Journal: The journal of physical chemistry. B

Article Title: Myelin-associated MAL and PLP are unusual among multi-pass transmembrane proteins in preferring ordered membrane domains

doi: 10.1021/acs.jpcb.0c03028

Figure Lengend Snippet: Shown are the raft affinity of multi-pass TMPs with varying number of TMHs. Raft affinity is shown in log scale, with 0 representing equal partitioning between the phases and positive and negative values representing enrichment in or depletion from the raft phase, respectively. Included in each panel is the value for LAT (a raft-preferring single pass TMD, black) and LDLR (a non-raft TMD, white). (A) 2-TM proteins. Three homologs of MOG from different species (h:human; m:mouse; r:rat) and both isoforms (alpha and beta) of the human protein are excluded from rafts, as is the unrelated CD36. (B) Two 4-TM proteins are highly raft-preferring, namely MAL and PLP. In contrast, several other members of the MARVEL family (BENE, MAL2, occludin, and synaptophysin) and other tetraspanin family proteins (CD9, CD81, CD82), as well as the neuronal membrane glycoprotein (GPM6a) are almost completely excluded. Two analogs of MAL (c:canine: h:human) were tested and both had significantly greater raft affinity than LAT. (C) All tested 6-TM and 7-TM proteins are raft excluded. Several proteins from the TRP family (TRPA1, TRPM8, TRPV1), Adenylyl Cyclase 9 (AC9), and G Protein-Coupled Receptors (DRD1, β2AR) were not significantly different from LDLR. (D) All tested proteins with 10 or more TMDs - PM scramblase TMEM16F and several transporters (NKCC1, KCNQ1 and GAT1) - were raft excluded. Data points represent the means of individual experiments of >10 vesicles each.

Article Snippet: Several multi-pass protein constructs were purchased from Addgene: mCherry-CD36-C10 (CD36, 55011), mCherry-Occludin-N-10 (Occludin, 55112), mCherry-CD9–10 (CD9, 55013), mCherry-CD81–10 (CD81, 55012), YFP-CD82 (CD82, 1819), pFCK(1.3)GFP-SynaptophysW (Synaptophysin, 27235), pEGFP-N-Drd1 (DRD1, 104358), Kv7.1/pcDNA3.1 (KCNQ1, 111452), pcDNA3.1(+)mGAT1–0-GFP (GAT1, 41662).

Techniques:

Journal: bioRxiv

Article Title: An evolutionary transcriptomics approach links CD36 to membrane remodeling in replicative senescence

doi: 10.1101/294512

Figure Lengend Snippet: Characterization of genes associated with replicative senescence. (A) The cumulative fraction plot of pLI (the probability of being loss-of-function intolerant) value of genes upregulated in old cells (YO, blue) and other protein-coding genes (gray). This measurement is obtained from ExAC database . (B) The cumulative fraction plot of b-value (the index of background selection between primate species) of genes in each category (Mc ). The color scheme is same as the previous panel. (C) X-axis shows the tolerance of genes in healthy human genomes (-log p- value of pLI), y-axis shows the log 2 fold change between young and old in BJ cells and the size of the bubble indicates the log 2 fold change between young and old in MRC-5 cells. The strength of the color shows the -log p-value of pLI. CD36 showed upregulation in both old BJ and MRC-5 cells and tolerance to loss-of-function mutations in healthy human genomes.

Article Snippet: FusionRed-CD36-C10 plasmid (#56104) was obtained from Addgene (deposited by the Davidson lab).

Techniques: Selection

Journal: bioRxiv

Article Title: An evolutionary transcriptomics approach links CD36 to membrane remodeling in replicative senescence

doi: 10.1101/294512

Figure Lengend Snippet: CD36 overexpression results in senescence-like phenotype. (A) Representative images of control and CD36 -overexpressing young MRC-5 cells. CD36 o verexpression was visualized via the FusionRed tag (scale bar = 10µm). (B-C) Measurement of β-galactosidase activity in control and CD36 overexpressing MRC-5 cells. A significant increase of SA-β-galactosidase positive cells was observed during CD36 overexpression for which representative images are shown (scale bar =100µm for control and center CD36 images, and 20µm for furthest to the right CD36 image). Data shown as means ± standard deviation, *** p < 0.001. (D) Representative western blot showing p16 levels in control and CD36 overexpressing young MRC-5 cells.α-tubulin was used as a loading control. We observe a modest increase in p16 levels. (E) Measurement of β-galactosidase activity in young MRC-5s that were treated with conditioned media. “Control” represents cells in standard growth medium, “Control_M” represents cells in medium from control wells during transfection, “+CD36_M1” represents cells in medium from CD36 overexpressing cells 48h after transfection, and “+CD36_M2” represents cells in medium from CD36 overexpressing cells 96h after transfection. An increase in SA-β-galactosidase positive cells was observed.

Article Snippet: FusionRed-CD36-C10 plasmid (#56104) was obtained from Addgene (deposited by the Davidson lab).

Techniques: Over Expression, Activity Assay, Standard Deviation, Western Blot, Transfection