Project description:Invadopodia are protrusive, F-actin-driven membrane structures that are thought to mediate basement membrane transmigration during development and tumor dissemination. An understanding of the mechanisms regulating invadopodia has been hindered by the difficulty of examining these dynamic structures in native environments. Using an RNAi screen and live-cell imaging of anchor cell (AC) invasion in Caenorhabditis elegans, we have identified UNC-60A (ADF/cofilin) as an essential regulator of invadopodia. UNC-60A localizes to AC invadopodia, and its loss resulted in a dramatic slowing of F-actin dynamics and an inability to breach basement membrane. Optical highlighting indicated that UNC-60A disassembles actin filaments at invadopodia. Surprisingly, loss of unc-60a led to the accumulation of invadopodial membrane and associated components within the endolysosomal compartment. Photobleaching experiments revealed that during normal invasion the invadopodial membrane undergoes rapid recycling through the endolysosome. Together, these results identify the invadopodial membrane as a specialized compartment whose recycling to form dynamic, functional invadopodia is dependent on localized F-actin disassembly by ADF/cofilin.
Project description:Natural resistance-associated macrophage protein 1 (Nramp1) is a divalent metal transporter expressed exclusively in phagocytic cells. We hypothesized that macrophage Nramp1 may participate in the recycling of iron acquired from phagocytosed senescent erythrocytes. To evaluate the role of Nramp1 in vivo, the iron parameters of WT and KO mice were analyzed after acute and chronic induction of hemolytic anemia. We found that untreated KO mice exhibited greater serum transferrin saturation and splenic iron content with higher duodenal ferroportin (Fpn) and divalent metal transporter 1 (DMT1) expression. Furthermore, hepatocyte iron content and hepcidin mRNA levels were dramatically lower in KO mice, indicating that hepcidin levels can be regulated by low-hepatocyte iron stores despite increased transferrin saturation. After acute treatment with the hemolytic agent phenylhydrazine (Phz), KO mice experienced a significant decrease in transferrin saturation and hematocrit, whereas WT mice were relatively unaffected. After a month-long Phz regimen, KO mice retained markedly increased quantities of iron within the liver and spleen and exhibited more pronounced splenomegaly and reticulocytosis than WT mice. After injection of (59)Fe-labeled heat-damaged reticulocytes, KO animals accumulated erythrophagocytosed (59)Fe within their liver and spleen, whereas WT animals efficiently recycled phagocytosed (59)Fe to the marrow and erythrocytes. These data imply that without Nramp1, iron accumulates within the liver and spleen during erythrophagocytosis and hemolytic anemia, supporting our hypothesis that Nramp1 promotes efficient hemoglobin iron recycling in macrophages. Our observations suggest that mutations in Nramp1 could result in a novel form of human hereditary iron overload.
Project description:β-Lactam antibiotics exploit the essentiality of the bacterial cell envelope by perturbing the peptidoglycan layer, typically resulting in rapid lysis and death. Many Gram-negative bacteria do not lyse but instead exhibit "tolerance," the ability to sustain viability in the presence of bactericidal antibiotics for extended periods. Antibiotic tolerance has been implicated in treatment failure and is a stepping-stone in the acquisition of true resistance, and the molecular factors that promote intrinsic tolerance are not well understood. Acinetobacter baumannii is a critical-threat nosocomial pathogen notorious for its ability to rapidly develop multidrug resistance. Carbapenem β-lactam antibiotics (i.e., meropenem) are first-line prescriptions to treat A. baumannii infections, but treatment failure is increasingly prevalent. Meropenem tolerance in Gram-negative pathogens is characterized by morphologically distinct populations of spheroplasts, but the impact of spheroplast formation is not fully understood. Here, we show that susceptible A. baumannii clinical isolates demonstrate tolerance to high-level meropenem treatment, form spheroplasts upon exposure to the antibiotic, and revert to normal growth after antibiotic removal. Using transcriptomics and genetic screens, we show that several genes associated with outer membrane integrity maintenance and efflux promote tolerance, likely by limiting entry into the periplasm. Genes associated with peptidoglycan homeostasis in the periplasm and cytoplasm also answered our screen, and their disruption compromised cell envelope barrier function. Finally, we defined the enzymatic activity of the tolerance determinants penicillin-binding protein 7 (PBP7) and ElsL (a cytoplasmic ld-carboxypeptidase). These data show that outer membrane integrity and peptidoglycan recycling are tightly linked in their contribution to A. baumannii meropenem tolerance. IMPORTANCE Carbapenem treatment failure associated with "superbug" infections has rapidly increased in prevalence, highlighting the urgent need to develop new therapeutic strategies. Antibiotic tolerance can directly lead to treatment failure but has also been shown to promote the acquisition of true resistance within a population. While some studies have addressed mechanisms that promote tolerance, factors that underlie Gram-negative bacterial survival during carbapenem treatment are not well understood. Here, we characterized the role of peptidoglycan recycling in outer membrane integrity maintenance and meropenem tolerance in A. baumannii. These studies suggest that the pathogen limits antibiotic concentrations in the periplasm and highlight physiological processes that could be targeted to improve antimicrobial treatment.
Project description:Cell migration is dependent on a series of integrated cellular events including the membrane recycling of the extracellular matrix receptor integrins. In this paper, we investigate the role of autophagy in regulating cell migration. In a wound-healing assay, we observed that autophagy was reduced in cells at the leading edge than in cells located rearward. These differences in autophagy were correlated with the robustness of MTOR activity. The spatial difference in the accumulation of autophagic structures was not detected in rapamycin-treated cells, which had less migration capacity than untreated cells. In contrast, the knockdown of the autophagic protein ATG7 stimulated cell migration of HeLa cells. Accordingly, atg3(-/-) and atg5(-/-) MEFs have greater cell migration properties than their wild-type counterparts. Stimulation of autophagy increased the co-localization of ?1 integrin-containing vesicles with LC3-stained autophagic vacuoles. Moreover, inhibition of autophagy slowed down the lysosomal degradation of internalized ?1 integrins and promoted its membrane recycling. From these findings, we conclude that autophagy regulates cell migration, a central mechanism in cell development, angiogenesis, and tumor progression, by mitigating the cell surface expression of ?1 integrins.
Project description:Heme-iron recycling from senescent red blood cells (erythrophagocytosis) accounts for the majority of total body iron in humans. Studies in cultured cells have ascribed a role for HRG1/SLC48A1 in heme-iron transport but the in vivo function of this heme transporter is unclear. Here we present genetic evidence in a zebrafish model that Hrg1 is essential for macrophage-mediated heme-iron recycling during erythrophagocytosis in the kidney. Furthermore, we show that zebrafish Hrg1a and its paralog Hrg1b are functional heme transporters, and genetic ablation of both transporters in double knockout (DKO) animals shows lower iron accumulation concomitant with higher amounts of heme sequestered in kidney macrophages. RNA-seq analyses of DKO kidney revealed large-scale perturbation in genes related to heme, iron metabolism and immune functions. Taken together, our results establish the kidney as the major organ for erythrophagocytosis and identify Hrg1 as an important regulator of heme-iron recycling by macrophages in the adult zebrafish.
Project description:Non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer, and the identification of the apoptotic process of NSCLC is vital for its treatment. Usually, both the expression level and the cell surface level of TNFRSF10B (TNF Receptor superfamily member 10B) will increase after treatment with some chemotherapeutic agents, which plays a critical role in the apoptosis induction. However, the exact molecular mechanism underlying TNFRSF10B regulation remains largely elusive. Here, we found that TNFRSF10B, along with a vesicular trafficking regulator protein, YIPF2, were upregulated after treatment with pemetrexed (PEM) in NSCLC cells. Besides, YIPF2 increased the surface level of TNFRF10B, while YIPF2 knockdown inhibited the upregulation of TNFRSF10B and its recycling to plasma membrane. In addition, RAB8 decreased the cell surface TNFRSF10B by promoting its removing from plasma membrane to cytoplasm. Furthermore, we found that YIPF2, RAB8 and TNFRSF10B proteins interacted physically with each other. YIPF2 could further inhibit the physical interaction between TNFRSF10B and RAB8, thereby suppressing the removing of TNFRSF10B from plasma membrane to cytoplasm mediated by RAB8 and maintaining its high level on cell surface. Finally, using bioinformatics database, the YIPF2-TNFRSF10B axis was confirmed to be associated with the malignant progression of lung cancer. Taken together, we show that YIPF2 promotes chemotherapeutic agent-mediated apoptosis via enhancing TNFRSF10B recycling to plasma membrane in NSCLC cells. These findings may be beneficial for the development of potential prognostic markers of NSCLC and may provide effective treatment strategy.
Project description:RNA-seq datasets on adult zebrafish kidneys and spleens, non-PHZ (phenylhydrazine) and PHZ treated, to compare the gene expression on Tü wild type and hrg1 knockout zebrafish.
Project description:The lysosome (or vacuole in yeast) is the central organelle responsible for cellular degradation and nutrient storage. Lysosomes receive cargo from the secretory, endocytic, and autophagy pathways. Many of these proteins and lipids are delivered to the lysosome membrane, and some are degraded in the lysosome lumen, whereas others appear to be recycled through unknown pathways. In this study, we identify the transmembrane autophagy protein Atg27 as a physiological cargo recycled from the vacuole. We reveal that Atg27 is delivered to the vacuole membrane and then recycled using a two-step recycling process. First, Atg27 is recycled from the vacuole to the endosome via the Snx4 complex and then from the endosome to the Golgi via the retromer complex. During the process of vacuole-to-endosome retrograde trafficking, Snx4 complexes assemble on the vacuolar surface and recognize specific residues in the cytoplasmic tail of Atg27. This novel pathway maintains the normal composition and function of the vacuole membrane.
Project description:The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune, and antibiotic insults. There is considerable evidence of mycomembrane plasticity during infection and in response to host-mimicking stresses. Since mycobacteria are resource and energy limited under these conditions, it is likely that remodeling has distinct requirements from those of the well-characterized biosynthetic program that operates during unrestricted growth. Unexpectedly, we found that mycomembrane remodeling in nutrient-starved, nonreplicating mycobacteria includes synthesis in addition to turnover. Mycomembrane synthesis under these conditions occurs along the cell periphery, in contrast to the polar assembly of actively growing cells, and both liberates and relies on the nonmammalian disaccharide trehalose. In the absence of trehalose recycling, de novo trehalose synthesis fuels mycomembrane remodeling. However, mycobacteria experience ATP depletion, enhanced respiration, and redox stress, hallmarks of futile cycling and the collateral dysfunction elicited by some bactericidal antibiotics. Inefficient energy metabolism compromises the survival of trehalose recycling mutants in macrophages. Our data suggest that trehalose recycling alleviates the energetic burden of mycomembrane remodeling under stress. Cell envelope recycling pathways are emerging targets for sensitizing resource-limited bacterial pathogens to host and antibiotic pressure.IMPORTANCE The glucose-based disaccharide trehalose is a stress protectant and carbon source in many nonmammalian cells. Mycobacteria are relatively unique in that they use trehalose for an additional, extracytoplasmic purpose: to build their outer "myco" membrane. In these organisms, trehalose connects mycomembrane biosynthesis and turnover to central carbon metabolism. Key to this connection is the retrograde transporter LpqY-SugABC. Unexpectedly, we found that nongrowing mycobacteria synthesize mycomembrane under carbon limitation but do not require LpqY-SugABC. In the absence of trehalose recycling, compensatory anabolism allows mycomembrane biosynthesis to continue. However, this workaround comes at a cost, namely, ATP consumption, increased respiration, and oxidative stress. Strikingly, these phenotypes resemble those elicited by futile cycles and some bactericidal antibiotics. We demonstrate that inefficient energy metabolism attenuates trehalose recycling mutant Mycobacterium tuberculosis in macrophages. Energy-expensive macromolecule biosynthesis triggered in the absence of recycling may be a new paradigm for boosting host activity against bacterial pathogens.
Project description:Cargo sorting and membrane carrier initiation in recycling endosomes require appropriately coordinated actin dynamics. However, the mechanism underlying the regulation of actin organization during recycling transport remains elusive. Here we report that the loss of PTRN-1/CAMSAP stalled actin exchange and diminished the cytosolic actin structures. Furthermore, we found that PTRN-1 is required for the recycling of clathrin-independent cargo hTAC-GFP The N-terminal calponin homology (CH) domain and central coiled-coils (CC) region of PTRN-1 can synergistically sustain the flow of hTAC-GFP We identified CYK-1/formin as a binding partner of PTRN-1. The N-terminal GTPase-binding domain (GBD) of CYK-1 serves as the binding interface for the PTRN-1 CH domain. The presence of the PTRN-1 CH domain promoted CYK-1-mediated actin polymerization, which suggests that the PTRN-1-CH:CYK-1-GBD interaction efficiently relieves autoinhibitory interactions within CYK-1. As expected, the overexpression of the CYK-1 formin homology domain 2 (FH2) substantially restored actin structures and partially suppressed the hTAC-GFP overaccumulation phenotype in ptrn-1 mutants. We conclude that the PTRN-1 CH domain is required to stimulate CYK-1 to facilitate actin dynamics during endocytic recycling.