Eukaryotic Research Articles

Emergent actin flows explain distinct modes of gliding motility

AbstractDuring host infection, Toxoplasma gondii and related unicellular parasites move using gliding, which differs fundamentally from other known mechanisms of eukaryotic cell motility. Gliding is thought to be powered by a thin layer of flowing filamentous (F)-actin sandwiched between the plasma membrane and a myosin-covered inner membrane complex. How this surface actin layer drives the various gliding modes observed in experiments—helical, circular, twirling and patch, pendulum or rolling—is unclear. Here we suggest that F-actin flows arise through self-organization and develop a continuum model of emergent F-actin flow within the confines provided by Toxoplasma geometry. In the presence of F-actin turnover, our model predicts the emergence of a steady-state mode in which actin transport is largely directed rearward. Removing F-actin turnover leads to actin patches that recirculate up and down the cell, which we observe experimentally for drug-stabilized actin bundles in live Toxoplasma gondii parasites. These distinct self-organized actin states can account for observed gliding modes, illustrating how different forms of gliding motility can emerge as an intrinsic consequence of the self-organizing properties of F-actin flow in a confined geometry.

Luminescent Pentacene‐Loaded Carbon Nanodots with Red‐Light Triggered Photothermal and Photosensitizing Properties

The development of multifunctional nanosystems for photo‐induced hyperthermia and photodynamic effect is a challenging topic in the research of advanced materials for application in biomedical field. Here, we report red‐luminescent carbon‐nanodots (CDs‐PNM/PTC) derived from entrapment of pentacene (PTC) in nanodots prepared from poly(N‐isopropylacrylamide) polymer (CDs‐PNM) by an easy and reagent‐free method. The CD‐PNM/PTC nanosystem was characterized by different techniques (UV‐Vis spectrophotometry, fluorescence, NMR, AFM). Molecular modelling investigations were performed to unveil stability, structures and energy of the CD‐PNM/PTC supramolecular adducts at 298 K and 315 K. The nanosized CDs–PNM/PTC exhibited excellent water‐dispersibility, good photothermal conversion efficiency and photosensitizing effect at 680 nm. No significant toxicity and eukaryotic cell uptake are features that open to potential applications in photothermal‐photodynamic treatments.

Transcriptome analysis reveals a new virulence-associated trimeric autotransporter responsible for Glaesserella parasuis autoagglutination.

Capsular polysaccharide is an important virulence factor of Glaesserella parasuis. An acapsular mutant displays multiple phenotype variations, while the underlying mechanism for these variations is unknown. In this study, we created an acapsular mutant by deleting the wza gene in the capsule locus. We then used transcriptome analysis to compare the gene expression profiles of the wza deletion mutant with those of the parental strain to understand the possible reasons for the phenotypic differences. The mutant Δwza, which has a deleted wza gene, secreted less polysaccharide and lost its capsule structure. The Δwza exhibited increased autoagglutination, biofilm formation and adherence to eukaryotic cells, while the complementary strain C-Δwza partially restored the phenotype. Transcriptome analysis revealed several differentially expressed genes (DEGs) in Δwza, including up-regulated outer membrane proteins and proteins involved in peptidoglycan biosynthesis, suggesting that wza deletion affects the cell wall homeostasis of G. parasuis. Transcriptome analysis revealed the contribution of non-coding RNAs in the regulation of DEGs. Moreover, a new virulence-associated trimeric autotransporter, VtaA31 is upregulated in Δwza. It is responsible for enhanced autoagglutination but not for enhanced biofilm formation and adherence to eukaryotic cells in Δwza. In conclusion, these data indicate that wza affects the expression of multiple genes, especially those related to cell wall synthesis. Furthermore, they provide evidence that vtaA31 is involved in the autoagglutination of G. parasuis.

Supplementation with ions enhances the efficiency of nucleic acid delivery with cell-penetrating peptides

The successful delivery of therapeutic nucleic acids (NAs) into eukaryotic cells is essential for numerous biomedical applications, including gene therapy, gene silencing, and genome editing. Cell-penetrating peptides (CPPs) have claimed significant attention as delivery vehicles due to their inherent ability to penetrate cellular membranes and efficiently transport cargo, including NAs, into the cells. However, further optimization and a deeper understanding of underlying mechanisms are necessary for such transfection methods. Previous studies have demonstrated that Ca2+ ions can significantly enhance NA delivery efficiency when included in transfection media or CPP/NA nanoparticles during preparation. Similar effects have been observed for Mg2+, but the impact of other ions in this context has not been thoroughly investigated. In this study, we supplemented the CPP/NA formulations with various inorganic biocompatible ions by introducing solutions of the respective salts to colloidal nanoparticles at the preparation stage. Our results indicated that supplementing the CPP/NA formulations with certain salt solutions enhanced the biological effect achieved with NAs while also influencing nanoparticle size, surface charge, complexation stability, and, to some extent, the internalization route. Our findings offer valuable insights for optimizing the formation of CPP nanoparticles to improve NA delivery efficiency.

Drug Combination Studies of Isoquinolinone AM12 with Curcumin or Quercetin: A New Combination Strategy to Synergistically Inhibit 20S Proteasome.

In the eukaryotic cells, the ubiquitin-proteasome system (UPS) plays a crucial role in the intracellular protein turnover. It is involved in several cellular functions such as the control of the regular cell cycle progression, the immune surveillance, and the homeostasis. Within the 20S proteasome barrel-like structure, the catalytic subunits, β1, β2 and β5, are responsible for different proteolytic activities: caspase-like (C-L), trypsin-like (T-L) and chymotrypsin-like (ChT-L), respectively. The β5 subunit is particularly targeted for its role in antitumor activity: the synthesis of β5 subunit inhibitors could be a promising strategy for the treatment of solid and hematologic tumors. In the present work, we performed two combination studies of AM12, a recently developed synthetic proteasome inhibitor, with curcumin and quercetin, two nutraceuticals endowed of many pharmacological properties. We measured the combination index (CI), applying the Chou and Talalay method, comparing the two studies, from 50% to 90% of proteasome inhibition. In the case of the combination AM12 + curcumin, an increasing synergism was observed from 50% to 90% of proteasome inhibition, while in the case of the combination AM12 + quercetin an additive effect was observed only from 50% to 70% of β5 subunit inhibition. These results suggest that combining AM12 with curcumin is a more promising strategy than combining it with quercetin for potential therapeutic applications, especially in treating tumors.

Evolution of the conformational dynamics of the molecular chaperone Hsp90

Hsp90 is a molecular chaperone of central importance for protein homeostasis in the cytosol of eukaryotic cells, with key functional and structural traits conserved from yeast to man. During evolution, Hsp90 has gained additional functional importance, leading to an increased number of interacting co-chaperones and client proteins. Here, we show that the overall conformational transitions coupled to the ATPase cycle of Hsp90 are conserved from yeast to humans, but cycle timing as well as the dynamics are significantly altered. In contrast to yeast Hsp90, the human Hsp90 is characterized by broad ensembles of conformational states, irrespective of the absence or presence of ATP. The differences in the ATPase rate and conformational transitions between yeast and human Hsp90 are based on two residues in otherwise conserved structural elements that are involved in triggering structural changes in response to ATP binding. The exchange of these two mutations allows swapping of the ATPase rate and of the conformational transitions between human and yeast Hsp90. Our combined results show that Hsp90 evolved to a protein with increased conformational dynamics that populates ensembles of different states with strong preferences for the N-terminally open, client-accepting states.

Super-resolution imaging reveals nucleolar encapsulation by single-stranded DNA.

In eukaryotic cell nuclei, specific sets of proteins gather in nuclear bodies and facilitate distinct genomic processes. The nucleolus, a nuclear body, functions as a factory for ribosome biogenesis by accumulating constitutive proteins, such as RNA polymerase I and nucleophosmin 1 (NPM1). Although in vitro assays have suggested the importance of liquid-liquid phase separation (LLPS) of constitutive proteins in nucleolar formation, how the nucleolus is structurally maintained with the intranuclear architecture remains unknown. This study revealed that the nucleolus is encapsulated by a single-stranded (ss)DNA-based molecular complex inside the cell nucleus. Super-resolution lattice-structured illumination microscopy (lattice-SIM) showed that there was a high abundance of ssDNA beyond the 'outer shell' of the nucleolus. Nucleolar disruption and the release of NPM1 were caused by in situ digestion of ssDNA, suggesting that ssDNA has a structural role in nucleolar encapsulation. Furthermore, we identified that ssDNA forms a molecular complex with histone H1 for nucleolar encapsulation. Thus, this study illustrates how an ssDNA-based molecular complex upholds the structural integrity of nuclear bodies to coordinate genomic processes such as gene transcription and replication.

The hnRNP A2B1 is important for the replication of SFTSV and other RNA viruses.

The heterogeneous nuclear ribonucleoprotein (hnRNP A2B1) is a key component of the hnRNP complex involving RNA modulation in eukaryotic cells and it has also been reported to be involved in the replication of the hepatitis E virus, influenza A virus, and hepatitis B virus. However, it is not clear whether the role of the hnRNP A2B1 in viral replication is conserved among RNA viruses and what is the mechanism of hnRNP A2B1 in RNA virus replication. In this study, we first used severe fever with thrombocytopenia syndrome virus (SFTSV), a tick-borne RNA virus that causes a severe viral hemorrhagic fever as well as other RNA viruses including VSV-GFP, SeV, EV71, and ZIKV to demonstrate that knockout hnRNPA2B1 gene inhibited viral RNA replication and overexpression of hnRNP A2B1 could restore the RNA levels of all tested RNA viruses. These results suggest that hnRNPA2B1 upregulation of viral replication is conserved among RNA viruses. Next, we demonstrated that hnRNP A2B1 was translocated from the nucleus to the cytoplasm under RNA virus infection including SFTSV, VSV-GFP, SeV, EV71, and ZIKV, suggesting translocation of hnRNP A2B1 from the nucleus to the cytoplasm is crucial for RNA virus replication. We then used SFTSV as a model to demonstrate the mechanism of hnRNP A2B1 in the promotion of RNA virus replication. We found that overexpression of SFTSV nucleoprotein can also cause hnRNP A2B1 translocation from the nucleus to the cytoplasm and that the SFTSV NP interacted with the RNA recognition motif 1 domain of hnRNP A2B1. We further demonstrated that the hnRNP A2B1 interacted with the 5' UTR of SFTSV RNA. In conclusion, we revealed that the hnRNP A2B1 upregulation of viral RNA replication is conserved among RNA viruses; the mechanism of hnRNP A2B1 in promotion of SFTSV viral RNA replication is that SFTSV NP interacted with the hnRNPA2B1 to retain it in the cytoplasm where the hnRNP A2B1 interacted with the 5' UTR of SFTSV RNA to promote the viral RNA replication.IMPORTANCESevere fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne RNA virus with a high mortality rate of up to 30%. In this study, we first used SFTSV as a model to demonstrate that the role of hnRNPA2B1 in viral replication is conserved in SFTSV. Then we used other RNA viruses, including VSV-GFP, SeV, EV71, and ZIKV, to repeat the experiment and demonstrated the same results as SFTSV in all tested RNA viruses. By knocking out the hnRNPA2B1 gene, SFTSV RNA replication was inhibited, and overexpression of hnRNPA2B1 restored RNA levels of SFTSV and other tested RNA viruses. We revealed a novel mechanism where the SFTSV nucleoprotein interacts with hnRNPA2B1, retaining it in the cytoplasm. This interaction promotes viral RNA replication by binding to the 5' UTR of SFTSV RNA. The findings suggest that targeting hnRNPA2B1 could be a potential strategy for developing broad-spectrum antiviral therapies, given its conserved role across different RNA viruses. This research provides significant insights into the replication mechanisms of RNA viruses and highlights potential targets for antiviral interventions.

Mechanisms and regulation of substrate degradation by the 26S proteasome.

The 26S proteasome is involved in degrading and regulating the majority of proteins in eukaryotic cells, which requires a sophisticated balance of specificity and promiscuity. In this Review, we discuss the principles that underly substrate recognition and ATP-dependent degradation by the proteasome. We focus on recent insights into the mechanisms of conventional ubiquitin-dependent and ubiquitin-independent protein turnover, and discuss the plethora of modulators for proteasome function, including substrate-delivering cofactors, ubiquitin ligases and deubiquitinases that enable the targeting of a highly diverse substrate pool. Furthermore, we summarize recent progress in our understanding of substrate processing upstream of the 26S proteasome by the p97 protein unfoldase. The advances in our knowledge of proteasome structure, function and regulation also inform new strategies for specific inhibition or harnessing the degradation capabilities of the proteasome for the treatment of human diseases, for instance, by using proteolysis targeting chimera molecules or molecular glues.

Cyclo(Pro-Tyr) elicits conserved cellular damage in fungi by targeting the [H+]ATPase Pma1 in plasma membrane domains

Bioactive metabolites play a crucial role in shaping interactions among diverse organisms. In this study, we identified cyclo(Pro-Tyr), a metabolite produced by Bacillus velezensis, as a potent inhibitor of Botrytis cinerea and Caenorhabditis elegans, two potential cohabitant eukaryotic organisms. Based on our investigation, cyclo(Pro-Tyr) disrupts plasma membrane polarization, induces oxidative stress and increases membrane fluidity, which compromises fungal membrane integrity. These cytological and physiological changes induced by cyclo(Pro-Tyr) may be triggered by the destabilization of membrane microdomains containing the [H+]ATPase Pma1. In response to cyclo(Pro-Tyr) stress, fungal cells activate a transcriptomic and metabolomic response, which primarily involves lipid metabolism and Reactive Oxygen Species (ROS) detoxification, to mitigate membrane damage. This similar response occurs in the nematode C. elegans, indicating that cyclo(Pro-Tyr) targets eukaryotic cellular membranes.

Mitophagy-associated programmed neuronal death and neuroinflammation.

Mitochondria are crucial organelles that play a central role in cellular metabolism and programmed cell death in eukaryotic cells. Mitochondrial autophagy (mitophagy) is a selective process where damaged mitochondria are encapsulated and degraded through autophagic mechanisms, ensuring the maintenance of both mitochondrial and cellular homeostasis. Excessive programmed cell death in neurons can result in functional impairments following cerebral ischemia and trauma, as well as in chronic neurodegenerative diseases, leading to irreversible declines in motor and cognitive functions. Neuroinflammation, an inflammatory response of the central nervous system to factors disrupting homeostasis, is a common feature across various neurological events, including ischemic, infectious, traumatic, and neurodegenerative conditions. Emerging research suggests that regulating autophagy may offer a promising therapeutic avenue for treating certain neurological diseases. Furthermore, existing literature indicates that various small molecule autophagy regulators have been tested in animal models and are linked to neurological disease outcomes. This review explores the role of mitophagy in programmed neuronal death and its connection to neuroinflammation.

Differential Mobility Spectrometry-Based Cardiolipin Analysis.

Cardiolipins (CL) are special lipids in many respects. First of all, CL are composed of four fatty acids linked by two phosphatidic acids, which provide CL a unique molecular structure. Secondly, in eukaryotic cells they are specific to a single organelle, mitochondria, where they are also synthetized. CL are one of the most abundant lipid classes in mitochondria, mainly localized in the inner membrane. They are key determinants of mitochondrial health and homeostasis by modulating membrane integrity and fluidity, mitochondrial shapes, and metabolic pathways. Disturbances in mitochondrial CL composition can lead to tissue malfunction and diseases. It is therefore important to develop analytical tools to study the mitochondrial lipidome, and more particularly the CL. The method described here allows the quantification of cardiolipins at the sum composition level in isolated mitochondria or in liver tissue by flow injection analysis coupled to differential mobility spectrometry (FIA-DMS), also known as DMS-based shotgun lipidomics.

Nonthermal fluctuations accelerate biomolecular motors

AbstractIntracellular transport is essential for maintaining cellular function. This process is driven by different mechanisms in prokaryotic and eukaryotic cells. In small prokaryotic cells, diffusion is the primary means of transport, while larger eukaryotic cells also rely on active transport by molecular motors such as kinesin and dynein. Recently, it has become evident that, in addition to diffusion based on thermal fluctuations (Brownian motion), which was conventionally considered a diffusion mechanism within living cells, nonthermal fluctuations generated by metabolic activities play a crucial role in intracellular diffusion. Similarly, while molecular motors have been proposed to exploit thermal fluctuations in the environment following the direct observation and manipulation of single molecules, they have also been reported to utilize nonthermal fluctuations in recent years. This review begins with a brief overview of the historical knowledge of diffusive intracellular transport, which has been extended from the thermal fluctuations to the nonthermal fluctuations generated by metabolic activity. It then introduces recent findings on how nonthermal fluctuations accelerate the motion of molecular motors and discusses future perspectives on the general effects of these fluctuations on molecules in living cells.

B-060 Prevalence of CCD and profilins antibodies in a seasonal allergy study

Abstract Background Allergen sources are complex and heterogeneous mixtures of proteins, which may contain from innocuous IgE-binding structures such as CCDs (cross-reactive carbohydrate determinants), which are not associated with symptoms, to a panallergen such as profilin, which is associated with multiple sensitizations to pollen and pollen-food-latex syndromes. CCDs are present in many plant and animal allergens and cause strong cross-reactivity due to the high similarity of their structure. They complicate the interpretation of positive in vitro diagnostic results because they do not associate symptomatology and, thats why, the determination of specific IgE antibodies against CCDs can provide useful information when positive IgE results are not consistent with the clinical picture. Profilins are ubiquitous proteins present in all eukaryotic cells and identified as allergens in pollen, latex and plant foods. The highly conserved structure explains the cross-reactivity of IgE antibodies against plant profilins and their designation as panallergens. As a panallergen, profilin is associated with multiple pollen sensitization and pollen-food-latex syndromes that the allergist must be aware of for accurate diagnosis and successful treatment of allergic diseases. We intend to evaluate the prevalence of these two markers with a new diagnostic tool, since profilins and CCDs are the substances most likely to cause in vitro cross-reactivity. Methods In our study 8,242 adults who presented rhinoconjunctivitis and visited their primary care center were included. The samples were analyzed in serum samples which were routinely processed in our laboratory with the EUROLINE Mediterranean Inhalation 2 (IgE) assay from Euroimmun® from February to July 2023, according to the manufacturer’s protocols. Patients with specific IgE levels ≥ 0.35 kU/L were identified as sensitized to allergen. Results A total of 8,242 patients were enrolled in this study, 5,173 women and 3,069 men, ranging in age from 15 to 65 years. Of these, 279 patients (3.39%) had antibodies against CCD, 570 (6.92%) antibodies against profilins and 183 (2.22%) had both. A higher prevalence of these antibodies was observed in men (CCD, n = 168, 5.47%; Profilins, n = 308, 10.04%) than in women (CCD, n = 111, 2.15%; Profilins, n = 262, 5.06%). Both markers were more frequently found in the presence of specific IgE antibodies to grass and weed pollens. Conclusions The possibility of detecting the presence of IgE antibodies against CCD and panallergenic profilins is useful to detect cross-reactivity in vitro, which could explain the differences found between laboratory results and patients' symptoms, helping the allergist to make a diagnosis with greater certainty and accuracy.

LYCHOS is a human hybrid of a plant-like PIN transporter and a GPCR

Lysosomes have crucial roles in regulating eukaryotic metabolism and cell growth by acting as signalling platforms to sense and respond to changes in nutrient and energy availability1. LYCHOS (GPR155) is a lysosomal transmembrane protein that functions as a cholesterol sensor, facilitating the cholesterol-dependent activation of the master protein kinase mechanistic target of rapamycin complex 1 (mTORC1)2. However, the structural basis of LYCHOS assembly and activity remains unclear. Here we determine several high-resolution cryo-electron microscopy structures of human LYCHOS, revealing a homodimeric transmembrane assembly of a transporter-like domain fused to a G-protein-coupled receptor (GPCR) domain. The class B2-like GPCR domain is captured in the apo state and packs against the surface of the transporter-like domain, providing an unusual example of a GPCR as a domain in a larger transmembrane assembly. Cholesterol sensing is mediated by a conserved cholesterol-binding motif, positioned between the GPCR and transporter domains. We reveal that the LYCHOS transporter-like domain is an orthologue of the plant PIN-FORMED (PIN) auxin transporter family, and has greater structural similarity to plant auxin transporters than to known human transporters. Activity assays support a model in which the LYCHOS transporter and GPCR domains coordinate to sense cholesterol and regulate mTORC1 activation.

The Archaeal Cell Cycle.

Since first identified as a separate domain of life in the 1970s, it has become clear that archaea differ profoundly from both eukaryotes and bacteria. In this review, we look across the archaeal domain and discuss the diverse mechanisms by which archaea control cell cycle progression, DNA replication, and cell division. While the molecular and cellular processes archaea use to govern these critical cell biological processes often differ markedly from those described in bacteria and eukaryotes, there are also striking similarities that highlight both unique and common principles of cell cycle control across the different domains of life. Since much of the eukaryotic cell cycle machinery has its origins in archaea, exploration of the mechanisms of archaeal cell division also promises to illuminate the evolution of the eukaryotic cell cycle.

Entangling roles of cholesterol-dependent interaction and cholesterol-mediated lipid phase heterogeneity in regulating listeriolysin O pore-formation.

Cholesterol-dependent cytolysins (CDCs) are the distinct class of β-barrel pore-forming toxins (β-PFTs) that attack eukaryotic cell membranes, and form large, oligomeric, transmembrane β-barrel pores. Listeriolysin O (LLO) is a prominent member in the CDC family. As documented for the other CDCs, membrane cholesterol is essential for the pore-forming functionality of LLO. However, it remains obscure how exactly cholesterol facilitates its pore formation. Here, we show that cholesterol promotes both membrane-binding and oligomerization of LLO. We demonstrate cholesterol not only facilitates membrane-binding, it also enhances the saturation threshold of LLO-membrane association, and alteration of the cholesterol-recognition motif in the LLO mutant (LLOT515G-L516G) compromises its pore-forming efficacy. Interestingly, such defect of LLOT515G-L516G could be rescued in the presence of higher membrane cholesterol levels, suggesting cholesterol can augment the pore-forming efficacy of LLO even in the absence of a direct toxin-cholesterol interaction. Furthermore, we find the membrane-binding and pore-forming abilities of LLOT515G-L516G, but not those of LLO, correlate with the cholesterol-dependent rigidity/ordering of the membrane lipid bilayer. Our data further suggest that the line tension derived from the lipid phase heterogeneity of the cholesterol-containing membranes could play a pivotal role in LLO function, particularly in the absence of cholesterol binding. Therefore, in addition to its receptor-like role, we conclude cholesterol can further facilitate the pore-forming, membrane-damaging functionality of LLO by asserting the optimal physicochemical environment in membranes. To the best of our knowledge, this aspect of the cholesterol-mediated regulation of the CDC mode of action has not been appreciated thus far.

Phloem sap from melon plants contains extracellular vesicles that carry active proteasomes which increase in response to aphid infestation.

The morphogenesis of higher plants requires communication among distant organs throughout vascular tissues (xylem and phloem). Numerous investigations have demonstrated that phloem also act as a distribution route for signalling molecules being observed that different macromolecules translocated by the sap, including nucleic acids and proteins, change under stress situations. The participation of extracellular vesicles (EVs) in this communication has been suggested, although little is known about their role. In fact, in the last decade, the presence of EVs in plants has originated a great controversy, where major concerns arose from their origin, isolation methods, and even the appropriate nomenclature for plant nanovesicles. Phloem sap exudates from melon plants, either aphid-free or infested with Aphis gossypii, were collected by stem incision. After sap concentration (Amicon), phloem EVs (PhlEVs) were isolated by size exclusion chromatography. PhlEVs were characterised using Nanoparticle Tracking Analysis, Transmission electron microscopy and proteomic analysis. Here we confirm the presence of EVs in phloem sap in vivo and the detection of changes in the particles/protein ratio and composition of PhlEVs in response to insect feeding, revealing the presence of typical defence proteins in their cargo as well as components of the proteasome complex. PhlEVs from infested plants showed lower particles/protein ratio and almost two times more proteolytic activity than PhlEVs from aphid-free plants. In both cases, such activity was inhibited in a dose-dependent manner by the proteasome inhibitor MG132. Our results suggest that plants may use this mechanism to prepare themselves to receive infectious agents and open up the possibility of an evolutionary conserved mechanism of defence against pathogens/stresses in eukaryotic organisms.

Eukaryotic cell size regulation and its implications for cellular function and dysfunction.

Depending on cell type, environmental inputs, and disease, the cells in the human body can have widely different sizes. In recent years, it has become clear that cell size is a major regulator of cell function. However, we are only beginning to understand how the optimization of cell function determines a given cell's optimal size. Here, we review currently known size control strategies of eukaryotic cells and the intricate link of cell size to intracellular biomolecular scaling, organelle homeostasis, and cell cycle progression. We detail the cell size-dependent regulation of early development and the impact of cell size on cell differentiation. Given the importance of cell size for normal cellular physiology, cell size control must account for changing environmental conditions. We describe how cells sense environmental stimuli, such as nutrient availability, and accordingly adapt their size by regulating cell growth and cell cycle progression. Moreover, we discuss the correlation of pathological states with misregulation of cell size and how for a long time this was considered a downstream consequence of cellular dysfunction. We review newer studies that reveal a reversed causality, with misregulated cell size leading to pathophysiological phenotypes such as senescence and aging. In summary, we highlight the important roles of cell size in cellular function and dysfunction, which could have major implications for both diagnostics and treatment in the clinic.

T6SS-associated Rhs toxin-encapsulating shells: Structural and bioinformatical insights into bacterial weaponry and self-protection.

Bacteria use the type VI secretion system (T6SS) to secrete toxins into pro- and eukaryotic cells via machinery consisting of a contractile sheath and a rigid tube. Rearrangement hotspot (Rhs) proteins represent one of the most common T6SS effectors. The Rhs C-terminal toxin domain displays great functional diversity, while the Rhs core is characterized by YD repeats. We elucidate the Rhs core structures of PAAR- and VgrG-linked Rhs proteins from Salmonella bongori and Advenella mimigardefordensis, respectively. The Rhs core forms a large shell of β-sheets with a negatively charged interior and encloses a large volume. The S.bongori Rhs toxin does not lead to ordered density in the Rhs shell, suggesting the toxin is unfolded. Together with bioinformatics analysis showing that Rhs toxins predominantly act intracellularly, this suggests that the Rhs core functions two-fold, as a safety feature for the producer cell and as delivery mechanism for the toxin.