Figure 1.
Coronin and actin regulatory factors are partitioned to the base of the bud during fission. (A) Representative images of COS-7 cells transfected with GFP-Rab7 (LE, magenta) and mCh-FAM21 (WASH complex, green). Magnified inset (5 × 5 μm) below shows time lapse of a representative fission event. Note: FAM21 signal localizes along entire length of bud, and signal is present on the post-fission bud. Block arrowhead indicates the bud neck, and line arrowhead indicates departing bud (Video 1). (B) Line scan analysis along the length of endosome bud in pre-fission frame from A shows FAM21 signal labels the length of the bud. Lines are shown in A adjacent to actual area measured so as not to obscure ROI. (C) Fission events as in A were scored for FAM21 enrichment at the bud neck in the pre-fission, fission, and post-fission frames. For post-fission frames, both the bud neck and departed bud were scored for positive FAM21 signal. Table shows data as percentage of fission events with enrichment at each stage (n = 37 fission events in 16 cells, performed in triplicate). Model indicates where enrichment was assessed at each stage of fission. (D–F) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and ARP3-mEm (ARP2/3 complex, green). Secondary inset shows departed bud that moved out of primary inset. In F, n = 24 fission events in 11 cells, performed in triplicate (Video 2). (G–I) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and α-actin-mNG (actin structure, green). For I, n = 19 fission events in 14 cells, performed in triplicate (Video 3). (J–L) As in A–C, for COS-7 cells transfected with GFP-Rab7 (LE, magenta) and COR1C Halo (green). For L, n = 36 fission events in 21 cells, performed in triplicate. Scale bars for whole cell = 5 μm; insets = 1 μm (Video 4). (M) Summary diagram of how actin recruitment and regulatory factors divide during fission.

Coronin and actin regulatory factors are partitioned to the base of the bud during fission. (A) Representative images of COS-7 cells transfected with GFP-Rab7 (LE, magenta) and mCh-FAM21 (WASH complex, green). Magnified inset (5 × 5 μm) below shows time lapse of a representative fission event. Note: FAM21 signal localizes along entire length of bud, and signal is present on the post-fission bud. Block arrowhead indicates the bud neck, and line arrowhead indicates departing bud (Video 1). (B) Line scan analysis along the length of endosome bud in pre-fission frame from A shows FAM21 signal labels the length of the bud. Lines are shown in A adjacent to actual area measured so as not to obscure ROI. (C) Fission events as in A were scored for FAM21 enrichment at the bud neck in the pre-fission, fission, and post-fission frames. For post-fission frames, both the bud neck and departed bud were scored for positive FAM21 signal. Table shows data as percentage of fission events with enrichment at each stage (n = 37 fission events in 16 cells, performed in triplicate). Model indicates where enrichment was assessed at each stage of fission. (D–F) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and ARP3-mEm (ARP2/3 complex, green). Secondary inset shows departed bud that moved out of primary inset. In F, n = 24 fission events in 11 cells, performed in triplicate (Video 2). (G–I) As in A–C, for COS-7 cells transfected with mCh-Rab7 (LE, magenta) and α-actin-mNG (actin structure, green). For I, n = 19 fission events in 14 cells, performed in triplicate (Video 3). (J–L) As in A–C, for COS-7 cells transfected with GFP-Rab7 (LE, magenta) and COR1C Halo (green). For L, n = 36 fission events in 21 cells, performed in triplicate. Scale bars for whole cell = 5 μm; insets = 1 μm (Video 4). (M) Summary diagram of how actin recruitment and regulatory factors divide during fission.

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