Partial depletion of gamma‐actin suppresses microtubule dynamics

Abstract

Actin and microtubule interactions are important for many cellular events, however these interactions are poorly described. Alterations in γ-actin are associated with diseases such as hearing loss and cancer. Functional investigations demonstrated that partial depletion of γ-actin affects cell polarity and induces resistance to microtubule-targeted agents. To determine whether γ-actin alterations directly affect microtubule dynamics, microtubule dynamic instability was analyzed in living cells following partial siRNA depletion of γ-actin. Partial depletion of γ-actin suppresses interphase microtubule dynamics by 17.5% due to a decrease in microtubule shortening rates and an increase in microtubule attenuation. γ-Actin partial depletion also increased distance-based microtubule catastrophe and rescue frequencies. In addition, knockdown of γ-actin delayed mitotic progression, partially blocking metaphase–anaphase transition and inhibiting cell proliferation. Interestingly, in the presence of paclitaxel, interphase microtubule dynamics were further suppressed by 24.4% in the γ-actin knockdown cells, which is comparable to 28.8% suppression observed in the control siRNA treated cells. Paclitaxel blocked metaphase–anaphase transition in both the γ-actin knockdown cells and the control siRNA cells. However, the extent of mitotic arrest was much higher in the control cells (28.4%), compared to the γ-actin depleted cells (8.5%). Therefore, suppression of microtubule dynamics by partial depletion of γ-actin is associated with marked delays in metaphase-anaphase transition and not mitotic arrest. This is the first demonstration that γ-actin can modulate microtubule dynamics by reducing the microtubule shortening rate, promoting paused/attenuated microtubules, and increasing transition frequencies suggesting a mechanistic link between γ-actin and microtubules. © 2013 Wiley Periodicals, Inc

Gamma-actin is involved in regulating centrosome function and mitotic progression in cancer cells

“…24 Consistent with this result, cell cycle analysis shows that g-actin knockdown reduces the ability of paclitaxel to induce G 2 /M cell cycle checkpoint compared to the control siRNA cells (Fig 2A). In order to investigate the process by which this occurred, the expression of several cell cycle markers in g-actin-depleted cells were examined, to determine whether these may be associated with the lack of mitotic arrest in paclitaxel-treated g-actin siRNA cells.…”

“…23,24 To gain an understanding of how partial depletion in g-actin can inhibit microtubuletargeted agent induced mitotic arrest, 24 we initially examined the localization and reorganization of b-actin and g-actin during mitosis following siRNA-mediated knockdown of g-actin in SH-EP transfected neuroblastoma cells. SH-EP cells were used in this study as they have good (~100%) transfection efficiency with siRNA 24 making them suitable for single cell imaging. In addition, successful siRNA-mediated knockdown of g-actin protein expression (~50%) in the SH-EP cell line has been previously demonstrated.…”

“…S4A, B and C). Thus, the aberrant expression of G 1 phase cell cycle markers accompanied by low levels of S, G 2 and M phase markers, would impede cell proliferation 24 and cause accumulation of cells in the G 0 /G 1 phase of the cell cycle as observed in the g-actin-depleted cells.…”

“…23 Microtubule-targeted agents such as the anti-cancer drug paclitaxel, induce mitotic arrest and subsequent cell death. In this regard, we have recently demonstrated that partial depletion of g-actin suppresses microtubule dynamics and also inhibits paclitaxel-induced mitotic arrest, 24 suggesting that g-actin may be playing an important role that impacts microtubule function during mitosis. To functionally dissect the role of g-actin in mitosis, we have used confocal microscopy and live cell imaging, coupled with g-actin siRNA knockdown.…”

Identification of Cancer-Targeted Tropomyosin Inhibitors and Their Synergy with Microtubule Drugs

“…Error bars represent AE SD. Ã , P < 0.05; ÃÃ , P < 0.01; ÃÃÃ , P < 0.001; Indeed, it has previously been demonstrated that reduction in the levels of functional gamma-actin can both suppress microtubule dynamics (29) and contribute to the development of cellular resistance to antimicrotubule agents (30). These observations taken together are consistent with a collaborative role for actin filaments with microtubules in G 2 -M progression.…”