Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion

Abstract

SummaryA balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol-and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipidraft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry.

Loss of functional MYO1C/myosin 1c, a motor protein involved in lipid raft trafficking, disrupts autophagosome-lysosome fusion

“…At the cellular level, ablating MYO1C function results in a redistribution of lipid-raft enriched domains from the plasma membrane to intracellular compartments and an accumulation of these membranes in the perinuclear region. 6 In addition, we also observed a very significant change in the morphology of late endocytic organelles in the form of enlarged swollen lysosomes following loss of MYO1C. 16 Here, we demonstrate that changes in cellular cholesterol trafficking in MYO1C-depleted cells cause a significant increase in total intracellular cholesterol content.…”

“…9 In addition, MYO1C is recruited onto highly dynamic lipid-raft enriched membrane tubules in the endocytic recycling pathway, where it is required for delivery and exocytosis of lipidraft enriched membranes at the plasma membrane. 6,10 This function is supported by the finding that MYO1C facilitates the exocytosis and delivery of several raftassociated cargoes such as SLC2A4/GLUT4, 11,12 AQP2/aquaporin 2, 13 KIRREL/NEPH1 14 as well as KDR/VEGFR2 15 to the cell surface. At the cellular level, ablating MYO1C function results in a redistribution of lipid-raft enriched domains from the plasma membrane to intracellular compartments and an accumulation of these membranes in the perinuclear region.…”

“…6 Since disturbance of the cellular lipid balance, distribution, and membrane composition have a strong impact on the autophagy pathway, 4,5 we investigated whether the depletion of functional MYO1C, either by siRNA-mediated knockdown (KD) or using the MYO1 inhibitor pentachloropseudilin (PCIP), 16 has an effect on autophagosome biogenesis or degradation. To assess whether MYO1C is required for autophagy progression, we quantified the number of LC3-positive autophagosomes under basal conditions by immunofluorescence microscopy in MYO1C KD cells (Fig.…”

“…Our previous work demonstrated that ablating MYO1C function not only causes a dramatic cellular redistribution of cholesterol-enriched lipid raft domains, 6 but also leads to a change in lysosomal morphology. 16 Inhibition of MYO1C expression or activity using either a SMARTpool of MYO1C-specific siRNA ( Fig.…”

“…Our previous work identified MYO1C, a member of class I myosins, as a key regulator of trafficking of lipid rafts from intracellular storage compartments to the plasma membrane. 6 MYO1C is a slow monomeric actin-based motor protein, adapted for sustained power mobility that links membrane cargo enriched in phospholipids, such as phosphatidylinositol 4,5-bisphosphate (PtdIns [4,5]P 2 ), to the actin cytoskeleton. Membrane targeting of MYO1C involves a putative PH-domain present in the short C-terminal tail.…”