*3.6. Microscopy Imaging*

Liquid-liquid phase separation (LLPS) experiments were performed using variable protein (wt Tau and Ac-Tau; 2.5–20 μM) and salt (0–250 mM NaCl) concentrations in 5 mM sodium phosphate buffer, pH 7.8. 10–20-μL drops were pipetted onto 35-mm glass bottom dishes (ibidi, Martinsried, Germany) and immediately monitored for droplet formation (with incubation time of 5 min). For heparin-induced LLPS experiments, heparin (8–25 kDa; Santa Cruz Biotech) was mixed with wt Tau or Ac-Tau at approximately 1:4 molar ratio (heparin:protein). LLPS experiments in the presence of crowding agent were carried out using 30 μM wt Tau or Ac-Tau in 10% PEG-8K, αβγ buffer (10 mM glycine, 10 mM acetate, 10 mM sodium phosphate, pH 7.5). Microscopy images were recorded at RT using an FL EVOS imaging system (Invitrogen).

#### *3.7. Thioflavin T (Th T) Aggregation Assay*

Heparin-induced wt Tau and Ac-Tau aggregation were detected following changes in Th T fluorescence using a Biotek Synergy H1 plate reader, employing 440 nm excitation and 480 nm emission wavelengths. Protein aggregation reactions were conducted using 20 μM wt Tau or Ac-Tau in 5 μM heparin, 10 μM Th T (GenDepot), 0.25 mM TCEP, 5 mM sodium phosphate, pH 7.8. Aggregation kinetics were monitored for ~24 h.

#### *3.8. Microtubule Assembly Assay*

The ability of wt Tau and Ac-Tau to promote microtubule assembly was investigated using fluorescence imaging. Rhodamine-labeled and unlabeled tubulin heterodimers (1:20 ratio; Cytoskeleton, Inc., Denver, CO, USA) were mixed with wt Tau or Ac-Tau (9 μM tubulin heterodimers and 27.5 μM Tau) in a final buffer condition of 0.2 mM MgCl2, 0.1 mM GTP, 50 μM EDTA, 9 mM sodium PIPES, pH 6.9. Microtubule formation was visually monitored using an FL EVOS fluorescence microscope (Invitrogen) starting from 10 min up to 18 h.

**Author Contributions:** J.C.F. and A.C.F. designed the experiments. J.C.F., K.J.C., and P.S.T. performed the microscopy imaging and molecular biology experiments. A.J. and S.Y.J. acquired and analyzed the mass spectrometry data. J.C.F., K.R.M., and A.C.F. wrote the manuscript.

**Funding:** This work was supported by laboratory startup funds from Baylor College of Medicine (J.C.F. and A.C.F.).

**Acknowledgments:** We thank Jin Wang for the use of the Biotek Synergy H1 instrument.

**Conflicts of Interest:** The authors declare no competing financial interests.
