RESUMEN
Pathogens use a vast number of strategies to alter host membrane dynamics. Targeting the host membrane machinery is important for the survival and pathogenesis of several extracellular, vacuolar, and cytosolic bacteria. Membrane manipulation promotes bacterial replication while suppressing host responses, allowing the bacterium to thrive in a hostile environment. This review provides a comprehensive summary of various strategies used by both extracellular and intracellular bacteria to hijack host membrane trafficking machinery. We start with mechanisms used by bacteria to alter the plasma membrane, delve into the hijacking of various vesicle trafficking pathways, and conclude by summarizing bacterial adaptation to host immune responses. Understanding bacterial manipulation of host membrane trafficking provides insights into bacterial pathogenesis and uncovers the molecular mechanisms behind various processes within a eukaryotic cell.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Membrana Celular/metabolismo , Células/microbiología , Interacciones Huésped-Patógeno/fisiología , Animales , Autofagia/fisiología , Proteínas Bacterianas/fisiología , Toxinas Bacterianas/farmacología , Transporte Biológico , Permeabilidad de la Membrana Celular , Células/ultraestructura , Citosol/microbiología , Endocitosis/fisiología , Humanos , Lisosomas/fisiología , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Fagosomas/fisiología , Transporte de Proteínas , Vacuolas/microbiología , Vacuolas/fisiologíaRESUMEN
Intracellular growth of Legionella pneumophila occurs in a replication vacuole constructed by host proteins that regulate vesicular traffic from the host endoplasmic reticulum (ER). This process is promoted by a combination of approximately 300 Icm/Dot translocated substrates (IDTS). One of these proteins, Ceg9, was previously identified in a screen for L. pneumophila IDTS that manipulate secretory traffic when overexpressed in yeast. Using ectopic expression of Ceg9 in mammalian cells, we demonstrate that Ceg9 interacts with isoforms of host reticulon 4 (Rtn4), a protein that regulates ER tubule formation. Binding occurs under conditions that prevent association with other known reticulon binding proteins, arguing that Ceg9 binding is stable. A tripartite complex was demonstrated among Rtn4, Ceg9, and atlastin 1, a previously characterized reticulon interacting partner. The binding of Ceg9 to Rtn4 was not due to bridging by atlastin 1 but resulted from the two interacting partners binding independently to reticulon. When Ceg9 is ectopically expressed in mammalian cells, it shows a localization pattern that is indistinguishable from that of Rtn4, perhaps due to interactions between and similar structural features of the two proteins. Consistent with Rtn4 playing a role in the formation of the Legionella-containing vacuole, it was recruited to almost 50% of the vacuoles within 20 min postinfection. Our studies suggest that L. pneumophila proteins interact with ER tubules at an early stage of replication vacuole formation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Interacciones Huésped-Parásitos/fisiología , Legionella pneumophila/patogenicidad , Enfermedad de los Legionarios/metabolismo , Proteínas de la Mielina/metabolismo , Animales , Western Blotting , Células COS , Chlorocebus aethiops , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Inmunoprecipitación , Legionella pneumophila/metabolismo , Espectrometría de Masas , Ratones , Proteínas Nogo , Reacción en Cadena de la Polimerasa , Transfección , VacuolasRESUMEN
INF2 is an unusual formin protein in that it accelerates both actin polymerization and depolymerization, the latter through an actin filament-severing activity. Similar to other formins, INF2 possesses a dimeric formin homology 2 (FH2) domain that binds filament barbed ends and is critical for polymerization and depolymerization activities. In addition, INF2 binds actin monomers through its diaphanous autoregulatory domain (DAD) that resembles a Wiskott-Aldrich syndrome protein homology 2 (WH2) sequence C-terminal to the FH2 that participates in both polymerization and depolymerization. INF2-DAD is also predicted to participate in an autoinhibitory interaction with the N-terminal diaphanous inhibitory domain (DID). In this work, we show that actin monomer binding to the DAD of INF2 competes with the DID/DAD interaction, thereby activating actin polymerization. INF2 is autoinhibited in cells because mutation of a key DID residue results in constitutive INF2 activity. In contrast, purified full-length INF2 is constitutively active in biochemical actin polymerization assays containing only INF2 and actin monomers. Addition of proteins that compete with INF2-DAD for actin binding (profilin or the WH2 from Wiskott-Aldrich syndrome protein) decrease full-length INF2 activity while not significantly decreasing activity of an INF2 construct lacking the DID sequence. Profilin-mediated INF2 inhibition is relieved by an anti-N-terminal antibody for INF2 that blocks the DID/DAD interaction. These results suggest that free actin monomers can serve as INF2 activators by competing with the DID/DAD interaction. We also find that, in contrast to past results, the DID-containing N terminus of INF2 does not directly bind the Rho GTPase Cdc42.
Asunto(s)
Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Unión Competitiva , Línea Celular Tumoral , Forminas , Humanos , Modelos Moleculares , Profilinas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
INF2 (inverted formin 2) is a formin protein with unusual biochemical characteristics. As with other formins, the formin homology 2 (FH2) domain of INF2 accelerates actin filament assembly and remains at the barbed end, modulating elongation. The unique feature of INF2 is its ability to sever filaments and enhance depolymerization, which requires the C-terminal region. Physiologically, INF2 acts in the secretory pathway and is mutated in two human diseases, focal and segmental glomerulosclerosis and Charcot-Marie-Tooth disease. In this study, we investigate the effects of mutating two FH2 residues found to be key in other formins: Ile-643 and Lys-792. Surprisingly, neither mutation abolishes barbed end binding, as judged by pyrene-actin and total internal reflection (TIRF) microscopy elongation assays. The I643A mutation causes tight capping of a subset of filaments, whereas K792A causes slow elongation of all filaments. The I643A mutation has a minor inhibitory effect on polymerization activity but causes almost complete abolition of severing and depolymerization activity. The K792A mutation has relatively small effects on polymerization, severing, and depolymerization. In cells, the K792A mutant causes actin accumulation around the endoplasmic reticulum to a similar extent as wild type, whereas the I643A mutant causes no measurable polymerization. The inability of I643A to induce actin polymerization in cells is explained by its inability to promote robust actin polymerization in the presence of capping protein. These results highlight an important point: it is dangerous to assume that mutation of conserved FH2 residues will have equivalent effects in all formins. The work also suggests that both mutations have effects on the mechanism of processive elongation.
Asunto(s)
Proteínas de Capping de la Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Microfilamentos/metabolismo , Mutación Missense , Proteínas de Capping de la Actina/genética , Citoesqueleto de Actina/genética , Actinas/genética , Sustitución de Aminoácidos , Línea Celular Tumoral , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Retículo Endoplásmico/genética , Forminas , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Humanos , Proteínas de Microfilamentos/genética , Estructura Terciaria de ProteínaRESUMEN
Intracellular pathogens manipulate host organelles to support replication within cells. For Legionella pneumophila, the bacterium translocates proteins that establish an endoplasmic reticulum (ER)-associated replication compartment. We show here that the bacterial Sde proteins target host reticulon 4 (Rtn4) to control tubular ER dynamics, resulting in tubule rearrangements as well as alterations in Rtn4 associated with the replication compartment. These rearrangements are triggered via Sde-promoted ubiquitin transfer to Rtn4, occurring almost immediately after bacterial uptake. Ubiquitin transfer requires two sequential enzymatic activities from a single Sde polypeptide: an ADP-ribosyltransferase and a nucleotidase/phosphohydrolase. The ADP-ribosylated moiety of ubiquitin is a substrate for the nucleotidase/phosphohydrolase, resulting in either transfer of ubiquitin to Rtn4 or phosphoribosylation of ubiquitin in the absence of a ubiquitination target. Therefore, a single bacterial protein drives a multistep biochemical pathway to control ubiquitination and tubular ER function independently of the host ubiquitin machinery.
Asunto(s)
Proteínas Bacterianas/metabolismo , Retículo Endoplásmico/metabolismo , Interacciones Huésped-Patógeno , Legionella pneumophila/fisiología , Ubiquitinación , ADP Ribosa Transferasas , Animales , Proteínas Bacterianas/genética , Células COS , Catálisis , Chlorocebus aethiops , Reordenamiento Génico , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/patogenicidad , Proteínas Nogo/genética , Ubiquitina/metabolismoRESUMEN
Mitochondrial fission is fundamentally important to cellular physiology. The dynamin-related protein Drp1 mediates fission, and interaction between mitochondrion and endoplasmic reticulum (ER) enhances fission. However, the mechanism for Drp1 recruitment to mitochondria is unclear, although previous results implicate actin involvement. Here, we found that actin polymerization through ER-localized inverted formin 2 (INF2) was required for efficient mitochondrial fission in mammalian cells. INF2 functioned upstream of Drp1. Actin filaments appeared to accumulate between mitochondria and INF2-enriched ER membranes at constriction sites. Thus, INF2-induced actin filaments may drive initial mitochondrial constriction, which allows Drp1-driven secondary constriction. Because INF2 mutations can lead to Charcot-Marie-Tooth disease, our results provide a potential cellular mechanism for this disease state.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Microfilamentos/metabolismo , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Dinámicas Mitocondriales , Citoesqueleto de Actina/ultraestructura , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Dinaminas , Retículo Endoplásmico/ultraestructura , Forminas , GTP Fosfohidrolasas/metabolismo , Humanos , Ratones , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/metabolismo , Células 3T3 NIH , Multimerización de Proteína , ARN Interferente Pequeño , Proteínas Recombinantes de Fusión/metabolismo , Tiazolidinas/farmacologíaRESUMEN
INF2 is a unique formin that can both polymerize and depolymerize actin filaments. Mutations in INF2 cause the kidney disease focal and segmental glomerulosclerosis. INF2 can be expressed as two C-terminal splice variants: CAAX and non-CAAX. The CAAX isoform contains a C-terminal prenyl group and is tightly bound to endoplasmic reticulum (ER). The localization pattern and cellular function of the non-CAAX isoform have not been studied. Here we find that the two isoforms are expressed in a cell type-dependent manner, with CAAX predominant in 3T3 fibroblasts and non-CAAX predominant in U2OS, HeLa, and Jurkat cells. Although INF2-CAAX is ER localized in an actin-independent manner, INF2-non-CAAX localizes in an actin-dependent meshwork pattern distinct from ER. INF2-non-CAAX is loosely attached to this meshwork, being extracted by brief digitonin treatment. Suppression of INF2-non-CAAX causes fragmentation of the Golgi apparatus. This effect is counteracted by treatment with the actin monomer-sequestering drug latrunculin B. We also find discrete patches of actin filaments in the peri-Golgi region, and these patches are reduced upon INF2 suppression. Our results suggest that the non-CAAX isoform of INF2 serves a distinct cellular function from that of the CAAX isoform.
Asunto(s)
Empalme Alternativo/fisiología , Aparato de Golgi/metabolismo , Proteínas de Microfilamentos/biosíntesis , Empalme Alternativo/efectos de los fármacos , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Forminas , Aparato de Golgi/genética , Células HeLa , Humanos , Células Jurkat , Ratones , Proteínas de Microfilamentos/genética , Células 3T3 NIH , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Tiazolidinas/farmacologíaRESUMEN
A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (K(d) < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , NADPH Deshidrogenasa/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Proteínas Portadoras/genética , Forminas , Humanos , Ratones , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Microtúbulos/genética , NADPH Deshidrogenasa/genética , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
In addition to its ability to accelerate filament assembly, which is common to formins, INF2 is a formin protein with the unique biochemical ability to accelerate actin filament depolymerization. The depolymerization activity of INF2 requires its actin monomer-binding WASP homology 2 (WH2) motif. In this study, we show that INF2 is peripherally bound to the cytoplasmic face of the endoplasmic reticulum (ER) in Swiss 3T3 cells. Both endogenous INF2 and GFP-fusion constructs display ER localization. INF2 is post-translationally modified by a C-terminal farnesyl group, and this modification is required for ER interaction. However, farnesylation is not sufficient for ER association, and membrane extraction experiments suggest that ionic interactions are also important. The WH2 motif also serves as a diaphanous autoregulatory domain (DAD), which binds to the N-terminal diaphanous inhibitory domain (DID), with an apparent dissociation constant of 1.1 muM. Surprisingly, the DID-DAD interaction does not inhibit the actin nucleation activity of INF2; however, it does inhibit the depolymerization activity. Point mutations to the DAD/WH2 inhibit both the DID-DAD interaction and depolymerization activity. Expression of GFP-INF2 containing these DAD/WH2 mutations causes the ER to collapse around the nucleus, with accumulation of actin filaments around the collapsed ER. This study is the first to show the association of an actin-assembly factor with the ER.