Molecular Mobility in Trehalose Loaded Mammalian Cells: Time-Resolved Fluorescence Anisotropy Measurements

2008 ◽  
Author(s):  
Nilay Chakraborty ◽  
Wesley Parker ◽  
Kevin E. Elliott ◽  
Stuart T. Smith ◽  
Patrick J. Moyer ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluid Phase ◽  
Water Soluble ◽  
Great Promise ◽  
Time Resolved ◽  
Intracellular Space ◽  
Membrane Pores ◽  
Fluid Phase Endocytosis ◽  
Membrane Bound ◽  
Cellular Compartments

Many preservation methods have utilized sugars such as trehalose as protectants against injury during cell preservation processing, especially during drying (1–5). As mammalian cells do not synthesize trehalose, research in the mammalian cell desiccation field has focused on the development of strategies to enable trehalose delivery into the intracellular milieu. Numerous techniques have been explored ranging from microinjection (2) to the creation or utilization of membrane pores (1,3). Fluid phase endocytosis has shown great promise as an effective strategy for non-invasively delivering water-soluble materials into the intracellular space (4, 5). In this technique trehalose is transported across the cell membrane in membrane-bound cellular compartments called endosomes. Cells incubated in cell culture medium containing trehalose have been shown to take up considerable amounts of trehalose by this technique (4, 5). How much of this trehalose actually become available for protection of biomolecules during the dehydration process has yet to be determined.

scholarly journals Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

2021 ◽  
Author(s):  
Farnaz Fekri ◽  
Ralph Christian Delos Santos ◽  
Raffi Karshafian ◽  
Costin N. Antonescu
Keyword(s):  
Drug Delivery ◽  
Fluid Phase ◽  
Acid Sphingomyelinase ◽  
Coated Pits ◽  
Drug Permeability ◽  
Membrane Pores ◽  
Signaling Mechanisms ◽  
Sheer Stress ◽  
Cell Plasma ◽  
Fluid Phase Endocytosis

Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

scholarly journals Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Zhou Yu ◽  
Lauren E Surface ◽  
Chong Yon Park ◽  
Max A Horlbeck ◽  
Gregory A Wyant ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mevalonate Pathway ◽  
Fluid Phase ◽  
Molecular Target ◽  
Solute Carrier Family ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Genome Wide ◽  
Fluid Phase Endocytosis ◽  
Solute Carrier

Nitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here, we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.

scholarly journals Fluorescence-Based Cell Viability Screening Assays Using Water-Soluble Oxygen Probes

2003 ◽  
Vol 8 (3) ◽  
pp. 264-272 ◽  
Author(s):  
James Hynes ◽  
Suzanne Floyd ◽  
Aleksi E. Soini ◽  
Rosemary O'Connor ◽  
Dmitri B. Papkovsky
Keyword(s):  
Cell Viability ◽  
Mammalian Cells ◽  
Cost Effective ◽  
Water Soluble ◽  
High Throughput Analysis ◽  
Time Resolved ◽  
Low Concentrations ◽  
Cell Line Cell ◽  
Plate Analysis ◽  
Soluble Oxygen

A simple luminescence-based assay for screening the viability of mammalian cells is described, based on the monitoring of cell respiration by means of a phosphorescent water-soluble oxygen probe that responds to changes in the concentration of dissolved oxygen by changing its emission intensity and lifetime. The probe was added at low concentrations (0.3 μM to 0.5 nM) to each sample containing a culture of cells in the wells of a standard 96-well plate. Analysis of oxygen consumption was initiated by applying a layer of mineral oil on top of each sample followed by monitoring of the phosphorescent signal on a prompt or time-resolved fluorescence plate reader. Rates of oxygen uptake could be determined on the basis of kinetic changes of the phosphorescence (initial slopes) and correlated with cell numbers (105 to 107 cells/mL for FL5.12 lymphoblastic cell line), cell viability, or drug/effector action using appropriate control samples. The assay is cell noninvasive, more simple, robust, and cost-effective than existing microplate-based cell viability assays; is compatible with existing instrumentation; and allows for high-throughput analysis of cell viability. ( Journal of Biomolecular Screening 2003:264-272)

scholarly journals Endosomes and Golgi vesicles in adsorptive and fluid phase endocytosis.

1984 ◽  
Vol 99 (4) ◽  
pp. 1379-1390 ◽  
Author(s):  
N K Gonatas ◽  
A Stieber ◽  
W F Hickey ◽  
S H Herbert ◽  
J O Gonatas
Keyword(s):  
Golgi Apparatus ◽  
Room Temperature ◽  
Fluid Phase ◽  
Intracellular Traffic ◽  
Pheochromocytoma Cells ◽  
Quantitative Studies ◽  
Fluid Phase Endocytosis ◽  
Morphometric Methods ◽  
Membrane Bound ◽  
Adsorptive Endocytosis

We studied with morphometric methods the endocytosis by pheochromocytoma cells of a conjugate of wheat germ agglutinin with ferritin (WGA-Ft) and of horseradish peroxidase (HRP). Quantitative studies indicated that WGA-Ft was cleared slowly from cell surfaces and that it was not recycled to the surface. Cells labeled with WGA-Ft for 15 min at room temperature were washed and incubated in medium containing HRP for 15 or 30 min at 37 degrees C. The greatest proportion of labeled vesicles and tubules contained only WGA-Ft (83.4% at 15 min and 85.3% at 30 min). A very small fraction of labeled vesicles and tubules contained only HRP (0.2% at 15 min and 0.9% at 30 min). Vesicles and tubules at the Golgi apparatus were labeled almost exclusively with WGA-Ft (97% at 15 min and 30 min); the rest had both labels. Most labeled lysosomes contained both labels (80.1% at 15 min and 80.8% at 30 min). Of the remainder more contained WGA-Ft alone (20% at 15 min and 10.9% at 30 min), then HRP alone (none at 15 min and 8.2% at 30 min). In contrast to the various and varying patterns of labeling with WGA-Ft and HRP of the other organelles studied, the vast majority of endosomes contained both markers (94.1% at 15 min and 100% at 30 min); the rest contained WGA-Ft only. These results demonstrate that endosomes are recipients of both fluid phase and adsorptive endocytosis markers; these findings are consistent with the hypothesis that endosomes mediate the sorting out and subsequent intracellular traffic of membrane bound and fluid phase markers. Cisterns of the Golgi apparatus did not contain WGA-Ft; in sharp contrast, when WGA-HRP was used, the cisterns of the Golgi apparatus consistently contained HRP.

Novel method for isolating mammalian cells defective in fluid-phase endocytosis

1992 ◽  
Vol 18 (6) ◽  
pp. 543-551 ◽  
Author(s):  
Ru-Hung Wang ◽  
Penelope A. Colbaugh ◽  
Peter Kuo ◽  
Mu-Yeh Bau ◽  
Lisa M. Poppe ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluid Phase ◽  
Fluid Phase Endocytosis ◽  
Novel Method

scholarly journals Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step.

1984 ◽  
Vol 4 (11) ◽  
pp. 2381-2388 ◽  
Author(s):  
G Barnes ◽  
W J Hansen ◽  
C L Holcomb ◽  
J Rine
Keyword(s):  
Mammalian Cells ◽  
Covalent Modification ◽  
Yeast Cells ◽  
Core Oligosaccharide ◽  
Early Step ◽  
Recessive Mutations ◽  
Membrane Bound ◽  
Essential Function ◽  
Cellular Compartments ◽  
Increased Activity

Asparagine-linked glycosylation is a form of covalent modification that distinguishes proteins that are either membrane bound or are in cellular compartments topologically outside of the cell from those proteins that remain soluble in the cytoplasm. This type of glycosylation occurs stepwise, with core oligosaccharide added in the endoplasmic reticulum and subsequent modifications occurring in the golgi. We used tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharide, to select for mutants that are resistant to this antibiotic. Genetic, biochemical, and physiological experiments led to the following conclusions. The synthesis of N-linked oligosaccharide is an essential function in cells. In contrast to mammalian cells, yeast cells do not transport tunicamycin by a glucosamine transport function. We identified a gene, ALG7, that is probably the structural gene for UDP-N-acetylglucosamine-1-P transferase, the enzyme inhibited by tunicamycin. Dominant mutations in this gene result in increased activity of the transferase and loss of the ability of the cell to sporulate. In addition, we identified another gene, TUN1, in which recessive mutations result in resistance to tunicamycin. The ALG7 and TUN1 genes both map on chromosome VII.

scholarly journals Incomplete pneumolysin oligomers form membrane pores

Open Biology ◽  
2014 ◽  
Vol 4 (4) ◽  
pp. 140044 ◽  
Author(s):  
Andreas F.-P. Sonnen ◽  
Jürgen M. Plitzko ◽  
Robert J. C. Gilbert
Keyword(s):  
Lipid Membranes ◽  
Electron Tomography ◽  
Electrical Conductance ◽  
Membrane Attack Complex ◽  
Water Soluble ◽  
Large Hole ◽  
Membrane Pores ◽  
Model Lipid Membranes ◽  
Membrane Bound ◽  
Pore Forming Proteins

Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.

Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step

1984 ◽  
Vol 4 (11) ◽  
pp. 2381-2388
Author(s):  
G Barnes ◽  
W J Hansen ◽  
C L Holcomb ◽  
J Rine
Keyword(s):  
Mammalian Cells ◽  
Covalent Modification ◽  
Yeast Cells ◽  
Core Oligosaccharide ◽  
Early Step ◽  
Recessive Mutations ◽  
Membrane Bound ◽  
Essential Function ◽  
Cellular Compartments ◽  
Increased Activity

Asparagine-linked glycosylation is a form of covalent modification that distinguishes proteins that are either membrane bound or are in cellular compartments topologically outside of the cell from those proteins that remain soluble in the cytoplasm. This type of glycosylation occurs stepwise, with core oligosaccharide added in the endoplasmic reticulum and subsequent modifications occurring in the golgi. We used tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharide, to select for mutants that are resistant to this antibiotic. Genetic, biochemical, and physiological experiments led to the following conclusions. The synthesis of N-linked oligosaccharide is an essential function in cells. In contrast to mammalian cells, yeast cells do not transport tunicamycin by a glucosamine transport function. We identified a gene, ALG7, that is probably the structural gene for UDP-N-acetylglucosamine-1-P transferase, the enzyme inhibited by tunicamycin. Dominant mutations in this gene result in increased activity of the transferase and loss of the ability of the cell to sporulate. In addition, we identified another gene, TUN1, in which recessive mutations result in resistance to tunicamycin. The ALG7 and TUN1 genes both map on chromosome VII.

scholarly journals Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

2021 ◽  
Author(s):  
Farnaz Fekri ◽  
Ralph Christian Delos Santos ◽  
Raffi Karshafian ◽  
Costin N. Antonescu
Keyword(s):  
Drug Delivery ◽  
Fluid Phase ◽  
Acid Sphingomyelinase ◽  
Coated Pits ◽  
Drug Permeability ◽  
Membrane Pores ◽  
Signaling Mechanisms ◽  
Sheer Stress ◽  
Cell Plasma ◽  
Fluid Phase Endocytosis

Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

scholarly journals A multicompartmental model of fluid-phase endocytosis in rabbit liver parenchymal cells

1989 ◽  
Vol 262 (2) ◽  
pp. 605-610 ◽  
Author(s):  
R Blomhoff ◽  
M S Nenseter ◽  
M H Green ◽  
T Berg
Keyword(s):  
Simulation Analysis ◽  
Mechanistic Model ◽  
Fluid Phase ◽  
Rabbit Liver ◽  
Liver Parenchymal ◽  
Lysosomal Compartment ◽  
Early Endosomes ◽  
Fluid Phase Endocytosis ◽  
Parenchymal Cells ◽  
Cellular Compartments

Fluid-phase endocytosis was studied in isolated rabbit liver parenchymal cells by using 125I-poly(vinylpyrrolidone) (PVP) as a marker. First, uptake of 125I-PVP by cells was determined. Also, cells were loaded with 125I-PVP for 20, 60 and 120 min, and release of marker was monitored for 120-220 min. Then we used the Simulation, Analysis and Modeling (SAAM) computer program and the technique of model-based compartmental analysis to develop a mechanistic model for fluid-phase endocytosis in these cells. To fit all data simultaneously, a model with three cellular compartments and one extracellular compartment was required. The three kinetically distinct cellular compartments are interpreted to represent (1) early endosomes, (2) a prelysosomal compartment equivalent to the compartment for uncoupling of receptor and ligand (CURL) and/or multivesicular bodies (MVB), and (3) lysosomes. The model predicts that approx. 80% of the internalized 125I-PVP was recycled to the medium from the early-endosome compartment. The apparent first-order rate constant for this recycling was 0.094 min-1, thus indicating that an average 125I-PVP molecule is recycled in 11 min. The model also predicts that recycling to the medium occurs from all three intracellular compartments. From the prelysosomal compartment, 40% of the 125I-PVP molecules are predicted to recycle to the medium and 60% are transferred to the lysosomal compartment. The average time for recycling from the prelysosomal compartment to the medium was estimated to be 66 min. For 125I-PVP in the lysosomal compartment, 0.3%/min was transferred back to the medium. These results, and the model developed to interpret the data, predict that there is extensive recycling of material endocytosed by fluid-phase endocytosis to the extracellular environment in rabbit liver parenchymal cells.

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