A role for the Golgi matrix protein giantin in ciliogenesis through control of the localization of dynein-2

The correct formation of primary cilia is central to the development and function of nearly all cells and tissues. Cilia grow from the mother centriole by extension of a microtubule core, the axoneme, which is then surrounded with a specialized ciliary membrane that is continuous with the plasma membrane. Intraflagellar transport moves particles along the length of the axoneme to direct assembly of the cilium and is also required for proper cilia function. The microtubule motor, cytoplasmic dynein-2 mediates retrograde transport along the axoneme from the tip to the base; dynein-2 is also required for some aspects of cilia formation. In most cells, the Golgi lies adjacent to the centrioles and key components of the cilia machinery localize to this organelle. Golgi-localized proteins have also been implicated in ciliogenesis and in intraflagellar transport. Here, we show that the transmembrane Golgi matrix protein giantin (GOLGB1) is required for ciliogenesis. We show that giantin is not required for the Rab11–Rabin8–Rab8 pathway that has been implicated in the early stages of ciliary membrane formation. Instead we find that suppression of giantin results in mis-localization of WDR34, the intermediate chain of dynein-2. Highly effective depletion of giantin or WDR34 leads to an inability of cells to form primary cilia. Partial depletion of giantin or of WDR34 leads to an increase in cilia length consistent with the concept that giantin acts through dynein-2. Our data implicate giantin in ciliogenesis through control of dynein-2 localization

Molecular investigations into Btk and wasp

Bruton's tyrosine kinase (Btk) is a modular non-receptor protein tyrosine kinase which when mutated results in the immunodeficiency X-linked agammaglobulinemia (XLA) in man and xid in mice. The disease is characterised by a block in the development of B cells and as a consequence, levels of serum immunoglobulin of all isotypes are reduced. Btk is therefore crucial for B cell development. It is also implicated in the transduction of signals in both developing and mature B cells. In humans, Btk has only been reported to be stimulated on ligation of the B cell receptor (BCR), and investigations in this thesis were performed in order to ascertain whether Btk activation is induced upon ligation of the cell surface receptors CD22 and CD38, as might be expected due to the nature of the XLA phenotype. The modular nature of the Btk protein led to investigations into ligands of these domains in order to determine possible in vitro ligands of Btk. This thesis presents studies of the SH3 domains of Btk, Itk and Tec, expressed as GST fusion proteins. The SH3 domains of all three proteins were seen to bind a similar set of proteins in B cell lysates including the proteins previously identified as Btk SH3 domain in vitro ligands, WASP and c-Cbl. Auto-phosphorylation of the Btk SH3 domain fusion protein led to altered ligand binding. A tyrosine phosphorylated protein observed to bind the phosphorylated but not the unphosphorylated Btk SH3 domain fusion protein was identified as another protein tyrosine kinase, Syk. The identification of WASP, mutated in patients suffering from another X-linked immunodeficiency, the Wiskott-Aldrich Syndrome (WAS), as an in vitro ligand of Btk, led to investigations into the expression of WASP in WAS patients. Results show that no patients with severe WAS in the study expressed WASP at detectable levels, irrespective of the nature or position of the mutation characterised in these patients

The hallmarks of cancer are also the hallmarks of wound healing

Many of the mechanisms that drive cancer development and progression also promote wound healing.</jats:p

Identification of a novel human BCL-X promoter and exon

BCL-XL is a key anti-apoptotic BCL-2 family protein that is widely expressed in human cancer cells and is induced in response to diverse survival signals. The translation initiation codon for BCL-XL is located in BCL-X exon II and previous analyses have indicated that BCL-XL RNAs initiate close to the start of exon II or additionally contain a non-coding first exon (exon IA) spliced to exon II. Using 5' RACE we have now identified a novel BCL-X non-coding exon (exon IB) which is spliced directly to exon II in place of exon IA. Exon IB-containing RNAs encoded BCL-XL and were detected in non-malignant lymphocytes and lymphoma cells from lymph node biopsies and were expressed at significant levels in cell lines derived from ovarian, colon and breast cancers. We identified two TATA-box sequences upstream of exon IB and demonstrated that surrounding genomic sequences contained strong promoter activity in lymphoma cells (approximately 300-fold active relative to controls). We have therefore identified a powerful new BCL-X promoter and a novel exon that contributes to BCL-XL expression

EphA receptors regulate prostate cancer cell dissemination through Vav2-RhoA mediated cell-cell repulsion

Metastatic prostate cancer cells display EphB receptor-mediated attraction when they contact stromal fibroblasts but EphA-driven repulsion when they contact one another. The impact of these ‘social’ interactions between cells during cancer cell invasion and the signalling mechanisms downstream of Eph receptors are unclear. Here we show that EphA receptors regulate prostate cancer cell dissemination in a 2D dispersal assay and in a 3D cancer cell spheroid assay. We show that EphA receptors signal via the exchange factor Vav2 to activate RhoA and that both Vav2 and RhoA are required for prostate cancer cell–cell repulsion. Furthermore, we find that in EphA2/EphA4, Vav2 or RhoA siRNA-treated cells, contact repulsion can be restored by partial microtubule destabilisation. We propose that EphA–Vav2–RhoA-mediated repulsion between contacting cancer cells at the tumour edge could enhance their local invasion away from the primary tumour