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.

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.

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.

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.