Amit Tuli

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity

By exploiting NK cell LROs (known as lytic granules) as a model, a new role is defined for Arl8b in regulating motility and exocytosis of lytic granules of NK cells. Not only lytic granules but also the MTOC is unable to polarize toward the immune synapse formed between the NK cell and its target in Arl8b-depleted NK cells.

The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes

Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLEKHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLEKHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLEKHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLEKHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLEKHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion.

MHC Class II Presentation Is Controlled by the Lysosomal Small GTPase, Arl8b

Dendritic cells (DCs) are specialized APCs with the ability to prime naive T cells. DCs first sample Ags from the environment and then orchestrate their processing and loading onto MHC class II (MHC II) Ag-presenting molecules in lysosomes. Once MHC II molecules have bound a peptide, the MHC II–peptide complex is delivered to the cell surface for presentation to CD4+ T cells. Regulation of Ag uptake via macropinocytosis and phagocytosis has been extensively studied, as well as trafficking in early endocytic vesicles notably regulated by the small GTPase Rab5 and its effectors. However, little is known about the regulators of Ag delivery from early endosomes to lysosomal compartments where the proper pH, proteases, MHC II, invariant chain, and HLA-DM reside, awaiting exogenous Ags for loading. In this article, we report the crucial role of the small GTPase ADP-ribosylation factor-like 8b (Arl8b) in MHC II presentation in DCs. We show for the first time, to our knowledge, that Arl8b localizes to MHC II compartments in DCs and regulates formation of MHC II–peptide complexes. Arl8b-silenced DCs display a defect in MHC II–Ag complex formation and its delivery to the cell surface during infection resulting in a defect in T cell recognition. Our results highlight the role of Arl8b as a trafficking regulator of the late stage of complex formation and MHC II presentation in DCs.

Regulation of major histocompatibility complex class i molecule expression on cancer cells by amyloid precursor-like protein 2

The three members of the amyloid precursor protein family in mammals [amyloid precursor protein, amyloid precursor-like protein 1, and amyloid precursor-like protein 2 (APLP2)] have been implicated in a large array of intracellular processes, which include development, transcription, apoptosis, metabolism, and the cell cycle. A series of studies by our laboratories has demonstrated that APLP2 is highly expressed by many cancer cell lines (with the highest expression in pancreatic cancer cell lines) and that it facilitates major histocompatibility complex (MHC) class I molecule endocytosis. This review focuses on this recently revealed function of APLP2 relevant to tumor immunology: that it acts as a novel regulator of MHC class I molecule surface expression.

Salmonella exploits the host endolysosomal tethering factor HOPS complex to promote its intravacuolar replication

Salmonella enterica serovar typhimurium extensively remodels the host late endocytic compartments to establish its vacuolar niche within the host cells conducive for its replication, also known as the Salmonella-containing vacuole (SCV). By maintaining a prolonged interaction with late endosomes and lysosomes of the host cells in the form of interconnected network of tubules (Salmonella-induced filaments or SIFs), Salmonella gains access to both membrane and fluid-phase cargo from these compartments. This is essential for maintaining SCV membrane integrity and for bacterial intravacuolar nutrition. Here, we have identified the multisubunit lysosomal tethering factor—HOPS (HOmotypic fusion and Protein Sorting) complex as a crucial host factor facilitating delivery of late endosomal and lysosomal content to SCVs, providing membrane for SIF formation, and nutrients for intravacuolar bacterial replication. Accordingly, depletion of HOPS subunits significantly reduced the bacterial load in non-phagocytic and phagocytic cells as well as in a mouse model of Salmonella infection. We found that Salmonella effector SifA in complex with its binding partner; SKIP, interacts with HOPS subunit Vps39 and mediates recruitment of this tethering factor to SCV compartments. The lysosomal small GTPase Arl8b that binds to, and promotes membrane localization of Vps41 (and other HOPS subunits) was also required for HOPS recruitment to SCVs and SIFs. Our findings suggest that Salmonella recruits the host late endosomal and lysosomal membrane fusion machinery to its vacuolar niche for access to host membrane and nutrients, ensuring its intracellular survival and replication.

Lysosome-Mediated Plasma Membrane Repair Is Dependent on the Small GTPase Arl8b and Determines Cell Death Type in Mycobacterium tuberculosis Infection

Mycobacterium tuberculosis is an extremely successful pathogen, and its success is widely attributed to its ability to manipulate the intracellular environment of macrophages. A central phenomenon of tuberculosis pathology enabling immune evasion is the capacity of virulent M. tuberculosis (H37Rv) to induce macrophage necrosis, which facilitates the escape of the mycobacteria from the macrophage and spread of infection. In contrast, avirulent M. tuberculosis (H37Ra) induces macrophage apoptosis, which permits Ag presentation and activation of adaptive immunity. Previously, we found that H37Rv induces plasma membrane microdisruptions, leading to necrosis in the absence of plasma membrane repair. In contrast, H37Ra permits plasma membrane repair, which changes the host cell death modality to apoptosis, suggesting that membrane repair is critical for sequestering the pathogen in apoptotic vesicles. However, mechanisms of plasma membrane repair induced in response to M. tuberculosis infection remain unknown. Plasma membrane repair is known to induce a Ca2+-mediated signaling, which recruits lysosomes to the area of damaged plasma membrane sites for its resealing. In this study, we found that the small GTPase Arl8b is required for plasma membrane repair by controlling the exocytosis of lysosomes in cell lines and in human primary macrophages. Importantly, we found that the Arl8b secretion pathway is crucial to control the type of cell death of the M. tuberculosis–infected macrophages. Indeed, Arl8b-depleted macrophages infected with avirulent H37Ra undergo necrotic instead of apoptotic cell death. These findings suggest that membrane repair mediated by Arl8b may be an important mechanism distinguishing avirulent from virulent M. tuberculosis–induced necrotic cell death.

ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling

ADP-ribosylation factor-like GTPase 11 (ARL11) is a cancer-predisposing gene that has remained functionally uncharacterized to date. In this study, we report that ARL11 is endogenously expressed in mouse and human macrophages and regulates their activation in response to lipopolysaccharide (LPS) stimulation. Accordingly, depletion of ARL11 impaired both LPS-stimulated pro-inflammatory cytokine production by macrophages and their ability to control intracellular replication of Salmonella. LPS-stimulated activation of extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) was substantially compromised in Arl11-silenced macrophages. In contrast, increased expression of ARL11 led to constitutive ERK1/2 phosphorylation, resulting in macrophage exhaustion. Finally, we found that ARL11 forms a complex with phospho-ERK in macrophages within minutes of LPS stimulation. Taken together, our findings establish ARL11 as a novel regulator of ERK signaling in macrophages, required for macrophage activation and immune function.