2.7. Quantification of Microtubule Stability

Immunofluorescence imaging was utilized to determine the morphology of microtubules. Log-phase cells were treated with 0, 3, or 5 mM of HA, or 0, 30, or 50 μM for AMA for 30 min shaking at 22 °C (log-phase cells were treated with cumene only on coverslips, see below). Then, 15 mL of cells were washed twice with Lo-Flo and resuspended to 4–8 × 10⁶ cells/mL. Further, 0.5 mL of washed cells were added directly to a coverslip in a 6-well plate and incubated for 30 min at room temperature with appropriate treatment (0, 0.3, or 0.6 mM for cumene; 0, 3, or 5 mM for HA, or 0, 30, or 50 μM for AMA). Cells were washed two times with 10 mM MES-NaOH, then fixed with 3% paraformaldehyde pH 6.0 for 30 min. The cells were quenched with 100 mM glycine in 1 × PBS for 5 min then permeabilized with 0.02% Triton X-100 for 5 min. After three quick 1 × PBS washes, cells were incubated in 0.045% fish gelatin and 0.5% BSA in 1 × PBS (PBG) for 1 h at room temperature. Mouse anti-tubulin (12G10, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) diluted 1:150 in PBG solution was added to the coverslips and incubated at 4 °C 12–18 h. Three 5 min washes with 1 × PBS were performed and the secondary antibody was added, AlexaFluor 488 goat alpha mouse IgG, (Life Tech, A11001, Thermo Fisher Scientific Grand Island, NY, USA) 1:250 in PBG solution for 1 h at 4 °C. The coverslips were kept in the dark from this point forward. Three final 5 min washes in 1 × PBS were performed then coverslips were mounted to glass slides with SlowFade Diamond (Invitrogen, Thermo Fisher Scientific Grand Island, NY, USA) and stored in the dark at 4 °C. Immunofluorescence imaging was performed on a Nikon A1R confocal on a Ti2-E inverted microscope (Nikon, Melville, NY, USA), settings included Galvano scanning with a pinhole of 26.82. A total of 61 z-sections, 0.1 um thick, were obtained. Image analysis was performed using NIS Elements AR 5.20.02 (Nikon, Melville, NY USA). Microtubules were categorized into intact, partial, spot, or dead based on microtubule morphology for at least 50 cells per treatment by two independent researchers (Figure 1). Intact refers to a complete microtubule complex extending throughout the cell, partial means less a complete complex and shorter microtubule filaments that do not reach periphery of the cell, spot indicates only the center of the microtubule organizing center is present with no radiating tubules, and dead represents cells where microtubules were depolymerized due to cellular death prior to fixation. Statistical analysis was performed using Chi Square analysis in GraphPad Prism version 6.07 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com (accessed on 12 August 2021)).

Microtubule Morphologies. (A) Fully intact microtubule structures with numerous tubules reaching toward the plasma membrane are characterized as intact. (B) A cytoskeleton that demonstrates an astral shape but does not extend to the plasma membrane is designated as partial. (C) Spot morphologies are where only the microtubule organizing center is present and (D) dead cells are when there is no detectable fluorescence (as shown here) or the plasma membrane is obviously no longer intact (data not shown). Scale bar is 5 μM.