Rab7l1 plays a role in regulating surface expression of toll like receptors and downstream signaling in activated macrophages.

Rab GTPases are known for controlling intracellular membrane traffic in a GTP-dependent manner. Rab7l1, belonging to family of Rab GTPases, is important for both endosomal sorting and retrograde transport. In our previous study, we identified a novel role of Rab7l1 in phagosome maturation. However, its role in regulating macrophage innate-effector signaling and cytokine response is not clearly understood. In this study, we have demonstrated that upon treatment of Rab7l1-knocked-down (Rab7l1-KD) THP-1 macrophages with lipopolysaccharide (LPS) and Pam3CSK4 has led to higher induction levels of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) as compared to the control cells that received scrambled shRNA. Similar results were observed in Rab7l1-KD RAW 264.7 and Balb/c peritoneal macrophages. The phospho-ERK 1/2 (extracellular signal-regulated kinase 1/2) and phospho-p38 MAPK (mitogen-activated protein kinase) levels, known to be responsible for higher induction of TNF-α and IL-10 respectively, were higher in Rab7l1-KD THP-1 macrophages which also displayed higher nuclear translocation of p50/p65 nuclear factor kappa B (NF-κB) upon stimulation with LPS. Surface expression levels of toll-like receptor 2 (TLR2), TLR4 and CD14 receptors were higher in Rab7l1-KD THP-1 macrophages as compared to the control cells. However, intracellular levels of these receptors were lower in Rab7l1-KD THP-1 macrophages as compared to the control group. Together, our study suggests that Rab7l1 has a role in regulating MAPK signaling and cytokine effector responses in macrophages by regulating the surface expression of membrane receptors.

Phagosome maturation and modulation of macrophage effector function by intracellular pathogens: target for therapeutics.

Macrophages are important cells that regulate various innate functions. Macrophages after engulfment of pathogens proceed for phagosome maturation and finally fuse with lysosomes to kill pathogens. Although pathogen degradation is one of the important functions of phagosomes, various immune-effector functions of macrophages are also dependent on the phagosome maturation process. This review discusses signaling processes regulating phagosome maturation as well as various effector functions of macrophages such as apoptosis, antigen presentation, autophagy and inflammasome that are dependent on the phagosome maturation process. It also discusses strategies adopted by various intracellular pathogens to counteract these functions to evade intracellular destruction mechanisms. These studies may give direction for the development of new therapeutics to control various intracellular infections.

Rabaptin5 acts as a key regulator for Rab7l1-mediated phagosome maturation process.

Phagosome maturation is an important innate defence mechanism of macrophages against pathogen infections. Phagosome-lysosome (P-L) fusion is a highly regulated process. Different RabGTPases are involved in P-L fusion. Rab7l1 is shown to regulate P-L fusion process. In this study, we demonstrate that Rabaptin5 is a guanine nucleotide exchange factor (GEF) for Rab7l1. We reveal that Rabaptin5 interacts with Rab7l1-GTP form and promotes its recruitment to phagosome. In the absence of Rabaptin5, localization of P-L markers like EEA1, Rab7, LAMP1 and LAMP2 was found to be poorer. Thus, our data suggest that Rabaptin5 works upstream to Rab7l1 and triggers Rab7l1 activation for further recruitment of P-L markers and downstream regulation of phagosomal maturation process.

Calcium Signaling Commands Phagosome Maturation Process.

Phagosome-lysosome (P-L) fusion is one of the central immune-effector responses of host. It is known that phagosome maturation process is associated with numerous signaling cascades and among these, important role of calcium (Ca2+) signaling has been realized recently. Ca2+ plays key roles in actin rearrangement, activation of NADPH oxidase and protein kinase C (PKC). Involvement of Ca2+ in these cellular processes directs phagosomal maturation process. Some of the intracellular pathogens have acquired the strategies to modulate Ca2+ associated pathways to block P-L fusion process. In this review we have described the mechanism of Ca2+ signals that influence P-L fusion by controlling ROS, actin and PKC signaling cascades. We have also discussed the strategies implemented by the intracellular pathogens to manipulate Ca2+ signaling to consequently subvert P-L fusion. A detail study of factors associated in manipulating Ca2+ signaling may provide new insights for the development of therapeutic tools for more effective treatment options against infectious diseases.

Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro.

Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.