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. 2009 Dec;20(24):5290-305.
doi: 10.1091/mbc.e08-10-1071.

Distinct Roles for CARMIL Isoforms in Cell Migration

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Free PMC article

Distinct Roles for CARMIL Isoforms in Cell Migration

Yun Liang et al. Mol Biol Cell. .
Free PMC article

Abstract

Molecular mechanisms for cell migration, especially how signaling and cytoskeletal systems are integrated, are not understood well. Here, we examined the role of CARMIL (capping protein, Arp2/3, and Myosin-I linker) family proteins in migrating cells. Vertebrates express three conserved genes for CARMIL, and we examined the functions of the two CARMIL genes expressed in migrating human cultured cells. Both isoforms, CARMIL1 and 2, were necessary for cell migration, but for different reasons. CARMIL1 localized to lamellipodia and macropinosomes, and loss of its function caused loss of lamellipodial actin, along with defects in protrusion, ruffling, and macropinocytosis. CARMIL1-knockdown cells showed loss of activation of Rac1, and CARMIL1 was biochemically associated with the GEF Trio. CARMIL2, in contrast, colocalized with vimentin intermediate filaments, and loss of its function caused a distinctive multipolar phenotype. Loss of CARMIL2 also caused decreased levels of myosin-IIB, which may contribute to the polarity phenotype. Expression of one CARMIL isoform was not able to rescue the knockdown phenotypes of the other. Thus, the two isoforms are both important for cell migration, but they have distinct functions.

Figures

Figure 1.
Figure 1.
CARMIL protein family sequence and expression analysis. (A) Domain architecture and sequence similarity among human CARMILs. CHD, CARMIL-homology domain; LRR, leucine-rich repeat; V, verprolin homology; CBR, capping protein-binding region (Bruck et al., 2006; Canton et al., 2006). (B) Alignment of CHD sequences. Residues identical to the majority in red, and similar ones in blue. (C) CARMIL family phylogenetic tree. Included proteins possess all three major domains: CHD, LRR, and CBR. Bootstrap analysis showed the branch points to be significant. Scale bar, the number of amino acid substitutions per site. Sequence accession numbers are in Materials and Methods. (D) Alignment of CBR sequences, performed as for the CHD sequences of B. (E) Expression of CARMIL and CP-alpha genes in cultured cells, determined by RT-PCR. Primers are listed in Supplemental Table S1. β-Actin was a positive control for loading. For CARMIL1, the primers flank a region present in CARMIL1a but not CARMIL1b, and the position of the single band is consistent with the predicted size of 443 base pairs for CARMIL1a. No band is seen at the position of 308 base pairs, predicted for CARMIL1b. For CARMIL2, the primers flank exon 37, which is present in CARMIL2a but not in CARMIL2b (Matsuzaka et al., 2004). The position of the upper band is consistent with the predicted size of 592 base pairs for CARMIL2a and that of the lower band with 511 base pairs, predicted for CARMIL2b. HT-1080 cells have more CARMIL2b than 2a. For the CP alpha subunit, isoforms alpha1 and alpha2 were detected, consistent with previous studies (Hart et al., 1997).
Figure 2.
Figure 2.
Knockdown of CARMIL1 or 2 inhibits cell migration during wound healing. (A) Knockdown of endogenous CARMIL1 protein. HT-1080 cells were transfected with vector, scrambled shRNA or shRNA targeting CARMIL1. Immunoblots of whole cell lysates were probed with anti-CARMIL1, as well as anti-CP and anti-actin. (B) Knockdown of endogenous CARMIL2 RNA. Cells were treated as in A, except with shRNA targeting CARMIL2, and RT-PCR was performed to assess the level of RNA expression. Serial twofold dilutions of the control RNA sample showed that the level of knockdown was between two- and fourfold (data not shown). (C) Cell migration during wound healing, monitored by time-lapse phase-contrast microscopy of HT-1080 cells. Images are frames from Movies 1–3. A monolayer of HT-1080 cells expressing scrambled, CARMIL1i, or CARMIL2 shRNA was scratched 3 d after transfection. Yellow lines show the boundary of the wound, the white arrow points in the direction of cell migration, and black arrowheads indicate protrusions and ruffles at the leading edge of cells. (D) Cell speed comparison, based on tracking nuclear position from frame to frame in time-lapse movies. At least 15 cells from two to three independent experiments were analyzed using Image J (http://rsb.info.nih.gov/ij/). Results plotted are mean and SEM; **p <0.001 and ***p < 0.0001.
Figure 3.
Figure 3.
Differential effects of CARMIL1 versus CARMIL2 knockdown on lamellipodium formation and cell polarity in HT-1080 cells. (A) Effects on lamellipodia revealed by fluorescence images of cells stained with coumarin-phalloidin. Lamellipodial regions are enlarged in insets. Arrowheads point to the leading edge of migrating cells. An RFP expression plasmid was cotransfected to identify transfected cells. Scale bar, 20 μm on all panels. (B) Scoring of the degree of polarity in cells, as a percentage of total cells. Polarity scored by blind observers as described (Sidani et al., 2007). n = >300 cells per group. Results plotted are mean and SEM from three experiments. Representative cells are shown. (C) Profile of fluorescent phalloidin intensity at the cell edge. The average fluorescence pixel intensity at each distance from the cell edge was determined by an Image J macro as described (Cai et al., 2007). The values were normalized to the highest value of the scrambled group. *p < 0.05 and **p < 0.001. n = >14 cells per group. (D) Effects on cell polarity revealed by time-lapse phase-contrast images, in frames from Movies 4–7. Black arrowheads indicate the leading edge of migrating cells, white arrowheads indicate sites of constriction, and white arrows indicate macropinosomes.
Figure 4.
Figure 4.
Effects of CARMIL1 versus 2 knockdown on lamellipodial dynamics and cell spreading. (A) Lamellipodial dynamics in frames from phase-contrast time-lapse Movies 8–10. Arrowheads indicate the cell edge. Scale bar, 20 μm. (B) Cell spreading, in frames from phase-contrast time-lapse Movies 11–13. White arrowheads, lamellipodia with features described in the text.
Figure 5.
Figure 5.
Effect of CARMIL1 knockdown on molecular markers and GTP-Rac1 activity. (A) Free barbed ends of actin filaments, assessed by incorporation of fluorescent actin in permeabilized cells. Scale bar, 20 μm on all panels. (B and C) Cells stained with antibodies to Arp2/3, cortactin, or VASP and costained with coumarin-phalloidin. Asterisks indicate transfected cells. Arrows in C indicate tips of filopodia. (D) Levels of GTP-Rac1 activity in cells spreading on fibronectin, over time in minutes. GTP-Rac1 was pulled down with PAK-PBD, and precipitates were analyzed by immunoblot.
Figure 6.
Figure 6.
Effect of CARMIL2 knockdown on polarity in migrating cells. (A) Fluorescence images of CARMIL2 knockdown cells stained for giantin of the Golgi complex and for microtubules. GFP-actin is a transfection marker. Arrows indicate the MTOC/Golgi. Insets show the relationship of microtubules with actin at the cell cortex. (B) MTOC polarization scored from images like those in A. Data are plotted as percent of cells ± SE of proportion; n = >100 cells per sample. (C) Cells stained with fluorescent antibodies for acetylated tubulin and γ-tubulin. Arrowheads indicate the centrosome. Scale bar, 20 μm.
Figure 7.
Figure 7.
Effect of CARMIL knockdown on Myosin-II. (A) Cells stained with anti-Myosin-IIA heavy chain and anti-paxillin Abs. Arrowheads, lamellipodia structures; arrows, focal adhesions. GFP-actin is a transfection marker. (B) Cells stained with anti-Myosin-IIB heavy chain and anti-Cortactin Abs. Arrows, Myosin-IIB localization; arrowheads, the leading edge of cells. Scale bar, 20 μm. (C) Expression of Myosin-II heavy chain isoforms in cells, analyzed by immunoblot.
Figure 8.
Figure 8.
Localization of CARMIL1 and 2. (A) HT-1080 cells expressing YFP, YFP-CARMIL1, or YFP-CARMIL2, stained with phalloidin-coumarin. Scale bar, 20 μm. (B) Cells cotransfected with RFP-CARMIL1 and YFP-CARMIL2 and then stained with anti-cortactin Abs. Arrowheads indicate areas of the leading edge where CARMIL1 and 2 localizations differ. Merge includes RFP-CARMIL1 and YFP-CARMIL2. (C) Sequential images of live cells expressing YFP-CARMIL1 or YFP-CARMIL2. Arrowheads point to macropinosome formation. Boxed regions are enlarged to show the detail. See Movies 14–15.
Figure 9.
Figure 9.
CARMIL2 colocalization with vimentin filaments. (A) Cells were treated with withaferin A (WFA) under conditions chosen for maximal collapse of vimentin filaments with minimal effects on microtubules. Fixed cells were stained for vimentin, microtubules, and filamentous actin. Scale bar, 20 μm. (B) Efficacy of vimentin knockdown determined by immunoblot. (C) Effects of vimentin knockdown on CARMIL2 localization. Control scrambled and vimentin-knockdown cells were cotransfected with YFP-CARMIL2 and stained with anti-vimentin antibody. Scale bar, 20 μm. (D) Fluorescence images showing colocalization of YFP-CARMIL2 with vimentin but not keratin in A549 cells. Control and WFA-treated cells are shown.
Figure 10.
Figure 10.
Cross-rescue expression analysis. CARMIL1 knockdown cells expressing CARMIL2, and vice versa. Cells are stained with coumarin-phalloidin. The characteristic phenotypes were not rescued in either case. In controls, each expression plasmid rescues its own knockdown phenotypes (Liang et al., 2009).

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