Cytoplasmic Environment Research Articles

Vesicular mechanisms of drug resistance in apicomplexan parasites.

SUMMARYVesicular mechanisms of drug resistance are known to exist across prokaryotes and eukaryotes. Vesicles are sacs that form when a lipid bilayer 'bends' to engulf and isolate contents from the cytoplasm or extracellular environment. They have a wide range of functions, including vehicles of communication within and across cells, trafficking of protein intermediates to their rightful organellar destinations, and carriers of substrates destined for autophagy. This review will provide an in-depth understanding of vesicular mechanisms of apicomplexan parasites, Plasmodium and Toxoplasma (that respectively cause malaria and toxoplasmosis). It will integrate mechanistic and evolutionarily insights gained from these and other pathogenic eukaryotes to develop a new model for plasmodial resistance to artemisinins, a class of drugs that have been the backbone of modern campaigns to eliminate malaria worldwide. We also discuss extracellular vesicles that present major vesicular mechanisms of drug resistance in parasite protozoa (that apicomplexans are part of). Finally, we provide a broader context of clinical drug resistance mechanisms of Plasmodium, Toxoplasma, as well as Cryptosporidium and Babesia, that are prominent members of the phyla, causative agents of cryptosporidiosis and babesiosis and significant for human and animal health.

Dendritic Platinum Nanoparticles Shielded by Pt‐S PEGylation as Intracellular Reactors for Bioorthogonal Uncaging Chemistry

Beyond their classical role as cytotoxics, Platinum (Pt) coordination complexes recently joined the selected group of transition metals capable of performing bioorthogonal reactions in living environments. To minimize their reactivity towards nucleophiles, which limit their catalytic performance, we investigated the use of Pt(0) with different forms, sizes and surface functionalization. We report herein the development of PEGylated Pt nanodendrites with the capacity to activate prodyes and prodrugs in cell culture and in vivo. Their dendritic morphology together with their surface shielding through Pt‐S‐bonded PEGylation synergistically contributed to create catalytic nanoreactors compatible with the highly‐crowded and reductive environment of the cell cytoplasm, thereby facilitating in situ bioorthogonal drug uncaging in cancer cells in 2D and 3D culture, including in microfluidic systems, and xenografted in zebrafish.

Dendritic Platinum Nanoparticles Shielded by Pt-S PEGylation as Intracellular Reactors for Bioorthogonal Uncaging Chemistry.

Beyond their classical role as cytotoxics, Platinum (Pt) coordination complexes recently joined the selected group of transition metals capable of performing bioorthogonal reactions in living environments. To minimize their reactivity towards nucleophiles, which limit their catalytic performance, we investigated the use of Pt(0) with different forms, sizes and surface functionalization. We report herein the development of PEGylated Pt nanodendrites with the capacity to activate prodyes and prodrugs in cell culture and in vivo. Their dendritic morphology together with their surface shielding through Pt-S-bonded PEGylation synergistically contributed to create catalytic nanoreactors compatible with the highly-crowded and reductive environment of the cell cytoplasm, thereby facilitating in situ bioorthogonal drug uncaging in cancer cells in 2D and 3D culture, including in microfluidic systems, and xenografted in zebrafish.

Multi-dimensional bio mass cytometry: simultaneous analysis of cytoplasmic proteins and metabolites on single cells.

Single-cell multi-dimensional analysis enables more profound biological insight, providing a comprehensive understanding of cell physiological processes. Due to limited cellular contents, the lack of protein and metabolite amplification ability, and the complex cytoplasmic environment, the simultaneous analysis of intracellular proteins and metabolites remains challenging. Herein, we proposed a multi-dimensional bio mass cytometry platform characterized by protein signal conversion and amplification through an orthogonal exogenous enzymatic reaction. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology was applied in the quantification of endogenous intracellular protein glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) through exogenous luciferase Nanoluc (Nluc). The simultaneous detection of GAPDH and hundreds of metabolites at the single-cell level was realized for the first time. Semiquantitative analysis of GAPDH together with single-cell metabolomes under S-nitrosoglutathione (GSNO)-induced oxidative stress was investigated. Bioinformatics analysis revealed 16 metabolites that correlated positively with GAPDH expression upon oxidative stress, including long-chain fatty acids (palmitoleic acid, myristic acid, etc.) and UDP-N-acetylglucosamine (UDP-GlcNAc). Potential synergetic functions of GAPDH and UDP-GlcNAc-mediated oxidative stress responses were also elucidated. Our work proposes a novel strategy for the simultaneous quantitative analysis of single-cell intracellular proteins and metabolites, deepens the understanding of inherent anti-oxidative stress response mechanisms, and provides the molecular fundamentals for the study of inherent biological processes.

Dengue virus and Zika virus alter endoplasmic reticulum-mitochondria contact sites to regulate respiration and apoptosis.

During infection, dengue virus (DENV) and Zika virus (ZIKV), two (ortho)flaviviruses of public health concern worldwide, induce alterations of mitochondria morphology to favor viral replication, suggesting a viral co-opting of mitochondria functions. Here, we performed an extensive transmission electron microscopy-based quantitative analysis to demonstrate that both DENV and ZIKV alter endoplasmic reticulum-mitochondria contact sites (ERMC). This correlated at the molecular level with an impairment of ERMC tethering protein complexes located at the surface of both organelles. Furthermore, virus infection modulated the mitochondrial oxygen consumption rate. Consistently, metabolomic and mitoproteomic analyses revealed a decrease in the abundance of several metabolites of the Krebs cycle and changes in the stoichiometry of the electron transport chain. Most importantly, ERMC destabilization by protein knockdown increased virus replication while dampening ZIKV-induced apoptosis. Overall, our results support the notion that flaviviruses hijack ERMCs to generate a cytoplasmic environment beneficial for sustained and efficient replication.

Motorless transport of microtubules along tubulin, RanGTP, and salt gradients

Microtubules are dynamic filaments that assemble spindles for eukaryotic cell division. As the concentration profiles of soluble tubulin and regulatory proteins are non-uniform during spindle assembly, we asked if diffusiophoresis - motion of particles under solute gradients - can act as a motorless transport mechanism for microtubules. We identify the migration of stable microtubules along cytoplasmic and higher concentration gradients of soluble tubulin, MgCl2, Mg-ATP, Mg-GTP, and RanGTP at speeds O(100) nm/s, validating the diffusiophoresis hypothesis. Using two buffers (BRB80 and CSF-XB), microtubule behavior under MgCl2 gradients is compared with negatively charged particles and analyzed with a multi-ion diffusiophoresis and diffusioosmosis model. Microtubule diffusiophoresis under gradients of tubulin and RanGTP is also compared with the charged particles and analyzed with a non-electrolyte diffusiophoresis model. Further, we find that tubulin and RanGTP display concentration dependent cross-diffusion that influences microtubule diffusiophoresis. Finally, using Xenopus laevis egg extract, we show that diffusiophoretic transport occurs in an active cytoplasmic environment.

Single-molecule diffusivity quantification in Xenopus egg extracts elucidates physicochemical properties of the cytoplasm.

The complex intracellular molecular environment is notably challenging to elucidate and recapitulate. Xenopus egg extracts provide a native yet manipulatable cytoplasm model. Through single-molecule microscopy, here we decipher the cytoplasmic environment and molecular interactions by examining the diffusion patterns of diverse proteins in Xenopus egg extracts with strategic manipulations. These experiments reveal an overwhelmingly negatively charged macromolecular environment with crosslinked meshworks, offering new insight into the inner workings of the cell.

Nucleophosmin: A Nucleolar Phosphoprotein Orchestrating Cellular Stress Responses.

Nucleophosmin (NPM1) is a key nucleolar protein released from the nucleolus in response to stress stimuli. NPM1 functions as a stress regulator with nucleic acid and protein chaperone activities, rapidly shuttling between the nucleus and cytoplasm. NPM1 is ubiquitously expressed in tissues and can be found in the nucleolus, nucleoplasm, cytoplasm, and extracellular environment. It plays a central role in various biological processes such as ribosome biogenesis, cell cycle regulation, cell proliferation, DNA damage repair, and apoptosis. In addition, it is highly expressed in cancer cells and solid tumors, and its mutation is a major cause of acute myeloid leukemia (AML). This review focuses on NPM1's structural features, functional diversity, subcellular distribution, and role in stress modulation.

Morphodynamical adaptation of the endolysosomal system to stress.

In eukaryotes, the spatiotemporal control of endolysosomal organelles is central to the maintenance of homeostasis. By providing an interface between the cytoplasm and external environment, the endolysosomal system is placed at the forefront of the response to a wide range of stresses faced by cells. Endosomes are equipped with a dedicated set of membrane-associated proteins that ensure endosomal functions as well as crosstalk with the secretory or the autophagy pathways. Morphodynamical processes operate through local spatialization of subdomains, enabling specific remodeling and membrane contact capabilities. Consequently, the plasticity of endolysosomal organelles can be considered a robust and flexible tool exploited by cells to cope with homeostatic deviations. In this review, we provide insights into how the cellular responses to various stresses (osmotic, UV, nutrient deprivation, or pathogen infections) rely on the adaptation of the endolysosomal system morphodynamics.

Computational Approaches to Predict Protein-Protein Interactions in Crowded Cellular Environments.

Investigating protein-protein interactions is crucial for understanding cellular biological processes because proteins often function within molecular complexes rather than in isolation. While experimental and computational methods have provided valuable insights into these interactions, they often overlook a critical factor: the crowded cellular environment. This environment significantly impacts protein behavior, including structural stability, diffusion, and ultimately the nature of binding. In this review, we discuss theoretical and computational approaches that allow the modeling of biological systems to guide and complement experiments and can thus significantly advance the investigation, and possibly the predictions, of protein-protein interactions in the crowded environment of cell cytoplasm. We explore topics such as statistical mechanics for lattice simulations, hydrodynamic interactions, diffusion processes in high-viscosity environments, and several methods based on molecular dynamics simulations. By synergistically leveraging methods from biophysics and computational biology, we review the state of the art of computational methods to study the impact of molecular crowding on protein-protein interactions and discuss its potential revolutionizing effects on the characterization of the human interactome.

Multifaceted role of HMGB1: From nuclear functions to cytoplasmic and extracellular signaling in inflammation and cancer-Review.

High-mobility group box 1 (HMGB1) is a highly conserved nuclear protein involved in key nuclear processes such as DNA repair, replication, and gene regulation. Beyond its established nuclear roles, HMGB1 has crucial functions in the cytoplasm and extracellular environment. When translocated to the cytoplasm, HMGB1 plays a role in autophagy, cell survival, and immune response modulation. In its extracellular form, HMGB1 acts as a damage-associated molecular patternmolecule, initiating inflammatory responses by interacting with receptors such as Receptor for advanced glycation endproductsand Toll-like receptors. Recent studies have shown its role in promoting tissue regeneration, wound healing, and angiogenesis, highlighting its dual role in both inflammation and tissue repair. Notably, the redox status of HMGB1 influences its function, with the reduced form promoting autophagy and the disulfide form driving inflammation. Dysregulation of HMGB1 contributes to the progression of various diseases, including cancer, where it influences tumor growth, metastasis, and resistance to therapy. This review provides an overview of the nuclear, cytoplasmic, and extracellular roles of HMGB1, discussing its involvement in nuclear homeostasis, rare genetic diseases, autophagy, inflammation, cancer progression, and tissue regeneration.

A zinc metal complex as an NIR emissive probe for real-time dynamics and in vivo embryogenic evolution of lysosomes using super-resolution microscopy.

Zinc (Zn) based fluorescent metal complexes have gained increasing attention due to their non-toxicity and high brightness with marked fluorescence quantum yield (QY). However, they have rarely been employed in super-resolution microscopy (SRM) to study live cells and in vivo dynamics of lysosomes. Here, we present an NIR emissive highly photostable Zn-complex as a multifaceted fluorescent probe for the long-term dynamical distribution of lysosomes in various cancerous and non-cancerous cells in live condition and in vivo embryogenic evolution in Caenorhabditis elegans (C. elegans). Apart from the normal fission, fusion, and kiss & run, the motility and the exact location of lysosomes at each point were mapped precisely. A notable difference in the lysosomal motility in the peripheral region between cancerous and non-cancerous cells was distinctly observed. This is attributed to the difference in viscosity of the cytoplasmic environment. On the other hand, along with the super-resolved structure of the smallest size lysosome (∼77 nm) in live C. elegans, the complete in vivo embryogenic evolution of lysosomes and lysosome-related organelles (LROs) was captured. We were able to capture the images of lysosomes and LROs at different stages of C. elegans, starting from a single cell and extending to a fully matured adult animal.

Structure Elucidation and Interaction Dynamics of MefA-MsrD Efflux Proteins in Streptococcus pneumoniae: Impact on Macrolide Susceptibility.

Macrolides are empirically used to treat bacterial community-acquired pneumonia (CAP). Streptococcus pneumoniae, being the major pathogen responsible for bacterial CAP with high mortality rates, express MefA-MsrD efflux pumps to hinder macrolide susceptibility. Despite its importance, the structural features of the efflux-protein complex and its impact on macrolide susceptibility have not yet been elucidated explicitly. Therefore, in the present study, combining homology, threading, and dynamics approaches, MefA and MsrD proteins in pathogenic S. pneumoniae were modeled. Both membrane (lipid-bilayer) and cytoplasmic (aqueous) environments were considered to simulate the MefA and MsrD proteins in their ideal cellular conditions followed by dynamics analyses. The simulated MefA structure represented a typical major facilitator superfamily protein structure with 13 transmembrane helices. MefA-MsrD interaction via clustering-based docking revealed low-energy conformers with stable intermolecular interactions. The higher clinical MIC value of azithromycin over erythromycin was reflected upon erythromycin eliciting stronger interactions (dissociation constant or ki = ∼52 μM) with the cytoplasmic ATP-binding MsrD than azithromycin (ki = ∼112 μM). The strong (binding energy = -132.1 ± 9.5 kcal/mol) and highly stable (root-mean-square fluctuation < 1.0 Å) physical association between MefA with MsrD was validated and was found to be unaffected by the antibiotic binding. Higher propensity of the macrolides to interact with MsrD than MefA established the importance of the former in macrolide susceptibility. Ours is probably the first report on the structural arrangements in the MefA-MsrD efflux complex and the macrolide susceptibility in S. pneumoniae. This study provides a novel lead for experimental explorations and efflux-pump inhibitor designs.

Self-Assembled Peptide Hydrogels in Regenerative Medicine.

Regenerative medicine is a complex discipline that is becoming a hot research topic. Skin, bone, and nerve regeneration dominate current treatments in regenerative medicine. A new type of drug is urgently needed for their treatment due to their high vulnerability to damage and weak self-repairing ability. A self-assembled peptide hydrogel is a good scaffolding material in regenerative medicine because it is similar to the cytoplasmic matrix environment; it promotes cell adhesion, migration, proliferation, and division; and its degradation products are natural and harmless proteins. However, fewer studies have examined the specific mechanisms of self-assembled peptide hydrogels in promoting tissue regeneration. This review summarizes the applications and mechanisms of self-assembled short peptide and peptide hydrogels in skin, bone, and neural healing to improve their applications in tissue healing and regeneration.

Potassium deficiency stress reduces Rubisco activity in Brassica napus leaves by subcellular acidification decreasing photosynthetic rate

Under potassium (K) deficiency photosynthetic carboxylation capacities are limited, affecting the photosynthetic rate of plants. However, it is not clear how ionic K within plants regulates carboxylation capacities. Therefore, the photosynthetic rate (A), ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) characteristics, and cytoplasmic pH of Brassica napus leaves with different K levels were measured to evaluate the effects of K on the carboxylation capacity by regulating subcellular pH. The results showed that biochemical limitation dominates the decrease of A. There was a close positive correlation between A and the Rubisco maximum carboxylation rate (Vcmax), which was closer than that between A and the maximum electron transport rate. The thresholds of leaf K concentrations causing decreased A, Vcmax, and Rubisco initial activity were consistent and close to 1.0% in the hydroponic experiments and 1.2% in the field experiments. K deficiency resulted in decreased Rubisco activity, which reduced carboxylation capacity. Moreover, the Rubisco initial activities in vitro with sufficient K supply or under K deficiency all were significantly reduced when the pH was decreased. The cytoplasmic pH was kept neutral at 7.5 under sufficient K supply, and decreased as the leaf K concentration declined below the threshold. Acidified cytoplasmic environment caused by K deficiency could not maintain the pH balance of the chloroplasts, leading to decreased Rubisco initial activity and photosynthetic capacity.

Cytosolic Interactome Protects Against Protein Unfolding in a Single Molecule Experiment.

Single molecule techniques are particularly well suited for investigating the processes of protein folding and chaperone assistance. However, current assays provide only a limited perspective on the various ways in which the cellular environment can influence the folding pathway of a protein. In this study, a single molecule mechanical interrogation assay is developed and used to monitor protein unfolding and refolding within a cytosolic solution. This allows to test the cumulative topological effect of the cytoplasmic interactome on the folding process. The results reveal a stabilization against forced unfolding for partial folds, which are attributed to the protective effect of the cytoplasmic environment against unfolding and aggregation. This research opens the possibility of conducting single molecule molecular folding experiments in quasi-biological environments.

The N-terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function

Due to unique features, proline residues may control protein structure and function. Here, we investigated the role of 52PPQ54 residues, indicated by the recently established experimental 3D structure of bovine herpesvirus 1-encoded UL49.5 protein as forming a characteristic proline hinge motif in its N-terminal domain. UL49.5 acts as a potent inhibitor of the transporter associated with antigen processing (TAP), which alters the antiviral immune response. Mechanisms employed by UL49.5 to affect TAP remain undetermined on a molecular level. We found that mutations in the 52PPQ54 region had a vast impact on its immunomodulatory function, increasing cell surface MHC class I expression, TAP levels, and peptide transport efficiency. This inhibitory effect was specific for UL49.5 activity towards TAP but not towards the viral glycoprotein M. To get an insight into the impact of proline hinge modifications on structure and dynamics, we performed all-atom and coarse-grained molecular dynamics studies on the native protein and PPQ mutants. The results demonstrated that the proline hinge sequence with its highly rigid conformation served as an anchor into the membrane. This anchor was responsible for the structural and dynamical behavior of the whole protein, constraining the mobility of the C-terminus, increasing the mobility of the transmembrane region, and controlling the accessibility of the C-terminal residues to the cytoplasmic environment. Those features appear crucial for TAP binding and inhibition. Our findings significantly advance the structural understanding of the UL49.5 protein and its functional regions and support the importance of proline motifs for the protein structure.

Maize cytolines as models to study the impact of different cytoplasms on gene expression under heat stress conditions

BackgroundCrops are under constant pressure due to global warming, which unfolds at a much faster pace than their ability to adapt through evolution. Agronomic traits are linked to cytoplasmic-nuclear genome interactions. It thus becomes important to understand the influence exerted by the organelles on gene expression under heat stress conditions and profit from the available genetic diversity. Maize (Zea mays) cytolines allow us to investigate how the gene expression changes under heat stress conditions in three different cytoplasmic environments, but each having the same nucleus. Analyzing retrograde signaling in such an experimental set-up has never been done before. Here, we quantified the response of three cytolines to heat stress as differentially expressed genes (DEGs), and studied gene expression patterns in the context of existing polymorphism in their organellar genomes.ResultsOur study unveils a plethora of new genes and GO terms that are differentially expressed or enriched, respectively, in response to heat stress. We report 19,600 DEGs as responding to heat stress (out of 30,331 analyzed), which significantly enrich 164 GO biological processes, 30 GO molecular functions, and 83 GO cell components. Our approach allowed for the discovery of a significant number of DEGs and GO terms that are not common in the three cytolines and could therefore be linked to retrograde signaling. Filtering for DEGs with a fold regulation > 2 (absolute values) that are exclusive to just one of the cytolines, we find a total of 391 up- and down-DEGs. Similarly, there are 19 GO terms with a fold enrichment > 2 that are cytoline-specific. Using GBS data we report contrasting differences in the number of DEGs and GO terms in each cytoline, which correlate with the genetic distances between the mitochondrial genomes (but not chloroplast) and the original nuclei of the cytolines, respectively.ConclusionsThe experimental design used here adds a new facet to the paradigm used to explain how gene expression changes in response to heat stress, capturing the influence exerted by different organelles upon one nucleus rather than investigating the response of several nuclei in their innate cytoplasmic environments.

Changes in seam number and location induce holes within microtubules assembled from porcine brain tubulin and in Xenopus egg cytoplasmic extracts.

Microtubules are tubes of about 25 nm in diameter that are critically involved in a variety of cellular functions, including motility, compartmentalization, and division. They are considered as pseudo-helical polymers whose constituent αβ-tubulin heterodimers share lateral homotypic interactions, except at one unique region called the seam. Here, we used a segmented sub-tomogram averaging strategy to reassess this paradigm and analyze the organization of the αβ-tubulin heterodimers in microtubules assembled from purified porcine brain tubulin in the presence of GTP and GMPCPP, and in Xenopus egg cytoplasmic extracts. We find that in almost all conditions, microtubules incorporate variable protofilament and/or tubulin subunit helical-start numbers, as well as variable numbers of seams. Strikingly, the seam number and location vary along individual microtubules, generating holes of one to a few subunits in size within their lattices. Together, our results reveal that the formation of mixed and discontinuous microtubule lattices is an intrinsic property of tubulin that requires the formation of unique lateral interactions without longitudinal ones. They further suggest that microtubule assembly is tightly regulated in a cytoplasmic environment.

Membrane component ergosterol builds a platform for promoting effector secretion and virulence in Magnaporthe oryzae.

The plasma membrane (PM) functions as a physical border between the extracellular and cytoplasmic environments that contribute to the interaction between host plants and pathogenic fungi. As a specific sterol constituent in the cell membrane, ergosterol plays a significant role in fungal development. However, the role of ergosterol in the infection of the rice blast fungus Magnaporthe oryzae remains unclear. In this study, we found that a sterol reductase, MoErg4, is involved in ergosterol biosynthesis and the regulation of plasma membrane integrity in M.oryzae. We found that defects in ergosterol biosynthesis disrupt lipid raft formation in the PM and cause an abnormal distribution of the t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein MoSso1, inhibiting its interaction with the v-SNARE protein MoSnc1. In addition, we found that MoSso1-MoSnc1 interaction is important for biotrophic interface complex development and cytoplasmic effector protein secretion. Our findings suggested that ergosterol-enriched lipid rafts constitute a platform for interactions among various SNARE proteins that are required for the development and pathogenicity of M.oryzae.