The overall goal of this procedure is to detect and quantify the pool of toxin which enters the cytosol. To accomplish this, the toxin is first bound to the surface of the hosted cell at four degrees Celsius. The unbound toxin is then removed and the cells are warmed to 37 degrees Celsius, which permits toxin movement into the endomembrane system, and subsequently the host cytosol dig tonin is then used to selectively perme the plasma membrane following cell permeation.
The cell extracts are partitioned into separate membrane and cytosolic fractions using differential centrifugation as a final step surface plasma resonance Abbreviate SBR is used to detect and quantify toxin entry into the cytosol through perfusion of the cytosol fraction over an SPR sensor coated with an antitoxin antibody. This method will help answer key questions in the field of toxin biology, such as the efficiency and kinetics of toxin entry into the cytosol and the quantity of toxin, which enters the cytosol and possible drug inhibition of toxin trafficking into the cytosol. Prior to the start of this protocol, prepare heli cells for cell intoxication as described in the written protocol following an overnight incubation of the hela cells.
Replace the culture medium with one milliliter DMEM containing 100 nanograms per milliliter of Ganglioside GM one and incubate for one hour at 37 degrees Celsius. This increases the number of binding sites for cholera toxin. As the toxins GM one receptor will interpolate into the plasma membrane of cells exposed to a GM one solution.
After a one hour incubation, remove the GM one containing medium and wash the cells twice with DMEM. Then add one milliliter of DMEM containing one microgram per milliliter of cholera toxin, and place the cells at four degrees Celsius for 30 minutes. Remove the toxin containing medium and wash the cells twice.
Then incubate the cells in one milliliter of DMEM at 37 degrees Celsius for the desired time. At the end of each chase period, wash the cells with PBS and then incubate each well with 250 microliters of 0.5 millimolar EDTA and PBS. Following a 10 minute incubation on ice, remove the cells from the six well plate by vigorous TRI with a P 1000 pipet men.
After one well of cells has been collected, combine it with the second and then third well from the same condition. Next place the combined cell suspension from the three replicate wells in a single micro centrifuge tube. Now spin the micro centrifuge tube in a tabletop micro centrifuge for five minutes.
At 5, 000 times G cell pellets of roughly equivalent size should be obtained for all conditions. Discard the supernatant and then resuspend the cell pellet in 100 microliters of a previously prepared and chilled 0.04%working solution of digit toin. Place the cell suspension on ice for 10 minutes using a tabletop micro centrifuge, spin the digit tone and permeable cells at 16, 000 times G for 10 minutes.
Transfer the cytosol containing supinate fraction to a fresh micro centrifuge tube and retain the organelle containing pellet fraction. SPR toxin standards are prepared at concentrations of one hundred ten one and 0.1 nanograms per milliliter in HCN buffer samples are prepared by diluting cytosol containing supinate fractions in HCN buffer to a final volume of one milliliter. Sample volumes of at least one milliliter necessary to ensure that air is purged from the 500 microliter sample loop before injection organelle containing pellet fractions are resuspended in one milliliter of HCN buffer containing 1%Triton X 100.
Addition of detergent is necessary to release the membrane encased pool of toxin. Also prepare parallel sets of cytosolic and organ L fractions for a control experiment to confirm the fidelity of the fractionation procedure, resuspend the cytosolic fraction in 20 microliters of four x sample buffer and the organ L fraction in 120 microliters of one x sample buffer resolve equivalent volumes of each fraction with sodium ESAL sulfate, poly acrylamide gel electrophoresis and probe by Western blood analysis to demonstrate the partitioning of a cytosolic protein in the supine fraction and a soluble resonant ER protein in the pellet fraction. To prepare the SPR slide first set a gold-plated glass slide with a self-assembled monolayer in the SPR instrument.
Activate the sensor slide by perfusing an E-D-C-N-H-S solution over the slide for 10 minutes at a flow rate of 41 microliters per minute. All subsequent perfusions, we use the same flow rate. Remove the E-D-C-N-H-S activation buffer by washing the plate for five minutes with PBS containing 0.05%2020.
The plate now contains reactive MI tethers due to uncapping by the E-D-C-N-H-S solution. Next perfuse an anti CTA antibody over the activated sensor slide at a dilution of one to 20, 000 in 20 millimolar sodium acetate for 15 minutes. An initial drop in the refractive index unit, abbreviated RIU, will be seen due to the pH change.
This will be followed by an increase in the RIU as the antibody is captured by the amide reactive tethers. On the sensor slide, remove the unbound antibody from the sensor slide with a five minute PBST wash. The RIU signal produced by the captured antibody will plateau and stabilize providing a new baseline signal.
As a final in slide preparation, PERFUSE one molar ethanolamine over the sensor slide for five minutes. This caps and inactivates any unbound tethers left on the sensor slide. The RIU signal will increase to establish a baseline reading perfuse PBST over the antibody coated sensor for five minutes.
Then perfuse an experimental sample or toxin standard over the sensor for 300 seconds. Remove the ligand from the buffer and perfuse PBST over the sensor for another 200 seconds. Any toxin remaining bound to the sensor is removed by a 102nd wash with PBST at pH 5.0.
This will return the signal to its initial baseline reading, thus allowing another sample to be processed on the same sensor. Before loading a new sample, push a small amount of air through the sample loop to expel any residual fluid from the prior sample. Following acquisition.
Data can be analyzed with the scrubber two software and the Igor software can be used for figure preparation. Pertussis toxin is an AB toxin that moves from the cell surface to the ER before its a chain. PTs one enters the cytosol.
The SPR based translocation assay could detect PTs one in the cytosol of intoxicated CHO cells. No signal was generated from the cytosol of unin intoxicated cells, which confirmed the anti PTs. One antibody did not cross-react with a component of the host cytosol.
The cytosol fraction from cells intoxicated in the presence of felden. A also failed to produce a positive signal, BFA prevents toxin transport to the ER translocation site, and thus H chain delivery to the cytosol. Chole toin is another AB type ER translocating toxin.
CTA one was detected in the cytosolic fraction from CT treated helo cells. This emphasized that the methodology works with multiple cell types and can be applied to any toxin for which an anti chain antibody is available. No signal was detected when the cytosolic fraction from CT treated cells was perfused over an SPR sensor coated with an anti CTB antibody, thus demonstrating that unlike the CT A one subunit, the cell binding CTB PENTAMER does not enter the cytosol saw.
Following profusion over an SPR sensor coated with an anti CTA antibody. The signal from the organelle fraction is off scale in comparison to the weaker signal from the cytosolic fraction. This is consistent with the known inefficiency of CT transport to the ER translocation site, which in turn limits the amount of toxin that can reach the cytosol.
The time-dependent accumulation of translocated cytosolic toxin can also be monitored. Following cell exposure to CT at four degrees Celsius. No toxin was detected in the cytosol 15 minutes after warming to 37 degrees Celsius.
A minor pool of cytosolic toxin was detected after 30 minutes at 37 degrees Celsius and progressively larger quantities of cytosolic toxin were detected after the 45 and 60 minute chase intervals. This demonstrates a continual long-term delivery of cell associated toxin to the hosted cytosol. The assay can also detect the inhibition of toxin translocation to the cytosol cells treated with 10%DMSO exhibited low levels of cytosol like CT one in comparison to the untreated intoxicated control cells unfolding of the toxin.
A chain is a prerequisite for translocation to the cytosol, so DMSO induced stabilization of CT A one accordingly prevented its movement from the ER to the cytosol. To quantify the DMSO induced block of toxin translocation, a standard curve generated from known concentrations of toxin was used to calculate a cytosolic cta, one concentration of 0.3 nanograms per milliliter for untreated cells, and 0.1 nanograms per milliliter for DMS O treated cells. The inhibition of CT A one unfolding by DMSO, thus generated a threefold reduction in the ER to cytosol translocation of CTA one.
Following this procedure, we'll be able to utilize other techniques such as intoxication assays to establish a direct link between the amount of toxin present in cytosol and toxin activity.
