Supplementary MaterialsSupplementary Document. nonlabeled Gag, we imaged Jurkat cells transfected with 1:1 Gag-GFP:SynGP by correlative SICM-FCM. SynGP is normally a codon-optimized artificial Gag-Pol that may type proteolytically matured HIV VLPs (17). SICM-FCM pictures revealed many membrane protrusions (Fig. 2= 39) and 140 nm (IQR = 160 to 125, n = 39), respectively, comparable to VLP sizes shaped on Jurkat cells transfected with pH2B-mPlum/Gag (and = 57) and 120 nm (IQR = 170 to 120, = 57), respectively, like the sizes of VLPs shaped in Jurkat cells transfected with pH2B-mPlum/Gag (got a half-life of 51 s when installed exponentially. Just like Gag-GFP, the looks of Vpr-GFP fluorescence at a detectable level preceded the formation of topographically detectable buds, indicating early recruitment of Vpr into VLPs (Fig. 3= 42) and diameters of 160 nm (IQR = 150 to 165, = 42) corresponding to microvilli, 2) finger-like protrusions (black arrow) corresponding to Idebenone dorsal filopodia or microvilli, and 3) membrane flaps corresponding to ruffles (cyan arrow). The proportion of stump, finger-like microvilli, and dorsal ruffles varied significantly from cell to cell (and = 42, six cells) and Gag-GFP transfected cells, individually for buds with (= 40) and without (= 47) corresponding Gag-GFP fluorescence spots (five cells) (two-sample test, * 0.05, ** 0.01). ((and = 40), that is, nearly twice the diameter of an average VLP, it was also Rabbit polyclonal to CDKN2A significantly lower compared to the heights of Gag-GFPCnegative protrusions (350 nm, IQR = 200 to 450, Idebenone = 47). The high variation in the measured heights suggested that these structures were microvilli going through active growth or retraction at the moment of fixation (22). The diameters of Gag-GFPCpositive and Cnegative protrusions were not significantly different (160 nm [IQR = 125 to 185, = 40] and 175 nm [IQR = 155 to 190, = 47], respectively). We identified a total of 312 Gag-GFP fluorescence spots and 343 topographically detected VLP buds, of which 96 overlapped (eight cells). Thus, 30% of the Gag-GFP fluorescence spots Idebenone correlated with budding structures and 28% of the topographically detected buds had associated fluorescence (Fig. 4and = 31) and width of 100 nm (IQR = 110 to 90, = 31). Both the height and width of the buds formed in Cos-7 cells were significantly lower ( 0.05) than those of VLPs formed by unlabeled Gag in Jurkat cells. The density of protrusions indicated that, similar to Jurkat cells, many VLPs remained adhered to the cell membrane after the completion of assembly. TEM images of Cos-7 cells transfected with SynGP also confirmed the formation of similar-looking VLPs in this cell type (= 19), significantly higher ( 0.001) than the Gag-GFPCpositive buds (90 Idebenone nm, IQR = 110 to 45, = 31), suggesting that the former could be microvilli (= 31) of Gag-GFPCpositive protrusions were significantly larger than the diameters of buds formed by unlabeled Gag ( 0.008), suggesting that, in Cos-7 cells, GFP tagging interferes with Gag-driven VLP formation. Confirming this, TEM images of immunolabeled cryosections of Cos-7 cells with 1:1 Gag-GFP:SynGP revealed the formation of flat and lightly curved Gag assemblies (= 84). The full-size Gag-GFPCpositive protrusions were also static (= 7). For comparison, cross-section profiles of lightly curved and fully developed VLPs are presented in = 31) and width of 103 nm (IQR = 115 to 90, = 31) ( 0.05) than in Jurkat cells. Protease treatment of cells with 100 g/mL Subtilisin A resulted in a rapid 64 13% (two Idebenone independent experiments) reduction in the number of protrusions, indicating that they represented VLPs that remained adhered to the cell surface after.