Cell Biology Research Articles

Specific sialylation of N-glycans and its novel regulatory mechanism.

Altered glycosylation is a common feature of cancer cells. Some subsets of glycans are found to be frequently enriched on the tumor cell surface and implicated in different tumor phenotypes. Among these, changes in sialylation have long been associated with metastatic cell behaviors such as invasion and enhanced cell survival. Sialylation typically exists in three prominent linkages: α2,3, α2,6, and α2,8, catalyzed by a group of sialyltransferases. The aberrant expression of all three linkages has been related to cancer progression. The increased α2,6 sialylation on N-glycans catalyzed by β-galactoside α2,6 sialyltransferase 1 (ST6Gal1) is frequently observed in many cancers. In contrast, functions of α2,3 sialylation on N-glycans catalyzed by at least three β-galactoside α2,3-sialyltransferases, ST3Gal3, ST3Gal4, and ST3Gal6 remain elusive due to a possibility of compensating for one another. In this minireview, we briefly describe functions of sialylation and recent findings that different α2,3 sialyltransferases specifically modify target proteins, as well as sialylation regulatory mechanisms vis a complex formation among integrin α3β1, Golgi phosphoprotein 3 (GOLPH3), phosphatidylinositol 4-kinase IIα (PI4KIIα), focal adhesion kinase (FAK) and sialyltransferase, which suggests a new concept for the regulation of glycosylation in cell biology.

MTORC1 Signaling in Brain Endothelial Progenitors Contributes to CCM Pathogenesis.

Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.

A novel induced pluripotent stem cell model of Schwann cell differentiation reveals NF2 - related gene regulatory networks of the extracellular matrix.

Schwann cells are vital to development and maintenance of the peripheral nervous system and their dysfunction has been implicated in a range of neurological and neoplastic disorders, including NF2 -related schwannomatosis. We developed a novel human induced pluripotent stem cell (hiPSC) model to study Schwann cell differentiation in health and disease. We performed transcriptomic, immunofluorescence, and morphological analysis of hiPSC derived Schwann cell precursors (SPCs) and terminally differentiated Schwann cells (SCs) representing distinct stages of development. To validate our findings, we performed integrated, cross-species analyses across multiple external datasets at bulk and single cell resolution. Our hiPSC model of Schwann cell development shared overlapping gene expression signatures with human amniotic mesenchymal stem cell (hAMSCs) derived SCs and in vivo mouse models, but also revealed unique features that may reflect species-specific aspects of Schwann cell biology. Moreover, we identified gene co-expression modules that are dynamically regulated during hiPSC to SC differentiation associated with ear and neural development, cell fate determination, the NF2 gene, and extracellular matrix (ECM) organization. By cross-referencing results between multiple datasets, we identified new genes potentially associated with NF2 expression. Our hiPSC model further provides a tractable platform for studying Schwann cell development in the context of human disease.

A Rapid Human Lung Tissue Dissociation Protocol Maximizing Cell Yield and Minimizing Cellular Stress.

The human lung is a complex organ comprised of diverse populations of epithelial, mesenchymal, vascular and immune cells, which gains even greater complexity during disease states. To effectively study the lung at a single cell level, a dissociation protocol that achieves the highest yield of viable cells of interest with minimal dissociation-associated protein or transcription changes key. Here, we detail a rapid collagenase-based dissociation protocol (Col-Short), which provides a high-yield single cell suspension suitable for a variety of downstream applications. Diseased human lung explants were obtained and dissociated through the Col-Short protocol and compared to four other dissociation protocols. Resulting single cell suspensions were then assessed with flow cytometry, differential staining, and quantitative real-time PCR to identify major hematopoietic and non-hematopoietic cell populations, as well as their activation states. We observed that the Col-Short protocol provides the greatest number of cells per gram of lung tissue with no reduction in viability when compared to previously described dissociation protocols. Col-Short had no observable surface protein marker cleavage as well as lower expression of protein activation markers and stress-related transcripts compared to four other protocols. The Col-Short dissociation protocol can be used as a rapid strategy to generate single cells for respiratory cell biology research.

MSphere of Influence: The power of observational research.

Katrina Velle is a cell biologist who uses microscopy to study amoebae. In this mSphere of Influence article, she reflects on how a classic paper on Listeria by Tilney and Portnoy made an impact on her by highlighting how much we can learn from simply looking at cells.

Mesenteric lymph nodes: a critical site for the up-regulatory effect of hUC-MSCs on Treg cells by producing TGF-β1 in colitis treatment

BackgroundMesenchymal stem cells (MSCs) demonstrate a wide range of therapeutic capabilities in the treatment of inflammatory bowel disease (IBD). The intraperitoneal injection of MSCs has exhibited superior therapeutic efficacy on IBD than intravenous injection. Nevertheless, the precise in vivo distribution of MSCs and their biological consequences following intraperitoneal injection remain inadequately understood. Additional studies are required to explore the correlation between MSCs distribution and their biological effects.MethodsFirst, the distribution of human umbilical cord MSCs (hUC-MSCs) and the numbers of Treg and Th17 cells in mesenteric lymph nodes (MLNs) were analyzed after intraperitoneal injection of hUC-MSCs. Subsequently, the investigation focused on the levels of transforming growth factor beta1 (TGF-β1), a key cytokine to the biology of both Treg and Th17 cells, in tissues of mice with colitis, particularly in MLNs. The study also delved into the impact of hUC-MSCs therapy on Treg cell counts in MLNs, as well as the consequence of TGFB1 knockdown hUC-MSCs on the differentiation of Treg cells and the treatment of IBD.ResultsThe therapeutic effectiveness of intraperitoneally administered hUC-MSCs in the treatment of colitis was found to be significant, which was closely related to their quick migration to MLNs and secretion of TGF-β1. The abundance of hUC-MSCs in MLNs of colitis mice is much higher than that in other organs even the inflamed sites of colon. Intraperitoneal injection of hUC-MSCs led to a significant increase in the number of Treg cells and a decrease in Th17 cells especially in MLNs. Furthermore, the concentration of TGF-β1, the key cytokine for Treg differentiation, were also found to be significantly elevated in MLNs after hUC-MSCs treatment. Knockdown of TGFB1 in hUC-MSCs resulted in a noticeable reduction of Treg cells in MLNs and the eventually failure of hUC-MSCs therapy in colitis.ConclusionsMLNs may be a critical site for the regulatory effect of hUC-MSCs on Treg/Th17 cells and the therapeutic effect on colitis. TGF-β1 derived from hUC-MSCs promotes local Treg differentiation in MLNs. This study will provide new ideas for the development of MSC-based therapeutic strategies in IBD patients.

An efficient in vivo-inducible CRISPR interference system for group A Streptococcus genetic analysis and pathogenesis studies.

While group A Streptococcus (GAS) remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference (CRISPRi) for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS and, together with SpyBrowse, represents a valuable resource for researchers in the GAS field.

IGF-1 Peptide Mimetic-functionalized Hydrogels Enhance MSC Survival and Immunomodulatory Activity.

Insulin-like growth factor 1 (IGF-1) plays a multifaceted role in stem cell biology and may promote proliferation, survival, migration, and immunomodulation for MSCs. In this study, we functionalized alginate hydrogels with integrin-binding and IGF-1 peptide mimetics to investigate their impact on MSC function. Embedding MSCs in these hydrogels enhanced their ability to reduce inflammatory cytokine production and promote anti-inflammatory gene expression in cells from degenerative human intervertebral discs exposed to proteins secreted by the MSC. This approach suggests a new way to retain and augment MSC functionality using IGF-1 peptide mimetics, offering an alternative to co-delivery of cells and high dose soluble growth factors for tissue repair and immune- system modulation.

Interview with Journal of Cell Science Editor Rob Parton

ABSTRACT Rob Parton is a Group Leader and Australian Research Council Laureate Fellow in the Institute for Molecular Bioscience and Deputy Director of the Centre for Microscopy and Microanalysis at the University of Queensland in Brisbane, Australia. He completed his doctorate at the University of Leicester in the UK. After using electron microscopy (EM) during his PhD to study trafficking of tetanus toxin in neurons, he was inspired to continue using advanced microscopy approaches to further our understanding of the fundamental cellular process of membrane trafficking. After several years fostering international collaborations as a postdoctoral fellow and junior Group Leader at the European Molecular Biology Laboratory in Heidelberg, Germany, his lab moved to the University of Queensland in 1996. In 2023, he was elected an Associate Member of the European Molecular Biology Organization (EMBO). Rob joined Journal of Cell Science as an Editor in March 2024 and brings to the journal his expertise in multiscale analysis of membrane function, membrane microdomains, lipid droplets and advanced microscopy techniques in cell biology. We spoke to Rob over Zoom to hear more about his career journey, the evolution of the membrane trafficking field and his advice for running a highly collaborative lab.

Enhancing amyloid beta inhibition and disintegration by natural compounds: A study utilizing spectroscopy, microscopy and cell biology

Amyloid proteins and peptides play a pivotal role in the etiology of various neurodegenerative diseases, including Alzheimer's disease (AD). Synthetically designed small molecules/ peptides/ peptidomimetics show promise towards inhibition of various kinds of amyloidosis. However, exploration of compounds isolated from natural extracts having such potential is lacking. Herein, we have investigated the repurposing of a traditional Indian medicine Lasunadya Ghrita (LG) in AD. LG is traditionally used to treat gut dysregulation and mental illnesses. Various extracts of LG were obtained, characterized, and analyzed for inhibition of Aβ aggregation. Biophysical studies show that the water extract of LG (LGWE) is more potent in inhibiting Aβ peptide aggregation and defibrillation of Aβ40/Aβ42 aggregates. NMR studies showed that LGWE binds to the central hydrophobic area and C-terminal residues of Aβ40/Aβ42, thereby modulating the aggregation, and reducing cell membrane damage. Additionally, LGWE rescues Aβ toxicity in neuronal SH-SY5Y cells evident from decreases in ROS generation, membrane leakage, cellular apoptosis, and calcium dyshomeostasis. Notably, LGWE is non-toxic to neuronal cells and mouse models. Our study thus delves into the mechanistic insights of a repurposed drug LGWE with the potential to ameliorate Aβ induced neuroinflammation.

High-Q WGM microcavity-based optofluidic sensor technologies for biological analysis.

High-quality-factor (Q) optical microcavities have attracted extensive interest due to their unique ability to confine light for resonant circulation at the micrometer scale. Particular attention has been paid to optical whispering-gallery mode (WGM) microcavities to harness their strong light-matter interactions for biological applications. Remarkably, the combination of high-Q optical WGM microcavities with microfluidic technologies can achieve a synergistic effect in the development of high-sensitivity optofluidic sensors for many emerging biological analysis applications, such as the detection of proteins, nucleic acids, viruses, and exosomes. They can also be utilized to investigate the behavior of living cells in human organisms, which may provide new technical solutions for studies in cell biology and biophysics. In this paper, we briefly review recent progress in high-Q microcavity-based optofluidic sensor technologies and their applications in biological analysis.

Cell biology: Converging paths to cohesion

Cohesin holds together the sister chromatids from DNA replication onwards. How cohesion is established has long remained a black box. Through recent studies, a model is emerging in which a replisome-cohesin encounter results in the establishment of cohesive linkages at sites of replication termination.

Subcutaneous checkpoint inhibition is equivalent to systemic delivery when combined with nelitolimod delivered via pressure-enabled drug delivery for depletion of intrahepatic myeloid-derived suppressor cells and control of liver metastases

BackgroundToll-like receptor 9 (TLR9) agonists induce inflammatory responses that promote the killing of infectious micro-organisms, cancer cells and develop adaptive immune responses. Their ability as immunomodulators to enhance the activity...

Approximate Bayesian computation for inferring Waddington landscapes fromsingle-cell data.

Single-cell technologies allow us to gain insights into cellular processes at unprecedented resolution. In stem cell and developmental biology snapshot data allow us to characterize how the transcriptional states of cells change between successive cell types. Here, we show how approximate Bayesian computation (ABC) can be employed to calibrate mathematical models against single-cell data. In our simulation study, we demonstrate the pivotal role of the adequate choice of distance measures appropriate for single-cell data. We show that for good distance measures, notably optimal transport with the Sinkhorn divergence, we can infer parameters for mathematical models from simulated single-cell data. We show that the ABC posteriors can be used (i) to characterize parameter sensitivity and identify dependencies between different parameters and(ii) to construct representations of the Waddington or epigenetic landscape, which forms a popular and interpretable representation of the developmental dynamics. In summary, these results pave the way for fitting mechanistic models of stem cell differentiation to single-cell data.

The people behind the papers - Paul Huber and Carole LaBonne.

The bromodomain and extra-terminal (BET) family of proteins reads epigenetic histone acetylation marks on the genome and regulates the transcriptional machinery. In their study, Carole LaBonne and colleagues reveal the role of BET protein activity in the maintenance of pluripotency and establishment of the neural crest in Xenopus laevis. To know more about their work, we spoke to the first author Paul Huber and the corresponding author Carole LaBonne, Developmental and Stem Cell Biologist at Northwestern University.

Impact of Matrix Gel Variations on Primary Culture of Microvascular Endothelial Cell Function.

The endothelium regulates crucial aspects of vascular function, including hemostasis, vasomotor tone, proliferation, immune cell adhesion, and microvascular permeability. Endothelial cells (ECs), especially in arterioles, are pivotal for flow distribution and peripheral resistance regulation. Investigating vascular endothelium physiology, particularly in microvascular ECs, demands precise isolation and culturing techniques. Freshly isolated ECs are vital for examining protein expression, ion channel behavior, and calcium dynamics. Establishing primary endothelial cell cultures is crucial for unraveling vascular functions and understanding intact microvessel endothelium roles. Despite the significance, detailed protocols and comparisons with intact vessels are scarce in microvascular research. We developed a reproducible method to isolate microvascular ECs, assessing substrate influence by cultivating cells on fibronectin and gelatin matrix gels. This comparative approach enhances our understanding of microvascular endothelial cell biology. Microvascular mesenteric ECs expressed key markers (VE-cadherin and eNOS) in both matrix gels, confirming cell culture purity. Under uncoated conditions, ECs were undetected, whereas proteins linked to smooth muscle cells and fibroblasts were evident. Examining endothelial cell (EC) physiological dynamics on distinct matrix substrates revealed comparable cell length, shape, and Ca2+ elevations in both male and female ECs on gelatin and fibronectin matrix gels. Gelatin-cultured ECs exhibited analogous membrane potential responses to acetylcholine (ACh) or adenosine triphosphate (ATP), contrasting with their fibronectin-cultured counterparts. In the absence of stimulation, fibronectin-cultured ECs displayed a more depolarized resting membrane potential than gelatin-cultured ECs. Gelatin-cultured ECs demonstrated electrical behaviors akin to intact endothelium from mouse mesenteric arteries, thus advancing our understanding of endothelial cell behavior within diverse microenvironments.

Neural-field-assisted transport-of-intensity phase microscopy: partially coherent quantitative phase imaging under unknown defocus distance

The transport-of-intensity equation (TIE) enables quantitative phase imaging (QPI) under partially coherent illumination by measuring the through-focus intensities combined with a linearized inverse reconstruction algorithm. However, overcoming its sensitivity to imaging settings remains a challenging problem because of the difficulty in tuning the optical parameters of the imaging system accurately and because of the instability to long-time measurements. To address these limitations, we propose and experimentally validate a solution called neural-field-assisted transport-of-intensity phase microscopy (NFTPM) by introducing a tunable defocus parameter into neural field. Without weak object approximation, NFTPM incorporates the physical prior of partially coherent image formation to constrain the neural field and learns the continuous representation of phase object without the need for training. Simulation and experimental results of HeLa cells demonstrate that NFTPM can achieve accurate, partially coherent QPI under unknown defocus distances, providing new possibilities for extending applications in live cell biology.

The unfolded protein response machinery in glioblastoma genesis, chemoresistance and as a druggable target.

The role of the unfolded protein response (UPR) has been progressively unveiled over the last decade and several studies have investigated its implication in glioblastoma (GB) development. The UPR restores cellular homeostasis by triggering the folding and clearance of accumulated misfolded proteins in the ER consecutive to endoplasmic reticulum stress. In case it is overwhelmed, it induces apoptotic cell death. Thus, holding a critical role in cell fate decisions. This article, reviews how the UPR is implicated in cell homeostasis maintenance, then surveils the evidence supporting the UPR involvement in GB genesis, progression, angiogenesis, GB stem cell biology, tumor microenvironment modulation, extracellular matrix remodeling, cell fate decision, invasiveness, and grading. Next, it concurs the evidence showing how the UPR mediates GB chemoresistance-related mechanisms. The UPR stress sensors IRE1, PERK, and ATF6 with their regulator GRP78 are upregulated in GB compared to lower grade gliomas and normal brain tissue. They are activated in response to oncogenes and are implicated at different stages of GB progression, from its genesis to chemoresistance and relapse. The UPR arms can be effectors of apoptosis as mediators or targets. Recent research has established the role of the UPR in GB pathophysiology and chemoresistance. Targeting its different sensors have shown promising in overcoming GB chomo- and radioresistance and inducing apoptosis.

Behaviors of nucleosomes with mutant histone H4s in euchromatic domains of living human cells.

Since Robert Feulgen first stained DNA in the cell, visualizing genome chromatin has been a central issue in cell biology to uncover how chromatin is organized and behaves in the cell. To approach this issue, we have developed single-molecule imaging of nucleosomes, a basic unit of chromatin, to unveil local nucleosome behavior in living cells. In this study, we investigated behaviors of nucleosomes with various histone H4 mutants in living HeLa cells to address the role of H4 tail acetylation, including H4K16Ac and others, which are generally associated with more transcriptionally active chromatin regions. We ectopically expressed wild-type (wt) or mutated H4s (H4K16 point; H4K5,8,12,16 quadruple; and H4 tail deletion) fused with HaloTag in HeLa cells. Cells that expressed wtH4-Halo, H4K16-Halo mutants, and multiple H4-Halo mutants had euchromatin-concentrated distribution. Consistently, the genomic regions of the wtH4-Halo nucleosomes corresponded to Hi-C contact domains (or topologically associating domains, TADs)with active chromatin marks (A-compartment). Utilizing single-nucleosome imaging, we found that none of the H4 deacetylation or acetylation mimicked H4 mutants altered the overall local nucleosome motion. This finding suggests that H4 mutant nucleosomes embedded in the condensed euchromatic domains with excess endogenous H4 nucleosomes cannot cause an observable change in the local motion. Interestingly, H4 with four lysine-to-arginine mutations displayed a substantial freely diffusing fraction in the nucleoplasm, whereas H4 with a truncated N-terminal tail was incorporated in heterochromatic regions as well as euchromatin. Our study indicates the power of single-nucleosome imaging to understand individual histone/nucleosome behavior reflecting chromatin environments in living cells.

Kinase-catalyzed biotinylation for discovery and validation of substrates to multispecificity kinases NME1 and NME2

Protein phosphorylation by kinases regulates mammalian cell functions, such as growth, division, and signal transduction. Among human kinases, NME1 and NME2 are associated with metastatic tumor suppression, but remain understudied due to the lack of tools to monitor their cellular substrates. In particular, NME1 and NME2 are multi-specificity kinases phosphorylating serine, threonine, histidine, and aspartic acid residues of substrate proteins, and the heat and acid sensitivity of phosphohistidine and phosphoaspartate complicates substrate discovery and validation. To provide new substrate monitoring tools, we established the γ-phosphate modified ATP analog, ATP-biotin, as a cosubstrate for phosphorylbiotinylation of NME1 and NME2 cellular substrates. Building upon this ATP-biotin compatibility, the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS) method enabled validation of a known substrate and the discovery of seven NME1 and three NME2 substrates. Given the paucity of methods to study kinase substrates, ATP-biotin and the K-BILDS method are valuable tools to characterize the roles of NME1 and NME2 in human cell biology.