scholarly journals The tubulin repertoire of Caenorhabditis elegans sensory neurons and its context‑dependent role in process outgrowth

2016 ◽  
Vol 27 (23) ◽  
pp. 3717-3728 ◽  
Author(s):  
Dean Lockhead ◽  
Erich M. Schwarz ◽  
Robert O’Hagan ◽  
Sebastian Bellotti ◽  
Michael Krieg ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Sensory Neurons ◽  
Cell Types ◽  
Genetic Dissection ◽  
Transcription Profiles ◽  
Tubulin Genes ◽  
Process Outgrowth ◽  
Context Dependent

Microtubules contribute to many cellular processes, including transport, signaling, and chromosome separation during cell division. They comprise αβ‑tubulin heterodimers arranged into linear protofilaments and assembled into tubes. Eukaryotes express multiple tubulin isoforms, and there has been a longstanding debate as to whether the isoforms are redundant or perform specialized roles as part of a tubulin code. Here we use the well‑characterized touch receptor neurons (TRNs) of Caenorhabditis elegans to investigate this question through genetic dissection of process outgrowth both in vivo and in vitro. With single‑cell RNA-seq, we compare transcription profiles for TRNs with those of two other sensory neurons and present evidence that each sensory neuron expresses a distinct palette of tubulin genes. In the TRNs, we analyze process outgrowth and show that four tubulins (tba‑1, tba‑2, tbb‑1, and tbb‑2) function partially or fully redundantly, whereas two others (mec‑7 and mec‑12) perform specialized, context‑dependent roles. Our findings support a model in which sensory neurons express overlapping subsets of tubulin genes whose functional redundancy varies among cell types and in vivo and in vitro contexts.

scholarly journals The tubulin repertoire of C. elegans sensory neurons and its context dependent role in process outgrowth

2016 ◽  
Author(s):  
Dean Lockhead ◽  
Erich M. Schwarz ◽  
Robert O'Hagan ◽  
Sebastian Bellotti ◽  
Michael Krieg ◽  
...  
Keyword(s):  
Sensory Neurons ◽  
Rna Seq ◽  
C Elegans ◽  
Cellular Processes ◽  
Tubulin Genes ◽  
Touch Receptor Neurons ◽  
Receptor Neurons ◽  
Process Outgrowth

AbstractMicrotubules contribute to many cellular processes, including transport, signaling, and chromosome separation during cell division (Kapitein and Hoogenraad, 2015). They are comprised of αβ-tubulin heterodimers arranged into linear protofilaments and assembled into tubes. Eukaryotes express multiple tubulin isoforms (Gogonea et al., 1999), and there has been a longstanding debate as to whether the isoforms are redundant or perform specialized roles as part of a tubulin code (Fulton and Simpson, 1976). Here, we use the well-characterized touch receptor neurons (TRNs) of Caenorhabditis elegans to investigate this question, through genetic dissection of process outgrowth both in vivo and in vitro. With single-cell RNA-seq, we compare transcription profiles for TRNs with those of two other sensory neurons, and present evidence that each sensory neuron expresses a distinct palette of tubulin genes. In the TRNs, we analyze process outgrowth and show that four tubulins (tba-1, tba-2, tbb-1, and tbb-2) function partially or fully redundantly, while two others (mec-7 and mec-12) perform specialized, context-dependent roles. Our findings support a model in which sensory neurons express overlapping subsets of tubulin genes whose functional redundancy varies between cell types and in vivo and in vitro contexts.Highlight SummaryMicrotubules contribute to key cellular processes and are composed of αβ-tubulin heterodimers. Neurons in C. elegans express cell type-specific isoforms in addition to a shared repertoire and rely on tubulins for neurite outgrowth. Isoform function varies between in vivo and in vitro contexts.AbbreviationsTRNsTouch Receptor NeuronsRNA-seqRNA sequencingRFGReceptive Field GapTPMTranscripts per MillionECMExtracellular Matrix,CVCoefficient of VariationConflict of InterestThe authors declare no conflicting financial interests.

scholarly journals Analysis of Herpes Simplex Virus ICP0 Promoter Function in Sensory Neurons during Acute Infection, Establishment of Latency, and Reactivation In Vivo

2003 ◽  
Vol 77 (22) ◽  
pp. 12319-12330 ◽  
Author(s):  
R. L. Thompson ◽  
May T. Shieh ◽  
N. M. Sawtell
Keyword(s):  
Reporter Gene ◽  
Sensory Neurons ◽  
Acute Infection ◽  
Cell Types ◽  
Staining Procedure ◽  
Native Protein ◽  
Promoter Function ◽  
Basal Expression

ABSTRACT We have begun an analysis of the functional architecture of the ICP0 promoter in neurons in vivo with the ultimate goal of determining how this gene is regulated during reactivation in vivo. Promoter/reporter mutants in which the Escherichia coli beta-galactosidase (β-Gal) gene was driven by various permutations of the ICP0 promoter were employed to permit the analysis of promoter function without the added complications that would arise due to inappropriate regulation of ICP0 protein levels. A whole-ganglion immunohistochemical staining procedure (N. M. Sawtell, J. Virol. 77:4127-4138, 2003) was used for direct comparisons of the expression of the promoter/reporter gene to expression of the native protein in the same cell. In this way, the expression of the putative wild-type promoter could be validated and results for mutant promoters could be compared to expression of the native gene. We found that a DNA fragment from bp −562 through the methionine start codon of the ICP0 gene contained all sequences required for properly regulated ICP0 expression in diverse cell types (including sensory neurons of the trigeminal ganglia [TG]) in vitro and in vivo, as indicated by colocalization of ICP0 and β-Gal. Truncation of the ICP0 promoter to bp −145 or −129 resulted in the loss of immediate-early (α) kinetics. The truncated promoters expressed high levels of the reporter gene with leaky late (γ1) kinetics in vitro and in some cell types in vivo. Unexpectedly, the truncated promoters did not express in TG neurons. Thus, TAATGARAT or other sequences upstream of bp −145 in the ICP0 promoter are required for basal expression of ICP0 in neurons but are not required for basal expression in other cells in vivo. There was a >95% concordance between reporter and native protein expression detected with the 562-bp promoter in neurons during the acute stage. However, this was not the case during reactivation from latency in vivo, as nearly twice as many neurons contained detectable β-Gal as contained detectable ICP0. This same 562-bp promoter/reporter cassette, when placed in the context of a latency-associated transcript (LAT) null mutant, resulted in >95% concordance of expression of β-Gal and ICP0 during reactivation in vivo. These last results strongly suggest that there is a posttranscriptional constraint on the expression of ICP0 protein during reactivation from latency and that this constraint is mediated by LAT.

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

scholarly journals Heme Oxygenase-1 Deficiency and Oxidative Stress: A Review of 9 Independent Human Cases and Animal Models

2021 ◽  
Vol 22 (4) ◽  
pp. 1514 ◽  
Author(s):  
Akihiro Yachie
Keyword(s):  
Oxidative Stress ◽  
Animal Models ◽  
Heme Oxygenase ◽  
Inflammatory Diseases ◽  
Cell Types ◽  
Pharmacological Intervention ◽  
Heme Oxygenase 1 ◽  
Inflammatory Reactions

Since Yachie et al. reported the first description of human heme oxygenase (HO)-1 deficiency more than 20 years ago, few additional human cases have been reported in the literature. A detailed analysis of the first human case of HO-1 deficiency revealed that HO-1 is involved in the protection of multiple tissues and organs from oxidative stress and excessive inflammatory reactions, through the release of multiple molecules with anti-oxidative stress and anti-inflammatory functions. HO-1 production is induced in vivo within selected cell types, including renal tubular epithelium, hepatic Kupffer cells, vascular endothelium, and monocytes/macrophages, suggesting that HO-1 plays critical roles in these cells. In vivo and in vitro studies have indicated that impaired HO-1 production results in progressive monocyte dysfunction, unregulated macrophage activation and endothelial cell dysfunction, leading to catastrophic systemic inflammatory response syndrome. Data from reported human cases of HO-1 deficiency and numerous studies using animal models suggest that HO-1 plays critical roles in various clinical settings involving excessive oxidative stress and inflammation. In this regard, therapy to induce HO-1 production by pharmacological intervention represents a promising novel strategy to control inflammatory diseases.

scholarly journals MyD88 Is Not Required for Muscle Injury-Induced Endochondral Heterotopic Ossification in a Mouse Model of Fibrodysplasia Ossificans Progressiva

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 630
Author(s):  
Huili Lyu ◽  
Cody M. Elkins ◽  
Jessica L. Pierce ◽  
C. Henrique Serezani ◽  
Daniel S. Perrien
Keyword(s):  
Heterotopic Ossification ◽  
Muscle Injury ◽  
Cell Types ◽  
Fibrodysplasia Ossificans Progressiva ◽  
Inflammatory Signaling ◽  
Conditional Deletion ◽  
Pathway Crosstalk ◽  
Multiple Cell

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1β or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

scholarly journals Adipose and Muscle Cell Co-Culture System: A Novel In Vitro Tool to Mimic the In Vivo Cellular Environment

Biology ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 6
Author(s):  
Palaniselvam Kuppusamy ◽  
Dahye Kim ◽  
Ilavenil Soundharrajan ◽  
Inho Hwang ◽  
Ki Choon Choi
Keyword(s):  
Drug Development ◽  
Culture System ◽  
Cell Types ◽  
Culture Cell ◽  
In Vitro Techniques ◽  
Culture Systems ◽  
Complex Interactions ◽  
Cellular Environment

A co-culture system allows researchers to investigate the complex interactions between two cell types under various environments, such as those that promote differentiation and growth as well as those that mimic healthy and diseased states, in vitro. In this paper, we review the most common co-culture systems for myocytes and adipocytes. The in vitro techniques mimic the in vivo environment and are used to investigate the causal relationships between different cell lines. Here, we briefly discuss mono-culture and co-culture cell systems and their applicability to the study of communication between two or more cell types, including adipocytes and myocytes. Also, we provide details about the different types of co-culture systems and their applicability to the study of metabolic disease, drug development, and the role of secretory factors in cell signaling cascades. Therefore, this review provides details about the co-culture systems used to study the complex interactions between adipose and muscle cells in various environments, such as those that promote cell differentiation and growth and those used for drug development.

scholarly journals Modelling the Human Placental Interface In Vitro—A Review

Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 884
Author(s):  
Marta Cherubini ◽  
Scott Erickson ◽  
Kristina Haase
Keyword(s):  
Drug Testing ◽  
Cell Types ◽  
In Vitro Models ◽  
Placental Development ◽  
Scientific Challenge ◽  
Induced Pluripotent ◽  
And Function ◽  
And Control

Acting as the primary link between mother and fetus, the placenta is involved in regulating nutrient, oxygen, and waste exchange; thus, healthy placental development is crucial for a successful pregnancy. In line with the increasing demands of the fetus, the placenta evolves throughout pregnancy, making it a particularly difficult organ to study. Research into placental development and dysfunction poses a unique scientific challenge due to ethical constraints and the differences in morphology and function that exist between species. Recently, there have been increased efforts towards generating in vitro models of the human placenta. Advancements in the differentiation of human induced pluripotent stem cells (hiPSCs), microfluidics, and bioprinting have each contributed to the development of new models, which can be designed to closely match physiological in vivo conditions. By including relevant placental cell types and control over the microenvironment, these new in vitro models promise to reveal clues to the pathogenesis of placental dysfunction and facilitate drug testing across the maternal–fetal interface. In this minireview, we aim to highlight current in vitro placental models and their applications in the study of disease and discuss future avenues for these in vitro models.

scholarly journals Caenorhabditis elegans as an in vivo Model to Assess Amphetamine Tolerance

2021 ◽  
pp. 1-9
Author(s):  
Dayana Torres Valladares ◽  
Sirisha Kudumala ◽  
Murad Hossain ◽  
Lucia Carvelli
Keyword(s):  
Caenorhabditis Elegans ◽  
Molecular Mechanisms ◽  
Drugs Of Abuse ◽  
Genetic Model ◽  
In Vivo Model ◽  
C Elegans ◽  
Hyperactivity Disorder ◽  
In Vitro Data

Amphetamine is a potent psychostimulant also used to treat attention deficit/hyperactivity disorder and narcolepsy. In vivo and in vitro data have demonstrated that amphetamine increases the amount of extra synaptic dopamine by both inhibiting reuptake and promoting efflux of dopamine through the dopamine transporter. Previous studies have shown that chronic use of amphetamine causes tolerance to the drug. Thus, since the molecular mechanisms underlying tolerance to amphetamine are still unknown, an animal model to identify the neurochemical mechanisms associated with drug tolerance is greatly needed. Here we took advantage of a unique behavior caused by amphetamine in <i>Caenorhabditis elegans</i> to investigate whether this simple, but powerful, genetic model develops tolerance following repeated exposure to amphetamine. We found that at least 3 treatments with 0.5 mM amphetamine were necessary to see a reduction in the amphetamine-induced behavior and, thus, to promote tolerance. Moreover, we found that, after intervals of 60/90 minutes between treatments, animals were more likely to exhibit tolerance than animals that underwent 10-minute intervals between treatments. Taken together, our results show that <i>C. elegans</i> is a suitable system to study tolerance to drugs of abuse such as amphetamines.

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