Figure 4.
Cap1/2 is not detected on actin cables but localizes to actin patches and the cytokinetic contractile ring (CAR). (A) Maximum intensity projections of Airyscan images of live small and medium budded cells expressing Cap2–3xmNeon (cyan). Cells were incubated with DMSO (carrier control) or the Arp2/3 inhibitor, CK666, to eliminate Arp2/3-generated patches. In DMSO-treated cells Cap2–3xNeon signal is observed in the cytosol, at Arp2/3-generated actin patches, and at the CAR at the mother-bud neck (white arrow). Upon CK666 treatment, patch associated Cap2–3xNeon signal is lost while the neck-associated signal (white arrow) is maintained, confirming that this signal is not from patches. (B) A TIRF image of a cell expressing Cap2–3xmNeon and a patch marker (Arc15-RFPmScarlet). White arrows indicate Cap2–3xmNeon puncta at the cell cortex not associated with Arc15-RFPmScarlet marked patches. (C) TIRF images of Cap2-3xNeon signal at the cell cortex in cells treated with DMSO, CK666 (to eliminate patches but not cables), or latrunculin B (LatB; to eliminate all F-actin). In the DMSO-treated cell, bright Cap2–3xMNeon signal is observed at patches (yellow arrows) in addition to dim Cap2–3xmNeon puncta (white arrows). Upon CK666 or LatB treatment, bright Cap2–3xmNeon signal is lost and only dim puncta remain. (D) Frequency distribution showing the percent of Cap2–3xmNeon puncta traveling at the indicated speeds in CK666 (no patches) and LatB (no cables or patches) treated cells (n = 5,549 tracks from 10 CK666 treated cells and 16,742 tracks from 10 LatB treated cells). There is no significant difference between particle speeds in CK666-treated or LatB-treated cells calculated by one-way ANOVA (P = 0.1071). Scale bar = 2 μm.

Cap1/2 is not detected on actin cables but localizes to actin patches and the cytokinetic contractile ring (CAR). (A) Maximum intensity projections of Airyscan images of live small and medium budded cells expressing Cap2–3xmNeon (cyan). Cells were incubated with DMSO (carrier control) or the Arp2/3 inhibitor, CK666, to eliminate Arp2/3-generated patches. In DMSO-treated cells Cap2–3xNeon signal is observed in the cytosol, at Arp2/3-generated actin patches, and at the CAR at the mother-bud neck (white arrow). Upon CK666 treatment, patch associated Cap2–3xNeon signal is lost while the neck-associated signal (white arrow) is maintained, confirming that this signal is not from patches. (B) A TIRF image of a cell expressing Cap2–3xmNeon and a patch marker (Arc15-RFPmScarlet). White arrows indicate Cap2–3xmNeon puncta at the cell cortex not associated with Arc15-RFPmScarlet marked patches. (C) TIRF images of Cap2-3xNeon signal at the cell cortex in cells treated with DMSO, CK666 (to eliminate patches but not cables), or latrunculin B (LatB; to eliminate all F-actin). In the DMSO-treated cell, bright Cap2–3xMNeon signal is observed at patches (yellow arrows) in addition to dim Cap2–3xmNeon puncta (white arrows). Upon CK666 or LatB treatment, bright Cap2–3xmNeon signal is lost and only dim puncta remain. (D) Frequency distribution showing the percent of Cap2–3xmNeon puncta traveling at the indicated speeds in CK666 (no patches) and LatB (no cables or patches) treated cells (n = 5,549 tracks from 10 CK666 treated cells and 16,742 tracks from 10 LatB treated cells). There is no significant difference between particle speeds in CK666-treated or LatB-treated cells calculated by one-way ANOVA (P = 0.1071). Scale bar = 2 μm.

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