Biochemistry and Pharmacology - Research Publications
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ItemNo Preview AvailableArf5-mediated regulation of mTORC1 at the plasma membrane.(American Society for Cell Biology (ASCB), 2023-04-01)The mechanistic target of rapamycin (mTOR) kinase regulates a major signaling pathway in eukaryotic cells. In addition to regulation of mTORC1 at lysosomes, mTORC1 is also localized at other locations. However, little is known about the recruitment and activation of mTORC1 at nonlysosomal sites. To identify regulators of mTORC1 recruitment to nonlysosomal compartments, novel interacting partners with the mTORC1 subunit, Raptor, were identified using immunoprecipitation and mass spectrometry. We show that one of the interacting partners, Arf5, is a novel regulator of mTORC1 signaling at plasma membrane ruffles. Arf5-GFP localizes with endogenous mTOR at PI3,4P2-enriched membrane ruffles together with the GTPase required for mTORC1 activation, Rheb. Knockdown of Arf5 reduced the recruitment of mTOR to membrane ruffles. The activation of mTORC1 at membrane ruffles was directly demonstrated using a plasma membrane-targeted mTORC1 biosensor, and Arf5 was shown to enhance the phosphorylation of the mTORC1 biosensor substrate. In addition, endogenous Arf5 was shown to be required for rapid activation of mTORC1-mediated S6 phosphorylation following nutrient starvation and refeeding. Our findings reveal a novel Arf5-dependent pathway for recruitment and activation of mTORC1 at plasma membrane ruffles, a process relevant for spatial and temporal regulation of mTORC1 by receptor and nutrient stimuli.
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ItemInteracting partners of Golgi-localized small G protein Arl5b identified by a combination of in vivo proximity labelling and GFP-Trap pull down(WILEY, 2022-09)The small G protein Arl5b is localised on the trans-Golgi network (TGN) and regulates endosomes-to-TGN transport. Here, we combined in vivo and in vitro techniques to map the interactive partners and near neighbours of Arl5b at the TGN, using constitutively active, membrane-bound Arl5b(Q70L)-GFP in stably expressing HeLa cells, and the proximity labelling techniques BioID and APEX2 in parallel with GFP-Trap pull down. From MS analysis, 22 Golgi proteins were identified; 50% were TGN-localised Rabs, Arfs and Arls. The scaffold/tethering factors ACBD3 (GCP60) and PIST (GOPC) were also identified, and we show that Arl5b is required for TGN recruitment of ACBD3. Overall, the combination of in vivo labelling and direct pull downs indicates a highly organised complex of small G proteins on TGN membranes.
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ItemForm and function of the Golgi apparatus: scaffolds, cytoskeleton and signalling(WILEY, 2019-09)In addition to the classical functions of the Golgi in membrane transport and glycosylation, the Golgi apparatus of mammalian cells is now recognised to contribute to the regulation of a range of cellular processes, including mitosis, DNA repair, stress responses, autophagy, apoptosis and inflammation. These processes are often mediated, either directly or indirectly, by membrane scaffold molecules, such as golgins and GRASPs which are located on Golgi membranes. In many cases, these scaffold molecules also link the actin and microtubule cytoskeleton and influence Golgi morphology. An emerging theme is a strong relationship between the morphology of the Golgi and regulation of a variety of signalling pathways. Here, we review the molecular regulation of the morphology of the Golgi, especially the role of the golgins and other scaffolds in the interaction with the microtubule and actin networks. In addition, we discuss the impact of the modulation of the Golgi ribbon in various diseases, such as neurodegeneration and cancer, to the pathology of disease.
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ItemRegulation of mTORC1 activity by the Golgi apparatus.(Faculty Opinions Ltd, 2021)Mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) is a major signalling kinase in cells that regulates proliferation and metabolism and is controlled by extrinsic and intrinsic signals. The lysosome has received considerable attention as a major hub of mTORC1 activation. However, mTOR has also been located to a variety of other intracellular sites, indicating the possibility of spatial regulation of mTORC1 signalling within cells. In particular, there have been numerous recent reports of mTORC1 activation associated with the Golgi apparatus. Here, we review the evidence for the regulation of mTORC1 signalling at the Golgi in mammalian cells. mTORC1 signalling is closely linked to the morphology of the Golgi architecture; a number of Golgi membrane tethers/scaffolds that influence Golgi architecture in mammalian cells that directly or indirectly regulate mTORC1 activation have been identified. Perturbation of the Golgi mTORC1 pathway arising from fragmentation of the Golgi has been shown to promote oncogenesis. Here, we highlight the potential mechanisms for the activation mTORC1 at the Golgi, which is emerging as a major site for mTORC1 signalling.
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ItemIntersectin-1 interacts with the golgin GCC88 to couple the actin network and Golgi architecture(AMER SOC CELL BIOLOGY, 2019-02-01)The maintenance of the Golgi ribbon relies on a dynamic balance between the actin and microtubule networks; however, the pathways controlling actin networks remain poorly defined. Previously, we showed that the trans-Golgi network (TGN) membrane tether/golgin, GCC88, modulates the Golgi ribbon architecture. Here, we show that dispersal of the Golgi ribbon by GCC88 is dependent on actin and the involvement of nonmuscle myosin IIA. We have identified the long isoform of intersectin-1 (ITSN-1), a guanine nucleotide exchange factor for Cdc42, as a novel Golgi component and an interaction partner of GCC88 responsible for mediating the actin-dependent dispersal of the Golgi ribbon. We show that perturbation of Golgi morphology by changes in membrane flux, mediated by silencing the retromer subunit Vps26, or in a model of neurodegeneration, induced by Tau overexpression, are also dependent on the ITSN-1-GCC88 interaction. Overall, our study reveals a role for a TGN golgin and ITSN-1 in linking to the actin cytoskeleton and regulating the balance between a compact Golgi ribbon and a dispersed Golgi, a pathway with relevance to pathophysiological conditions.
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ItemGolgi Dynamics: The Morphology of the Mammalian Golgi Apparatus in Health and Disease(FRONTIERS MEDIA SA, 2019-07-03)In vertebrate cells the Golgi consists of individual stacks fused together into a compact ribbon structure. The function of the ribbon structure of the Golgi has only begun to be appreciated (De Matteis et al., 2008; Gosavi and Gleeson, 2017; Wei and Seemann, 2017). Recent advances have identified a role for the Golgi in the regulation of a broad range of cellular processes and of particular interest is that the modulation of the Golgi ribbon is associated with regulation of a number of signaling pathways (Makhoul et al., 2018). Various cell responses, such as inflammation, and various disorders and diseases, including neurodegeneration and cancer, are associated with the loss of the Golgi ribbon and the appearance of a dispersed or semi-dispersed Golgi. Often the dispersed Golgi is referred to as a "fragmented" morphology. However, the description of a dispersed Golgi ribbon as "fragmented" is inadequate as it does not accurately define the morphological state of the Golgi. This issue is particularly relevant as there are an increasing number of reports describing Golgi fragmentation under physiological and pathological conditions. Knowledge of the precise Golgi architecture is relevant to an appreciation of the functional status of the Golgi apparatus and the underlying molecular mechanism for the contribution of the Golgi to different cellular processes. Here we propose a classification to define the various morphological states of the non-ribbon architecture of the Golgi in mammalian cells as a guide to more precisely define the relationship between the morphological and functional status of this organelle.