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. Morphological changes were accompanied by upregulation of beta-oxidation proteins and depletion of muscle glycogen and lipid stores. Muscle lipid stores were also decreased in Colon-26 adenocarcinoma mouse muscle samples, and expression of the beta-oxidation gene CPT1A was negatively associated with muscle quality in cachectic patients. Mechanistically, mitochondrial defects result from reduced muscle insulin signalling, downstream of tumour-secreted insulin growth factor binding protein (IGFBP) homologue ImpL2. Strikingly, muscle-specific inhibition of Forkhead box O (FOXO), mitochondrial fusion, or beta-oxidation in tumour-bearing animals preserved muscle integrity. Finally, dietary supplementation with nicotinamide or lipids, improved muscle health in tumour-bearing animals. Overall, our work demonstrates that muscle FOXO, mitochondria dynamics/beta-oxidation and lipid utilisation are key regulators of muscle wasting in cancer cachexia.

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. The polarity protein, Scribble cooperates with E-cadherin for sequential roles in daughter positioning. First Scribble stabilises E-cadherin at the mitotic cortex as well as the retraction fibres, to mediate spindle orientation. Second, Scribble re-locates to the junction between the two daughters to allow a new E-cadherin-based-interface to form between them, influencing the width of the nascent daughter-daughter junction and subsequent cell positioning. Thus, E-cadherin and Scribble dynamically relocate to different intracellular sites during cell division to orient the mitotic spindle and control placement of the daughter cells after cell division. This article has an associated First Person interview with the first author of the paper.

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. Here, we assessed the functional relationship between Notch and ACD in mouse T cell development. To attain immunological specificity, developing T cells must pass through a pivotal stage termed ß-selection, which involves Notch signalling and ACD. We assessed functional interactions between Notch1 and ACD during ß-selection through direct presentation of Notch ligands, DL1 and DL4, and pharmacological inhibition of Notch signalling. Contrary to prevailing models, we demonstrate that Notch signalling controls the distribution of Notch1 itself and cell fate determinants, α-adaptin and Numb. Furthermore, Notch and CXCR4 signalling cooperated to drive polarity during division. Thus, Notch signalling directly orchestrates ACD, and Notch1 is differentially inherited by sibling cells.This article has an associated First Person interview with the first author of the paper.

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. Despite convincing epidemiological data, the biology that underpins this protection remains unclear. Parity-induced protection has been postulated to be due to a decrease in mammary stem cells (MaSCs); however, reports to date have provided conflicting data. METHODS: We have completed rigorous functional testing of repopulating activity in parous mice using unfractionated and MaSC (CD24midCD49fhi)-enriched populations. We also developed a novel serial transplant method to enable us to assess self-renewal of MaSC following pregnancy. Lastly, as each pregnancy confers additional BCa protection, we subjected mice to multiple rounds of pregnancy to assess whether additional pregnancies impact MaSC activity. RESULTS: Here, we report that while repopulating activity in the mammary gland is reduced by parity in the unfractionated gland, it is not due to a loss in the classically defined MaSC (CD24+CD49fhi) numbers or function. Self-renewal was unaffected by parity and additional rounds of pregnancy also did not lead to a decrease in MaSC activity. CONCLUSIONS: Our data show instead that parity impacts on the stem-like activity of cells outside the MaSC population.

Asymmetric proteasome segregation as a mechanism for unequal partitioning of the transcription factor T-bet during T lymphocyte division.

Polarized segregation of proteins in T cells is thought to play a role in diverse cellular functions including signal transduction, migration, and directed secretion of cytokines. Persistence of this polarization can result in asymmetric segregation of fate-determining proteins during cell division, which may enable a T cell to generate diverse progeny. Here, we provide evidence that a lineage-determining transcription factor, T-bet, underwent asymmetric organization in activated T cells preparing to divide and that it was unequally partitioned into the two daughter cells. This unequal acquisition of T-bet appeared to result from its asymmetric destruction during mitosis by virtue of concomitant asymmetric segregation of the proteasome. These results suggest a mechanism by which a cell may unequally localize cellular activities during division, thereby imparting disparity in the abundance of cell fate regulators in the daughter cells.

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. Here, we introduce lineage variability maps which describe the pattern of second-order variation in lineage trees. These maps can be undirected graphs of the partial correlations between every lineal position, or directed graphs showing the dynamics of bifurcated patterns in each subtree. We show how to infer these graphical models for lineages of any depth from sample sizes of only a few pedigrees. This required developing the generalized spectral analysis for a binary tree, the natural framework for describing tree-structured variation. When tested on pedigrees from C. elegans expressing a marker for pharyngeal differentiation potential, the variability maps recover essential features of the known lineage map. When applied to highly-variable pedigrees monitoring cell size in T lymphocytes, the maps show that most of the phenotype is set by the founder naive T cell. Lineage variability maps thus elevate the concept of the lineage map to the population level, addressing questions about the potency and dynamics of cell lineages and providing a way to quantify the progressive restriction of cell fate with increasing depth in the tree.

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

In T cell development, a pivotal decision-making stage, termed ß-selection, integrates a TCRß checkpoint to coordinate survival, proliferation and differentiation to an αß T cell. Here, we review how transcriptional regulation coordinates fate determination in early T cell development to enable ß-selection. Errors in this transcription control can trigger T cell acute lymphoblastic leukaemia. We describe how the ß-selection checkpoint goes awry in leukaemic transformation.

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. In this review, we will discuss how advances in imaging are changing the way we view immune activation and control using T cells as the model immune system. We will describe how imaging has transformed our knowledge of molecular and signalling events in T-cell activation, and the impact of these molecular events on the behaviour of T cells.

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. When challenged with antigen-expressing virus or tumor, Lgl-1-deficient mice displayed altered T-cell responses. This manifested in a stronger antiviral and antitumor effector CD8(+) T-cell response, the latter resulting in enhanced control of MC38-OVA tumors. These results reveal a novel role for Lgl-1 in the regulation of virus-specific T-cell responses and antitumor immunity.

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. We proposed that small molecule labels might enable activator-dependent STORM if the reporter or activator were linked to separate small molecules that bound within 3.5 nm of each other. This would greatly increase the labeling density and therefore improve resolution. We tested various mixtures of phalloidin- or mCling-conjugated fluorophore to demonstrate this feasibility. The specific activation was dependent on the choice of activator, its density, a matching activating laser and its power. In addition to providing an effective means of multi-channel 3D STORM imaging, this method also provides information about the local proximity between labels, potentially enabling super-resolved mapping of the conformation of the labeled structures.

Cutting edge: DNAX accessory molecule 1-deficient CD8+ T cells display immunological synapse defects that impair antitumor immunity.

DNAX accessory molecule 1 (DNAM-1) is expressed on all CD8(+) T cells and promotes their activation and effector function. DNAM-1 interacts with LFA-1, a critical molecule for immunological synapse formation between T cells and APCs, and for cytotoxic killing of target cells. Mice that lack DNAM-1 display abnormal T cell responses and antitumor activity; however, the mechanism involved is unclear. In this article, we show that DNAM-1 deficiency results in reduced proliferation of CD8(+) T cells after Ag presentation and impaired cytotoxic activity. We also demonstrate that DNAM-1-deficient T cells show reduced conjugations with tumor cells and decreased recruitment of both LFA-1 and lipid rafts to the immunological synapse, which correlates with reduced tumor cell killing in vitro. This synapse defect may explain why DNAM-1-deficient mice cannot clear tumors in vivo, and highlights the importance of DNAM-1 and the immunological synapse in T cell-mediated antitumor immunity.

Polarization of excitation light influences molecule counting in single-molecule localization microscopy.

Single-molecule localization microscopy has been widely applied to count the number of biological molecules within a certain structure. The percentage of molecules that are detected significantly affects the interpretation of data. Among many factors that affect this percentage, the polarization state of the excitation light is often neglected or at least unstated in publications. We demonstrate by simulation and experiment that the number of molecules detected can be different from -40 up to 100% when using circularly or linearly polarized excitation light. This is determined mainly by the number of photons emitted by single fluorescent molecule, namely the choice of fluorescence proteins, and the background noise in the system, namely the illumination scheme. This difference can be further exaggerated or mitigated by various fixation methods, magnification, and camera settings We conclude that the final choice between circularly or linearly polarized excitation light should be made experimentally, based on the signal to noise ratio of the system.

TACTICS, an interactive platform for customized high-content bioimaging analysis.

We describe a modular MATLAB® Toolbox named TACTICS for time-lapse image analysis that meets several requirements not generally offered by currently available software packages: (i) the ability to assess quality of extracted imaging information by directly linking data end points to the original position, (ii) massively parallel analysis of each parameter, for flow cytometry-like assessment of possible relationships between parameters within sub-populations of the images, (iii) options for user control of the tracking such as an interface to restrict the analysis region, (iv) manual correction of automated processes and (v) user interfaces for post-tracking analysis that is linked to the original images, including options to view cell pedigrees and normalized polarization ratios based on fluorescence ratiometric measurements.

Asymmetric segregation and self-renewal of hematopoietic stem and progenitor cells with endocytic Ap2a2.

The stem cell-intrinsic model of self-renewal via asymmetric cell division (ACD) posits that fate determinants be partitioned unequally between daughter cells to either activate or suppress the stemness state. ACD is a purported mechanism by which hematopoietic stem cells (HSCs) self-renew, but definitive evidence for this cellular process remains open to conjecture. To address this issue, we chose 73 candidate genes that function within the cell polarity network to identify potential determinants that may concomitantly alter HSC fate while also exhibiting asymmetric segregation at cell division. Initial gene-expression profiles of polarity candidates showed high and differential expression in both HSCs and leukemia stem cells. Altered HSC fate was assessed by our established in vitro to in vivo screen on a subcohort of candidate polarity genes, which revealed 6 novel positive regulators of HSC function: Ap2a2, Gpsm2, Tmod1, Kif3a, Racgap1, and Ccnb1. Interestingly, live-cell videomicroscopy of the endocytic protein AP2A2 shows instances of asymmetric segregation during HSC/progenitor cell cytokinesis. These results contribute further evidence that ACD is functional in HSC self-renewal, suggest a role for Ap2a2 in HSC activity, and provide a unique opportunity to prospectively analyze progeny from HSC asymmetric divisions.

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. Next, we provide an image segmentation and centroid tracking-based analysis pipeline for automated quantification of a large number of microscopy images. These protocols together offer comprehensive approaches to investigate mitochondrial dynamics in developing T cells, enabling a deeper understanding of their metabolic processes.

Divergent lymphocyte signalling revealed by a powerful new tool for analysis of time-lapse microscopy.

We describe a new approach for interactive analysis of time-lapse microscopy, and apply this approach to elucidating whether polarity regulation is conserved between epithelial cells and lymphocytes. A key advantage of our analysis platform, 'TACTICS', is the capacity to visualize individual data points in the context of large data sets, similar to standard approaches in flow cytometry. Scatter plots representing microscopic parameters or their derivations such as polarity ratios are linked to the original data such that clicking on each dot enables a link to images and movies of the corresponding cell. Similar to flow cytometric analysis, subsets of the data can be gated and reanalyzed to explore the relationships between different parameters. TACTICS was used to dissect the regulation of polarization of the cell fate determinant, Numb, in migrating lymphocytes. We show here that residues of Numb that are phosphorylated by atypical protein kinase C (aPKC) to mediate apicobasal polarity in epithelial cells are not required for polarization of Numb in T cells, indicating that the role of aPKC is not conserved between lymphocytes and epithelia.

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

A critical stage of T cell development is ß-selection; at this stage, the T cell receptor ß (TCRß) chain is generated, and the developing T cell starts to acquire antigenic specificity. Progression through ß-selection is assisted by low-affinity interactions between the nascent TCRß 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. E-cadherin mediates cell-cell interactions and influences cell fate in many developmental systems. In developing T cells, E-cadherin contributed to the formation of an immunological synapse and the alignment of the mitotic spindle with the polarity axis during division, which facilitated subsequent T cell development. Collectively, these data suggest that E-cadherin facilitates interactions with the thymic niche to coordinate the ß-selection stage of T cell development.

Stepwise progression of ß-selection during T cell development involves histone deacetylation.

During T cell development, the first step in creating a unique T cell receptor (TCR) is genetic recombination of the TCRß chain. The quality of the new TCRß is assessed at the ß-selection checkpoint. Most cells fail this checkpoint and die, but the coordination of fate at the ß-selection checkpoint is not yet understood. We shed new light on fate determination during ß-selection using a selective inhibitor of histone deacetylase 6, ACY1215. ACY1215 disrupted the ß-selection checkpoint. Characterising the basis for this disruption revealed a new, pivotal stage in ß-selection, bookended by up-regulation of TCR co-receptors, CD28 and CD2, respectively. Within this "DN3bPre" stage, CD5 and Lef1 are up-regulated to reflect pre-TCR signalling, and their expression correlates with proliferation. These findings suggest a refined model of ß-selection in which a coordinated increase in expression of pre-TCR, CD28, CD5 and Lef1 allows for modulating TCR signalling strength and culminates in the expression of CD2 to enable exit from the ß-selection checkpoint.

Asymmetric cell division of T cells upon antigen presentation uses multiple conserved mechanisms.

Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naive CD8(+) T cells undergoing initial division while attached to dendritic cells during Ag presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with APCs provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the APC. The cue from the APC is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes, and orientation of the mitotic spindle during division is orchestrated by the partner of inscuteable/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division.

Interplay of polarity proteins and GTPases in T-lymphocyte function.

Polarity refers to the asymmetric distribution of different cellular components within a cell and is central to many cell functions. In T-cells, polarity regulates the activation, migration, and effector function of cytotoxic T-cells (CTLs) during an immune response. The regulation of asymmetric cell division by polarity proteins may also dictate CTL effector and memory differentiation following antigen presentation. Small GTPases, along with their associated polarity and adaptor proteins, are critical for mediating the polarity changes necessary for T-cell activation and function, and in turn, are regulated by guanine exchange factors (GEFS) and GTPase activating proteins (GAPS). For example, a novel GEF, dedicator of cytokinesis 8 (DOCK8) was recently identified as a regulator of immune cell function and mutations in DOCK8 have been detected in patients with severe combined immunodeficiency. Both B and T-cells from DOCK8 mutant mice form defective immunological synapses and have abnormal functions, in addition to impaired immune memory development. This paper will discuss the interplay between polarity proteins and GTPases, and their role in T-cell function.