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. 2020 Feb 28;21(5):1656.
doi: 10.3390/ijms21051656.

Beta3-Tubulin is Critical for Microtubule Dynamics, Cell Cycle Regulation, and Spontaneous Release of Microvesicles in Human Malignant Melanoma Cells (A375)

Mohammed O Altonsy  1   2 , Anutosh Ganguly  1   3   4 , Matthias Amrein  5 , Philip Surmanowicz  1 , Shu Shun Li  3 , Gilles J Lauzon  1 , P Régine Mydlarski  1
Affiliations

Beta3-Tubulin is Critical for Microtubule Dynamics, Cell Cycle Regulation, and Spontaneous Release of Microvesicles in Human Malignant Melanoma Cells (A375)

Mohammed O Altonsy et al. Int J Mol Sci. .

Abstract

Microtubules (MTs), microfilaments, and intermediate filaments, the main constituents of the cytoskeleton, undergo continuous structural changes (metamorphosis), which are central to cellular growth, division, and release of microvesicles (MVs). Altered MTs dynamics, uncontrolled proliferation, and increased production of MVs are hallmarks of carcinogenesis. Class III beta-tubulin (β3-tubulin), one of seven β-tubulin isotypes, is a primary component of MT, which correlates with enhanced neoplastic cell survival, metastasis and resistance to chemotherapy. We studied the effects of β3-tubulin gene silencing on MTs dynamics, cell cycle, and MVs release in human malignant melanoma cells (A375). The knockdown of β3-tubulin induced G2/M cell cycle arrest, impaired MTs dynamics, and reduced spontaneous MVs release. Additional studies are therefore required to elucidate the pathophysiologic and therapeutic role of β3-tubulin in melanoma.

Keywords: melanoma; microtubules; microvesicles; β3-tubulin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
β3-tubulin knockdown reduces the numbers of microvesicles (MVs) spontaneously released by A375 melanoma cells under the standard culture conditions. (A) Western blots of β3-tubulin and GAPDH loading control (panel A, left), and RT-PCR quantification of β3-tubulin mRNA expression normalized to the CT values of RPL19 (panel A, right). (B) Naïve, control (ctrl)−, and β3-tubulin siRNA-treated cells were immunostained with antibodies specific for β3-tubulin (green) and MAA (orange), DAPI (blue) was used as nuclear DNA counterstain, original magnification ×630 (bar, 20 μm). (C) MTT assay shows the effect of siRNA treatment on the cellular viability of A375 cells. Flow cytometric contour plots (D) and quantification (E) of MVs collected from the culture media of naïve, ctrl-, and β3-tubulin-siRNA treated cells and immunostained with an MAA antibody or isotype control, plots illustrate the percentage of positively stained MVs (D, red square gated events). Super-resolution microscopy with maximum intensity projection of a confocal stack micrographs of A375 cells (F), or MVs collected from culture media (G), stained with Alexa fluor 488-conjugated-WGA, red rectangles, in F and G, show randomly selected areas for higher magnification (right enlarged micrographs), illustrating ring-like microvesicles of varying sizes (200–1000 nm) with optically clear lumen. Original magnification ×1000, scale bar 5 μm (left graphs) or 1 μm (magnified right graphs). Statistical significance was determined between different groups using an ANOVA with Tukey’s correction for multiple comparisons. n = 5, *** p < 0.001 versus naïve cells.
Figure 2
Figure 2
β3-tubulin knockdown suppresses microtubules (MTs) dynamics and disrupts cell cycle in A375 cells. (Panel (A), right) Micrographs of EGFP-MAP-4-labeled MTs in naïve, ctrl-, and β3-tubulin siRNA-treated A375 cells. Images were acquired every 5 s and show MTs +end growth (arrows) and shortening (arrowheads). Original magnification ×1000 (bar, 5 μm). (panel (A) left) Traces of ten representative MTs +end displacements (growth and shortening) measured over a period of 75 s. Quantification of dynamic growth and shortening (panel (B), left), and pause frequency (panel (B), middle) and dynamicity (panel (B), right). (C) Flow cytometric histograms (top panels), and cell cycle growth phase distribution analysis (bottom panel) demonstrate the effect of β3-tubulin knockdown on cell cycle in A375 cells. Statistical significance between naïve, ctrl-, and β3-tubulin-siRNA groups was determined using an ANOVA with Tukey’s correction for multiple comparisons. n = 10 (MTs analysis) or = 6 (cell cycle analysis), *** p < 0.001, and * p < 0.05 versus naïve cells.
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