Autophagy coordinates chondrocyte development and early joint formation in zebrafish

Autophagy is a catabolic process responsible for the removal of waste and damaged cellular components by lysosomal degradation. It plays a key role in fundamental cell processes, including ER stress mitigation, control of cell metabolism, and cell differentiation and proliferation, all of which are essential for cartilage cell (chondrocyte) development and survival, and for the formation of cartilage. Correspondingly, autophagy dysregulation has been implicated in several skeletal disorders such as osteoarthritis and osteoporosis. To test the requirement for autophagy during skeletal development in zebrafish, we generated an atg13 CRISPR knockout zebrafish line. This line showed a complete loss of atg13 expression, and restricted autophagic activity in vivo. In the absence of autophagy, chondrocyte maturation was accelerated, with chondrocytes exhibiting signs of premature hypertrophy. Focussing on the jaw element, autophagy disruption affected joint articulation causing restricted mouth opening. This gross behavioural phenotype corresponded with a failure to thrive, and death in homozygote atg13 nulls within 17 days. Taken together, our results are consistent with autophagy contributing to the timely regulation of chondrocyte maturation and for extracellular matrix formation.

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Skeletal defects in VEGF120/120 mice reveal multiple roles for VEGF in skeletogenesis

Development ◽

10.1242/dev.129.8.1893 ◽

2002 ◽

Vol 129 (8) ◽

pp. 1893-1904 ◽

Cited By ~ 14

Author(s):

Elazar Zelzer ◽

William McLean ◽

Yin-Shan Ng ◽

Naomi Fukai ◽

Anthony M. Reginato ◽

...

Keyword(s):

Blood Vessels ◽

Skeletal Development ◽

Surrounding Tissue ◽

Ossification Center ◽

Direct Role ◽

Osteoblastic Activity ◽

Chondrocyte Maturation ◽

Skeletal Defects

Angiogenesis is an essential component of skeletal development and VEGF signaling plays an important if not pivotal role in this process. Previous attempts to examine the roles of VEGF in vivo have been largely unsuccessful because deletion of even one VEGF allele leads to embryonic lethality before skeletal development is initiated. The availability of mice expressing only the VEGF120 isoform (which do survive to term) has offered an opportunity to explore the function of VEGF during embryonic skeletal development. Our study of these mice provides new in vivo evidence for multiple important roles of VEGF in both endochondral and intramembranous bone formation, as well as some insights into isoform-specific functions. There are two key differences in vascularization of developing bones between wild-type and VEGF120/120 mice. VEGF120/120 mice have not only a delayed recruitment of blood vessels into the perichondrium but also show delayed invasion of vessels into the primary ossification center, demonstrating a significant role of VEGF at both an early and late stage of cartilage vascularization. These findings are the basis for a two-step model of VEGF-controlled vascularization of the developing skeleton, a hypothesis that is supported by the new finding that VEGF is expressed robustly in the perichondrium and surrounding tissue of cartilage templates of future bones well before blood vessels appear in these regions. We also describe new in vivo evidence for a possible role of VEGF in chondrocyte maturation, and document that VEGF has a direct role in regulating osteoblastic activity based on in vivo evidence and organ culture experiments.

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Solving the mechanisms responsible for organelle behavior in vivo by same-cell correlative light and electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120473 ◽

1992 ◽

Vol 50 (1) ◽

pp. 14-15

Author(s):

Conly L. Rieder

Keyword(s):

Electron Microscopy ◽

Quantitative Analysis ◽

Light Microscopy ◽

Living Cell ◽

Light And Electron Microscopy ◽

Time Behavior ◽

Dynamic Interactions ◽

Positive Identification ◽

Cellular Components

The behavior of many cellular components, and their dynamic interactions, can be characterized in the living cell with considerable spatial and temporal resolution by video-enhanced light microscopy (video-LM). Indeed, under the appropriate conditions video-LM can be used to determine the real-time behavior of organelles ≤ 25-nm in diameter (e.g., individual microtubules—see). However, when pushed to its limit the structures and components observed within the cell by video-LM cannot be resolved nor necessarily even identified, only detected. Positive identification and a quantitative analysis often requires the corresponding electron microcopy (EM).

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(Preprint)

10.2196/preprints.12282 ◽

2018 ◽

Author(s):

Dr Malathi Dayalan ◽

Dr Sudeshna Sharma ◽

Dr Shweta Poovani ◽

Dr Saher Altaf

Keyword(s):

Myofascial Pain ◽

Mouth Opening ◽

Masticatory Muscles ◽

Matched Pair ◽

Masticatory Muscle ◽

Muscle Disorders ◽

Occlusal Contact ◽

Group I ◽

Group Ii

BACKGROUND Masticatory system is a complex functional unit, primarily engaged in chewing, swallowing and breathing functions, and some parts are involved in taste recognition and determination of food consistency. Sophisticated functional performances of speech and emotional expressions are specifically human qualities. Irregularities in occlusion appears to be the precipitating factor in the pathogenesis of myofascial pain dysfunction syndrome. Tek- Scan III records the bite length, number, distribution, timing, duration and the relative force of each tooth contact. It also records the sequence of occlusal contacts in terms of time and the associated force with each occlusal contact. The aim of this study was to treat masticatory muscle disorders with occlusal equilibration, and compare the efficacy of treatment outcomes between selective grinding and stabilization splints using Tek-Scan III. OBJECTIVE Objective of this study was to compare the efficacy of occlusal equilibration achieved through selective griding and stabilization splints using Tek-Scan III. METHODS In this in vivo study, 40 patients with masticatory muscle disorders were selected based on the inclusion and exclusion criteria. The occlusal discrepancies were analyzed using Tek-Scan III. The selected 40 subjects were then randomly divided into 2 groups based on the treatment they recieved; Group I – Selective grinding group (20) and Group II – Stabilization splint group (20). Comparison of pre-treatment and post treatment results were evaluated in terms of pain, mouth opening, left and right side force percentage as recorded through Tek-Scan III and reduction of disclusion time. Statistical analysis was carried out with Kolmogorov Smirnov test, Wilcoxon matched pair test and Mann-Whitney U test. RESULTS Wilcoxon matched pairs test demonstrated that there was statistically significant results ( p = 0.0007) in both the groups for reduction of disclusion time, elimination of pain and improved mouth opening. Patients in Group I showed better results as compared to Group II in terms of disclusion time, pain and mouth opening. CONCLUSIONS Occlusal equilibration brought about by reducing the disclusion time using the Tek- Scan III reduced the symptoms of pain in masticatory muscles. Patients in group I (Selective grinding) however showed better results when compared to patients in group II (Stabilization splints).

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Metabolic Signatures of Cryptosporidium parvum-Infected HCT-8 Cells and Impact of Selected Metabolic Inhibitors on C. parvum Infection under Physioxia and Hyperoxia

Biology ◽

10.3390/biology10010060 ◽

2021 ◽

Vol 10 (1) ◽

pp. 60

Author(s):

Juan Vélez ◽

Zahady Velasquez ◽

Liliana M. R. Silva ◽

Ulrich Gärtner ◽

Klaus Failing ◽

...

Keyword(s):

Host Cell ◽

Cryptosporidium Parvum ◽

Cell Metabolism ◽

Lactate Production ◽

Glucose Consumption ◽

Host Cells ◽

Metabolic Inhibitors ◽

Low Oxygen ◽

Metabolic Signatures

Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C.parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1–11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium, C. parvum infection led to a massive increase in glucose consumption and lactate production. Together, these results point to the important role of both glycolysis and glutaminolysis during C. parvum intracellular replication. Referring to obtained metabolic signatures, we targeted glycolysis as well as glutaminolysis in C. parvum-infected host cells by using the inhibitors lonidamine [inhibitor of hexokinase, mitochondrial carrier protein (MCP) and monocarboxylate transporters (MCT) 1, 2, 4], galloflavin (lactate dehydrogenase inhibitor), syrosingopine (MCT1- and MCT4 inhibitor) and compound 968 (glutaminase inhibitor) under hyperoxic and physioxic conditions. In line with metabolic signatures, all inhibitors significantly reduced parasite replication under both oxygen conditions, thereby proving both energy-related metabolic pathways, glycolysis and glutaminolysis, but also lactate export mechanisms via MCTs as pivotal for C. parvum under in vivo physioxic conditions of mammals.

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Activation of MAT2A-RIP1 signaling axis reprograms monocytes in gastric cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001364 ◽

2021 ◽

Vol 9 (2) ◽

pp. e001364

Author(s):

Yan Zhang ◽

Hui Yang ◽

Jun Zhao ◽

Ping Wan ◽

Ye Hu ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Metabolism ◽

Vital Role ◽

Methionine Metabolism ◽

Promoter Regions ◽

Normal Tissues ◽

Methionine Cycle

BackgroundThe activation of tumor-associated macrophages (TAMs) facilitates the progression of gastric cancer (GC). Cell metabolism reprogramming has been shown to play a vital role in the polarization of TAMs. However, the role of methionine metabolism in function of TAMs remains to be explored.MethodsMonocytes/macrophages were isolated from peripheral blood, tumor tissues or normal tissues from healthy donors or patients with GC. The role of methionine metabolism in the activation of TAMs was evaluated with both in vivo analyses and in vitro experiments. Pharmacological inhibition of the methionine cycle and modulation of key metabolic genes was employed, where molecular and biological analyses were performed.ResultsTAMs have increased methionine cycle activity that are mainly attributed to elevated methionine adenosyltransferase II alpha (MAT2A) levels. MAT2A modulates the activation and maintenance of the phenotype of TAMs and mediates the upregulation of RIP1 by increasing the histone H3K4 methylation (H3K4me3) at its promoter regions.ConclusionsOur data cast light on a novel mechanism by which methionine metabolism regulates the anti-inflammatory functions of monocytes in GC. MAT2A might be a potential therapeutic target for cancer cells as well as TAMs in GC.

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Inhibition of Glycolysis Suppresses Cell Proliferation and Tumor Progression In Vivo: Perspectives for Chronotherapy

International Journal of Molecular Sciences ◽

10.3390/ijms22094390 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4390

Author(s):

Jana Horváthová ◽

Roman Moravčík ◽

Miroslava Matúšková ◽

Vladimír Šišovský ◽

Andrej Boháč ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Progression ◽

Umbilical Vein ◽

Cell Metabolism ◽

High Rate ◽

Ki 67 ◽

Immunodeficient Mice ◽

Inhibition Of Glycolysis

A high rate of glycolysis is considered a hallmark of tumor progression and is caused by overexpression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Therefore, we analyzed the possibility of inhibiting tumor and endothelial cell metabolism through the inhibition of PFKFB3 by a small molecule, (E)-1-(pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one (PFK15), as a promising therapy. The effects of PFK15 on cell proliferation and apoptosis were analyzed on human umbilical vein endothelial cells (HUVEC) and the human colorectal adenocarcinoma cell line DLD1 through cytotoxicity and proliferation assays, flow cytometry, and western blotting. The results showed that PFK15 inhibited the proliferation of both cell types and induced apoptosis with decreasing the Bcl-2/Bax ratio. On the basis of the results obtained from in vitro experiments, we performed a study on immunodeficient mice implanted with DLD1 cells. We found a reduced tumor mass after morning PFK15 treatment but not after evening treatment, suggesting circadian control of underlying processes. The reduction in tumor size was related to decreased expression of Ki-67, a marker of cell proliferation. We conclude that inhibition of glycolysis can represent a promising therapeutic strategy for cancer treatment and its efficiency is circadian dependent.

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Autophagy-Related Protein ATG8 Has a Noncanonical Function for Apicoplast Inheritance in Toxoplasma gondii

mBio ◽

10.1128/mbio.01446-15 ◽

2015 ◽

Vol 6 (6) ◽

Cited By ~ 43

Author(s):

Maude F. Lévêque ◽

Laurence Berry ◽

Michael J. Cipriano ◽

Hoa-Mai Nguyen ◽

Boris Striepen ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Growth Conditions ◽

Model Organisms ◽

Secondary Endosymbiosis ◽

Related Protein ◽

Content Type ◽

Superresolution Microscopy ◽

Animal Pathogens ◽

Cellular Components ◽

Catabolic Process

ABSTRACT Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. This mechanism involves the formation of a double-membrane structure called the autophagosome that sequesters cellular components to be degraded. To orchestrate this process, yeasts and animals rely on a conserved set of autophagy-related proteins (ATGs). Key among these factors is ATG8, a cytoplasmic protein that is recruited to nascent autophagosomal membranes upon the induction of autophagy. Toxoplasma gondii is a potentially harmful human pathogen in which only a subset of ATGs appears to be present. Although this eukaryotic parasite seems able to generate autophagosomes upon stresses such as nutrient starvation, the full functionality and biological relevance of a canonical autophagy pathway are as yet unclear. Intriguingly, in T. gondii, ATG8 localizes to the apicoplast under normal intracellular growth conditions. The apicoplast is a nonphotosynthetic plastid enclosed by four membranes resulting from a secondary endosymbiosis. Using superresolution microscopy and biochemical techniques, we show that TgATG8 localizes to the outermost membrane of this organelle. We investigated the unusual function of TgATG8 at the apicoplast by generating a conditional knockdown mutant. Depletion of TgATG8 led to rapid loss of the organelle and subsequent intracellular replication defects, indicating that the protein is essential for maintaining apicoplast homeostasis and thus for survival of the tachyzoite stage. More precisely, loss of TgATG8 led to abnormal segregation of the apicoplast into the progeny because of a loss of physical interactions of the organelle with the centrosomes. IMPORTANCE By definition, autophagy is a catabolic process that leads to the digestion and recycling of eukaryotic cellular components. The molecular machinery of autophagy was identified mainly in model organisms such as yeasts but remains poorly characterized in phylogenetically distant apicomplexan parasites. We have uncovered an unusual function for autophagy-related protein ATG8 in Toxoplasma gondii: TgATG8 is crucial for normal replication of the parasite inside its host cell. Seemingly unrelated to the catabolic autophagy process, TgATG8 associates with the outer membrane of the nonphotosynthetic plastid harbored by the parasite called the apicoplast, and there it plays an important role in the centrosome-driven inheritance of the organelle during cell division. This not only reveals an unexpected function for an autophagy-related protein but also sheds new light on the division process of an organelle that is vital to a group of important human and animal pathogens.

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RNA viruses and the mitogenic Raf/MEK/ERK signal transduction cascade

Biological Chemistry ◽

10.1515/bc.2008.145 ◽

2008 ◽

Vol 389 (10) ◽

Cited By ~ 64

Author(s):

Stephan Pleschka

Keyword(s):

Signal Transduction ◽

Rna Viruses ◽

Rna Virus ◽

Cell Metabolism ◽

Mitogen Activated Protein Kinase ◽

Erk Signaling ◽

Control Center ◽

Signal Transduction Cascade ◽

Host Interaction ◽

Differentiation And Proliferation

AbstractThe Raf/MEK/ERK signal transduction cascade belongs to the mitogen-activated protein kinase (MAPK) cascades. Raf/MEK/ERK signaling leads to stimulus-specific changes in gene expression, alterations in cell metabolism or induction of programmed cell death (apoptosis), and thus controls cell differentiation and proliferation. It is induced by extracellular agents, including pathogens such as RNA viruses. Many DNA viruses are known to induce cellular signaling via this pathway. As these pathogens partly use the DNA synthesis machinery for their replication, they aim to drive cells into a proliferative state. In contrast, the consequences of RNA virus-induced Raf/MEK/ERK signaling were less clear for a long time, but since the turn of the century the number of publications on this topic has rapidly increased. Research on this virus/host-interaction will broaden our understanding of its relevance in viral replication. This important control center of cellular responses is differently employed to support the replication of several important human pathogenic RNA viruses including influenza, Ebola, hepatitis C and SARS corona viruses.

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Considerations when investigating lncRNA function in vivo

eLife ◽

10.7554/elife.03058 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 222

Author(s):

Andrew R Bassett ◽

Asifa Akhtar ◽

Denise P Barlow ◽

Adrian P Bird ◽

Neil Brockdorff ◽

...

Keyword(s):

Normal Cell ◽

Regulatory Elements ◽

Genomic Locus ◽

Cellular Processes ◽

Cell Processes ◽

Non Coding Rnas ◽

Per Se ◽

Dna Regulatory Elements

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.

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The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis

eLife ◽

10.7554/elife.00947 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 226

Author(s):

Liang Ge ◽

David Melville ◽

Min Zhang ◽

Randy Schekman

Keyword(s):

Autophagosome Formation ◽

Functional Studies ◽

A Cell ◽

Intermediate Compartment ◽

Membrane Isolation ◽

Necessary And Sufficient ◽

Organelle Membrane ◽

Catabolic Process

Autophagy is a catabolic process for bulk degradation of cytosolic materials mediated by double-membraned autophagosomes. The membrane determinant to initiate the formation of autophagosomes remains elusive. Here, we establish a cell-free assay based on LC3 lipidation to define the organelle membrane supporting early autophagosome formation. In vitro LC3 lipidation requires energy and is subject to regulation by the pathways modulating autophagy in vivo. We developed a systematic membrane isolation scheme to identify the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) as a primary membrane source both necessary and sufficient to trigger LC3 lipidation in vitro. Functional studies demonstrate that the ERGIC is required for autophagosome biogenesis in vivo. Moreover, we find that the ERGIC acts by recruiting the early autophagosome marker ATG14, a critical step for the generation of preautophagosomal membranes.

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