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1.
J Cell Biol ; 221(11)2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36165850

RESUMO

Phosphoinositides are pivotal regulators of vesicular traffic and signaling during phagocytosis. Phagosome formation, the initial step of the process, is characterized by local membrane remodeling and reorganization of the actin cytoskeleton that leads to formation of the pseudopods that drive particle engulfment. Using genetically encoded fluorescent probes, we found that upon particle engagement a localized pool of PtdIns(3,4)P2 is generated by the sequential activities of class I phosphoinositide 3-kinases and phosphoinositide 5-phosphatases. Depletion of this locally generated pool of PtdIns(3,4)P2 blocks pseudopod progression and ultimately phagocytosis. We show that the PtdIns(3,4)P2 effector Lamellipodin (Lpd) is recruited to nascent phagosomes by PtdIns(3,4)P2. Furthermore, we show that silencing of Lpd inhibits phagocytosis and produces aberrant pseudopodia with disorganized actin filaments. Finally, vasodilator-stimulated phosphoprotein (VASP) was identified as a key actin-regulatory protein mediating phagosome formation downstream of Lpd. Mechanistically, our findings imply that a pathway involving PtdIns(3,4)P2, Lpd, and VASP mediates phagocytosis at the stage of particle engulfment.


Assuntos
Actinas , Macrófagos , Fagocitose , Fosfatidilinositóis , Actinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Corantes Fluorescentes , Macrófagos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fagossomos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo
2.
Nat Cell Biol ; 24(5): 708-722, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35484249

RESUMO

Despite their low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival in host organisms. We demonstrate that PtdIns(3,4)P2 is a major product generated in host cells by the effectors of the enteropathogenic bacteria Salmonella and Shigella. Pharmacological, gene silencing and heterologous expression experiments revealed that, remarkably, the biosynthesis of PtdIns(3,4)P2 occurs independently of phosphoinositide 3-kinases. Instead, we found that the Salmonella effector SopB, heretofore believed to be a phosphatase, generates PtdIns(3,4)P2 de novo via a phosphotransferase/phosphoisomerase mechanism. Recombinant SopB is capable of generating PtdIns(3,4,5)P3 and PtdIns(3,4)P2 from PtdIns(4,5)P2 in a cell-free system. Through a remarkable instance of convergent evolution, bacterial effectors acquired the ability to synthesize 3-phosphorylated phosphoinositides by an ATP- and kinase-independent mechanism, thereby subverting host signalling to gain entry and even provoke oncogenic transformation.


Assuntos
Fosfatos de Fosfatidilinositol , Fosfatidilinositóis , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Salmonella , Transdução de Sinais
3.
Front Cell Dev Biol ; 9: 626136, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33614656

RESUMO

The dynamic re-organization of cellular membranes in response to extracellular stimuli is fundamental to the cell physiology of myeloid and lymphoid cells of the immune system. In addition to maintaining cellular homeostatic functions, remodeling of the plasmalemma and endomembranes endow leukocytes with the potential to relay extracellular signals across their biological membranes to promote rolling adhesion and diapedesis, migration into the tissue parenchyma, and to ingest foreign particles and effete cells. Phosphoinositides, signaling lipids that control the interface of biological membranes with the external environment, are pivotal to this wealth of functions. Here, we highlight the complex metabolic transitions that occur to phosphoinositides during several stages of the leukocyte lifecycle, namely diapedesis, migration, and phagocytosis. We describe classical and recently developed tools that have aided our understanding of these complex lipids. Finally, major downstream effectors of inositides are highlighted including the cytoskeleton, emphasizing the importance of these rare lipids in immunity and disease.

4.
Nat Cell Biol ; 21(10): 1234-1247, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570833

RESUMO

Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.


Assuntos
Retículo Endoplasmático/metabolismo , Monócitos/metabolismo , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/genética , Transdução de Sinais , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Sistemas CRISPR-Cas , Retículo Endoplasmático/ultraestrutura , Edição de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Monócitos/ultraestrutura , Fagocitose , Fagossomos/ultraestrutura , Cultura Primária de Células , Proteólise , Células RAW 264.7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
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