MAL2 and rab17 selectively redistribute invadopodia proteins to laterally-induced protrusions in hepatocellular carcinoma cells

Abstract

MAL2 (myelin and lymphocyte protein 2) and rab17 have been identified as hepatocellular carcinoma tumor suppressors. However, little is known how their functions in hepatic polarized protein sorting/trafficking translate into how they function in the epithelial-to-mesenchymal transition and/or the mesenchymal-to-epithelial transition in metastases. To investigate this, we expressed MAL2 and rab17 alone or together in hepatoma-derived Clone 9 cells (that lack endogenous MAL2 and rab17). Like MAL2, we found that rab17 expression led to the formation of actin- and cholesterol-dependent protrusions that correlated to its anti-oncogenic properties. MAL2 or rab17 selectively promoted the redistribution of invadopodia proteins to the protrusion tips that correlated with decreased matrix degradation. MAL2-mediated redistribution required a putative EVH1 recognition motif whereas rab17-mediated redistribution was GTP dependent. We also determined that MAL2 and rab17 interaction was GTP dependent, but not dependent on the MAL2 EVH1 recognition motifs, and that protrusions formed by their combined expression shared features of those induced by either alone. Finally, we report that MAL2 or rab17 can redirect trafficking of newly synthesized membrane proteins from the Golgi to the induced protrusions and that the EVH1 recognition motif was required in MAL2 and that rab17-mediated trafficking was GTP dependent.

FIGURE 1:

rab17 expression promotes protrusion formation that is GTP dependent and correlates with decreased migration. (A) A diagram of rab17 is shown indicating the GTP-bound/Q77L and GDP-bound/N132I mutations. All constructs were FLAG-tagged at the N-terminus. (B) Lysates from uninfected, wild type (WT), Q77L and N132I rab17-expressing cells were immunoblotted for rab17 or tubulin (as a loading control) as indicated. Molecular weight markers are indicated on the left in kDa. The 25 kDa and sumoylated 40 kDa rab17 (sumo-rab17) species are indicated with arrows. The asterisk marks a minor 36 kDa cross-reactive species. (C) Monolayers of uninfected cells or cells expressing wild-type or mutant rab17 as indicated were scratched and wound healing was imaged at 0 h (a–d) and 18 h (e–h) postscratch. White lines indicate the edges of the scratch gap. The boxes indicated in e–h were enlarged (i–l). Arrowheads in panels j and k indicate protrusions at the scratch edge in cells expressing wild-type or Q77L rab17. (D) The scratch gap area remaining after the indicated times of recovery was determined relative to the area of the initial scratch gap in cells as indicated. Values are expressed as the average ± SEM from at least three independent experiments; *** p ≤ 0.001. (E) A diagram of the cytosolic, N-terminal amino acid sequence of MAL2 is shown. The two putative proline-rich EVH1 recognition sites selected for mutational analyses are underlined. The amino acids in these regions were replaced with alanines. All constructs were FLAG-tagged at the C-terminus. (F) Lysates from uninfected, wild-type (WT), VPPPP and FPAP MAL2-expressing cells were immunoblotted for MAL2. Molecular weight markers are indicated on the left in kDa. MAL2 was detected at 19 kDa (the predicted molecular weight), 25 kDa and as a diffuse set of bands ranging from 30–50 kDa (marked with a bracket).  (G) Uninfected cells were labeled with actin and cells expressing wild-type, VPPPP or FPAP MAL2 were stained for MAL2. Arrowheads point to MAL2 detected in protrusion tips. Bar = 10 µm. (H) From micrographs, the percent of total cells showing either protrusion form was calculated and plotted for the indicated conditions. Values are expressed as the average ± SEM from at least three independent experiments; *** p ≤ 0.001. (I) Uninfected cells were labeled with actin and cells expressing FLAG-tagged wild-type, Q77L or N132I rab17 were labeled for FLAG. Arrowheads point to rab17 detected in protrusion tips. (J) From micrographs, the percent of total cells showing either protrusion form was calculated and plotted for the indicated conditions. Values are expressed as the average ± SEM from at least three independent experiments; *** p ≤ 0.001.

FIGURE 2:

MAL2 or rab17 expression promotes decreased matrix degradation, migration, and invasion. (A) Uninfected cells (a and b) and cells expressing MAL2 (c and d), VPPPP (e and f) or FPAP (g and h) MAL2 were labeled for actin (a, c, e, and g). Inverted images of FITC-labeled gelatin are shown in panels b, d, f, and h. (B) From micrographs, the contact area for each cell was measured and the percent of total cell contact area degraded was calculated. Values are expressed as the average ± SEM from at least three independent experiments; *** p ≤ 0.001. (C) Uninfected cells and cells expressing rab17, Q77L, or N132I rab17 as indicated were labeled for actin (a, c, e, and g). Inverted images of FITC-labeled gelatin are shown in panels b, d, f, and h. Bar = 10 µm. (D) From micrographs, the contact area for each cell was measured and the percent of total cell contact area degraded was calculated. Values are expressed as the average ± SEM from at least three independent experiments; *** p ≤ 0.001. (E–H) Uninfected cells or cells expressing wild-type or mutant rab17 were seeded in serum-free medium onto filters in Boyden chambers in the absence (E and F) or presence of matrigel (G and H). After 24 h, the nuclei of cells that had traversed the filter were labeled with DAPI and visualized (E and G). The number of migrating/invading rab17-expressing cells was determined relative to the number of uninfected cells and plotted as the percent of control (F and H).

FIGURE 3:

Tks5 redistributes from ventral puncta to protrusion tips in cells only expressing MAL2 while vinculin remains in focal adhesions in MAL2- or rab17-expressing cells. (A) Uninfected cells were colabeled for Tks5 and FLAG as indicated. An enlarged merged image is shown of the boxed area indicated in panel c. Cells expressing MAL2 or rab17 were labeled for Tks5 as indicated. In f and i, merged images are shown of the boxed area indicated in panels e and h. Bar = 10 µm. (B and C) Cells were infected with the indicated wild-type or mutant MAL2 or rab17 species. Cells were scored for the number of puncta per cell (B) or the number of protrusions positive for the indicated marker (C). In C, the values are plotted as the percent of total protrusion tips positive for each marker. Values are expressed as the average ± SEM from at least three independent experiments; ***p ≤ 0.001. (D) Uninfected cells were colabeled for vinculin and FLAG as indicated. An enlarged merged image is shown of the boxed area indicated in panel c. Cells expressing MAL2 or rab17 were labeled for vinculin as indicated. In f and i, merged images are shown of the boxed area indicated in panels e and h. (E and F) Cells were infected with the indicated wild-type or mutant MAL2 or rab17 species. Cells were scored for the number of protrusions positive for the indicated marker in MAL2-expressing cells (E) or rab17-expressing cells (F). In E and F, the values are plotted as the percent of total protrusion tips positive for vinculin. Values are expressed as the average ± SEM from at least three independent experiments; ***p ≤ 0.001.

FIGURE 4:

Mena and VASP, but not talin or α5β1 integrin, redistribute to the tips of MAL2-induced protrusions. (A) Uninfected or MAL2-expressing cells were labeled for Mena (a–d), VASP (e–h), talin (i–l), or α5β1 integrin (m–p). MAL2-expressing cells were also labeled for MAL2. The boxed regions in each of the panels are shown at higher magnification. Merged images are shown for cells expressing MAL2 (d, h, l, p). Bar = 10 µm. (B) Cells were scored for the number of protrusions positive for the indicated marker. The values are plotted as the percent of total protrusion tips positive for each marker. Values are expressed as the average ± SEM from at least three independent experiments; ***p ≤ 0.001. (C) Lysates from cells expressing MAL2 were incubated with FLAG antibodies to immunoprecipitate MAL2. The total lysate (lys), unbound (UB), bound (B) and control bound fractions (those that contained no IgG or no lysate as indicated) were immunoblotted for MAL2, Tks5, Mena, VASP, talin, or α5β1 integrin as indicated. Arrowheads point to the two prominent MAL2 immunoreactive species. (D) Lysates of uninfected cells or cells expressing wild-type or mutant MAL2 were immunoblotted for MAL2, rab17, Tks5, Mena, VASP, talin, or α5β1 integrin as indicated. Tubulin served as the loading control.

FIGURE 5:

Talin and α5β1 integrin, but not Mena or VASP, redistribute to the tips of rab17-induced protrusions. (A) Uninfected or rab17-expressing cells were labeled for Mena (a–d), VASP (e–h), talin (i–l), or α5β1 integrin (m–p). rab17-expressing cells were also labeled for rab17. The boxed regions in each of the panels are shown at higher magnification. Merged images are shown for cells expressing rab17 (d, h, l, p). Bar = 10 µm. (B) Cells were scored for the number of protrusions positive for the indicated marker. The values are plotted as the percent of total protrusion tips positive for each marker. Values are expressed as the average ± SEM from at least three independent experiments; ***p ≤ 0.001. (C) Lysates from cells expressing rab17 were incubated with FLAG antibodies to immunoprecipitate rab17. The total lysate (lys), unbound (UB), bound (B) and control bound fractions (those that contained no IgG or no lysate as indicated) were immunoblotted for rab17, Tks5, Mena, talin, or α5β1 integrin as indicated. Arrowheads point to the two rab17 immunoreactive species. (D) Lysates of uninfected cells or cells expressing wild-type or mutant rab17 were immunoblotted for MAL2, rab17, Tks5, Mena, VASP, talin, or α5β1 integrin as indicated. Tubulin served as the loading control.

FIGURE 6:

Mena and talin redistribute from invadopodia to the induced protrusions. (A) Uninfected cells or cells expressing wild-type, VPPPP or FPAP mutant MAL2 were labeled with Mena and phalloidin as indicated. (B) Cells were scored for the number of ventral puncta per cell (labeled with phalloidin) that were positive for Mena. The values are plotted as the percent of total ventral puncta that were Mena positive (upper graph). Cells were scored for the number of protrusions positive for the indicated marker. The values are plotted as the percent of total protrusion tips positive for each marker (lower graph). Values are expressed as the average ± SEM from at least three independent experiments; ***p ≤ 0.001. (C) Uninfected cells or cells expressing wild-type, GTP-bound/Q77L or GDP-bound N132I rab17 were labeled with talin and phalloidin as indicated. Bar = 10 µm. (D) Cells were scored for the number of ventral puncta per cell (labeled with phalloidin) that were positive for talin. The values are plotted as the percent of total ventral puncta that were talin positive (upper graph). Cells were scored for the number of protrusions positive for the indicated marker. The values are plotted as the percent of total protrusion tips positive for each marker (lower graph). Values are expressed as the average ± SEM from at least three independent experiments; ***p ≤ 0.001.

FIGURE 7:

MAL2 and rab17 interactions are GTP dependent, but EVH1 recognition independent and their coexpression induces protrusions that share features of those induced by MAL2 or rab17 expression alone. (A) Cells were processed for proximity ligation assay analysis and nuclei labeled with DAPI. Cells processed without antibody (-IgG) are shown (a). Cells coexpressing MAL2 and pIgA-R that were labeled for MAL2 and pIgA-R (b) served as a positive control for the assay while cells expressing MAL2 that were labeled for ZEB1 served as a negative control (c). Cells coexpressing MAL2 and rab17 (d), Q77L rab17 (e) or N132I rab17 (f) were labeled for MAL2 and rab17. Cells coexpressing rab17 and VPPPP MAL2 (g) or FPAP MAL2 (h) were labeled for MAL2 and rab17. Bar = 10 µm. (B) From micrographs, the puncta per cell were counted and the averages plotted for the indicated combinations. Values are expressed as the average ± SEM from at least three independent experiments; *** p ≤ 0.001. (C) Cells coexpressing either MAL2 and rab17 (a and b), MAL2 and Q77L rab17 (c and d) or MAL2 and N132I rab17 (e and f) were labeled for MAL2 and rab17. Arrowheads point to MAL2 and rab17 colocalizing in protrusion tips (a–d) or the cell surface with no protrusions (e and f). Bar = 10 µm. (D) Cells coexpressing either rab17 and VPPPP MAL2 (a and b) or rab17 and FPAP MAL2 (c and d) were labeled for MAL2 and rab17. Arrowheads point to rab17 and VPPPP MAL2 colocalizing in protrusion tips (a and b) or to protrusions positive for rab17 only (c and d). Bar = 10 µm. (E) From micrographs, the number of cells positive for short or long protrusions was counted. The percent of total cells showing either protrusion form was calculated and plotted for the indicated combinations. Values are expressed as the average ± SEM from at least three independent experiments; ***p ≤ 0.001. (F) Uninfected Hep3B cells or Hep3B cells expressing GDP-bound/N132I rab17 were labeled for actin. (G) From micrographs, the number of Hep3B cells positive for short or long protrusions was counted as in E. Values are expressed as the average ± SEM from at least three independent experiments; ***p ≤ 0.001.

FIGURE 8:

MAL2 and rab17 coexpression impairs matrix degradation, migration, and invasion and redistributes Tks5. (A) Uninfected cells (a and b), cells coexpressing MAL2 and rab17 (c and d), coexpressing MAL2 and N132I (e and f) or coexpressing rab17 and FPAP MAL2 were labeled for actin (a, c, e, g). Inverted images of FITC-labeled gelatin are shown in panels b, d, f, and h. Bar = 10 µm. (B) From micrographs, the contact area for each cell was measured and the percent of total cell contact area degraded was calculated. Values are expressed as the average ± SEM from at least three independent experiments; *** p ≤ 0.001. (C) Uninfected cells or cells coexpressing MAL2 and rab17 were seeded in serum-free medium onto filters in Boyden chambers in the absence (a and b) or presence of matrigel (c and d). After 24 h, the nuclei of cells that had traversed the filter were labeled with DAPI and visualized. (D) The number of migrating/invading rab17-expressing cells were determined relative to the number of uninfected cells and plotted as the percent of control. (E) Cells coexpressing MAL2 and rab17 were labeled for Tks5 and MAL2 (a and b) or Tks5 and rab17 (c and d). The boxed regions indicated in panels a or c were enlarged and merged images are shown (c and d). (F) From micrographs, the total number of Tks5-positive puncta per cell were counted and the averages plotted. Cells were also scored for the number of protrusions positive for Tks5. The values are plotted as the percent of total protrusion tips positive for Tks5. Values are expressed as the average ± SEM from at least three independent experiments; *** p ≤ 0.001. (G) Cells coexpressing MAL2 and N132I (a and b) were labeled for Tks5 and rab17. The boxed region indicated in panel a was enlarged and the merged image shown (b). Cells coexpressing rab17 and FPAP MAL2 (c and d) were labeled for Tks5 and rab17. The boxed region indicated in panel c was enlarged and merged images are shown (d). (H) From micrographs, the total number of Tks5-positive puncta per cell were counted and the averages plotted. Cells were also scored for the number of protrusions positive for Tks5. The values are plotted as the percent of total protrusion tips positive for Tks5. Values are expressed as the average ± SEM from at least three independent experiments; *** p ≤ 0.001.

FIGURE 9:

Mena, VASP, talin, and α5β1 integrin redistribute to MAL2 and rab17-induced protrusion tips. (A–D) Cells were coinfected with MAL2 and rab17 and labeled for Mena (A), VASP (B), talin (C), or α5β1 integrin (D). Enlarged merged images are shown of each marker with MAL2 or rab17 as indicated. Bar = 10 µm. (E) From micrographs, cells were scored for the number of protrusions positive for the indicated marker. The values are plotted as the percent of total protrusion tips positive for each marker. Values are expressed as the average ± SEM from at least three independent experiments; ***p ≤ 0.001. (F) Lysates of uninfected cells or cells expressing wild-type MAL2, rab17 or their respective mutants were immunoblotted for MAL2, rab17, Tks5, Mena, VASP, talin, or α5β1 integrin as indicated. Tubulin served as the loading control.

FIGURE 10:

MAL2 and rab17 expression routes cargo to protrusion tips. (A) Uninfected cells (a) or cells infected with pIgA-R were labeled for pIgA-R (b). Arrows mark pIgA-R present in the Golgi. (B) Cells coinfected with pIgA-R and wild-type (a and b) or mutant FPAP MAL2 (c and d) were labeled for MAL2 and pIgA-R. Arrows mark pIgA-R in MAL2 induced protrusions (a and b) or remaining at the Golgi in FPAP MAL2 expressing cells (c and d). (C) Cells coinfected with pIgA-R and wild-type (a and b) or N132I rab17 (c and d) were labeled for rab17 and pIgA-R. Arrows mark pIgA-R in rab17-induced protrusions (a and b) or remaining at the Golgi in N132I expressing cells (c and d). Bar = 10 µm. (D) Uninfected cells (a and b) or cells expressing MAL2 (c–f) were incubated at 19°C for 6 h. The cells were returned to 37°C for 1 h and labeled for Mena and MAL2 as indicated. Arrows mark Mena or MAL2 accumulated in the Golgi (c and e) or chased to the induced protrusions (d and f). (E) Uninfected cells (a and b) or cells expressing rab17 (c–f) were incubated at 19°C for 6 h. The cells were returned to 37°C for 1 h and labeled for α5β1 integrin or rab17 as indicated. Arrows mark α5β1 integrin (c) or rab17 accumulated in the Golgi (e) or chased to the induced protrusions (d and f).