ABSTRACT
A multifunctionalized graphene oxide (GO)-based carrier with conjugation of aminated-polyethylene glycol (PEG-diamine), octaarginine (R8), and folic acid (FA), which also contains chloroquine (CQ), a lysosomotropic agent, is introduced. The cellular uptake mechanisms and intracellular targeting of FA-functionalized nanocarriers are examined. The localized releases of CQ and siRNA intracellular delivery are evaluated. Microencapsulation of the nanocarrier complexed with genes in layer-by-layer coating of alginate microbeads is also investigated. The covalently coconjugated FA with PEG and R8 provides a stable formulation with increased cellular uptake compared to FA-free carrier. The CQ-equipped nanocarrier shows a 95% release of CQ at lysosomal pH. The localized release of the drug inside the lysosomes is verified which accelerates the cargo discharge into cytoplasm.
Subject(s)
Chloroquine , Graphite , Chloroquine/pharmacology , Drug Carriers , Folic Acid , Polyethylene Glycols , RNA, Small Interfering/geneticsABSTRACT
Historically, studies of intracellular membrane trafficking have focused on the secretory and endocytic pathways and their major organelles. However, these pathways are also directly implicated in the biogenesis and function of other important intracellular organelles, the best studied of which are peroxisomes and lipid droplets. There is a large recent body of work on these organelles, which have resulted in the introduction of new paradigms regarding the roles of membrane trafficking organelles. In this review, we discuss the roles of membrane trafficking in the life cycle of lipid droplets. This includes the complementary roles of lipid phase separation and proteins in the biogenesis of lipid droplets from endoplasmic reticulum (ER) membranes, and the attachment of mature lipid droplets to membranes by lipidic bridges and by more conventional protein tethers. We also discuss the catabolism of neutral lipids, which in part results from the interaction of lipid droplets with cytosolic molecules, but with important roles for both macroautophagy and microautophagy. Finally, we address their eventual demise, which involves interactions with the autophagocytotic machinery. We pay particular attention to the roles of small GTPases, particularly Rab18, in these processes.
Subject(s)
Autophagy , Endoplasmic Reticulum/metabolism , Intracellular Membranes/metabolism , Lipid Droplets/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Biological Transport , HumansABSTRACT
The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain-containing guanine nucleotide exchange factor GBF1 (Golgi brefeldin A resistant guanine nucleotide exchange factor 1). This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have atomic-level knowledge of Arf1 activation by Sec7 domain-containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used fluorescence recovery after photobleaching data and kinetic Monte Carlo simulation to assess the behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest that Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting that GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1 and suggests that GBF1/E794K is stabilized on the membrane independently of complex formation.
Subject(s)
ADP-Ribosylation Factor 1/metabolism , COP-Coated Vesicles/metabolism , Guanine Nucleotide Exchange Factors/metabolism , ADP-Ribosylation Factor 1/physiology , ADP-Ribosylation Factors/metabolism , COP-Coated Vesicles/physiology , Coat Protein Complex I/metabolism , Endocytosis , Endoplasmic Reticulum/metabolism , Fluorescence Recovery After Photobleaching/methods , Golgi Apparatus/metabolism , Guanine Nucleotide Exchange Factors/physiology , HeLa Cells , Humans , Kinetics , Monomeric GTP-Binding Proteins/metabolism , Monte Carlo Method , Protein Binding , Protein TransportABSTRACT
The biological identity of nanoparticles (NPs) is established by their interactions with a wide range of biomolecules around their surfaces after exposure to biological media. Understanding the true nature of the biomolecular corona (BC) in its native state is, therefore, essential for its safe and efficient application in clinical settings. The fundamental challenge is to visualize the biomolecules within the corona and their relationship/association to the surface of the NPs. Using a synergistic application of cryo-electron microscopy, cryo-electron tomography, and three-dimensional reconstruction, we revealed the unique morphological details of the biomolecules and their distribution/association with the surface of polystyrene NPs at a nanoscale resolution. The analysis of the BC at a single NP level and its variability among NPs in the same sample, and the discovery of the presence of nonspecific biomolecules in plasma residues, enable more precise characterization of NPs, improving predictions of their safety and efficacies.
Subject(s)
Cryoelectron Microscopy/methods , Electron Microscope Tomography/methods , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Plasma/chemistry , Polystyrenes/chemistry , Computer Simulation , Humans , Imaging, Three-Dimensional/methods , Microscopy, Electron, Transmission/methods , Protein Corona/chemistry , Reproducibility of ResultsABSTRACT
Here, we report on an electrochemical biosensor based on core-shell structure of gold nano/micro-islands (NMIs) and electropolymerized imprinted ortho-phenylenediamine (o-PD) for detection of heart-fatty acid binding protein (H-FABP). The shape and distribution of NMIs (the core) were tuned by controlled electrodeposition of gold on a thin layer of electrochemically reduced graphene oxide (ERGO). NMIs feature a large active surface area to achieve a low detection limit (2.29 fg mL-1, a sensitivity of 1.34 × 1013 µA mM-1) and a wide linear range of detection (1 fg mL-1 to 100 ng mL-1) in PBS. Facile template H-FABP removal from the layer (the shell) in less than 1 min, high specificity against interference from myoglobin and troponin T, great stability at ambient temperature, and rapidity in detection of H-FABP (approximately 30 s) are other advantages of this biomimetic biosensor. The electrochemical measurements in human serum, human plasma, and bovine serum showed acceptable recovery (between 91.1 ± 1.7 and 112.9 ± 2.1%) in comparison with the ELISA method. Moreover, the performance of the biosensor in clinical serum showed lower detection time and limit of detection against lateral flow assay (LFA) rapid test kits, as a reference method. Ultimately, the proposed biosensor based on the core-shell structure of gold NMIs and MIP opens interesting avenues in the detection of proteins with low cost, high sensitivity and significantstability for clinical applications.
Subject(s)
Biosensing Techniques , Molecular Imprinting , Animals , Cattle , Gold , Humans , Islands , Molecularly Imprinted PolymersABSTRACT
Arf proteins are small Ras-family GTPases which recruit clathrin and COPI coats to Golgi membranes and regulate components of the membrane trafficking machinery. It is believed membrane association and activity of Arfs is coupled to GTP binding, with GTP hydrolysis required for vesicle uncoating. In humans, four Arf proteins (Arf1, Arf3, Arf4 and Arf5) are Golgi-associated. Conflicting reports have suggested that HA-GFP-tagged Class II ARFs (Arf4 and Arf5) are recruited to membrane independently of the brefeldin A sensitive exchange factor GBF1, suggesting regulation fundamentally different from the Class I Arfs (Arf1, Arf3), or alternately that the GTPase cycle of GFP-tagged Class II Arfs is similar to other Arfs. We show that these results depend on the fluorescent tag, with Arf4-HA-GFP tag resistant to brefeldin, but Arf4-GFP acting similarly to Arf1-GFP in brefeldin-sensitivity and photobleach assays. Arf4-HA-GFP could be partially reverted to the behavior of Arf4-GFP by mutation of two aspartic acids in the HA tag to alanine. Our results, which indicate a high sensitivity of Arf4 to tagging, can explain the discrepancies between previous studies. We discuss the implications of this study for future work with tagged Arfs.
Subject(s)
ADP-Ribosylation Factors/metabolism , Golgi Apparatus/metabolism , Guanine Nucleotide Exchange Factors/metabolism , ADP-Ribosylation Factor 1/metabolism , Brefeldin A/metabolism , HeLa Cells , HumansABSTRACT
Alzheimer's disease (AD) is a multifactorial neurodegenerative disease which leads to progressive dysfunction of cognition, memory and learning in elderly people. Common therapeutic agents are not only inadequate to suppress the progression of AD pathogenesis but also produce deleterious side effects; hence, development of alternative therapies is required to specifically suppress complications of AD. The current review provides a commentary on conventional as well as novel therapeutic approaches with an emphasis on stem cell and nano-based therapies for improvement and management of AD pathogenesis. According to our overview of the current literature, AD is a multi-factorial disorder with various pathogenic trajectories; hence, a multifunctional strategy to create effective neuroprotective agents is required to treat this disorder.
Subject(s)
Alzheimer Disease/pathology , Cell- and Tissue-Based Therapy/methods , Neurodegenerative Diseases/pathology , Alzheimer Disease/therapy , Animals , Humans , Neurodegenerative Diseases/therapyABSTRACT
Joint replacement and bone reconstructive surgeries are on the rise globally. Current strategies for implants and bone regeneration are associated with poor integration and healing resulting in repeated surgeries. A multidisciplinary approach involving basic biological sciences, tissue engineering, regenerative medicine and clinical research is required to overcome this problem. Considering the nanostructured nature of bone, expertise and resources available through recent advancements in nanobiotechnology enable researchers to design and fabricate devices and drug delivery systems at the nanoscale to be more compatible with the bone tissue environment. The focus of this review is to present the recent progress made in the rationale and design of nanomaterials for tissue engineering and drug delivery relevant to bone regeneration.
Subject(s)
Bone Regeneration/physiology , Nanostructures/chemistry , Animals , Biocompatible Materials/chemistry , Bone Regeneration/genetics , Bone and Bones/cytology , Humans , Nanotechnology/methods , Tissue Engineering/methodsABSTRACT
Rab18 is a small GTPase associated with lipid droplets and other membranes. While it likely has multiple functions on lipid droplets, one proposed function is regulation of lipolysis. Previous work has concentrated on regulation of autophagy; however, in this study, we provide evidence that Rab18 plays a role upstream of the cytosolic lipolytic enzyme adipose triglyceride lipase (ATGL) and that recruitment of ATGL by Rab18 is mediated by elements of the Arf/GBF1 machinery. We find that Arf4-GFP is accumulated on the subset of lipid droplets associated with Rab18, and that this association is lost within 5â¯min upon treatment with 5⯵g/ml of the drug brefeldin A, which targets GBF1 and other Sec7-domain containing Arf exchange factors. ATGL-GFP is also recruited to lipid droplets, but is lost more slowly after treatment with 5⯵g/ml brefeldin A, with significant loss from lipid droplets after 1â¯h treatment, and almost complete loss from lipid droplets 4â¯h after brefeldin A treatment. Upon overexpression of the dominant negative GDP-locked cerulean-Rab18-S22â¯N, GFP-ATGL and Arf4 are lost from the surface of lipid droplets similarly to BFA treatment. This study establishes, for the first time, an essential role for Rab18 in recruiting ATGL to lipid droplets.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Lipase/metabolism , Lipolysis/physiology , rab GTP-Binding Proteins/metabolism , ADP-Ribosylation Factors/metabolism , Brefeldin A/pharmacology , Green Fluorescent Proteins/metabolism , Guanosine Triphosphate/metabolism , HeLa Cells , Humans , Lipid Droplets/drug effects , Lipid Droplets/metabolism , Models, Biological , Protein Binding/drug effects , Recombinant Fusion Proteins/metabolismABSTRACT
Saliva aids in digestion, lubrication, and protection of the oral cavity against dental caries and oropharyngeal infections. Reduced salivary secretion, below an adequate level to sustain normal oral functions, is unfortunately experienced by head and neck cancer patients treated with radiotherapy and by patients with Sjögren's syndrome. No disease-modifying therapies exist to date to address salivary gland hypofunction (xerostomia, dry mouth) because pharmacotherapies are limited by the need for residual secretory acinar cells, which are lost at the time of diagnosis, whereas novel platforms such as cell therapies are yet immature for clinical applications. Autologous salivary gland primary cells have clinical utility as personalized cell therapies, if they could be cultured to a therapeutically useful mass while maintaining their in vivo phenotype. Here, we devised a serum-free scalable suspension culture system that grows partially digested human salivary tissue filtrates composing of acinar and ductal cells attached to their native extracellular matrix components while retaining their 3D in vivo spatial organization; we have coined these salivary spheroids as salivary functional units (SFU). The proposed SFU culture system was sub-optimal, but we have found that the cells could still survive and grow into larger salivary spheroids through cell proliferation and aggregation for 5 to 10 days within the oxygen diffusion rates in vitro. In summary, by using a less disruptive cell isolation procedure as the starting point for primary cell culture of human salivary epithelial cells, we demonstrated that aggregates of cells remained proliferative and continued to express acinar and ductal cell-specific markers.
Subject(s)
Cell Culture Techniques/methods , Cell Differentiation , Epithelial Cells/cytology , Models, Biological , Salivary Glands/cytology , Suspensions , Acinar Cells/cytology , Aquaporin 5/metabolism , Basement Membrane/metabolism , Cell Aggregation , Cell Proliferation , Cell Survival , Culture Media, Serum-Free , Extracellular Matrix/metabolism , Gene Expression Regulation , Humans , Phenotype , Salivary Glands/ultrastructure , Spheroids, Cellular/cytology , Spheroids, Cellular/metabolismABSTRACT
Rab18 is one of the small number of conserved Rab proteins which have been traced to the last eukaryotic common ancestor. It is found in organisms ranging from humans to trypanosomes, and localizes to multiple organelles, including most notably endoplasmic reticulum and lipid droplets. In humans, absence of Rab18 leads to a severe illness known as Warburg-Micro syndrome. Despite this evidence that Rab18 is essential, its role in cells remains mysterious. However, recent studies identifying effectors and interactors of Rab18, are now shedding light on its mechanism of action, suggesting functions related to organelle tethering and to autophagy. In this review, we examine the variety of roles proposed for Rab18 with a focus on new evidence giving insights into the molecular mechanisms it utilizes. Based on this summary of our current understanding, we identify priority areas for further research.
Subject(s)
Endoplasmic Reticulum/metabolism , Lipid Droplets/metabolism , Lipid Metabolism , rab GTP-Binding Proteins/metabolism , Abnormalities, Multiple/genetics , Abnormalities, Multiple/metabolism , Animals , Autophagy/genetics , Cataract/congenital , Cataract/genetics , Cataract/metabolism , Cornea/abnormalities , Cornea/metabolism , Humans , Hypogonadism/genetics , Hypogonadism/metabolism , Intellectual Disability/genetics , Intellectual Disability/metabolism , Microcephaly/genetics , Microcephaly/metabolism , Models, Biological , Mutation , Optic Atrophy/genetics , Optic Atrophy/metabolism , rab GTP-Binding Proteins/geneticsABSTRACT
Senescent cells undergo structural and functional changes that affect essentially every aspect of cell physiology. To date, the impact of senescence on the cytoskeleton is poorly understood. This study evaluated the cytoskeleton in two independent cellular models of kidney epithelium senescence. Our work identified multiple senescence-related alterations that impact microtubules and filamentous actin during interphase. Both filamentous systems reorganized profoundly when cells became senescent. As such, microtubule stability increased during senescence, making these filaments more resistant to disassembly in the cold or by nocodazole. Microtubule stabilization was accompanied by enhanced α-tubulin acetylation on lysine 40 and the depletion of HDAC6, the major deacetylase for α-tubulin lysine 40. Rho-associated kinase Rock1 is an upstream regulator that modulates key properties of the cytoplasmic cytoskeleton. Our research shows that Rock1 concentrations were reduced significantly in senescent cells, and we revealed a mechanistic link between microtubule stabilization and Rock1 depletion. Thus, Rock1 overexpression partially restored the cold sensitivity of microtubules in cells undergoing senescence. Additional components relevant to microtubules were affected by senescence. Specifically, we uncovered the senescence-related loss of the microtubule nucleating protein γ-tubulin and aberrant formation of γ-tubulin foci. Concomitant with the alterations of microtubule and actin filaments, senescent cells displayed functional changes. In particular, cell migration was impaired significantly in senescent cells. Taken together, our study identified new senescence-associated deficiencies of the microtubule and actin cytoskeleton, provided insights into the underlying molecular mechanisms and demonstrated functional consequences that are important to the physiology and function of renal epithelial cells.
Subject(s)
Actin Cytoskeleton/metabolism , Cellular Senescence , Microtubules/metabolism , Tubulin/metabolism , Actin Cytoskeleton/drug effects , Animals , Cell Line , Epithelial Cells/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Kidney Tubules, Proximal/cytology , Microscopy, Confocal , Microtubules/drug effects , Nocodazole/pharmacology , Swine , Tubulin Modulators/pharmacology , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolismABSTRACT
Determination of lipid droplet (LD) volume has depended on direct measurement of the diameter of individual LDs, which is not possible when LDs are small or closely apposed. To overcome this problem, we describe a new method in which a volume-fluorescence relationship is determined from automated analysis of calibration samples containing well-resolved LDs. This relationship is then used to estimate total cellular droplet volume in experimental samples, where the LDs need not be individually resolved, or to determine the volumes of individual LDs. We describe quantitatively the effects of various factors, including image noise, LD crowding, and variation in LD composition on the accuracy of this method. We then demonstrate this method by utilizing it to address a scientifically interesting question, to determine the density of green fluorescent protein (GFP)-tagged Perilipin-Adipocyte-Tail (PAT) proteins on the LD surface. We find that PAT proteins cover only a minority of the LD surface, consistent with models in which they primarily serve as scaffolds for binding of regulatory proteins and enzymes, but inconsistent with models in which their major function is to sterically block access to the droplet surface.
Subject(s)
Image Processing, Computer-Assisted/methods , Lipid Droplets/ultrastructure , Microscopy, Confocal/methods , Perilipins/analysis , Cell Culture Techniques/methods , Fluorescence Resonance Energy Transfer/methods , Green Fluorescent Proteins/analysis , HeLa Cells , Hep G2 Cells , Humans , Lipid Droplets/chemistry , SoftwareABSTRACT
Retinoid X receptor (RXR) occupies a central position within the nuclear receptor superfamily, serving as an obligatory partner to numerous other nuclear receptors, including vitamin D receptor (VDR). In the current study, we examined whether phosphorylation of RXRα at serine 260 affects VDR/RXR and VDR interacting protein (DRIP) 205 coactivator recruitment, interactions, and binding of the VDR/human RXRα (hRXRα)/DRIP205 complex to chromatin. Serine 260 is a critical amino acid on the hRXRα that is located in close spatial proximity to regions of coactivator and corepressor interactions. Using fluorescence resonance energy transfer and immunofluorescence studies, we showed that the physical interaction between hRXRα and DRIP205 coactivator was impaired in human keratinocytes with the ras oncogene (HPK1Aras) or transfected with the wild-type hRXRα. Furthermore, the nuclear colocalization of VDR/DRIP205, hRXRα/DRIP205, and VDR/hRXRα/DRIP205 complex binding to chromatin is impaired in the HPK1Aras cells when compared with the normal human keratinocytes (HPK1A cells). However, transfection with the nonphosphorylatable hRXRα (S260A) mutant or treatment with the mitogen-activated protein kinase (MAPK) inhibitor UO126 rescued their nuclear localization, interaction, and binding of the complex to chromatin in the HPK1Aras cells. In summary, we have demonstrated, using highly specific intracellular tagging methods in live and fixed cells, important alterations of the vitamin D signaling system in cancer cells in which the ras-raf-MAPK system is activated, suggesting that specific inhibition of this commonly activated pathway could be targeted therapeutically to enhance vitamin D efficacy.
Subject(s)
Keratinocytes/metabolism , Mediator Complex/metabolism , Neoplasms/drug therapy , Receptors, Calcitriol/metabolism , Retinoid X Receptors/metabolism , Vitamin D/therapeutic use , Cell Nucleus/chemistry , Cell Transformation, Neoplastic , Chromatin/metabolism , DNA/metabolism , Genes, ras , Humans , Keratinocytes/ultrastructure , Mediator Complex/analysis , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Phosphorylation , Receptors, Calcitriol/analysis , Retinoid X Receptors/analysis , Serine/metabolism , Signal TransductionABSTRACT
SUN proteins participate in diverse cellular activities, many of which are connected to the nuclear envelope. Recently, the family member SUN1 has been linked to novel biological activities. These include the regulation of nucleoli, intranuclear compartments that assemble ribosomal subunits. We show that SUN1 associates with nucleoli in several mammalian epithelial cell lines. This nucleolar localization is not shared by all cell types, as SUN1 concentrates at the nuclear envelope in ganglionic neurons and non-neuronal satellite cells. Database analyses and Western blotting emphasize the complexity of SUN1 protein profiles in different mammalian cells. We constructed a STRING network which identifies SUN1-related proteins as part of a larger network that includes several nucleolar proteins. Taken together, the current data highlight the diversity of SUN1 proteins and emphasize the possible links between SUN1 and nucleoli.
ABSTRACT
The plasma membrane of mammalian cells undergoes constitutive endocytosis, endocytic sorting, and recycling, which delivers nutrients to the lysosomes. The receptors, along with membrane lipids, are normally returned to the plasma membrane to sustain this action. It is not known, however, whether this process is influenced by metabolic conditions. Here we report that endocytic recycling requires active mechanistic target of rapamycin (aka mammalian target of rapamycin) (mTORC1), a master metabolic sensor. Upon mTORC1 inactivation, either by starvation or by inhibitor, recycling receptors and plasma membrane lipids, such as transferrin receptors and sphingomyelin, are delivered to the lysosomes. This lysosomal targeting is independent of canonical autophagy: both WT and Atg5-/- mouse embryonic fibroblasts responded similarly. Furthermore, we identify hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), an endosomal sorting complexes required for transport (ESCORT-0) component, as a downstream target of mTORC1. Hrs requires mTORC1 activity to maintain its protein expression level. Silencing Hrs without decreasing mTORC1 activity is sufficient to target transferrin and sphingomyelin to the lysosomes. It is thus evident that the canonical recycling pathway is under the regulation of mTORC1 and likely most predominant in proliferating cells where mTORC1 is highly active.
Subject(s)
Embryo, Mammalian/metabolism , Endocytosis/physiology , Fibroblasts/metabolism , Lysosomes/metabolism , Multiprotein Complexes/metabolism , Sphingomyelins/metabolism , TOR Serine-Threonine Kinases/metabolism , Transferrin/metabolism , Animals , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 5/metabolism , Biological Transport, Active/physiology , Cell Proliferation/physiology , Cells, Cultured , Embryo, Mammalian/cytology , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Fibroblasts/cytology , Lysosomes/genetics , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Knockout , Multiprotein Complexes/genetics , Sphingomyelins/genetics , TOR Serine-Threonine Kinases/genetics , Transferrin/geneticsABSTRACT
Human retinoid X receptor α (hRXRα) plays a critical role in DNA binding and transcriptional activity through heterodimeric association with several members of the nuclear receptor superfamily, including the human vitamin D receptor (hVDR). We previously showed that hRXRα phosphorylation at serine 260 through the Ras-Raf-MAPK ERK1/2 activation is responsible for resistance to the growth inhibitory effects of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the biologically active metabolite of vitamin D3 To further investigate the mechanism of this resistance, we studied intranuclear dynamics of hVDR and hRXRα-tagged constructs in living cells together with endogenous and tagged protein in fixed cells. We find that hVDR-, hRXRα-, and hVDR-hRXRα complex accumulate in the nucleus in 1α,25(OH)2D3-treated HPK1A cells but to a lesser extent in HPK1ARas-treated cells. Also, by using fluorescence resonance energy transfer (FRET), we demonstrate increased interaction of the hVDR-hRXRα complex in 1α,25(OH)2D3-treated HPK1A but not HPK1ARas cells. In HPK1ARas cells, 1α,25(OH)2D3-induced nuclear localization and interaction of hRXRα are restored when cells are treated with the MEK1/2 inhibitor UO126 or following transfection of the non-phosphorylatable hRXRα Ala-260 mutant. Finally, we demonstrate using fluorescence loss in photobleaching and quantitative co-localization with chromatin that RXR immobilization and co-localization with chromatin are significantly increased in 1α,25(OH)2D3-treated HPK1ARas cells transfected with the non-phosphorylatable hRXRα Ala-260 mutant. This suggests that hRXRα phosphorylation significantly disrupts its nuclear localization, interaction with VDR, intra-nuclear trafficking, and binding to chromatin of the hVDR-hRXR complex.
Subject(s)
Calcitriol/pharmacology , Cell Nucleus/metabolism , Keratinocytes/metabolism , Retinoid X Receptor alpha/metabolism , Active Transport, Cell Nucleus/drug effects , Amino Acid Substitution , Cell Line, Transformed , Cell Nucleus/genetics , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Mutation, Missense , Phosphorylation/drug effects , Phosphorylation/genetics , Retinoid X Receptor alpha/genetics , Serine , ras Proteins/genetics , ras Proteins/metabolismABSTRACT
A number of pathogens for which there are no effective treatments infect the cells via endocytosis. Once in the endosomes, the pathogens complete their life cycle by overriding normal lysosomal functions. Recently, our laboratory identified the lysosomal targeting signal of prosaposin, which is recognized by the sorting receptor "sortilin". Based on this evidence, we tested whether the antimicrobial peptide ß-Defensin linked to the targeting sequence of prosaposin (ßD-PSAP) could be redirected from its secretory pathway to the endolysosomal compartment. To this effect, ßD-PSAP was transfected into COS-7 cells. The sub-cellular distribution of ßD-PSAP was analyzed by confocal microscopy and differential centrifugation. Confocal microscopy demonstrated that ßD-PSAP overlaid with the lysosomal marker LAMP1, indicating that the construct reached endosomes and lysosomes. Differential centrifugation also showed that ßD-PSAP was in the lysosomal fractions. In addition, our binding inhibition assay demonstrated that ßD-PSAP bound specifically to sortilin. Similarly, the delivery of ßD-PSAP was abolished after overexpressing a truncated sortilin. These results indicate that the prosaposin C-terminus and D/C-domain (prosaposin targeting sequence) was an effective "guidance system" to redirect ßD-PSAP to the endolysosomal compartment. In the future, this and other fusion proteins with antimicrobial properties will be assembled to our "biotic vehicle" to target pathogens growing within these compartments.
Subject(s)
Anti-Infective Agents/pharmacology , Drug Delivery Systems/methods , Lysosomes/drug effects , Pharmaceutical Vehicles , beta-Defensins/pharmacology , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Anti-Infective Agents/administration & dosage , COS Cells , Chlorocebus aethiops , Endosomes/metabolism , Humans , Lysosomal-Associated Membrane Protein 1/metabolism , Protein Binding/drug effects , Saposins/metabolism , Subcellular Fractions/metabolism , beta-Defensins/administration & dosageABSTRACT
Lipid droplets are the major organelle for intracellular storage of triglycerides and cholesterol esters. Various methods have been attempted for automated quantitation of fluorescently stained lipid droplets using either thresholding or watershed methods. We find that thresholding methods deal poorly with clusters of lipid droplets, whereas watershed methods require a smoothing step that must be optimized to remove image noise. We describe here a novel three-stage hybrid method for automated segmentation and quantitation of lipid droplets. In this method, objects are initially identified by thresholding. They are then tested for circularity to distinguish single lipid droplets from clusters. Clusters are subjected to a secondary watershed segmentation. We provide a characterization of this method in simulated images. Additionally, we apply this method to images of fixed cells containing stained lipid droplets and GFP-tagged proteins to provide a proof-of-principle that this method can be used for colocalization studies. The circularity measure can additionally prove useful for the identification of inappropriate segmentation in an automated way; for example, of non-cellular material. We will make the programs and source code available to the community under the Gnu Public License. We believe this technique will be of interest to cell biologists for light microscopic studies of lipid droplet biology.
Subject(s)
Image Processing, Computer-Assisted/methods , Lipid Droplets/ultrastructure , Microscopy, Confocal/methods , Single-Cell Analysis/methods , Fluorescent Dyes/analysis , HeLa Cells , Humans , SoftwareABSTRACT
ArfGAPs are known to be involved in cargo sorting in COPI transport. However, the role of ArfGAPs in post-Golgi membrane traffic has not been defined. To determine the function of ArfGAPs in post-Golgi traffic, we used small interfering RNA to examine each of 25 ArfGAPs for effects on cation-independent mannose 6-phosphate receptor (CIMPR) localization. We found that downregulation of ArfGAP3 resulted in the peripheral localization of CIMPR. The effect was specific for ArfGAP3 and dependent on its GAP activity, because the phenotype was rescued by ArfGAP3 but not by ArfGAP1, ArfGAP2, or the GAP domain mutants of ArfGAP3. ArfGAP3 localized to the trans-Golgi network and early endosomes. In cells with reduced expression of ArfGAP3, Cathepsin D maturation was slowed and its secretion was accelerated. Also retrograde transport from the endosomes to the trans-Golgi network of endogenous CIMPR, but not truncated CIMPR lacking the luminal domain, was perturbed in cells with reduced expression of ArfGAP3. Furthermore the exit of epidermal growth factor receptor (EGFR) from the early endosomes and degradation of EGFR after EGF stimulation was slowed in cells with reduced expression of ArfGAP3. ArfGAP3 associates with Golgi-localized, γ-ear-containing, ADP-ribosylation factor binding proteins (GGAs), and ArfGAP3 knockdown reduces membrane association of GGAs. A possible mechanism explaining our results is that ArfGAP3 regulates transport from early endosomes to late endosomes. We suggest a model in which ArfGAP3 regulates Golgi association of GGA clathrin adaptors.