Cell Biological Mechanisms Research Articles

Thermosynechococcus switches the direction of phototaxis by a c-di-GMP-dependent process with high spatial resolution.

Many cyanobacteria, which use light as an energy source via photosynthesis, show directional movement towards or away from a light source. However, the molecular and cell biological mechanisms for switching the direction of movement remain unclear. Here, we visualized type IV pilus-dependent cell movement in the rod-shaped thermophilic cyanobacterium Thermosynechococcus vulcanus using optical microscopy at physiological temperature and light conditions. Positive and negative phototaxis were controlled on a short time scale of 1 min. The cells smoothly moved over solid surfaces towards green light, but the direction was switched to backward movement when we applied additional blue light illumination. The switching was mediated by three photoreceptors, SesA, SesB, and SesC, which have cyanobacteriochrome photosensory domains and synthesis/degradation activity of the bacterial second messenger cyclic dimeric GMP (c-di-GMP). Our results suggest that the decision-making process for directional switching in phototaxis involves light-dependent changes in the cellular concentration of c-di-GMP. Direct visualization of type IV pilus filaments revealed that rod-shaped cells can move perpendicular to the light vector, indicating that the polarity can be controlled not only by pole-to-pole regulation but also within-a-pole regulation. This study provides insights into previously undescribed rapid bacterial polarity regulation via second messenger signalling with high spatial resolution.

Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division

Asymmetric stem cell division (ASCD) is a key mechanism in development, cancer, and stem cell biology. Drosophila neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic mechanisms. Here, we show that the extrinsic axon guidance cues Netrins, secreted by a glial niche surrounding larval brain neural stem cell lineages, regulate NB ASCD. Netrin-Frazzled/DCC signaling modulates, through Abelson kinase, Robo1 signaling threshold levels in Drosophila larval brain neural stem and progenitor cells of NBII lineages. Unbalanced Robo1 signaling levels induce ectopic NBs and progenitor cells due to failures in the ASCD process. Mechanistically, Robo1 signaling directly impinges on the intrinsic ASCD machinery, such as aPKC, Canoe/Afadin, and Numb, through the small GTPases Rac1 and Cdc42, which are required for the localization in mitotic NBs of Par-6, a Cdc42 physical partner and a core component of the Par (Par-6-aPKC-Par3/Bazooka) apical complex.

Antigen-derived peptides engage the ER stress sensor IRE1α to curb dendritic cell cross-presentation.

Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER), bind to major histocompatibility type I (MHC-I) protein complexes, and are transported to the cell surface for cross-presentation. DCs can exhibit activation of the ER stress sensor IRE1α without ER stress, but the underlying mechanism remains obscure. Here, we show that antigen-derived hydrophobic peptides can directly engage ER-resident IRE1α, masquerading as unfolded proteins. IRE1α activation depletes MHC-I heavy-chain mRNAs through regulated IRE1α-dependent decay (RIDD), curtailing antigen cross-presentation. In tumor-bearing mice, IRE1α disruption increased MHC-I expression on tumor-infiltrating DCs and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1α inhibition synergized with anti-PD-L1 antibody treatment to cause tumor regression. Our findings identify an unexpected cell-biological mechanism of antigen-driven IRE1α activation in DCs, revealing translational potential for cancer immunotherapy.

Spatial regulation of endosomes in growing dendrites

Many membrane proteins are highly enriched in either dendrites or axons. This non-uniform distribution is a critical feature of neuronal polarity and underlies neuronal function. The molecular mechanisms responsible for polarized distribution of membrane proteins has been studied for some time and many answers have emerged. A less well studied feature of neurons is that organelles are also frequently non-uniformly distributed. For instance, EEA1-positive early endosomes are somatodendritic whereas synaptic vesicles are axonal. In addition, some organelles are present in both axons and dendrites, but not distributed uniformly along the processes. One well known example are lysosomes which are abundant in the soma and proximal dendrite, but sparse in the distal dendrite and the distal axon. The mechanisms that determine the spatial distribution of organelles along dendrites are only starting to be studied. In this review, we will discuss the cell biological mechanisms of how the distribution of diverse sets of endosomes along the proximal-distal axis of dendrites might be regulated. In particular, we will focus on the regulation of bulk homeostatic mechanisms as opposed to local regulation. We posit that immature dendrites regulate organelle motility differently from mature dendrites in order to spatially organize dendrite growth, branching and sculpting.

A dominant negative variant of RAB5B disrupts maturation of surfactant protein B and surfactant protein C

Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.

Low-Cost Devices for Three-Dimensional Cell Aggregation, Real-Time Monitoring Microscopy, Microfluidic Immunostaining, and Deconvolution Analysis.

The wide use of 3D-organotypic cell models is imperative for advancing our understanding of basic cell biological mechanisms. For this purpose, easy-to-use enabling technology is required, which should optimally link standardized assessment methods to those used for the formation, cultivation, and evaluation of cell aggregates or primordial tissue. We thus conceived, manufactured, and tested devices which provide the means for cell aggregation and online monitoring within a hanging drop. We then established a workflow for spheroid manipulation and immune phenotyping. This described workflow conserves media and reagent, facilitates the uninterrupted tracking of spheroid formation under various conditions, and enables 3D-marker analysis by means of 3D epifluorescence deconvolution microscopy. We provide a full description of the low-cost manufacturing process for the fluidic devices and microscopic assessment tools, and the detailed blueprints and building instructions are disclosed. Conclusively, the presented compilation of methods and techniques promotes a quick and barrier-free entry into 3D cell biology.

VEGF-Trap Modulates Retinal Inflammation in the Murine Oxygen-Induced Retinopathy (OIR) Model.

Anti-Vascular Endothelial Growth Factor (VEGF) agents are the first-line treatment for retinal neovascular diseases, which represent the most prevalent causes of acquired vision loss world-wide. VEGF-Trap (Aflibercept, AFL), a recombinant decoy receptor recognizing ligands of both VEGFR-1 and -2, was recently reported to be highly efficient in improving visual acuity and preserving retinal anatomy in individuals affected by diabetic macular edema. However, the precise molecular and cell biological mechanisms underlying the beneficial effects of this novel tool have yet to be elucidated. Using the mouse oxygen-induced retinopathy (OIR) model as a surrogate of retinopathies with sterile post-ischemic inflammation, such as late proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), and diabetic macular edema (DME), we provide evidence that AFL modulates inflammation in response to hypoxia by regulating the morphology of microglial cells, a parameter commonly used as a proxy for changes in their activation state. We show that AFL administration during the hypoxic period of OIR leads to an increased number of ramified Iba1+ microglial cells/macrophages while subsequently limiting the accumulation of these cells in particular retinal layers. Our results suggest that, beyond its well-documented beneficial effects on microvascular regeneration, AFL might exert important modulatory effects on post-ischemic retinal inflammation.

Emerging Piezo1 signaling in inflammation and atherosclerosis; a potential therapeutic target.

Purpose of Review: Atherosclerosis is the principal cause of cardiovascular diseases (CVDs) which are the major cause of death worldwide. Mechanical force plays an essential role in cardiovascular health and disease. To bring the awareness of mechanosensitive Piezo1 role in atherosclerosis and its therapeutic potentials we review recent literature to highlight its involvement in various mechanisms of the disease.Recent Findings: Recent studies reported Piezo1 channel as a sensor, and transducer of various mechanical forces into biochemical signals, which affect various cellular activities such as proliferation, migration, apoptosis and vascular remodeling including immune/inflammatory mechanisms fundamental phenomenon in atherogenesis.Summary: Numerous evidences suggest Piezo1 as a player in different mechanisms of cell biology, including immune/inflammatory and other cellular mechanisms correlated with atherosclerosis. This review discusses mechanistic insight about this matter and highlights the drugability and therapeutic potentials consistent with emerging functions Piezo1 in various mechanisms of atherosclerosis. Based on the recent works, we suggest Piezo1 as potential therapeutic target and a valid candidate for future research. Therefore, a deeper exploration of Piezo1 biology and translation towards the clinic will be a novel strategy for treating atherosclerosis and other CVDs.

Low-Dose Fluvoxamine Modulates Endocytic Trafficking of SARS-CoV-2 Spike Protein: A Potential Mechanism for Anti-COVID-19 Protection by Antidepressants.

Commonly prescribed antidepressants may be associated with protection against severe COVID-19. The mechanism of their action in this context, however, remains unknown. Here, I investigated the effect of an antidepressant drug fluvoxamine on membrane trafficking of the SARS-CoV-2 spike protein and its cell host receptor ACE2 in HEK293T cells. A sub-therapeutic concentration (80 nM) of fluvoxamine rapidly upregulated fluid-phase endocytosis, resulting in enhanced accumulation of the spike-ACE2 complex in enlarged early endosomes. Diversion of endosomal trafficking provides a simple cell biological mechanism consistent with the protective effect of antidepressants against COVID-19, highlighting their therapeutic and prophylactic potential.

Neurological symptoms associated with cancer treatment

Die onkologische Behandlung ist biomarkerbasierter, molekular maßgeschneiderter und effektiver geworden. Aufbauend auf der zunehmenden Entschlüsselung zellbiologischer und molekularer Mechanismen steigt auch die Zahl zielgerichteter medikamentöser Therapien. Es steigt zudem die Zahl der Langzeitüberlebenden. Eine neuro(onko)logische Betreuung wird immer wichtiger, nicht nur wegen vermehrter direkter tumorbedingter Symptome – wie etwa der höheren Inzidenz einer Metastasierung in das Zentralnervensystem –, sondern weil im Zuge dieser modernen onkologischen systemischen Therapieformen ein breites Spektrum therapieassoziierter neurologischer Symptome auftritt, die einer sorgfältigen und raschen neurologischen/neuroonkologischen Evaluation und Therapiekonzeption bedürfen. Das Ziel dieses Artikels ist es, das Bewusstsein für die häufigsten therapieassoziierten neurologischen Symptome zu schärfen.

CRISPR/Cas9-mediated knockout of the DCL2 and DCL4 genes in Nicotiana benthamiana and its productivity of recombinant proteins.

DCL2 and DCL4 genes in Nicotiana benthamiana plants were successfully edited using the CRISPR/Cas9 system. Recently, plants have been utilized for recombinant protein production similar to other expression systems, i.e., bacteria, yeast, insect, and mammal cells. However, insufficient amounts of recombinant proteins are often produced in plant cells. The repression of RNA silencing within plant cells could improve production levels of recombinant protein because RNA silencing frequently decomposes mRNAs from transgenes. In this study, the genes dicer-like protein 2 and 4 (NbDCL2 and NbDCL4) were successfully edited to produce double-knockout transgenic Nicotiana benthamiana plants (dcl2dcl4 plants) using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. A transient green fluorescent protein (GFP) gene expression assay revealed that the dcl2dcl4 plants accumulated higher amounts of GFP and GFP mRNA than wild type (WT) and RNA-dependent RNA polymerase 6-knockout N. benthamiana plants (ΔRDR6 plants). Small RNA sequencing also showed that dcl2dcl4 plants accumulated lower amounts of small interfering RNAs (siRNAs) against the GFP gene than WT plants. The dcl2dcl4 plants might also produce higher amounts of human fibroblast growth factor 1 (FGF1) than WT and ΔRDR6 plants. These observations appear to reflect differences between DCLs and RDR6 in plant cell biological mechanisms. These results reveal that dcl2dcl4 plants would be suitable as platform plants for recombinant protein production.

Neuronal activity regulates the nuclear proteome to promote activity-dependent transcription.

The formation and plasticity of neuronal circuits relies on dynamic activity-dependent gene expression. Although recent work has revealed the identity of important transcriptional regulators and of genes that are transcribed and translated in response to activity, relatively little is known about the cell biological mechanisms by which activity alters the nuclear proteome of neurons to link neuronal stimulation to transcription. Using nucleus-specific proteomic mapping in silenced and stimulated neurons, we uncovered an understudied mechanism of nuclear proteome regulation: activity-dependent proteasome-mediated degradation. We found that the tumor suppressor protein PDCD4 undergoes rapid stimulus-induced degradation in the nucleus of neurons. We demonstrate that degradation of PDCD4 is required for normal activity-dependent transcription and that PDCD4 target genes include those encoding proteins critical for synapse formation, remodeling, and transmission. Our findings highlight the importance of the nuclear proteasome in regulating the activity-dependent nuclear proteome and point to a specific role for PDCD4 as a regulator of activity-dependent transcription in neurons.

N-myc Downstream-Regulated Gene 2 (NDRG2) Function as a Positive Regulator of Apoptosis: A New Insight into NDRG2 as a Tumor Suppressor

N-myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor gene that increases tumor sensitivity to anticancer drugs, slows tumor progression, and inhibits metastasis. NDRG2 is suppressed in various aggressive tumor positions, whereas NDRG2 expression is associated with patient prognosis, such as an improved survival rate. In this review, we summarize the tumor suppressor mechanism of NDRG2 and provide information on the function of NDRG2 concerning the susceptibility of cells to apoptosis. NDRG2 increases the susceptibility to apoptosis in various physiological environments of cells, such as development, hypoxia, nutrient deprivation, and cancer drug treatment. Although the molecular and cell biological mechanisms of NDRG2 have not been fully elucidated, we provide information on the mechanisms of NDRG2 in relation to apoptosis in various environments. This review can assist the design of research regarding NDRG2 function and suggests the potential of NDRG2 as a molecular target for cancer patients.

Drosophila Rab39 Attenuates Lysosomal Degradation

Lysosomal degradation, the common destination of autophagy and endocytosis, is one of the most important elements of eukaryotic metabolism. The small GTPases Rab39A and B are potential new effectors of this pathway, as their malfunction is implicated in severe human diseases like cancer and neurodegeneration. In this study, the lysosomal regulatory role of the single Drosophila Rab39 ortholog was characterized, providing valuable insight into the potential cell biological mechanisms mediated by these proteins. Using a de novo CRISPR-generated rab39 mutant, we found no failure in the early steps of endocytosis and autophagy. On the contrary, we found that Rab39 mutant nephrocytes internalize and degrade endocytic cargo at a higher rate compared to control cells. In addition, Rab39 mutant fat body cells contain small yet functional autolysosomes without lysosomal fusion defect. Our data identify Drosophila Rab39 as a negative regulator of lysosomal clearance during both endocytosis and autophagy.

The multicellular interplay of microglia in health and disease: lessons from leukodystrophy.

ABSTRACTMicroglia are highly dynamic cells crucial for developing and maintaining lifelong brain function and health through their many interactions with essentially all cellular components of the central nervous system. The frequent connection of microglia to leukodystrophies, genetic disorders of the white matter, has highlighted their involvement in the maintenance of white matter integrity. However, the mechanisms that underlie their putative roles in these processes remain largely uncharacterized. Microglia have also been gaining attention as possible therapeutic targets for many neurological conditions, increasing the demand to understand their broad spectrum of functions and the impact of their dysregulation. In this Review, we compare the pathological features of two groups of genetic leukodystrophies: those in which microglial dysfunction holds a central role, termed ‘microgliopathies’, and those in which lysosomal or peroxisomal defects are considered to be the primary driver. The latter are suspected to have notable microglia involvement, as some affected individuals benefit from microglia-replenishing therapy. Based on overlapping pathology, we discuss multiple ways through which aberrant microglia could lead to white matter defects and brain dysfunction. We propose that the study of leukodystrophies, and their extensively multicellular pathology, will benefit from complementing analyses of human patient material with the examination of cellular dynamics in vivo using animal models, such as zebrafish. Together, this will yield important insight into the cell biological mechanisms of microglial impact in the central nervous system, particularly in the development and maintenance of myelin, that will facilitate the development of new, and refinement of existing, therapeutic options for a range of brain diseases.

Compensatory expression of NRF2-dependent antioxidant genes is required to overcome the lethal effects of Kv11.1 activation in breast cancer cells and PDOs

Potassium channels are important regulators of cellular homeostasis and targeting these proteins pharmacologically is unveiling important mechanisms in cancer cell biology. Here we demonstrate that pharmacological stimulation of the Kv11.1 potassium channel activity results in mitochondrial reactive oxygen species (ROS) production and fragmentation in breast cancer cell lines and patient-derived organoids independent of breast cancer subtype. mRNA expression profiling revealed that Kv11.1 activity significantly altered expression of genes controlling the production of ROS and endoplasmic-reticulum (ER) stress. Characterization of the transcriptional signature of breast cancer cells treated with Kv11.1 potassium channel activators strikingly revealed an adaptive response to the potentially lethal augmentation of ROS by increasing Nrf2-dependent transcription of antioxidant genes. Nrf2 in this context was shown to promote survival in breast cancer, whereas knockdown of Nrf2 lead to Kv11.1-induced cell death. In conclusion, we found that the Kv11.1 channel activity promotes oxidative stress in breast cancer cells and that suppression of the Nrf2-mediated anti-oxidant survival mechanism strongly sensitized breast cancer cells to a lethal effect of pharmacological activation of Kv11.1.

Causal interactions from proteomic profiles: Molecular data meet pathway knowledge

SummaryWe present a computational method to infer causal mechanisms in cell biology by analyzing changes in high-throughput proteomic profiles on the background of prior knowledge captured in biochemical reaction knowledge bases. The method mimics a biologist's traditional approach of explaining changes in data using prior knowledge but does this at the scale of hundreds of thousands of reactions. This is a specific example of how to automate scientific reasoning processes and illustrates the power of mapping from experimental data to prior knowledge via logic programming. The identified mechanisms can explain how experimental and physiological perturbations, propagating in a network of reactions, affect cellular responses and their phenotypic consequences. Causal pathway analysis is a powerful and flexible discovery tool for a wide range of cellular profiling data types and biological questions. The automated causation inference tool, as well as the source code, are freely available at http://causalpath.org.

Upregulation of APP endocytosis by neuronal aging drives amyloid-dependent synapse loss.

Neuronal aging increases the risk of late-onset Alzheimer's disease. During normal aging, synapses decline, and β-amyloid (Aβ) accumulates intraneuronally. However, little is known about the underlying cell biological mechanisms. We studied neuronal aging using normal-aged brain and aged mouse primary neurons that accumulate lysosomal lipofuscin and show synapse loss. We identified the upregulation of amyloid precursor protein (APP) endocytosis as a neuronal aging mechanism that potentiates APP processing and Aβ production in vitro and in vivo. The increased APP endocytosis may contribute to the early endosome enlargement observed in the aged brain. Mechanistically, we showed that clathrin-dependent APP endocytosis requires F-actin and that clathrin and endocytic F-actin increase with neuronal aging. Finally, Aβ production inhibition reverts synaptic decline in aged neurons, whereas Aβ accumulation, promoted by endocytosis upregulation in younger neurons, recapitulates aging-related synapse decline. Overall, we identify APP endocytosis upregulation as a potential mechanism of neuronal aging and, thus, a novel target to prevent late-onset Alzheimer's disease. This article has an associated First Person interview with the first author of the paper.

Comparison of Global DNA Methylation Patterns in Human Melanoma Tissues and Their Derivative Cell Lines

Simple SummaryCancer cell lines are a defined population of cells, originally sourced from tumour tissue, that can be maintained in culture for an extended period of time. They are a critical laboratory-based model, and are frequently used to unravel mechanisms of cancer cell biology. In all cells, gene activity is in part regulated by DNA methylation, the epigenetic process by which methyl groups are added to DNA. In this study, we demonstrate that at a global level, DNA methylation profiles are globally well conserved, but we identify specific sites that are consistently more methylated in tumour-derived cell lines compared to the original tumour tissue. The genes associated with these common differentially methylated regions are involved in important cellular processes and are strongly enriched for epigenetic mechanisms associated with suppression of gene activity. This study provides a valuable resource for identifying false positives due to cell culture and for better interpretation of future cancer epigenetics studies.DNA methylation is a heritable epigenetic mark that is fundamental to mammalian development. Aberrant DNA methylation is an epigenetic hallmark of cancer cells. Cell lines are a critical in vitro model and very widely used to unravel mechanisms of cancer cell biology. However, limited data are available to assess whether DNA methylation patterns in tissues are retained when cell lines are established. Here, we provide the first genome-scale sequencing-based methylation map of metastatic melanoma tumour tissues and their derivative cell lines. We show that DNA methylation profiles are globally conserved in vitro compared to the tumour tissue of origin. However, we identify sites that are consistently hypermethylated in cell lines compared to their tumour tissue of origin. The genes associated with these common differentially methylated regions are involved in cell metabolism, cell cycle and apoptosis and are also strongly enriched for the H3K27me3 histone mark and PRC2 complex-related genes. Our data indicate that although global methylation patterns are similar between tissues and cell lines, there are site-specific epigenomic differences that could potentially impact gene expression. Our work provides a valuable resource for identifying false positives due to cell culture and for better interpretation of cancer epigenetics studies in the future.

On chip detection of glial cell-derived neurotrophic factor secreted from dopaminergic cells under magnetic stimulation

Glial cell-derived neurotrophic factor (GDNF) is a small protein potently promoting the survival of dopaminergic and motor neurons. GDNF can be secreted from different types of cells including the dopaminergic neural cell line, N27. N27 cells, a rat dopaminergic neural cell line, is regarded as a suitable in vitro model for Parkinson's disease (PD) research. For PD treatment, transcranial magnetic stimulation (TMS), a noninvasive therapeutic method, showed beneficial clinical effects, but the mechanism for its benefit is not understood. Because GDNF is a potent neurotrophic factor, it is of great value to evaluate if GDNF secretion from N27 cells can be affected by magnetic stimulation (MS). However, the current methods for detecting GDNF are time-consuming and expensive. In this paper we outline the detection of GDNF secretion from N27 cells by ultrasensitive nanopore thin film sensors (nanosensor) for the first time. As low as 2 pg/mL GDNF can be readily detected by the nanosensor. Furthermore, we show that MS can promote GDNF secretion from N27 cells. Specifically, the GDNF concentration in N27 cell-conditioned media under MS treatment shows statistically significant increase up to 2-fold after 5 days in vitro in comparison with the control. This nanosensor along with the in vitro PD model N27 cells provides a low-cost, easy-to-use, sensitive approach for studying potential cell biological mechanisms of the clinical benefits of MS on PD.