Macropinocytosis is an actin-dependent mode of nonselective endocytosis that mediates the uptake of extracellular fluid-phase cargoes. It is now well recognized that tumor cells exploit macropinocytosis to internalize macromolecules that can be catabolized and used to support cell growth and proliferation under nutrient-limiting conditions. Therefore, the identification of molecular mechanisms that control macropinocytosis is fundamental to the understanding of the metabolic adaptive landscape of tumor cells. Here, we report that the acetyl-CoA-producing enzyme, ATP citrate lyase (ACLY), is a key regulator of macropinocytosis and describes a heretofore-unappreciated association of ACLY with the actin cytoskeleton. The cytoskeletal tethering of ACLY is required for the spatially defined acetylation of heterodimeric actin capping protein, which we identify as an essential mediator of the actin remodeling events that drive membrane ruffling and macropinocytosis. Furthermore, we identify a requirement for mitochondrial-derived citrate, an ACLY substrate, for macropinocytosis, and show that mitochondria traffic to cell periphery regions juxtaposed to plasma membrane ruffles. Collectively, these findings establish a mode of metabolite compartmentalization that supports the spatiotemporal modulation of membrane-cytoskeletal interactions required for macropinocytosis by coupling regional acetyl-CoA availability with dynamic protein acetylation.

Although the phagocytic activity of macrophages has long been studied, the involvement of microtubules in the process is not well understood. In this study, we improved the fixation protocol and revealed a dynamically rearranging microtubule network in macrophages, consisting of a basal meshwork, thick bundles at the cell edge, and astral microtubules. Some astral microtubules extended beneath the cell cortex and continued to form bundles at the cell edge. These microtubule assemblies were mutually exclusive of actin accumulation during membrane ruffling. Although the stabilization of microtubules with paclitaxel did not affect the resting stage of the macrophages, it reduced the phagocytic activity and membrane ruffling of macrophages activated with serum-MAF, which induced rapid phagocytosis. In contrast, the destabilization of microtubules with nocodazole enhanced membrane ruffling and the internalization of phagocytic targets suggesting an inhibitory effect of the microtubule network on the remodeling of the actin network. Meanwhile, the microtubule network was necessary for phagosome maturation. Our detailed analyses of cytoskeletal filaments suggest a phagocytosis control system involving Ca2+ influx, the destabilization of microtubules, and activation of actin network remodeling, followed by the translocation and acidification of phagosomes on the microtubule bundles.

A mechanosensitive ion channel, Piezo1 induces non-selective cation flux in response to various mechanical stresses. However, the biological interpretation and underlying mechanisms of cells resulting from Piezo1 activation remain elusive. This study elucidates Piezo1-mediated Ca2+ influx driven by channel activation and cellular behavior using novel Förster Resonance Energy Transfer (FRET)-based biosensors and single-cell imaging analysis. Results reveal that extracellular Ca2+ influx via Piezo1 requires intact caveolin, cholesterol, and cytoskeletal support. Increased cytoplasmic Ca2+ levels enhance PKA, ERK, Rac1, and ROCK activity, which have the potential to promote cancer cell survival and migration. Furthermore, we demonstrate that Piezo1-mediated Ca2+ influx upregulates membrane ruffling, a characteristic feature of cancer cell metastasis, using spatiotemporal image correlation spectroscopy. Thus, our findings provide new insights into the function of Piezo1, suggesting that Piezo1 plays a significant role in the behavior of cancer cells.

Alzheimer\'s disease (AD) is known by characteristic features, extracellular burden of amyloid-β and intracellular neuronal Tau. Microglia, the innate immune cell of the brain has the ability to clear the burden of accumulated proteins via phagocytosis. But the excessive proinflammatory cytokine production, altered cellular signaling and actin remodeling hampers the process of migration and phagocytosis by microglia. Actin remodeling is necessary to initiate the chemotactic migration of microglia towards the target and engulf it. The formation of lamellipodia, filopodia, membrane ruffling and rapid turnover of F-actin is necessary to sense the extracellular target by the cells. Omega-3 fatty acids, are known to impose anti-inflammatory phenotype of microglia by enhancing its ability for migration and phagocytosis. But the role of omega-3 fatty acids in cellular actin remodeling, which is the basis of cellular functions such as migration and phagocytosis, is not well understood. Here, we have focused on the effect of dietary supplement of α-linolenic acid (ALA) on extracellular Tau internalization and assisted actin polymerization for the process. ALA is found to induce membrane ruffling and phagocytic cup formation along with cytoskeletal rearrangement. ALA also enhances the localization of Arp2/3 complex at the leading edge and its colocalization with F-actin to induce the actin polymerization. The excessive actin polymerization might help the cell to protrude forward and perform its migration. The results suggest that dietary supplement of ALA could play a neuroprotective role and slow down the AD pathology.

Macropinocytosis, an evolutionarily conserved endocytic mechanism that mediates non-specific fluid-phase uptake, is potently upregulated by various oncogenic pathways. It is now well appreciated that high macropinocytic activity is a hallmark of many human tumors, which use this adaptation to scavenge extracellular nutrients for fueling cell growth. In the context of the nutrient-scarce tumor microenvironment, this process provides tumor cells with metabolic flexibility. However, dependence on this scavenging mechanism also illuminates a potential metabolic vulnerability. As such, there is a great deal of interest in understanding the molecular underpinnings of macropinocytosis. In this review, we will discuss the most recent advances in characterizing macropinocytosis: the pathways that regulate it, its contribution to the metabolic fitness of cancer cells, and its therapeutic potential.

Membrane ruffling plays an important role in the directed cell migration and escape of tumor cells from the monolayer. Metastasis suppressor 1 (MTSS1), also known as missing in metastasis, has been implicated in cell morphology, motility, metastasis, and development. Here, the dynamic interaction proteins associated with MTSS1 and involved in membrane ruffling were determined by cross-linking and mass spectrometry analysis. We identified α-actinin 4 (ACTN4) as an interacting protein and confirmed a direct interaction between MTSS1 and ACTN4. Moreover, co-expression of MTSS1 in fibroblasts recruited cytoplasmic ACTN4 to the cell periphery, at which point ruffling became thick and rigid. In MCF-7 cells, MTSS1 knockdown did not show an obvious effect on the cell shape or the distribution of endogenous ACTN4; however, ACTN4 overexpression transformed cell morphology from an epidermal- to a fibroblast-like shape, and further MTSS1 depletion significantly increased the ratio of fibroblast cells exhibiting prominent ruffling. Furthermore, biochemical data suggested that MTSS1 cross-linking with ACTN4 induced the formation of actin fiber bundles into more organized structures in vitro. These data indicated that MTSS1 might recruit cytoplasmic ACTN4 to the cell periphery and regulate cytoskeleton dynamics to restrict its performance in membrane ruffling.

Macropinocytosis is a unique pathway of endocytosis characterised by the nonspecific internalisation of large amounts of extracellular fluid, solutes and membrane in large endocytic vesicles known as macropinosomes. Macropinocytosis is important in a range of physiological processes, including antigen presentation, nutrient sensing, recycling of plasma proteins, migration and signalling. It has become apparent in recent years from the study of specialised cells that there are multiple pathways of macropinocytosis utilised by different cell types, and some of these pathways are triggered by different stimuli. Understanding the physiological function of macropinocytosis requires knowledge of the regulation and fate of the macropinocytosis pathways in a range of cell types. Here, we compare the mechanisms of macropinocytosis in different primary and immortalised cells, identify the gaps in knowledge in the field and discuss the potential approaches to analyse the function of macropinocytosis in vivo.

p130 Crk-associated substrate (Cas) is an adaptor protein associating with many other signaling proteins and regulates a various biological processes including cell adhesion, migration, and growth factor stimulation. However, the exact functional role of Cas in growth factor signaling pathway was poorly understood. Here we investigated the role of Cas and its domains in the effects of insulin, EGF, and IGF-1 on c-Jun gene expression, DNA synthesis, cytoskeletal reorganization. We found that microinjection of anti-Cas antibody and C-terminal domain of Cas (Cas-CT) specifically inhibited EGF-induced, but not insulin- or IGF-1-induced, c-Jun expression. Cell cycle progression and cytoskeleton reorganization induced by insulin and EGF, but not by IGF-1, were inhibited by microinjected anti-Cas and Cas-CT. In contrast, microinjection of the substate domain (Cas-SD) of Cas did not have any inhibitory effects. These results revealed that the Cas-CT is differentially implicated in insulin and EGF-mediated, but not IGF-1-mediated, c-Jun expression, DNA synthesis and membrane ruffling.

Salmonella causes disease in humans and animals ranging from mild self-limiting gastroenteritis to potentially life-threatening typhoid fever. Salmonellosis remains a considerable cause of morbidity and mortality globally, and hence imposes a huge socio-economic burden worldwide. A key property of all pathogenic Salmonella strains is the ability to invade non-phagocytic host cells. The major determinant of this invasiveness is a Type 3 Secretion System (T3SS), a molecular syringe that injects virulence effector proteins directly into target host cells. These effectors cooperatively manipulate multiple host cell signaling pathways to drive pathogen internalization. Salmonella does not only rely on these injected effectors, but also uses several other T3SS-independent mechanisms to gain entry into host cells. This review summarizes our current understanding of the methods used by Salmonella for cell invasion, with a focus on the host signaling networks that must be coordinately exploited for the pathogen to achieve its goal.

APPL1- and RAB5-positive signaling endosomes play a crucial role in the activation of AKT in response to extracellular stimuli. Myosin VI (MYO6) and two of its cargo adaptor proteins, GIPC and TOM1/TOM1L2, localize to these peripheral endosomes and mediate endosome association with cortical actin filaments. Loss of MYO6 leads to the displacement of these endosomes from the cell cortex and accumulation in the perinuclear space. Depletion of this myosin not only affects endosome positioning, but also induces actin and lipid remodeling consistent with endosome maturation, including accumulation of F-actin and the endosomal lipid PI(3)P. These processes acutely perturb endosome function, as both AKT phosphorylation and RAC-dependent membrane ruffling were markedly reduced by depletion of either APPL1 or MYO6. These results place MYO6 and its binding partners at a central nexus in cellular signaling linking actin dynamics at the cell surface and endosomal signaling in the cell cortex.