high expression of Spike however leads to the total disruption of normal ER structure.pic.twitter.com/8eYIxhBoC8
Biological Scientist @SalkInstitute @Scripps_Ocean dabbling in Bio DataScience, Neuroscience, Cephalopods, AI&HPC using Nonlinear Dynamics & Molecular Biology
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high expression of Spike however leads to the total disruption of normal ER structure.pic.twitter.com/8eYIxhBoC8
Another piece of evidence that demonstrates plasma membrane presence of spike is this experiment. Express spike with GFP in some cells, express hACE2 with TdTomato in other cells. Mix. Cells fuse to create massive syncytia (cells sharing cytoplasm after fusion)pic.twitter.com/qcLGW3hhdf
because you are mixing GFP and TdTomato the cells will look yellow under the fluorescent microscope when the ratio of both FPs are balanced. The syncytia can become quite massive. This is good evidence of plasma membrane spike protein not just ER.pic.twitter.com/G5PcHQFWi8
Surprisingly this was not completely lethal and cells could be stably infected with a lentiviral vector construct. You can get this vector from @Addgene that we deposited some time ago. https://www.addgene.org/141347/ over time however you will lose spike expression.pic.twitter.com/u6vypOKDuG
you can see large areas of fused syncytia. Ultimately some will round up and become large spheres as you can see happening in this image. Observe also the extensive fusionpic.twitter.com/qX2Xat0eFP
at higher magnification you can see multiple nuclei but in some cases they seem to degenerate. This fusion happens in normal cell culture conditions so does not require low pH.pic.twitter.com/o7s91y7ng6
In immunofluorescence you can clearly see that not only you have multiple nuclei in syncytia but the nuclei themselves fuse. So you have plasma membrane fusion events, ER fusion events and nuclear fusion events.pic.twitter.com/5FOglpdJ8m
If you look more carefully however, it appears to be that the majority of nuclear fusion is only of the outer nuclear membrane. You can still see spike separating nuclei by IF.pic.twitter.com/TBhuc4bhML
Working with syncytia is difficult. Syncytia are fragile and cannot be FACS sorted. However they will not pass a FACS mesh filter so you can quantify the loss of spike expressing cells. D614G spike had a small but statistically significant (p<0.02) 11% greater loss of GFP+ cells.pic.twitter.com/0qJdLVPHlK
when comparing Spike 614D and Spike 614G when pseudotyped with GFP expressing lentiviral vectors we observed that the #SARSCoV2 D614G mutant was ~5X more infectious.pic.twitter.com/UBBYi7ILkp
If you want a lentiviral vector expressing the SARS-CoV2 Spike glycoprotein with the D614G mutation, it should be available through @Addgene soon. This is brought to you by a generous donation of @DanielleFong and the W.M. Keck foundation *End of Thread*pic.twitter.com/zEPQMtqK1J
Thanks for sharing this plasmid! If anyone is looking to use this, you can sign up for email alerts for new plasmids from the lab here: https://www.addgene.org/Gerald_Pao/
This is good thank you.
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