Vimentin filaments support extension of tubulin-based microtentacles in detached breast tumor cells

Abstract

Solid tumor metastasis often involves detachment of epithelial carcinoma cells into the vasculature or lymphatics. However, most studies of cytoskeletal rearrangement in solid tumors focus on attached cells. In this study, we report for the first time that human breast tumor cells produce unique tubulin-based protrusions when detached from extracellular matrix. Tumor cell lines of high metastatic potential show significantly increased extension and frequency of microtubule protrusions, which we have termed tubulin microtentacles. Our previous studies in nontumorigenic mammary epithelial cells showed that such detachment-induced microtentacles are enriched in detyrosinated alpha-tubulin. However, amounts of detyrosinated tubulin were similar in breast tumor cell lines despite varying microtentacle levels. Because detyrosinated alpha-tubulin associates strongly with intermediate filament proteins, we examined the contribution of cytokeratin and vimentin filaments to tumor cell microtentacles. Increased microtentacle frequency and extension correlated strongly with loss of cytokeratin expression and up-regulation of vimentin, as is often observed during tumor progression. Moreover, vimentin filaments coaligned with microtentacles, whereas cytokeratin did not. Disruption of vimentin with PP1/PP2A-specific inhibitors significantly reduced microtentacles and inhibited cell reattachment to extracellular matrix. Furthermore, expression of a dominant-negative vimentin mutant disrupted endogenous vimentin filaments and significantly reduced microtentacles, providing specific genetic evidence that vimentin supports microtentacles. Our results define a novel model in which coordination of vimentin and detyrosinated microtubules provides structural support for the extensive microtentacles observed in detached tumor cells and a possible mechanism to promote successful metastatic spread.

Fig. 1. Microtentacles in detached human breast tumor cell lines

(A) Human mammary epithelial cells (MCF10A) and breast tumor cell lines transfected with GFP-membrane (GFP-Mem) and plated over low-attachment wells display morphologically different extensive membrane microtentacles 30min following detachment (black arrows). Invasive cell lines (HCC1395, Hs578t, MDA-MB-436, MDA-MB-157) exhibit long, flexible microtentacles while non-invasive lines (MCF10A, ZR75-1, Bt-20, SkBr3, MDA-MB-468) display short, rigid microtentacles. (B) Treatment with actin-depolymerizing agent Latrunculin-A (LA, 5μM) enhanced the flexibility and length of observed microtentacles. (C) Populations of live, suspended cells were scored blindly for two or more microtentacles longer than the cell radius. Each bar represents the mean +S.D. for three experiments in which at least 100 single, GFP-Mem⁺ cells were counted. Vimentin-expressing, invasive cell lines had a statistically-significant higher frequency then non-vimentin expressing, non-invasive cell lines (P<0.05, t-test, black asterisks). (D) Western blot expression profile of Glu-tubulin, α-tubulin, PAN-cytokeratin, and vimentin indicates that cells with higher microtentacle frequencies express elevated vimentin, lower cytokeratin and Glu-tubulin remains largely unchanged.

Fig. 2. Vimentin, not cytokeratin, is localized to membrane microtentacles

(A) MCF10A and MDA-436 were suspended for 30min media containing 5μM Latrunculin-A. Cells were fixed in 0.5% glutaraldehyde while in suspension and gently spun onto glass coverslips coated with 1% poly(ethyleneimine) (PEI) solution. Cells were fluorescently stained for vimentin or PAN cytokeratin (CytoK). Microtentacles stain positively for vimentin but not for cytokeratin. Microtentacles can be seen both by phase-contrast (black arrow) and fluorescence (white arrow).

Fig. 3. Vimentin aligns with Glu-tubulin in membrane microtentacles

MCF10A and MDA-436 were suspended for 30min in (A,B) DMEM or (C) media containing 5μM Latrunculin-A. Cells were fixed in 0.5% glutaraldehyde while in suspension and gently spun onto glass coverslips coated with 1% poly(ethyleneimine) (PEI) solution. (A) Immunostaining indicates that both vimentin and Glu-tubulin align in membrane microtentacles. (B) A confocal maximum intensity projection (MIP) of an MDA-MB-436 cell computationally flattens the 3-dimensional structure of the detached cell to allow visualization of microtentacles in different imaging planes. This image and one that has been deconvolved (Slidebook) show clear localization of vimentin in microtentacles (arrows). (C) MDA-MB-436 display significantly longer microtentacles compared to MCF10A. Microtentacles can be seen both by phase-contrast (black arrow) and fluorescence (white arrow).

Fig. 4. Inhibitors of PP1/PP2A disrupt vimentin without toxicity

Disruption of vimentin intermediate filaments following 1h treatment in PP1/PP2A inhibitors is observed in attached MCF10A and MDA-MB-436 seeded on glass coverslips. (A) MCF10A and MDA-MB-436 cells grown on glass coverslips were left untreated in growth media (A, E); vehicle (0.002% EtOH) treated in DMEM (B, F); 5nM calyculin-A (Cal-A) (C,G) ; or 1μM okadaic acid (OKA) (D, H). Vimentin is heterogeneously expressed in MCF10A while homogeneously expressed in MDA-MB-436 (inset depicts reference DNA stain). Treatment with PP1/PP2A inhibitors causes vimentin fragmentation and retraction to the perinuclear region compared to untreated and vehicle treated cells (white arrows). (B) Cell lysates of MCF10A and MDA-MB-436 treated over for 1h, 3h, and 6hr in Vehicle (0.002% EtOH), OKA (1μM), Cal-A (5nM), and positive control treatment cycloheximide (CHX, 30μg/ml) were immunobloted for PARP cleavage to show minimal degree of apoptosis caused by PP1/PP2A inhibitor treatment. PARP cleavage is observed in the MCF10A positive control but no significant cleavage in presence of PP1/PP2A inhibitors. (C) XTT viability assay shows minimal loss of cell viability via necrosis up to 24h in presence of PP1/PP2A inhibitor treatment.

Fig. 5. Vimentin disassembly with PP1/PP2A inhibitors reduces microtentacles and attachment

(A) Transiently transfected GFP-Mem MCF10A and MDA-MB-436 were left untreated (■) or pretreated for 1h in OKA (1μM, ) or Cal-A (5nM, ) then suspended in DMEM −/+ LA (5μM) in combination with respective pretreatment. Blind microtentacle counts were performed following 30min suspension. Each bar represents the +S.D. for three experiments in which at least 100 GFP-Mem⁺ cells were counted. Pretreatment with both inhibitors and subsequent suspension in the same treatment show a statistically significant reduction in microtentacles in a cell population compared to the untreated counterpart (P<0.05, t-test, black asterisks). Suspension of cells in the presence of LA also show decrease in microtentacle frequency in combination with PP1/PP2A inhibitors (P<0.05, t-test, black asterisks). (B) DIC timecourse of transiently attached MDA-MB-436 displaying motile membrane microtentacles captured over 30min timecourse to observe effects of PP1/PP2A inhibitors on microtentacles (white arrows). (C) Disruption of vimentin by PP1/PP2A inhibitors affects cell attachment to surfaces. MCF10A and MDA-MB-436 were left untreated () or pretreated for 1h in Cal-A (5nM, ) or OKA (1μM, ). Cells were detached and suspended over uncoated tissue culture plates or laminin coated plates in the presence of the respective PP1/PP2A inhibitor pretreatment. Reattachment efficiency was measured by XTT viability at 24h post-detachment. Values represent mean +S.D. between triplicate wells of the % cells attached in each well. Three independent experiments were performed showing similar attachment curves.

Fig. 6. Dominant negative vimentin (Vim-DN) disrupts filaments and reduces microtentacles

(A) GFP-tagged human vimentin (GFP-Vim) human vimentin was restricted with Xho I and religated with a unique Xho I site in the C-terminal multiple cloning site to generate a dominant negative form (Vim-DN). The final Vim-DN construct preserves 134 a.a. of the full-length vimentin (466 a.a.) to include the head and highly conserved region of the rod 1A domain. Western blot analysis of Hs578t transfected with the GFP-Vim and GFP-Vim-DN indicates the expression of the truncated GFP-Vim-DN was recognized by both an N-terminal vimentin antibody and GFP antibody [inset - A: GFP Vim (85kD), B: Endo Vim (58kD), C: GFP-Vim-DN (43kD)]. (B) Vimentin expressing cell lines (Hs578t, HCC1395, MDA-MB-436) were transfected with full length vimentin (GFP-Vim) and dominant-negative (GFP-Vim-DN). Matched panels showing localization of the expressed protein (GFP) and endogenous vimentin (Endo Vim) indicate that GFP-Vim-DN disrupts endogenous vimentin in transfected cells (white arrows) while surrounding untransfected cells retain their IF network (white arrowheads). Expression of full-length GFP-Vim overlaid with the endogenous IF network indicates that the N-terminal GFP tag Vim is not disruptive. (C) Cells co-transfected with GFP-Vim or GFP-Vim-DN and membrane-targeted RFP (RFP-mem) were suspended in DMEM and blind microtentacle counts were performed following 30min suspension. Each bar represents the +S.D. for at least three independent experiments in which a minimum of 100 RFP-Mem⁺ cells were counted. Expression of the GFP-Vim-DN shows a statistically significant reduction in microtentacles compared to GFP-Vim transfection (P<0.001, t-test, black asterisks).