Browsing by Subject "Vacuole"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    The Dually Localized EF-Hand Domain-Containing Protein TgEFP1 Regulates the Lytic Cycle of Toxoplasma gondii
    (MDPI, 2022-05-21) Dave, Noopur; LaFavers, Kaice; Arrizabalaga, Gustavo; Pharmacology and Toxicology, School of Medicine
    The propagation of the obligate intracellular parasite Toxoplasma gondii is tightly regulated by calcium signaling. However, the mechanisms by which calcium homeostasis and fluxes are regulated in this human pathogen are not fully understood. To identify Toxoplasma’s calcium homeostasis network, we have characterized a novel EF-hand domain-containing protein, which we have named TgEFP1. We have determined that TgEFP1 localizes to a previously described compartment known as the plant-like vacuole or the endosomal-like compartment (PLV/ELC), which harbors several proteins related to ionic regulation. Interestingly, partial permeabilization techniques showed that TgEFP1 is also secreted into the parasitophorous vacuole (PV), within which the parasite divides. Ultrastructure expansion microscopy confirmed the unusual dual localization of TgEFP1 at the PLV/ELC and the PV. Furthermore, we determined that the localization of TgEFP1 to the PV, but not to the PLV/ELC, is affected by disruption of Golgi-dependent transport with Brefeldin A. Knockout of TgEFP1 results in faster propagation in tissue culture, hypersensitivity to calcium ionophore-induced egress, and premature natural egress. Thus, our work has revealed an interplay between the PV and the PLV/ELC and a role for TgEFP1 in the regulation of calcium-dependent events.
  • Thumbnail Image
    Item
    Effects of Collagen Gel Stiffness on Cdc42 Activities of Endothelial Colony Forming Cells during Early Vacuole Formation
    (2013-08-14) Kim, Seung Joon; Na, Sungsoo; Xie, Dong; Li, Jiliang
    Recent preclinical reports have provided evidence that endothelial colony forming cells (ECFCs), a subset of endothelial progenitor cells, significantly improve vessel formation, largely due to their robust vasculogenic potential. While it has been known that the Rho family GTPase Cdc42 is involved in this ECFC-driven vessel formation process, the effect of extracellular matrix (ECM) stiffness on its activity during vessel formation is largely unknown. Using a fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor, we examined the spatio-temporal activity of Cdc42 of ECFCs in three-dimensional (3D) collagen matrices with varying stiffness. The result revealed that ECFCs exhibited an increase in Cdc42 activity in a soft (150 Pa) matrix, while they were much less responsive in a rigid (1 kPa) matrix. In both soft and rigid matrices, Cdc42 was highly activated near vacuoles. However, its activity is higher in a soft matrix than that in a rigid matrix. The observed Cdc42 activity was closely associated with vacuole formation. Soft matrices induced higher Cdc42 activity and faster vacuole formation than rigid matrices. However, vacuole area is not dependent on the stiffness of matrices. Time courses of Cdc42 activity and vacuole formation data revealed that Cdc42 activity proceeds vacuole formation. Collectively, these results suggest that matrix stiffness is critical in regulating Cdc42 activity in ECFCs and its activation is an important step in early vacuole formation.
  • Thumbnail Image
    Item
    mTORC1 contributes to ER stress induced cell death
    (2012-12) Babcock, Justin Thomas; Quilliam, Lawrence; Atkinson, Simon; Nakshatri, Harikrishna; Wek, Ronald C.
    Patients with the genetic disorder tuberous sclerosis complex (TSC) suffer from neoplastic growths in multiple organ systems. These growths are the result of inactivating mutations in either the TSC1 or TSC2 tumor suppressor genes, which negatively regulate the activity of mammalian target of rapamycin complex 1(mTORC1). There is currently no cure for this disease; however, my research has found that cells harboring TSC2-inactivating mutations derived from a rat model of TSC are sensitive to apoptosis induced by the clinically approved proteasome inhibitor, bortezomib, in a manner dependent on their high levels of mTORC1 activation. We see that bortezomib induces the unfolded protein response (UPR) in our cell model of TSC, resulting in cell death via apoptosis. The UPR is induced by accumulation of unfolded protein in the endoplasmic reticulum (ER) which activates the three branches of this pathway: Activating transcription factor 6 (ATF6) cleavage, phosphorylation of eukaryotic initiation factor 2α (eIF2α), and the splicing of X-box binding protein1 (XBP1) mRNA. Phosphorylation of eIF2α leads to global inhibition of protein synthesis, preventing more unfolded protein from accumulating in the ER. This phosphorylation also induces the transcription and translation of ATF4 and CCAAT-enhancer binding protein homologous protein (CHOP). Blocking mTORC1 activity in these cells using the mTORC1 inhibitor, rapamycin, prevented the expression of ATF4 and CHOP at both the mRNA and protein level during bortezomib treatment. Rapamycin treatment also reduced apoptosis induced by bortezomib; however, it did not affect bortezomib-induced eIF2α phosphorylation or ATF6 cleavage. These data indicate that rapamycin can repress the induction of UPR-dependent apoptosis by suppressing the transcription of ATF4 and CHOP mRNAs. In addition to these findings, we find that a TSC2-null angiomyolipoma cell line forms vacuoles when treated with the proteasome inhibitor MG-132. We found these vacuoles to be derived from the ER and that rapamycin blocked their formation. Rapamycin also enhanced expansion of the ER during MG-132 stress and restored its degradation by autophagy. Taken together these findings suggest that bortezomib might be used to treat neoplastic growths associated with TSC. However, they also caution against combining specific cell death inducing agents with rapamycin during chemotherapy.
  • Thumbnail Image
    Item
    Recruitment and function of ORP1L on the Coxiella burnetii parasitophorous vacuole
    (2017-12-07) Justis, Anna Victoria; Gilk, Stacey D.; Spinola, Stanley M.; Nelson, David; Arrizabalaga, Gustavo A.; Harrington, Maureen A.
    Coxiella burnetii, the zoonotic agent of human Q fever and chronic endocarditis, is an obligate intracellular bacterial pathogen. The Coxiella intracellular niche, a large, lysosome-like parasitophorous vacuole (PV), is essential for bacterial survival and replication. There is growing evidence that host cell cholesterol trafficking plays a critical role in PV development and maintenance, prompting an examination of the role of cholesterol-binding host protein ORP1L (Oxysterol binding protein-Related Protein 1, Long) during infection. ORP1L is a multi-functional cholesterol-binding protein involved in late endosome/lysosome (LEL) trafficking, formation of membrane contact sites between LEL and the endoplasmic reticulum (ER), and cholesterol transfer from LEL to the ER. ORP1L localizes to the PV at novel membrane contact sites between the ER and the PV membrane. Ectopically expressed ORP1L in Coxiella-infected cells localizes to the PV membrane early during infection, before significant PV expansion and independent of other PV-localized proteins. Further, the N-terminal ORP1L Ankyrin repeats are both necessary and sufficient for PV localization, suggesting that protein-protein interactions, and not protein-lipid interactions, are primarily involved in PV association. Coxiella employs a Type IVB Secretion System (T4BSS) to translocate effector proteins into the host cytoplasm and manipulate various cellular functions. ORP1L is not found on the PV of a Coxiella mutant lacking a functional T4BSS, indicating a secreted bacterial protein is likely responsible for ORP1L recruitment. We identified a Coxiella mutant with a transposon insertion in CBU_0352 that exhibits a 50% decrease in ORP1L recruitment, suggesting that Coxiella CBU_0352 interacts directly or indirectly with ORP1L. Finally, we found that ORP1L depletion using siRNA alters PV dynamics, resulting in smaller yet more fusogenic Coxiella PVs. Together, these data suggest that ORP1L is specifically recruited to the PV, where it plays a novel role in Coxiella PV development and interactions between the PV and the host cell.