Fig 1.
Tau 4R2N and Full Tau protein purification.
(A) Alternative splicing scheme of Tau 4R2N and Full Tau and specific domains. NTR: N-Terminal Region, PRR: Proline-Rich Region, MTBR: Microtubule-Binding Region, CTR: C-Terminal Region (B) Coomassie brilliant blue staining of purification of both proteins showing control of both expressions without and with induction of expression using 0.5mM IPTG. (C) Western Blot using the antibody that recognizes human Tau (HT7) indicating bands from induction are corresponding Tau. Both B and C panels are electrophoresis from same purifications. Unadjusted and uncropped images of blots and gels are found on S2 File.
Fig 2.
Microtubule binding capacity of Full Tau isoform.
(A) Scheme of the interaction of human Tau isoforms to second polymerization cycle of mouse microtubules (see Methods). (B) Electron microscopy images of second polymerization cycle of mouse brain microtubules in the absence (control) or presence of Tau 4R2N or Full Tau isoform. Circles indicate thin MTs. (C) Coomassie brilliant blue staining showing tubulin percentage after centrifugation of the two cycle-polymerization MTs. SN: Supernatant, PLL: Pellet (D) Western Blot analyses of human Tau bound to microtubules after copolymerization, in the second polymerization cycle of mouse microtubules, detected by anti-human Tau antibody HT7. Unadjusted and uncropped images of blots and gels are found on Data Availability. (E) Quantification of the ratio between Tau 4R2N and Full Tau Western Blot signal intensity in C. Quantitative analysis shows the mean ± SEM. * p < 0.05; ** p < 0.01 using one-way ANOVA; post-hoc Turkeys’s test, followed by Student’s t-test for comparisons. Sample size: n = 3.
Fig 3.
Aggregation capacity of T42 and Full Tau isoforms.
(A) Representative images of electron microscopy of Tau aggregates obtained upon in vitro incubation of purified T42 and Full Tau extracts from bacteria in presence of heparin to promote aggregate formation. Filaments marked with white arrows ends. Scale bars show 400nm. (B) Area of the amorphous polymers found on Full Tau in vitro aggregation analysis. (C) Length distribution of the fibrillar polymers formed by Tau 4R2N. (D) Number of amorphous aggregates grouped by area intervals. (E) Number of fibrillar polymers of Tau 4R2N grouped by length intervals. (F) Western Blot of Tau isoforms present in soluble or pellet fractions after centrifugation. SN: Supernatant, PLL: Pellet Unadjusted and uncropped images of blots and gels are found on Data Availability. (G) Quantitative analysis of pelleted proteins. (H) Quantitative ratio of Tau 4R2Nand Full Tau protein. Quantitative analysis shows the mean ± SEM. * p < 0.05; ** p < 0.01 using unpaired; 2-tailed t test. Sample size: n = 3.
Fig 4.
Tridimensional structure prediction of Tau 4R2N and Full Tau.
Microtubule Binding Domain Exons’ 9, 10, 11, 12 are represented in red, the rest of exons in white. Both are the predictions with the highest confidence (highest pLDDT) among the five predictions made by AF2. Images were taken at different degrees of rotation about the “y” axis for complete visualization. Exon 8 is highlighted in green. MTBD is colorized in red. Scheme of proteins structure highlighting differences in both structure and size.
Fig 5.
Proliferation and viability of HEK 293T cells following lentiviral infection with Tau 4R2N and Full Tau.
(A) Cellular proliferation rate, assessed through images taken 24 hours post-transduction and after 48 hours of growth. Scale bar: 40 µm. (B) Quantification of cellular proliferation based on the newly occupied area by cells. Sample size: n = 3 fields per replicate (n = 3). (C) Calcein viability assay comparing cell death between the negative control and transduction controls for each protein. Scale bar: 100 µm (D) Quantitative analysis of viability, measure from the data shown in (C). Sample size: n = 3 fields per replicate (n = 3). (E) Representative images of the calcein viability assay in cells overexpressing Tau 4R2N and Full Tau, along with their respective transduction controls (C. Empty-Tau 4R2N and C. Empty-Full Tau). Same scale as (E). (F) Quantification of cellular viability the data shown in (E). Sample size: n = 3 fields per replicate (n = 3). Quantitative data are presented as mean ± SEM. Statistical significance: *p < 0.05; **p < 0.01, determined by one-way ANOVA followed by Tukey’s post-hoc test, with additional comparisons conducted using Student’s t-test. Sample size: n = 5 fields per replicate (n = 3).