Endosomal Recycling Compartment Research Articles

Tryptophan-based motifs in the LLP3 region of the HIV-1 envelope glycoprotein cytoplasmic tail direct trafficking to the endosomal recycling compartment and mediate particle incorporation.

The HIV-1 envelope glycoprotein (Env) is an essential component of the virus and has an exceedingly long cytoplasmic tail (CT). Previous studies have suggested that trafficking signals in the CT interact with host factors to regulate the incorporation of Env into particles. One particular area of interest is termed lentiviral lytic peptide 3 (LLP3), as small deletions in this region have been shown to disrupt Env incorporation. In this study, we identify a small region within LLP3 that regulates how Env associates with cellular recycling compartments. Mutants that reduced or eliminated Env from the recycling compartment also reduced Env incorporation into particles. These findings emphasize the importance of two tryptophan motifs in LLP3 for the incorporation of Env into particles and provide additional support for the idea that the CT interacts with host recycling pathways to determine particle incorporation.

Keynote Lecture 1A non-canonical pathway of cross-presentation disables self/non-self discrimination

The classic pathway of MHC class I (MHC-I) presentation by dendritic cells (DC) is critical to initiate immunity against microbial pathogens. For viruses that do not infect DC or when the antigen presentation functions of infected DC have been compromised, cross-presentation ensures the mobilization of an antiviral CD8 T cell response. Cross-presentation is an adaptation of the classical pathway of MHC-I presentation that DC specialize in to present peptides derived from extracellular antigens by MHC-I molecules. Understanding the mechanisms and regulation of cross-presentation has direct implications to the urgent need for developing T cell vaccines against intracellular pathogens such as HIV, Mycobacterium tuberculosis and malaria. We have shown that the cross-presentation of particulate antigens is highly dependent on Toll-like receptor (TLR) signaling in phagosomes. We reported that MHC-I molecules accumulate in endosomal recycling compartments (ERC) in DC and serve to supply phagosomes with MHC-I molecules for loading with peptides during cross-presentation. Mechanistically, MyD88-dependent TLR signals drive IκB-kinase (IKK)2-mediated phosphorylation of phagosome-associated synaptosome associated protein 23 (SNAP23). Phospho-SNAP23 stabilizes SNARE complexes between the ERC and phagosomes and orchestrates their fusion. As a result, ERC-resident MHC-I molecules are delivered specifically to phagosomes containing TLR ligands favoring the cross-presentation of microbial over self peptides. I will review this pathway and present unpublished data showing how subcellular mislocalization of MHC-I molecules under specific circumstances disrupts the TLR-mediated control of cross-presentation and disables the ability to discriminate self from non-self peptides.

Early Endosomal Vps34-Derived Phosphatidylinositol-3-Phosphate Is Indispensable for the Biogenesis of the Endosomal Recycling Compartment.

Phosphatidylinositol-3-phosphate (PI3P), a major identity tag of early endosomes (EEs), provides a platform for the recruitment of numerous cellular proteins containing an FYVE or PX domain that is required for PI3P-dependent maturation of EEs. Most of the PI3P in EEs is generated by the activity of Vps34, a catalytic component of class III phosphatidylinositol-3-phosphate kinase (PI3Ks) complex. In this study, we analyzed the role of Vps34-derived PI3P in the EE recycling circuit of unperturbed cells using VPS34-IN1 (IN1), a highly specific inhibitor of Vps34. IN1-mediated PI3P depletion resulted in the rapid dissociation of recombinant FYVE- and PX-containing PI3P-binding modules and endogenous PI3P-binding proteins, including EEA1 and EE sorting nexins. IN1 treatment triggered the rapid restructuring of EEs into a PI3P-independent functional configuration, and after IN1 washout, EEs were rapidly restored to a PI3P-dependent functional configuration. Analysis of the PI3P-independent configuration showed that the Vps34-derived PI3P is not essential for the pre-EE-associated functions and the fast recycling loop of the EE recycling circuit but contributes to EE maturation toward the degradation circuit, as previously shown in Vps34 knockout and knockdown studies. However, our study shows that Vps34-derived PI3P is also essential for the establishment of the Rab11a-dependent pathway, including recycling cargo sorting in this pathway and membrane flux from EEs to the pericentriolar endosomal recycling compartment (ERC). Rab11a endosomes of PI3P-depleted cells expanded and vacuolized outside the pericentriolar area without the acquisition of internalized transferrin (Tf). These endosomes had high levels of FIP5 and low levels of FIP3, suggesting that their maturation was arrested before the acquisition of FIP3. Consequently, Tf-loaded-, Rab11a/FIP5-, and Rab8a-positive endosomes disappeared from the pericentriolar area, implying that PI3P-associated functions are essential for ERC biogenesis. ERC loss was rapidly reversed after IN1 washout, which coincided with the restoration of FIP3 recruitment to Rab11a-positive endosomes and their dynein-dependent migration to the cell center. Thus, our study shows that Vps34-derived PI3P is indispensable in the recycling circuit to maintain the slow recycling pathway and biogenesis of the ERC.

Restoring Oat Nanoparticles Mediated Brain Memory Function of Mice Fed Alcohol by Sorting Inflammatory Dectin-1 Complex Into Microglial Exosomes.

Microglia modulate pro-inflammatory and neurotoxic activities. Edible plant-derived factors improve brain function. Current knowledge of the molecular interactions between edible plant-derived factors and the microglial cell is limited. Here an alcohol-induced chronic brain inflammation model is used to identify that the microglial cell is the novel target of oat nanoparticles (oatN). Oral administration of oatN inhibits brain inflammation and improves brain memory function of mice that are fed alcohol. Mechanistically, ethanol activates dectin-1 mediated inflammatory pathway. OatN is taken up by microglial cells via β-glucan mediated binding to microglial hippocalcin (HPCA) whereas oatN digalactosyldiacylglycerol (DGDG) prevents assess of oatN β-glucan to dectin-1. Subsequently endocytosed β-glucan/HPCA is recruited in an endosomal recycling compartment (ERC) via interaction with Rab11a. This complex then sequesters the dectin-1 in the ERC in an oatN β-glucan dependent manner and alters the location of dectin-1 from Golgi to early endosomes and lysosomes and increases exportation of dectin-1 into exosomes in an Rab11a dependent manner. Collectively, these cascading actions lead to preventing the activation of the alcoholic induced brain inflammation signing pathway(s). This coordinated assembling of the HPCA/Rab11a/dectin-1 complex by oral administration of oatN may contribute to the prevention of brain inflammation.

Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection.

Cytomegalovirus (CMV) infection initiates massive rearrangement of cytoplasmic organelles to generate assembly compartment (AC). The earliest events, the establishment of the preAC, are initiated in the early phase as an extensive reorganization of early endosomes (EEs), endosomal recycling compartment (ERC), trans-Golgi network (TGN), and the Golgi. Here, we demonstrate that dynamin inhibitors (Dynasore, Dyngo-4a, MiTMAB, and Dynole-34-2) block the establishment of the preAC in murine CMV (MCMV) infected cells. In this study, we extensively analyzed the effect of Dynasore on the Golgi reorganization sequence into the outer preAC. We also monitored the development of the inner preAC using a set of markers that define EEs (Rab5, Vps34, EEA1, and Hrs), the EE-ERC interface (Rab10), the ERC (Rab11, Arf6), three layers of the Golgi (GRASP65, GM130, Golgin97), and late endosomes (Lamp1). Dynasore inhibited the pericentriolar accumulation of all markers that display EE-ERC-TGN interface in the inner preAC and prevented Golgi unlinking and dislocation to the outer preAC. Furthermore, in pulse-chase experiments, we demonstrated that the presence of dynasore only during the early phase of MCMV infection (4–14 hpi) is sufficient to prevent not only AC formation but also the synthesis of late-phase proteins and virion production. Therefore, our results indicate that dynamin-2 acts as a part of the machinery required for AC generation and rearrangement of EE/ERC/Golgi membranes in the early phase of CMV infection.

Arf GTPases Are Required for the Establishment of the Pre-Assembly Compartment in the Early Phase of Cytomegalovirus Infection.

Shortly after entering the cells, cytomegaloviruses (CMVs) initiate massive reorganization of cellular endocytic and secretory pathways, which results in the forming of the cytoplasmic virion assembly compartment (AC). We have previously shown that the formation of AC in murine CMV- (MCMV) infected cells begins in the early phase of infection (at 4–6 hpi) with the pre-AC establishment. Pre-AC comprises membranes derived from the endosomal recycling compartment, early endosomes, and the trans-Golgi network, which is surrounded by fragmented Golgi cisterns. To explore the importance of Arf GTPases in the biogenesis of the pre-AC, we infected Balb 3T3 cells with MCMV and analyzed the expression and intracellular localization of Arf proteins in the early phases (up to 16 hpi) of infection and the development of pre-AC in cells with a knockdown of Arf protein expression by small interfering RNAs (siRNAs). Herein, we show that even in the early phase, MCMVs cause massive reorganization of the Arf system of the host cells and induce the over-recruitment of Arf proteins onto the membranes of pre-AC. Knockdown of Arf1, Arf3, Arf4, or Arf6 impaired the establishment of pre-AC. However, the knockdown of Arf1 and Arf6 also abolished the establishment of infection. Our study demonstrates that Arf GTPases are required for different steps of early cytomegalovirus infection, including the establishment of the pre-AC.

Endosomal Phosphatidylinositol-3-Phosphate-Associated Functions Are Dispensable for Establishment of the Cytomegalovirus Pre-Assembly Compartment but Essential for the Virus Growth.

Murine cytomegalovirus (MCMV) initiates the stepwise establishment of the pre-assembly compartment (pre-AC) in the early phase of infection by the expansion of the early endosome (EE)/endosomal recycling compartment (ERC) interface and relocation of the Golgi complex. We depleted Vps34-derived phosphatidylinositol-3-phosphate (PI(3)P) at EEs by VPS34-IN1 and inhibited PI(3)P-associated functions by overexpression of 2xFYVE- and p40PX PI(3)P-binding modules to assess the role of PI(3)P-dependent EE domains in the pre-AC biogenesis. We monitored the accumulation of Rab10 and Evectin-2 in the inner pre-AC and the relocation of GM130-positive cis-Golgi organelles to the outer pre-AC by confocal microscopy. Although PI(3)P- and Vps34-positive endosomes build a substantial part of pre-AC, the PI(3)P depletion and the inhibition of PI(3)P-associated functions did not prevent the establishment of infection and progression through the early phase. The PI(3)P depletion in uninfected and MCMV-infected cells rapidly dispersed PI(3)P-bond proteins and reorganized EEs, including ablation of EE-to-ERC transport and relocation of Rab11 endosomes. The PI(3)P depletion one hour before pre-AC initiation and overexpression of 2xFYVE and p40PX domains neither prevented Rab10- and Evectin-2 accumulation, nor Golgi unlinking and relocation. These data demonstrate that PI(3)P-dependent functions, including the Rab11-dependent EE-to-ERC route, are dispensable for pre-AC initiation. Nevertheless, the virus growth was drastically reduced in PI(3)P-depleted cells, indicating that PI(3)P-associated functions are essential for the late phase of infection.

TAP dysfunction in dendritic cells enables noncanonical cross-presentation for T cell priming.

Classic MHC-I presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8+ T cells. Priming CD8+ T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells (DCs). We found that protective CD8+ T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment (ERC) of MHC-I and as such impaired Toll-like receptor-regulated cross-presentation. Instead, MHC-I accumulated in ER-Golgi intermediate compartments (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and ERC-dependent cross-presentation are impaired in DCs, cell-autonomous non-canonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8+ T cell priming.

Cytomegalovirus Generates Assembly Compartment in the Early Phase of Infection by Perturbation of Host-Cell Factors Recruitment at the Early Endosome/Endosomal Recycling Compartment/Trans-Golgi Interface.

Beta-herpesviruses develop a unique structure within the infected cell known as an assembly compartment (AC). This structure, as large as the nucleus, is composed of host-cell-derived membranous elements. The biogenesis of the AC and its contribution to the final stages of beta-herpesvirus assembly are still unclear. In this study, we performed a spatial and temporal analysis of the AC in cells infected with murine CMV (MCMV), a member of the beta-herpesvirus family, using a panel of markers that characterize membranous organelle system. Out of 64 markers that were analyzed, 52 were cytosolic proteins that are recruited to membranes as components of membrane-shaping regulatory cascades. The analysis demonstrates that MCMV infection extensively reorganizes interface between early endosomes (EE), endosomal recycling compartment (ERC), and the trans-Golgi network (TGN), resulting in expansion of various EE-ERC-TGN intermediates that fill the broad area of the inner AC. These intermediates are displayed as over-recruitment of host-cell factors that control membrane flow at the EE-ERC-TGN interface. Most of the reorganization is accomplished in the early (E) phase of infection, indicating that the AC biogenesis is controlled by MCMV early genes. Although it is known that CMV infection affects the expression of a large number of host-cell factors that control membranous system, analysis of the host-cell transcriptome and protein expression in the E phase of infection demonstrated no sufficiently significant alteration in expression levels of analyzed markers. Thus, our study demonstrates that MCMV-encoded early phase function targets recruitment cascades of host cell-factors that control membranous flow at the EE-ERC-TGN interface in order to initiate the development of the AC.

Effect of Pharmacological Inhibition of the Catalytic Activity of Phosphatases of Regenerating Liver in Early T Cell Receptor Signaling Dynamics and IL-2 Production.

We have previously shown the delivery of phosphatase of regenerating liver-1 (PRL-1) to the immunological synapse (IS) and proposed a regulatory role of the catalytic activity of PRLs (PRL-1, PRL-2 and PRL-3) in antigen-induced IL-2 production. Nonetheless, the expression in T cells and delivery to the IS of the highly homologous PRL-3, as well as the role of the catalytic activity of PRLs in antigen-induced early signaling, has not been investigated. Here, the expression of PRL-3 protein was detected in primary CD4 T cells and in the CD4 T cell line Jurkat (JK), in which an overexpressed GFP-PRL-3 fluorescent fusion protein trafficked through the endosomal recycling compartment and co-localized with PLCγ1 signaling sites at the IS. Pharmacological inhibition was used to compare the role of the catalytic activity of PRLs in antigen-induced early signaling and late IL-2 production. Although the phosphatase activity of PRLs was not critical for early signaling triggered by antigen, it seemed to regulate signaling dynamics and was necessary for proper IL-2 production. We propose that enzymatic activity of PRLs has a higher significance for cytokine production than for early signaling at the IS. However, further research will be necessary to deeply understand the regulatory role of PRLs during lymphocyte activation and effector function.

Protein kinase C regulates ErbB3 turnover

ErbB3, which belongs to the epidermal growth factor receptor (EGFR) or ErbB family of receptor tyrosine kinases, is involved in progression of several human cancers and a tight regulation of its expression is crucial. An important mechanism for regulation of ErbB proteins is endocytosis and we recently showed that ErbB3, contrary to other ErbB proteins, like EGFR and ErbB2, is constitutively internalized and degraded. Several studies show that protein kinase C (PKC) can regulate the activation, localization and stability of EGFR and ErbB2. Activation of PKC causes their down-regulation from the plasma membrane, but instead of being degraded the receptors accumulate in an endosomal recycling compartment. Since little is known about possible connections between ErbB3 and PKC, we have in the present study investigated effects PKC activity has on ErbB3 stability and intracellular trafficking. While PKC inhibition tends to increase ErbB3 degradation, activation of PKC causes ErbB3 stabilization. The stabilization was not due to inhibited internalization, on the contrary we find that expression of ErbB3 at the plasma membrane is reduced upon PMA-induced PKC activation. However, while endocytosed ErbB3 under normal conditions and upon PKC inhibition is found in early endosomal antigen 1 (EEA1) positive early endosomes and lysosomal-associated membrane protein 1 (LAMP1) positive late endosomes/lysosomes, indicating that it follows the classic degradative pathway, ErbB3 localizes to EEA1 and LAMP1 negative compartments upon PMA-induced activation of PKC. Altogether this shows that PKC regulates the stability of ErbB3, and knockdown experiments show that PKCδ is essential in this process. A likely explanation is that PKC regulates endosomal sorting of ErbB3 and that activated PKC sorts ErbB3 away from the degradative pathway.

Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R.

Development of physiologic cardiac hypertrophy has primarily been ascribed to the IGF-1 and its receptor, IGF-1 receptor (IGF-1R), and subsequent activation of the protein kinase B (Akt) pathway. However, regulation of endosome-mediated recycling and degradation of IGF-1R during physiologic hypertrophy has not been investigated. In a physiologic hypertrophy model of treadmill-exercised mice, we observed that levels of tumor susceptibility gene 101 (Tsg101), a key member of the endosomal sorting complex required for transport, were dramatically elevated in the heart compared with sedentary controls. To determine the role of Tsg101 on physiologic hypertrophy, we generated a transgenic (TG) mouse model with cardiac-specific overexpression of Tsg101. These TG mice exhibited a physiologic-like cardiac hypertrophy phenotype at 8 wk evidenced by: 1) the absence of cardiac fibrosis, 2) significant improvement of cardiac function, and 3) increased total and plasma membrane levels of IGF-1R and increased phosphorylation of Akt. Mechanistically, we identified that Tsg101 interacted with family-interacting protein 3 (FIP3) and IGF-1R, thereby stabilizing FIP3 and enhancing recycling of IGF-1R. In vitro, adenovirus-mediated overexpression of Tsg101 in neonatal rat cardiomyocytes resulted in cell hypertrophy, which was blocked by addition of monensin, an inhibitor of endosome-mediated recycling, and by small interfering RNA-mediated knockdown (KD) of FIP3. Furthermore, cardiac-specific KD of Tsg101 showed a significant reduction in levels of endosomal recycling compartment members (Rab11a and FIP3), IGF-1R, and Akt phosphorylation. Most interestingly, Tsg101-KD mice failed to develop cardiac hypertrophy after intense treadmill training. Taken together, our data identify Tsg101 as a novel positive regulator of physiologic cardiac hypertrophy through facilitating the FIP3-mediated endosomal recycling of IGF-1R.-Essandoh, K., Deng, S., Wang, X., Jiang, M., Mu, X., Peng, J., Li, Y., Peng, T., Wagner, K.-U., Rubinstein, J., Fan, G.-C. Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R.

Cytomegaloviruses Exploit Recycling Rab Proteins in the Sequential Establishment of the Assembly Compartment.

Cytomegaloviruses (CMV) reorganize membranous system of the cell in order to develop a virion assembly compartment (VAC). The development starts in the early (E) phase of infection with the reorganization of the endosomal system and the Golgi and proceeds to the late phase until newly formed virions are assembled and released. The events in the E phase involve reorganization of the endosomal recycling compartment (ERC) in a series of cellular alterations that are mostly unknown. In this minireview, we discuss the effect of murine CMV infection on Rab proteins, master regulators of membrane trafficking pathways, which in the cascades with their GEFs and GAPs organize the flow of membranes through the ERC. Immunofluorescence analyzes of murine CMV infected cells suggest perturbations of Rab cascades that operate at the ERC. Analysis of cellular transcriptome in the course of both murine and human CMV infection demonstrates the alteration in expression of cellular genes whose products are known to build Rab cascades. These alterations, however, cannot explain perturbations of the ERC. Cellular proteome data available for human CMV infected cells suggests the potential role of RabGAP downregulation at the end of the E phase. However, the very early onset of the ERC alterations in the course of MCMV infection indicates that CMVs exploit Rab cascades to reorganize the ERC, which represents the earliest step in the sequential establishment of the cVAC.

Reinterpretation of the localization of the ATP binding cassette transporter ABCG1 in insulin-secreting cells and insights regarding its trafficking and function.

The ABC transporter ABCG1 contributes to the regulation of cholesterol efflux from cells and to the distribution of cholesterol within cells. We showed previously that ABCG1 deficiency inhibits insulin secretion by pancreatic beta cells and, based on its immunolocalization to insulin granules, proposed its essential role in forming granule membranes that are enriched in cholesterol. While we confirm elsewhere that ABCG1, alongside ABCA1 and oxysterol binding protein OSBP, supports insulin granule formation, the aim here is to clarify the localization of ABCG1 within insulin-secreting cells and to provide added insight regarding ABCG1’s trafficking and sites of function. We show that stably expressed GFP-tagged ABCG1 closely mimics the distribution of endogenous ABCG1 in pancreatic INS1 cells and accumulates in the trans-Golgi network (TGN), endosomal recycling compartment (ERC) and on the cell surface but not on insulin granules, early or late endosomes. Notably, ABCG1 is short-lived, and proteasomal and lysosomal inhibitors both decrease its degradation. Following blockade of protein synthesis, GFP-tagged ABCG1 first disappears from the ER and TGN and later from the ERC and plasma membrane. In addition to aiding granule formation, our findings raise the prospect that ABCG1 may act beyond the TGN to regulate activities involving the endocytic pathway, especially as the amount of transferrin receptor is increased in ABCG1-deficient cells. Thus, ABCG1 may function at multiple intracellular sites and the plasma membrane as a roving sensor and modulator of cholesterol distribution, membrane trafficking and cholesterol efflux.

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C560-649) and examined the consequences on Env trafficking and incorporation into particles. FIP1C560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane.IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes trapped inside the cell within the endosomal recycling compartment. Intracellular trapping resulted in a loss of envelope protein on released particles and a corresponding loss of infectivity. Mutations of specific trafficking motifs in the envelope protein tail prevented its trapping in the recycling compartment. These results establish that trafficking to the endosomal recycling compartment is an essential step in HIV envelope protein particle incorporation.

Landmarks of endosomal remodeling in the early phase of cytomegalovirus infection

Cytomegaloviruses (CMVs) extensively rearrange the cellular membrane system to develop assembly compartment (AC), but the earliest events in this process are poorly characterized. Here, we demonstrate that murine CMV (MCMV) infection restrains endosomal trafficking of cargo molecules that travel along the recycling (TfR and MHC-I) and the late endosomal (EGFR, M6PR, Lamp1) circuit. Internalized cargo accumulates in Arf6-, Rab5-, Rab22A-, and Rab11-positive and Rab35-, Rab8-, and Rab10-negative juxtanuclear endosomes, suggesting the disruption of Arf/Rab regulatory cascade at the stage of sorting endosomes and the endosomal recycling compartment. Rearrangement of the endosomal system is initiated by an MCMV-encoded function very early in the infection. Our study, thus, establishes a set of landmarks of endosomal remodeling in the early phase of MCMV-infection which coincide with the Golgi rearrangement, suggesting that these perturbations are the earliest membrane reorganizations that may represent an initial step in the biogenesis of the AC.

Thioether-stapled macrocyclic inhibitors of the EH domain of EHD1

Recycling of receptors from the endosomal recycling compartment to the plasma membrane is a critical cellular process, and recycling is particularly important for maintaining invasiveness in solid tumors. In this work, we continue our efforts to inhibit EHD1, a critical adaptor protein involved in receptor recycling. We applied a diversity-oriented macrocyclization approach to produce cyclic peptides with varied conformations, but that each contain a motif that binds to the EH domain of EHD1. Screening these uncovered several new inhibitors for EHD1’s EH domain, the most potent of which bound with a Kd of 3.1μM. Several of the most potent inhibitors were tested in a cellular assay that measures extent of vesicle recycling. Inhibiting EHD1 could potentially slow cancer invasiveness and metastasis, and these cyclic peptides represent the most potent inhibitors of EHD1 to date.

Tsg101 Regulates Rab11FIP3‐Mediated Endosomal Recycling of IGF‐1R in Physiological Cardiac Hypertrophy

BackgroundDevelopment of physiological cardiac hypertrophy has primarily been ascribed to the insulin‐like growth factor 1 (IGF‐1) and its receptor, IGF‐1R and subsequent activation of the Akt pathway. Previous studies have demonstrated the effects of IGF‐1 on both pathological and physiological remodeling whereas IGF‐1R has consistently been linked to only physiological heart growth. Thus, the balance between de novo synthesis and recycling or degradation of IGF‐1R through the endosomal system is critical for physiological heart growth. However, regulation of endosome‐mediated recycling and degradation of IGF‐1R during physiological hypertrophy has not been investigated.Methods & ResultsIn the current study, we sought to determine the role of endosome‐mediated recycling and degradation of IGF‐1R on physiological cardiac hypertrophy. Male FVB/n mice (7–8 weeks old) underwent intense treadmill exercise training for 1 week to induce physiological hypertrophy. We observed that mediators of recycling (Rab11a, Rab11FIP3) and degradation (Rab7) of endosomes were enhanced in exercise‐trained mice. Of importance, levels of tumor susceptibility gene 101 (Tsg101), a key member of the Endosomal Sorting Complex required for Transport (ESCRT), were dramatically elevated in hearts of exercise‐trained mice, compared to controls (n=9, p<0.01). To determine the role of Tsg101 on physiological hypertrophy, we generated a transgenic mice model with cardiac‐specific overexpression of Tsg101. These transgenic mice exhibited a physiological hypertrophy‐like phenotype at 8 weeks, evidenced by the absence of cardiac fibrosis, significant improvement of cardiac function, increased total and plasma membrane levels of IGF‐1R and increased phosphorylation of Akt. Mechanistically, we identified that Tsg101 interacted with IGF‐1R and Rab11FIP3, which constitutively binds to Rab11a at endosomal recycling compartments to initiate recycling of endosomes. In vitro, adenovirus‐mediated overexpression of Tsg101 in neonatal rat cardiomyocytes resulted in increased cardiomyocyte size, which was blocked by addition of monensin, an inhibitor of Rab11/FIP3‐endosome recycling. Furthermore, cardiac‐specific knockdown of Tsg101 showed a significant reduction in levels of Rab11a, Rab11FIP3, IGF‐1R and Akt phosphorylation. Most interestingly, Tsg101‐knockdown mice failed to develop cardiac hypertrophy after intense treadmill training.ConclusionOur data identify Tsg101 as a novel regulator of physiological cardiac hypertrophy through facilitating the Rab11FIP3‐mediated endosomal recycling of IGF‐1R.Support or Funding InformationNIH 2R01HL‐087861

Molecular Mechanisms of V-SNARE Function in Secretory Granule Exocytosis

Insulin is released by regulated exocytosis and docking of insulin granules at the plasma membrane, followed by the assembly of the secretory machinery is required for insulin release. Newly formed granules require a complex maturation process to become release-ready and the formation of these fusion competent mature secretory granules involves a series of discrete and unique events comprised of granule sorting, maturation, docking, priming, and finally Ca2+-dependent fusion. Soluble NSF attachment receptor (SNARE) proteins are the conserved core machinery required for all intracellular membrane fusion events. While SNAREs specifically control fusion, vesicular SNAREs (v-SNAREs) perform additional roles that are still elusive. In secretory cell types, v-SNAREs are pivotal for targeting vesicles to different locations, mediating both sorting and membrane protein distribution and reductions in v-SNAREs result in defective regulated exocytosis. Using total internal reflection fluorescence (TIRF) microscopy, we studied the role of VAMP8 in secretory granule exocytosis in pancreatic beta cells. We found that VAMP8 associates with early, late and recycling endosome compartment, rather than insulin containing granules. However, VAMP8 traffics and fuses at the plasma membrane specifically with the recycling endosomal compartment. VAMP8 expression compromised secretory granule fusion, whereas VAMP2 expression facilitated granule fusion, delineating a VAMP8-dependent fusion step between recycling endosomes and the plasma membrane. Truncation of the VAMP8 C-terminal region resulted in attenuated exocytosis and a change in secretion kinetics. Moreover, secreting insulin measurements are negatively correlated with increased VAMP8 copy number in human islets. We conclude that VAMP8 vesicles play an important role in granule genesis and sorting of essential membrane proteins regulating insulin secretion. Additionally, the TMD of VAMP8 may be necessary for initiating fusion upon stimulation and appears to have some direct interaction on insulin secretion. This work should fill critical gaps in our understanding of regulated secretion and the treatment of type-2 diabetes.

Iodixanol Gradient Centrifugation to Separate Components of the Low-Density Membrane Fraction from 3T3-L1 Adipocytes.

We optimized a set of fractionation techniques to facilitate the isolation of subcellular compartments containing insulin-sensitive glucose transporter isoform 4 (GLUT4), which is mobilized from GLUT4 storage vesicles (GSVs) in fat and muscle cells in response to insulin. In the absence of insulin, GLUT4 undergoes a continuous cycle of GSV formation and fusion with other compartments. Full membrane fractionation of 3T3-L1 adipocytes produces a low-density membrane fraction that contains both the constitutive recycling pool (the endosomal recycling compartments) and the insulin-sensitive pool (the GSVs). These two pools can be separated based on density using iodixanol gradient centrifugation, described here.