A Pipeline for Dynamic Analysis of Mitochondrial Content in Developing T Cells: Bridging the Gap Between High-Throughput Flow Cytometry and Single-Cell Microscopy Analysis.

Analyzing the dynamics of mitochondrial content in developing T cells is crucial for understanding the metabolic state during T cell development. However, monitoring mitochondrial content in real-time needs a balance of cell viability and image resolution. In this chapter, we present experimental protocols for measuring mitochondrial content in developing T cells using three modalities: bulk analysis via flow cytometry, volumetric imaging in laser scanning confocal microscopy, and dynamic live-cell monitoring in spinning disc confocal microscopy.

Mitochondrial fusion and altered beta-oxidation drive muscle wasting in a Drosophila cachexia model.

Cancer cachexia is a tumour-induced wasting syndrome, characterised by extreme loss of skeletal muscle. Defective mitochondria can contribute to muscle wasting; however, the underlying mechanisms remain unclear. Using a Drosophila larval model of cancer cachexia, we observed enlarged and dysfunctional muscle mitochondria.

Scribble and E-cadherin cooperate to control symmetric daughter cell positioning by multiple mechanisms.

The fate of the two daughter cells is intimately connected to their positioning, which is in turn regulated by cell junction remodelling and orientation of the mitotic spindle. How multiple cues are integrated to dictate the ultimate positioning of daughters is not clear. Here, we identify novel mechanisms of regulation of daughter positioning in single MCF10A cells.

E-cadherin in developing murine T cells controls spindle alignment and progression through β-selection.

A critical stage of T cell development is β-selection; at this stage, the T cell receptor β (TCRβ) chain is generated, and the developing T cell starts to acquire antigenic specificity. Progression through β-selection is assisted by low-affinity interactions between the nascent TCRβ chain and peptide presented on stromal major histocompatibility complex and cues provided by the niche. In this study, we identify a cue within the developing T cell niche that is critical for T cell development.

Establishing a multiplex imaging panel to study T cell development in the thymus in mouse.

Multiplexed immunohistochemistry enables analysis of cellular and signaling events in the context of an intact organ. Here, we describe protocols for applying multiplexed immunohistochemistry to the mouse thymus. In particular, we describe how to identify cells at the specific differentiation stage known as β-selection, and to monitor pre-TCR signaling and the cellular response at that stage.

The use of the Performance Diagnostic Checklist-Human Services in development of interventions to increase fidelity.

Children with Autism Spectrum Disorder (ASD) and other Developmental Disabilities (DD) often have deficits in social, play, and language which often require substantial support to develop the skills. Caregivers and educators are often tasked with developing these skills and working to transfer those acquired skill sets across settings and people (i.e.

Developing T cells form an immunological synapse for passage through the β-selection checkpoint.

The β-selection checkpoint of T cell development tests whether the cell has recombined its genomic DNA to produce a functional T cell receptor β (TCRβ). Passage through the β-selection checkpoint requires the nascent TCRβ protein to mediate signaling through a pre-TCR complex. In this study, we show that developing T cells at the β-selection checkpoint establish an immunological synapse in in vitro and in situ, resembling that of the mature T cell.

Parity reduces mammary repopulating activity but does not affect mammary stem cells defined as CD24 + CD29/CD49fhi in mice.

Background: Breast cancer (BCa) mortality is decreasing with early detection and improvement in therapies. The incidence of BCa, however, continues to increase, particularly estrogen-receptor-positive (ER +) subtypes. One of the greatest modifiers of ER + BCa risk is childbearing (parity), with BCa risk halved in young multiparous mothers.

A new role for Notch in the control of polarity and asymmetric cell division of developing T cells.

A fundamental question in biology is how single cells can reliably produce progeny of different cell types. Notch signalling frequently facilitates fate determination. Asymmetric cell division (ACD) often controls segregation of Notch signalling by imposing unequal inheritance of regulators of Notch.

An integrated transcriptional switch at the β-selection checkpoint determines T cell survival, development and leukaemogenesis.

In T cell development, a pivotal decision-making stage, termed β-selection, integrates a TCRβ checkpoint to coordinate survival, proliferation and differentiation to an αβ T cell. Here, we review how transcriptional regulation coordinates fate determination in early T cell development to enable β-selection. Errors in this transcription control can trigger T cell acute lymphoblastic leukaemia.

Maps of variability in cell lineage trees.

New approaches to lineage tracking have allowed the study of differentiation in multicellular organisms over many generations of cells. Understanding the phenotypic variability observed in these lineage trees requires new statistical methods. Whereas an invariant cell lineage, such as that for the nematode Caenorhabditis elegans, can be described by a lineage map, defined as the pattern of phenotypes overlaid onto the binary tree, a traditional lineage map is static and does not describe the variability inherent in the cell lineages of higher organisms.

Context-Specific Mechanisms of Cell Polarity Regulation.

Cell polarity is an essential process shared by almost all animal tissues. Moreover, cell polarity enables cells to sense and respond to the cues provided by the neighboring cells and the surrounding microenvironment. These responses play a critical role in regulating key physiological processes, including cell migration, proliferation, differentiation, vesicle trafficking and immune responses.

Chitosan-coated amyloid fibrils increase adipogenesis of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have the potential to revolutionize medicine due to their ability to differentiate into specific lineages for targeted tissue repair. Development of materials and cell culture platforms that improve differentiation of either autologous or allogenic stem cell sources into specific lineages would enhance clinical utilization of MCSs. In this study, nanoscale amyloid fibrils were evaluated as substrate materials to encourage viability, proliferation, multipotency, and differentiation of MSCs.

In vitro tracking and intracellular protein distribution in immunology.

New imaging techniques have enabled major advances in understanding how immune reactions are initiated, coordinated and controlled. Imaging methods, which were previously mostly descriptive and supplementary to more quantitative approaches, have now reached sufficient precision and throughput that they are becoming integral to almost all aspects of immunology research. Imaging methodologies that increase the resolution and sensitivity of detection, alongside an ever-expanding range of fluorescent reporters of molecular and cellular activity, and vastly improved analysis methods, have all facilitated this transformation.

Imaging Asymmetric T Cell Division.

Asymmetric cell division (ACD) controls cell fate decisions in model organisms such as Drosophila and C. elegans and has recently emerged as a mediator of T cell fate and hematopoiesis. The most appropriate methods for assessing ACD in T cells are still evolving.

The Asymmetric Cell Division Regulators Par3, Scribble and Pins/Gpsm2 Are Not Essential for Erythroid Development or Enucleation.

Erythroid enucleation is the process by which the future red blood cell disposes of its nucleus prior to entering the blood stream. This key event during red blood cell development has been likened to an asymmetric cell division (ACD), by which the enucleating erythroblast divides into two very different daughter cells of alternate molecular composition, a nucleated cell that will be removed by associated macrophages, and the reticulocyte that will mature to the definitive erythrocyte. Here we investigated gene expression of members of the Par, Scribble and Pins/Gpsm2 asymmetric cell division complexes in erythroid cells, and functionally tested their role in erythroid enucleation in vivo and ex vivo.

Dense small molecule labeling enables activator-dependent STORM by proximity mapping.

Stochastic optical reconstruction microscopy (STORM) enables high-resolution imaging, but multi-channel 3D imaging is problematic because of chromatic aberrations and alignment errors. The use of activator-dependent STORM in which spectrally distinct activators can be coupled with a single reporter can circumvent such issues. However, the standard approach of linking activators and reporters to a single antibody molecule is hampered by low labeling density and the large size of the antibody.

Polarity and asymmetric cell division in the control of lymphocyte fate decisions and function.

Polarity is important in several lymphocyte processes including lymphocyte migration, formation of the immunological synapse, and asymmetric cell division (ACD). While lymphocyte migration and immunological synapse formation are relatively well understood, the role of lymphocyte ACD is less clear. Recent advances in measuring polarity enable more robust analyses of asymmetric cell division.

Calcium Signaling Is Required for Erythroid Enucleation.

Although erythroid enucleation, the property of erythroblasts to expel their nucleus, has been known for 7ore than a century, surprisingly little is known regarding the molecular mechanisms governing this unique developmental process. Here we show that similar to cytokinesis, nuclear extrusion requires intracellular calcium signaling and signal transduction through the calmodulin (CaM) pathway. However, in contrast to cytokinesis we found that orthochromatic erythroblasts require uptake of extracellular calcium to enucleate.

A Chemical Screening Approach to Identify Novel Key Mediators of Erythroid Enucleation.

Erythroid enucleation is critical for terminal differentiation of red blood cells, and involves extrusion of the nucleus by orthochromatic erythroblasts to produce reticulocytes. Due to the difficulty of synchronizing erythroblasts, the molecular mechanisms underlying the enucleation process remain poorly understood. To elucidate the cellular program governing enucleation, we utilized a novel chemical screening approach whereby orthochromatic cells primed for enucleation were enriched ex vivo and subjected to a functional drug screen using a 324 compound library consisting of structurally diverse, medicinally active and cell permeable drugs.

Lethal giant larvae-1 deficiency enhances the CD8(+) effector T-cell response to antigen challenge in vivo.

Lethal giant larvae-1 (Lgl-1) is an evolutionary conserved protein that regulates cell polarity in diverse lineages; however, the role of Lgl-1 in the polarity and function of immune cells remains to be elucidated. To assess the role of Lgl-1 in T cells, we generated chimeric mice with a hematopoietic system deficient for Lgl-1. Lgl-1 deficiency did not impair the activation or function of peripheral CD8(+) T cells in response to antigen presentation in vitro, but did skew effector and memory T-cell differentiation.

Asymmetric cell division during T cell development controls downstream fate.

During mammalian T cell development, the requirement for expansion of many individual T cell clones, rather than merely expansion of the entire T cell population, suggests a possible role for asymmetric cell division (ACD). We show that ACD of developing T cells controls cell fate through differential inheritance of cell fate determinants Numb and α-Adaptin. ACD occurs specifically during the β-selection stage of T cell development, and subsequent divisions are predominantly symmetric.