Figure 1. Septin filaments interface with RSF and TA stress fibers in the leading lamellae of renal epithelia. (A) Confocal images of SEPT2 (green), actin stress fibers (phalloidin; red), and FAs (paxillin; white) in HGF-treated (16 h) MDCK epithelia. White arrows point to RSFs that connect with the TA. (B) 3D rendering of the lamellar region outlined in A. Arrows point to SEPT2 fibers that localize on FA-anchored RSFs. Bars, 1 µm. (C) Quantification of SEPT2 colocalization with F-actin (green) and vice versa (red). Error bars indicate SEM. (D) SIM images show ventral and dorsal optical sections from the lamellar region of an HGF-treated MDCK cell. The arrowhead points to SEPT9 localization on the dorsal segment of an RSF that connects with the TA. (E) SIM images show an HGF-treated MDCK cell stained for F-actin (phalloidin; red), SEPT2 (green), and paxillin (white). FA-anchored RSF (1) and TA filaments (2) are shown in higher magnification. Arrowheads point to SEPT2 localization on and between actin filaments. (F) Still frames show FA formation (paxillin-mCherry; red) and subsequent recruitment of SEPT2-YFP (green). Arrows point to septin accumulation at the distal end of a nascent focal adhesion. (G) TIRF microscopy kymograph shows recruitment of SEPT2-YFP (green) to a nascent RSF (ABP140-ABD-mCherry, red). (H) Inverted monochrome images show frames from spinning disk confocal time-lapse microscopy of HGF-treated MDCK cells expressing ABP140-ABD-mCherry and SEPT2-YFP. A subset of TA filaments and an RSF were pseudo-colored red and superimposed onto the inverted monochrome image of the SEPT2-YFP channel. SEPT2-YFP elements recruited to the junction of the RSF with the TA were pseudo-colored green. The overlay image shows the pseudocolored stress fibers and SEPT2 outlined in the inverted monochrome frames. The arrow points to the RSF end and its junction point with the TA.
Figure 1.

Septin filaments interface with RSF and TA stress fibers in the leading lamellae of renal epithelia. (A) Confocal images of SEPT2 (green), actin stress fibers (phalloidin; red), and FAs (paxillin; white) in HGF-treated (16 h) MDCK epithelia. White arrows point to RSFs that connect with the TA. (B) 3D rendering of the lamellar region outlined in A. Arrows point to SEPT2 fibers that localize on FA-anchored RSFs. Bars, 1 µm. (C) Quantification of SEPT2 colocalization with F-actin (green) and vice versa (red). Error bars indicate SEM. (D) SIM images show ventral and dorsal optical sections from the lamellar region of an HGF-treated MDCK cell. The arrowhead points to SEPT9 localization on the dorsal segment of an RSF that connects with the TA. (E) SIM images show an HGF-treated MDCK cell stained for F-actin (phalloidin; red), SEPT2 (green), and paxillin (white). FA-anchored RSF (1) and TA filaments (2) are shown in higher magnification. Arrowheads point to SEPT2 localization on and between actin filaments. (F) Still frames show FA formation (paxillin-mCherry; red) and subsequent recruitment of SEPT2-YFP (green). Arrows point to septin accumulation at the distal end of a nascent focal adhesion. (G) TIRF microscopy kymograph shows recruitment of SEPT2-YFP (green) to a nascent RSF (ABP140-ABD-mCherry, red). (H) Inverted monochrome images show frames from spinning disk confocal time-lapse microscopy of HGF-treated MDCK cells expressing ABP140-ABD-mCherry and SEPT2-YFP. A subset of TA filaments and an RSF were pseudo-colored red and superimposed onto the inverted monochrome image of the SEPT2-YFP channel. SEPT2-YFP elements recruited to the junction of the RSF with the TA were pseudo-colored green. The overlay image shows the pseudocolored stress fibers and SEPT2 outlined in the inverted monochrome frames. The arrow points to the RSF end and its junction point with the TA.

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