Membrane Protrusion Formation Mediated by Rho/ROCK Signalling and Modulation of Chloride Flux

Abstract

33Membrane protrusion is an important structural property associated with various cellular functions. The 34 pentaspan membrane protein Prominin-1 (Prom1/CD133) is known to be localised to the protrusions and 35 plays a pivotal role in migration and the determination of cellular morphology; however, the underlying 36 mechanisms have been elusive. Here, we demonstrate that Prom1 is sufficient to trigger membrane 37 protrusion formation. Overexpression of Prom1 in the RPE-1 cells triggers multiple long cholesterol-38 enriched protrusions, independently from actin and tubulin polymerisation. For this protrusion formation, 39 the five amino acid stretch located at the carboxyl cytosolic region is essential. Moreover, the small GTPase 40Rho and its effector kinase ROCK are essential for this protrusion formation, and the intersection point of 41 active Rho and Prom1 is where the protrusion formation initiates. Importantly, Prom1 causes the chloride42 ion efflux induced by calcium ion uptake, and protrusion formation is closely associated with the chloride 43 efflux activity. Altogether, this study has elucidated that Prom1 plays critical roles for the membrane 44 morphology and chloride ion flux.48 curvature and invagination. 49Cilia, cytonemes and microvilli are representative protrusions (1). Cilia contain microtubules and 50 act as antennae for physical stimuli or extracellular signal molecules. Cytonemes, which comprise actin, are 51 presumed to transport the signal molecules distant from the cell body. Microvilli, which are often formed at 52 the luminal membrane in the intestine, are also membrane protrusions rich in cholesterol (2), and are formed 53 to widen the cell surface and to efficiently incorporate extracellular materials into the body. In general, these 54 protrusions transduce essential information into the cells in order to decide the cell response to these stimuli. 55Therefore, the mechanisms for cell shape regulation is one of the central questions of cell biology. 56In the vertebrate retina, the photoreceptor cell has a long cell shape, and is divided into different 57 functional compartments. Among these compartments, the discs, which are responsible for initial light 58 perception, are continuously formed in the outer segment. Disc formation commences with the curvature of 59 the membrane at the adjacent region of the connecting cilium, which is then separated from the cell 60 membrane to form microvesicles in the photoreceptor cell (3). 61Prominin-1 (CD133, Prom1) encodes a pentaspan transmembrane glycoprotein, highly expressed 62 in the retina, kidney, and testis (4). Prom1 is localised at the connecting cilium and in the outer segment (5), 63 and is recognised as a crucial gene for the homeostasis of photoreceptor cells (5). The loss of Prom1 function 64 leads to photoreceptor degeneration (6-8). In pedigrees with mutations in the Prom1 gene, individuals 65 carrying the homologous mutation suffer from inherited macular dystrophies termed as Stargardt's disease 66 and retinitis pigmentosa (RP)...