbp35 and Cfd1 are the components of the cytosolic FeS assembly (CIA) pathway, and HCF101 is involved in FeS assembly of photosystem I in plastids of plants (chHCF101). The AbpC protein operates in Bacteria and Archaea.

Anti-CRISPR AcrIF9 functions by inducing the CRISPR-Cas complex to bind DNA non-specifically.

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity.

The two-component system ActJK is involved in acid stress tolerance and symbiosis in Sinorhizobium meliloti.

The nitrogen-fixing α-proteobacterium Sinorhizobium meliloti genome codifies at least 50 response regulator (RR) proteins mediating different and, in many cases, unknown processes. RR-mutant library screening allowed us to identify genes potentially implicated in survival to acid conditions. actJ mutation resulted in a strain with reduced growth rate under mildly acidic conditions as well as a lower capacity to tolerate a sudden shift to lethal acidic conditions compared with the parental strain.

Aethionema arabicum genome annotation using PacBio full-length transcripts provides a valuable resource for seed dormancy and Brassicaceae evolution research.

Aethionema arabicum is an important model plant for Brassicaceae trait evolution, particularly of seed (development, regulation, germination, dormancy) and fruit (development, dehiscence mechanisms) characters. Its genome assembly was recently improved but the gene annotation was not updated. Here, we improved the Ae.

Establishing Live-Cell Single-Molecule Localization Microscopy Imaging and Single-Particle Tracking in the Archaeon .

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and are indispensable tools for the investigation of cellular biology and biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology.

Postmitotic expansion of cell nuclei requires nuclear actin filament bundling by α-actinin 4.

The actin cytoskeleton operates in a multitude of cellular processes including cell shape and migration, mechanoregulation, and membrane or organelle dynamics. However, its filamentous properties and functions inside the mammalian cell nucleus are less well explored. We previously described transient actin assembly at mitotic exit that promotes nuclear expansion during chromatin decondensation.

Design of a MAPK signalling cascade balances energetic cost versus accuracy of information transmission.

Cellular processes are inherently noisy, and the selection for accurate responses in presence of noise has likely shaped signalling networks. Here, we investigate the trade-off between accuracy of information transmission and its energetic cost for a mitogen-activated protein kinase (MAPK) signalling cascade. Our analysis of the pheromone response pathway of budding yeast suggests that dose-dependent induction of the negative transcriptional feedbacks in this network maximizes the information per unit energetic cost, rather than the information transmission capacity itself.

Functional Modules of Minimal Cell Division for Synthetic Biology.

Cellular reproduction is one of the fundamental hallmarks of life. Therefore, the development of a minimal division machinery capable of proper genome condensation and organization, mid-cell positioning and segregation in space and time, and the final septation process constitute a fundamental challenge for synthetic biology. It is therefore important to be able to engineer such modules for the production of artificial minimal cells.

Cytoskeletal and Actin-Based Polymerization Motors and Their Role in Minimal Cell Design.

Life implies motion. In cells, protein-based active molecular machines drive cell locomotion and intracellular transport, control cell shape, segregate genetic material, and split a cell in two parts. Key players among molecular machines driving these various cell functions are the cytoskeleton and motor proteins that convert chemical bound energy into mechanical work.

DNA Segregation in Natural and Synthetic Minimal Systems.

Faithful segregation of replicated genomes to dividing daughter cells is a major hallmark of cellular life and needs to be part of the future design of the robustly proliferating minimal cell. So far, the complexity of eukaryotic chromosome segregation machineries has limited their applicability to synthetic systems. Prokaryotic plasmid segregation machineries offer promising alternative tools for bottom-up synthetic biology, with the first three-component DNA segregation system being reconstituted a decade ago.