Cdc42 localization and cell polarity depend on membrane traffic - PubMed (original) (raw)
Cdc42 localization and cell polarity depend on membrane traffic
Naël Osmani et al. J Cell Biol. 2010.
Abstract
Cell polarity is essential for cell division, cell differentiation, and most differentiated cell functions including cell migration. The small G protein Cdc42 controls cell polarity in a wide variety of cellular contexts. Although restricted localization of active Cdc42 seems to be important for its distinct functions, mechanisms responsible for the concentration of active Cdc42 at precise cortical sites are not fully understood. In this study, we show that during directed cell migration, Cdc42 accumulation at the cell leading edge relies on membrane traffic. Cdc42 and its exchange factor βPIX localize to intracytosplasmic vesicles. Inhibition of Arf6-dependent membrane trafficking alters the dynamics of Cdc42-positive vesicles and abolishes the polarized recruitment of Cdc42 and βPIX to the leading edge. Furthermore, we show that Arf6-dependent membrane dynamics is also required for polarized recruitment of Rac and the Par6-aPKC polarity complex and for cell polarization. Our results demonstrate influence of membrane dynamics on the localization and activation of Cdc42 and consequently on directed cell migration.
Figures
Figure 1.
GFP-Cdc42 localizes to plasma membrane and intracellular vesicles of migrating cells. (A) GFP-Cdc42 (top left) and GFP (top right) fluorescence in migrating primary astrocytes. (bottom left) Higher magnification view of the boxed region shows accumulation of GFP-Cdc42 at the leading edge. (bottom right) Fluorescence intensity of GFP-Cdc42 (blue) measured along three lines (as indicated) drawn across the leading edge (blue) or side edge (green) is shown. Fluorescence intensity of GFP measured along three lines drawn across the leading edge of the GFP-expressing cell is shown in red. Cell–cell contact areas with increased Cdc42 intensity have been disregarded. (B) GFP-Cdc42 fluorescence in a nonmigrating confluent astrocyte (left) and image showing the maximum projection of
Video 3
(right). (C) Images showing the maximum projection of
Video 2
. A higher magnification of the boxed area is shown on the right. Directed movement of small Cdc42-positive vesicles appears as white lines along the length of the protrusion. Large immobile Cdc42-positive vesicles are indicated with asterisks. (D) Still images from Video 2 showing the retrograde (black arrow) and anterograde movement (white arrows) of Cdc42-positive vesicles in the cell protrusion. (E) GM130 staining (red) in GFP-Cdc42 (green)–expressing cells. (F) EEA1 staining (red) in GFP-Cdc42 (green)–expressing cells showing the endosomal EEA1 marker on Cdc42-positive vesicles (arrowheads). Images are representative of at least 100 cells from three independent experiments. The dashed line indicates the direction of the wound. Bars, 10 µm.
Figure 2.
Arf6 colocalizes with Cdc42 and regulates Cdc42 dynamics and localization. (A) Colocalization of GFP-Cdc42 (green) and CFP-Arf6 (red) in a migrating astrocyte. Higher magnification images of boxed areas 1 and 2 are shown on the left and right, respectively. CFP-Arf6 and GFP-Cdc42 colocalized (arrowheads) in discrete areas at the leading edge plasma membrane (box 1) and on intracellular vesicles (box 2). (B) Fluorescence intensities of GFP-Cdc42 (green) and CFP-Arf6 (red) measured along the line shown in A (box 1). (C) GFP-Cdc42 recruitment to intracytoplasmic vesicles in astrocytes microinjected with the indicated constructs or nucleofected with the indicated siRNA. (D) Maximum projection of
Video 4
. (bottom) A higher magnification of the boxed area is shown. (E) Recruitment of GFP-Cdc42 to the leading edge of migrating astrocytes after microinjection with the indicated constructs or nucleofection with the indicated siRNA. (F) GFP-Cdc42 fluorescence images in cells microinjected with the indicated constructs or nucleofected with the indicated siRNA. (right) Higher magnification images of the leading edges (boxed areas) are shown. Dotted lines indicate the direction of the wound. Data are shown as mean ± SEM of three to six independent experiments totalizing >200 cells. Bars, 10 µm.
Figure 3.
Arf6 controls the localization of the Cdc42-GEF βPIX and Cdc42 activation upon wounding. (A) Wound-induced Cdc42 activation in cells nucleofected with the indicated siRNA. (left) Western blots showing a representative experiment. (right) Histogram showing the quantitative measurement of Cdc42 activity. Values are normalized by the Cdc42 activity observed in the control condition (siCTL, time 0). **, P = 0.01. (B) Colocalization of GFP-Cdc42 (green) and HA-βPIX (red) in a migrating astrocyte. (C) Magnified view of the front edge area of the cell shown in B. Arrowheads point to regions showing colocalization of GFP-Cdc42 and HA-βPIX. Fluorescence intensities of GFP-Cdc42 (green) and HA-βPIX (red) along the line drawn in the image show the colocalization of Cdc42 and βPIX at the leading edge and on some of the Cdc42-positive vesicles. Large Cdc42-positive vesicles (asterisks) are usually not stained with the anti-βPIX antibody. (D) βPIX recruitment to the leading edge of astrocytes nucleofected with the indicated siRNA. (left) The percentage of cells with a leading edge accumulation of βPIX. (right) Representative images of βPIX immunofluorescence in cells nucleofected with the indicated siRNA. Higher magnification views of the boxed regions are shown. Dotted lines indicate the direction of the wound. Data are shown as mean ± SEM of three independent experiments totalizing >300 cells. Bars, 10 µm.
Figure 4.
Arf6 controls the Cdc42-mediated polarity signaling pathway and cell orientation during migration. Astrocytes were nucleofected with the indicated siRNA or microinjected with the indicated constructs. (A) Immunostaining of Par6 and aPKC in migrating astrocytes 4 h after wounding. (right) Higher magnification views of the boxed regions are shown. Graphs plotting the fluorescence intensity of Par6 and aPKC measured along lines drawn perpendicularly to the leading edge of three different siCTL (blue)- and siArf6-2 (red)–transfected cells are shown below the corresponding images. Histogram showing the percentage of wound-edge cells with a leading edge accumulation of Par6 (black bars) and aPKC (gray bars). (B) Immunostaining of Thr410-phosphorylated aPKC in control or Arf6-depleted astrocytes 4 h after wounding. (right) Higher magnification views of the boxed areas are shown. The histogram shows the percentage of cells with an accumulation of phospho-aPKC at the leading edge. (C) Immunostaining of APC (red) and tubulin (green) in migrating astrocytes 8 h after wounding. (right) Histogram shows the percentage of wound-edge cells with APC clusters at microtubule plus ends. (D) Fluorescence images of the microtubules (green), centrosome (red), and nucleus (blue) in nucleofected migrating astrocytes 8 h after wounding. (E) Centrosome and Golgi reorientation 8 h after wounding. (F) Immunostaining of tubulin in migrating astrocytes 16 h after wounding. Data are shown as mean ± SEM of three independent experiments totalizing >300 cells. The dotted lines indicate the direction of the wound. Bars, 10 µm.
Figure 5.
Arf6-dependent membrane traffic and wound-induced integrin engagement control Cdc42 activation at the wound edge. Cdc42 accumulation and activation at the wound edge result from wound-induced integrin signaling (Etienne-Manneville and Hall, 2001) and Arf6-dependent vesicular delivery of Cdc42 and its GEF βPIX. We propose that βPIX interaction with Scrib (Osmani et al., 2006) and phosphorylation by Src (Feng et al., 2006) at the wound edge promotes βPIX GEF activity toward Cdc42 and thus leads to the generation of a Cdc42-mediated polarity signal that involves Par6, aPKC, and APC (Etienne-Manneville and Hall, 2003).
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