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. 2011 Nov 29;108(48):19228-33.
doi: 10.1073/pnas.1117011108. Epub 2011 Nov 14.

Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing

Chun-Chun Li  1 , Jean-Cheng Kuo, Clare M Waterman, Ryoiti Kiyama, Joel Moss, Martha Vaughan
Affiliations

Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing

Chun-Chun Li et al. Proc Natl Acad Sci U S A. .

Abstract

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates class I ADP ribosylation factors (ARFs) by accelerating the replacement of bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Among proteins that interact with BIG1, kinesin family member 21A (KIF21A), a plus-end-directed motor protein, moves cargo away from the microtubule-organizing center (MTOC) on microtubules. Because KANK1, a protein containing N-terminal KN, C-terminal ankyrin-repeat, and intervening coiled-coil domains, has multiple actions in cells and also interacts with KIF21A, we explored a possible interaction between it and BIG1. We obtained evidence for a functional and physical association between these proteins, and found that the effects of BIG1 and KANK1 depletion on cell migration in wound-healing assays were remarkably similar. Treatment of cells with BIG1- or KANK1-specific siRNA interfered significantly with directed cell migration and initial orientation of Golgi/MTOC toward the leading edge, which was not mimicked by KIF21A depletion. Although colocalization of overexpressed KANK1 and endogenous BIG1 in HeLa cells was not clear microscopically, their reciprocal immunoprecipitation (IP) is compatible with the presence of small percentages of each protein in the same complexes. Depletion or overexpression of BIG1 protein appeared not to affect KANK1 distribution. Our data identify actions of both BIG1 and KANK1 in regulating cell polarity during directed migration; these actions are consistent with the presence of both BIG1 and KANK1 in dynamic multimolecular complexes that maintain Golgi/MTOC orientation, differ from those that might contain all three proteins (BIG1, KIF21A, and KANK1), and function in directed transport along microtubules.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Immunoprecipitation of KANK1 with BIG1. (A and B) Samples of Input (2.5% of total) and 25% of proteins from IP with antibodies against BIG1, BIG2, or KANK1, or control IgG from extracts of HeLa cells (1 mg) were separated by SDS/PAGE before reaction of Western blots with indicated antibodies. (A and B are enlarged in Fig. S1.) (C) Samples (50%) of proteins precipitated with anti-HA antibodies or control IgG from 200 μg of extracts prepared 24 h after transfection of cells with HA-KIF21A (HA-21A) or empty vector (EV) were used for Western blotting with indicated antibodies. Input (20 μg) was 10% of amount used for IP. (D) Cells transfected with 100 nM nontargeted (NT) or KANK1-specific siRNA or 100 or 150 nM KIF21A-specific siRNA or with vehicle alone (Mock) were lysed 48 h later. Proteins precipitated with antibodies against BIG1 were analyzed by Western blotting and densitometric quantification. Amounts of proteins from BIG1 IP in three experiments expressed relative to that of the same protein in Mock cells (=100%) are means ± SEM *P < 0.005 vs. Mock. Arrow: protein band. Arrowhead: position of 160-kDa marker.
Fig. 2.
Fig. 2.
Endogenous BIG1 and overexpressed KANK1 in HeLa cells. After transfection (24 h) with cDNA encoding untagged KANK1, cells were fixed, reacted with rabbit anti-BIG1 and mouse anti-KANK1 antibodies, and prepared for confocal immunofluorescence microscopy. In three Z-stack images of 1-μm planes (upper, middle, and lower), overexpressed KANK1 (red) did not apparently coincide with or alter the distribution of endogenous BIG1 (green), which is scattered in punctate collections through cytoplasm of all cells, with greatest perinuclear concentration in the upper plane. (Scale bar, 10 μm.)
Fig. 3.
Fig. 3.
Effects of BIG1, BIG2, KANK1, or KIF21A depletion on wound healing. (A) Cells were lysed 48 h after transfection with nontargeted (NT), BIG1, BIG2, KANK1, or KIF21A siRNA or vehicle alone (Mock) and amounts of proteins quantified by densitometry of Western blots. (B) Images of cells at 0 and 6 h after wounding as in A. (Scale bar, 70 μm.) (C) Covered area is the difference between wound areas 0 and 6 h after wounding. Data are means ± SEM of values from five experiments. **P < 0.005 *; P < 0.05, (two-tailed t test) for difference from cells transfected with nontargeted siRNA. (D) Migration paths of five representative cells at wound edges during 6-h assays (B). Initiation of cell migration = 0. (E–G) Motility characteristics in B and C were calculated for individual cells as described in Materials and Methods. Data are means ± SEM of values from three experiments for at least 120–150 cells in each group. **P < 0.005; *P < 0.05, (ANOVA test) for difference from cells transfected with vehicle alone (Mock).
Fig. 4.
Fig. 4.
Depletion of BIG1 or KANK1 interfered with cell polarization during wound healing. Confluent monolayers of HeLa cells transfected 48 h before with indicated siRNA or vehicle alone (Mock), as in Fig.3, were wounded and fixed 6 h later for staining with DAPI and anti-GM130 (A) or anti-γ-tubulin antibodies (B) and confocal immunofluorescence microscopy. Arrowheads indicate GM130 (A) or γ-tubulin (B). (Scale bar, 10 μm.) (C) Percentage of wound-edge cells with Golgi or γ-tubulin structures in forward-facing 120° sector between nucleus and wound was recorded for at least 100 cells of each population for Golgi localization and 30 cells for γ-tubulin in each experiment. Data are means ± SEM of values from six experiments. **P < 0.02.
Fig. 5.
Fig. 5.
Effects of BIG1, BIG2, KANK1, or KIF21A depletion on intracellular microtubule and actin morphology 6 h after wounding. HeLa cells treated as in Figs. 3 and 4 were fixed 6 h after wounding and reacted with anti-α-tubulin antibodies to mark microtubules (A) or Alexa Fluor 488-conjugated phalloidin for F-actin (B). Patterns of microtubules and F-actin were altered in cells depleted of any of the four proteins, but effects on F-actin were most obvious and prominent in cells treated with BIG1 or KANK1 siRNA. (Scale bar, 10 μm.) Arrowheads indicate wound-edge membrane.
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