The Role of the Rac1 Activator STEF/Tiam2 in the Regulation of Front-Rear Polarity and Migration

Abstract

Directed cell migration has a critical role in many physiological process including wound healing, immune function and development, however aberrant migration can underpin a number of pathologies, including cancer. The capacity to migrate and invade surrounding tissues is a hallmark of the transition from benign to malignant tumours, which underlies the development of metastases. As such, understanding the processes that regulate motility is a crucial area of cancer research. The establishment of front-rear polarity is a pre-requisite for mesenchymal cell migration, which involves both morphological changes and re-organisation of the sub-cellular organelles. During the acquisition of the polarised phenotype the microtubule organising centre (MTOC) is retained at the cell centroid, positioned between the nucleus and the leading edge, with co-incident rearward movement of the nucleus. Additionally, the nucleus is re-oriented, undergoing a rotation in the plane of the substratum to align the longitudinal nuclear axis with the cellular axis of polarity.The Rac1 selective guanine nucleotide exchange factor (GEF) STEF/Tiam2 has been previously demonstrated to have important roles in neuronal polarisation and morphology. In addition, STEF-mediated activation of Rac1 has been shown to be required for optimal directed cell migration through the regulation of microtubule targeting for efficient focal adhesion disassembly. In this study, it is confirmed that depletion of STEF reduces the capacity for migration in a number of different cell-types. Single-cell tracking experiments revealed that down-regulation of STEF significantly impairs migrational persistence. This loss of directionality was indicative of a defect in front-rear polarity, and it was subsequently demonstrated that depletion of STEF significantly inhibits nuclear re-orientation in response to wounding. This crucial role of STEF in the regulation of nuclear re-orientation is supported by its pronounced localisation at the nuclear envelope, a novel observation for a Rac1 GEF.Importantly, it has been demonstrated that STEF interacts with two key components of the nuclear positioning machinery; Nesprin-2G and Non-muscle myosin IIB (NMMIIB). Nesprin-2G is localised in the outer nuclear envelope and is a key structural component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, required to couple the nucleus to the cytoskeleton for efficient nuclear rotation and anchoring. It has been demonstrated that Nesprin-2G depletion dramatically affects STEF; resulting in a reduction in the localisation of STEF at the nuclear envelope, an increase in the mobility of STEF, and a moderate reduction in total STEF expression levels. This indicates an important role for Nesprin-2G in anchoring STEF at the nuclear envelope to generate localised Rac1 activity.STEF has also been shown to interact with the actin motor protein NMMIIB, a key component of the acto-myosin perinuclear actin cage, which is responsible for constraining nuclear morphology and anchoring the nucleus in the correct orientation. This study has also identified that down-regulation of STEF results in an increase in nuclear height in front-rear polarised cells and that unpolarised nuclei in STEF-depleted wound-edge cells display a reduction in actin cable formation. These results collaboratively suggest an important role for STEF in regulating the perinuclear cytoskeleton, particularly the formation and maintenance of the perinuclear actin cage.

Date of Award	31 Dec 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Adam Hurlstone (Supervisor) & Angeliki Malliri (Supervisor)