Citation

  • Authors: Porciello N. et al.
  • Year: 2022
  • Journal: J Biol Chem
  • Applications: in vitro / DNA / PEIpro
  • Cell type: Lenti-X293T

Method

Production of lentiviral particles Lentiviruses were generated using the packaging cell lines Lenti-X293T. The culture medium was exchanged with RPMI supplemented with 10 % FBS just prior to transfection. Lenti-X293T at 80 % confluence were transfected using PEIpro (Polyplus) according to the manufacturer’s instructions. The packaging plasmids pVSVG and pSPAX2 were mixed with the lentivirus expression vectors containing the gene of interest. PEIpro solution was added to the plasmids mix and immediately vortexed, left 15 min at RT and then added dropwise to the cells by gently swirling the plate. Supernatant containing lentiviral particles was collected after 48 h and filtered through a 0.45 μm sterile filter (Sartorius Stedim). Lentivirus supernatants were concentrated with PEG-itTM (SBI) concentration kit according to the manufacturer’s instruction. Briefly, lentiviral supernatants were mixed with Virus Precipitation Solution (SBI) to a final concentration of 1X Virus Precipitation Solution and incubated overnight at 4 ⁰C followed by a centrifugation at 1,500 x g for 30 min at 4 ⁰C. Pellets containing lentivirus particles were re-suspended in 1/100 of the volume of the original cell culture using cold RPMI. Aliquots were immediately frozen in cryogenic vials at - 80 ⁰C and stored until use. Aliquots of each lentivirus batch were routinely pre-tested by serial dilution titration. Frozen aliquots were thawed only once and used immediately with minimal loss of virus titre as determined by FCM.

Abstract

Theoretical work suggests that collective spatiotemporal behaviour of integral membrane proteins (IMPs) should be modulated by boundary lipids sheathing their membrane anchors. Here, we show evidence for this prediction whilst investigating the mechanism for maintaining a steady amount of the active form of IMP Lck kinase (LckA) by Lck trans-autophosphorylation regulated by the phosphatase CD45. We used super-resolution microscopy, flow cytometry, and pharmacological and genetic perturbation to gain insight into the spatiotemporal context of this process. We found that LckA is generated exclusively at the plasma membrane, where CD45 maintains it in a ceaseless dynamic equilibrium with its unphosphorylated precursor. Steady LckA shows linear dependence, after an initial threshold, over a considerable range of Lck expression levels. This behaviour fits a phenomenological model of trans-autophosphorylation that becomes more efficient with increasing LckA. We then challenged steady LckA formation by genetically swapping the Lck membrane anchor with structurally divergent ones, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45 that were expected to drastically modify Lck boundary lipids. We observed small but significant changes in LckA generation, except for the CD45 transmembrane domain that drastically reduced LckA due to its excessive lateral proximity to CD45. Comprehensively, LckA formation and maintenance can be best explained by lipid bilayer critical density fluctuations rather than liquid-ordered phase-separated nanodomains, as previously thought, with "like/unlike" boundary lipids driving dynamical proximity and remoteness of Lck with itself and with CD45.

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