QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online March 12, 2004
doi: 10.1242/10.1242/jcs.00986

This Article Summary Full Text Full Text (PDF) Movies Movies Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Finkelstein, E. Articles by Bulinski, J. C. Search for Related Content PubMed PubMed Citation Articles by Finkelstein, E. Articles by Bulinski, J. C. Social Bookmarking                         What's this?

Roles of microtubules, cell polarity and adhesion in electric-field-mediated motility of 3T3 fibroblasts

Erik Finkelstein^1,*, Winston Chang^1,*, P.-H. Grace Chao³, Dorota Gruber¹, Audrey Minden¹, Clark T. Hung³ and J. Chloë Bulinski^1,2,

¹ Department of Biological Sciences, Columbia University, New York, NY 10027, USA
² Department of Anatomy and Cell Biology, and Department of Pathology, Colleges of Arts and Sciences, and Physicians and Surgeons
³ Department of Biomedical Engineering, School of Engineering and Applied Sciences, Columbia University, New York, NY 10027, USA

View larger version (26K): [in a new window]   Fig. 1. Exposure to EFs increases directional migration of 3T3 fibroblasts. (A) Measurement of migration speed and directed velocity of sparse cells. EF chambers were oriented with the cathode down (=270°); cells traveled distance, d, at migration angle, , relative to the horizontal. Speed was calculated as d/t, where t is elapsed time. Directional velocity was calculated as speedxsin(), as shown. For these measurements, N20 cells each; three experiments per voltage. (B) Voltage dependence of migration speed. Sparse 3T3 cells placed in EFs for 1.5 hours were significantly increased in average motility speed (P<0.05) only at EFs >2 V cm-1. (C) Voltage dependence of migration direction. Motility angle was plotted as sin() for each EF strength. Notice that, for sparse cells moving towards the cathode, sin()=-1; towards anode, sin()=+1; randomly, sin()=0. Directionality of motility increased significantly (P<0.05) at EFs 2 V cm-1. (D) EF increases average migration speed. Histograms document speed of individual, sparse cells in a 1.5-hour run in 0 V cm-1 and 6 V cm-1 EF. Although speeds are heterogeneous, the 6 V cm-1 EF increased average speed (arrows) roughly threefold. (E) EF increases average migration speed and directs motility cathodally. Polar plot shows displacement from the origin of individual sparse 3T3 cells during a 1.5-hour run with no electric field (circles) or 6 V cm-1 EF (triangles). The EF increased directionality, speed and proportion of cells that translocated significantly and decreased the proportion of cells remaining inside a 10-µm-radius circle.

View larger version (79K): [in a new window]   Fig. 2. Microfilaments, not microtubules, are required for EF-mediated migration. (A) Inhibiting dynamic microfilaments blocks motility. Micrographs document positions of numbered cells initially (0 hours; a,c) and after 2 hours (b,d), EF exposure (6 V cm-1), with (a,b) or without (c,d) cytochalasin B (2 µM, 1.5 hours pretreatment). Cytochalasin completely abrogated motility, as quantified in (C) [see supplementary movies of drug-treated Cyto_6V_2hr and control cells Sparse_6V_2hr (http://jcs.biologists.com/supplemental/)]. The array of actin-containing structures visualized with anti-actin immunofluorescence (e,g) and rhodamine phalloidin (f,h) is shown with (e,f) and without (g,h) cytochalasin B. Notice that, although abundant microfilament structures remain in drug-treated cells (e,f), brightly labeled actin arc-like assemblies at cathode-facing edges are present only in control cells (g,h); compare thick white arrows in (e,g). EF direction shown by (+) and (-), and migration direction by thin arrows. Scale bars, 20 µm (phase-contrast) and 10 µm (immunofluorescence). (B) Inhibiting MTs slows EF-mediated motility. Micrographs show positions of numbered cells initially (0 hours; a,c) and after 2 hours (b,d), EF exposure (6 V cm-1), with (a,b) or without (c,d) Colcemid pretreatment (15 µM, 2 hours). Breakdown of all cellular MTs (see anti-tubulin immunofluorescence, e,f) slows but does not stop motility [quantified in Fig. 2C; compare supplementary movies of drug-treated (Colcemid_6V_2hr) and control (Sparse_6V_2hr) cells (http://jcs.biologists.com/supplemental/)]. Notice that the alignment perpendicular to motility direction is blocked by Colcemid (compare a,c, to b,d). EF direction shown by (+) and (-), and migration direction by thin arrows. Scale bars, 20 µm (phase-contrast) and 10 µm (immunofluorescence). (C) EF-mediated motility requires microfilaments, not MTs. Average migration speed and directional motility [speedxsin(); Fig. 1A] in the EF (6 V cm-1; 2 hours) was quantified in cells treated to inhibit microfilaments (Cytochalasin B) or MTs (Colcemid), and compared with untreated cells (Control). Unlike cells lacking dynamic microfilaments, which showed no movement, cells lacking MTs displayed EF-mediated directional motility that was merely decreased in speed relative to controls.

View larger version (68K): [in a new window]   Fig. 3. The MTOC and stable MT subset do not reorient during EF motility, unlike wound-healing motility. (A) The total MT array (anti-ß-tubulin; a,c) and stable MT subset (detected via enrichment in detyrosinated tubulin; Glu, b,d) did not become polarized during EF-mediated motility (a,b), in contrast to wound-healing motility without applied EF (no EF; c,d), in which Glu MTs reoriented towards the wound edge. Glu MT labeling also allowed the detection of the MTOC (arrowheads, b,d). Migration direction is to the left in all micrographs; the cathode (-) (a,b) and wound edge (c,d) are shown with thin white arrows. Scale bar (d), 10 µm. (B) The proportion of cells with an oriented MTOC [i.e. within the cathode-facing quadrant (dotted lines)] was indistinguishable from the 25% expected for a random distribution. Similarly, 5.1% of cells in the EF showed a Glu MT array oriented towards the migration direction (i.e. with >75% of Glu MTs facing the migration direction), indistinguishable from controls (8.3%). Notice that, in the EF, only a small proportion of sparse cells had Glu MTs oriented in any direction. MTs are depicted as thin lines, Glu MTs as thick lines and the MTOC as a dot.

View larger version (59K): [in a new window]   Fig. 4. Migration of monolayer cells exposed to wound-healing and EF signals. (A) EF application after wounding hastens initiation and biases motility direction of monolayer cells. Wounded 3T3 cell monolayers were placed in the indicated EFs as rapidly as practical after wounding (30 minutes) with the length of wounds oriented perpendicular to the EF. Cell movements in a 1.5 hour interval (i.e. 0.5-2.0 hours after wounding) with the EF (towards cathode) and against the EF (towards anode) were plotted as absolute values of directional velocity. Notice that EFs increased initial speed toward the cathode significantly (P<0.05, marked with , for comparing 2 V cm-1 and 0 V cm-1). EFs also biased motility towards the cathode (P<0.05, marked with * for comparing cathode- and anode-directed velocities at both EF strengths). EFs >2 V cm-1 were not used; they caused cells to round up and motility was compromised. More than 20 cells were quantified per direction, and three experiments per condition. (B) Cells in monolayers simultaneously wounded and placed in EFs migrate preferentially towards the cathode. 3T3 cell monolayers placed in a 0.6 V cm-1 EF 30 minutes after wounding migrated towards the anode (+) and cathode (-) during a 3-hour recording; black line traces the initial wound edges. Notice that cells migrated farther with than against the EF (A). Scale bar, 40 µm. Also, see supplementary movie of wound-healing migration Wound_2V_3hr (http://jcs.biologists.com/supplemental/). (C) Applying EFs to pre-polarized cells directionally biases wound-healing motility. Confluent 3T3 cell monolayers were wounded and incubated for 2.5 hours to allow reorientation of cytoskeletal components toward the wound edge and were then observed for 1.5 hours (i.e. 3.0-4.5 hours after wounding) with EFs indicated. Directional velocities show that wound-healing speed of cells toward the cathode was unaffected by applied voltage (0.6 V cm-1 and 2.0 V cm-1 were equivalent to 0 V cm-1, P>0.05), but speed toward the anode was decreased by the EF (P<0.05 at 0.6 V cm-1). Statistical significance, and *, shown as in (A). (D) MT cytoskeleton reorients toward wound edge with and against the EF. 3T3 cell monolayers treated as in (C) were placed in an EF (0.6 V cm-1) 0.5-2.5 hours after wounding. (a,b) Glu and (c,d) total tubulin antibody staining of cells migrating against (a,c) or with (b,d) the EF shows that cells oriented MTOCs (white arrows) and stable Glu MTs towards the wound edge, regardless of their directional velocity (C) and of whether they faced the cathode or anode. Short black arrows show the wound edge for each pair of micrographs; long black arrow shows the EF direction in a-d. Scale bar, 10 µm.

View larger version (63K): [in a new window]   Fig. 5. Detyrosinated MTs decrease EF-mediated motility. (A) NO2Tyr inhibits Glu MT elaboration. Incubation of 3T3 cells with (NO2Tyr; a,c) or without (control, b,d) NO2Tyr, which inhibits the formation of Glu MTs (a,b) without altering the total MT network (c,d) or the stable, acetylated MTs (Chang et al., 2002) (not shown). Scale bar, 10 µm. (B) Cells lacking Glu MTs migrate more rapidly in the EF. Average motility speed (µm hour-1) of NO2Tyr-treated 3T3 cells in a 6 V cm-1 EF (1.5 hours) was significantly greater than control cells (marked with *; P<0.05). At least 20 cells were quantified in each of three experiments.

View larger version (36K): [in a new window]   Fig. 6. Increased spreading time alters morphology and speed of cells migrating in response to an EF. 3T3 cells plated on acid-washed glass coverslips at T=0 were allowed to attach and spread for 1-24 hours. (A) Morphology of cells undergoing EF-mediated motility is altered by spreading time. Micrographs excerpted from time-lapse images of cells migrating after spreading for 1 hour (a) show that cells are less flat than after 3 hours (b) and 24 hours (c). The cathode (-) and 40 µm bar (a) are indicated. (B) Increased spreading time alters cell footprint and substratum contact area. TIRF (a-c) and corresponding immunofluorescence (a'-c') micrographs of cells spread for 1 hour (a,a'), 3 hours (b,b') and 24 hours (c,c'). TIRF micrographs (a-c) were used to quantify the cell surface area directly contacting substratum. Immunofluorescence micrographs (a'-c') were used to show that cell surface area (`footprint') and proportion of cells with asymmetric lamellipodia both increased with spreading time (more than 12 cells in each of two experiments). Scale bar, 10 µm. (C) Increased spreading time alters adhesion strength. The number of cells per field remaining after centrifugation (1500 g or 6400 g) was compared with the control (not centrifuged) for cells plated 1 hour, 3 hours and 24 hours before fixation. Centrifugation detached >80% of 1-hour-plated cells but did not significantly detach 3-hour- or 24-hour-plated cells. (D) EF-motility speed is altered by spreading time. Average speeds (µm hour-1) of cells plated for specified times were quantified from time-lapse recordings of EF motility (6 V cm-1; 1.5 hours); speeds were normalized to the 24-hour-plated cells (100%) (at least 18 cells in each of three experiments). Migration of 1-hour-plated cells (*) was significantly faster than 3-hour- and 24-hour-plated cells (P<0.05); the latter two were indistinguishable.

View larger version (42K): [in a new window]   Fig. 7. Altering PAK4 expression alters EF motility. (A) Morphology of fibroblasts with altered PAK4 levels. Phase-contrast images of 3T3 cells stably transfected with PAK4 (a) or empty vector (b) show that most PAK4 transfectants were flatter and more bipolar than controls, in which 20-25% of cells appear spherical before EF application. Primary fibroblasts from mouse embryos whose PAK4 expression was knocked out by homologous recombination (PAK4-/-; c) and PAK4-expressing heterozygotes (PAK4+/-; d) were morphologically indistinguishable. Scale bar, 40 µm. (B) Altered PAK4 expression alters EF-motility speed. EF-motility speed of PAK4 transfectants (2 hours; 6 V cm-1) was significantly greater than control 3T3 cells (P<0.05, marked with *). Embryonic fibroblasts from PAK4-null mice (PAK4-/-) migrated significantly less rapidly than fibroblasts from PAK4-heterozygous littermates (PAK4+/-) (P<0.05). At least 17 cells were tested in each of three experiments for each comparison.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter