A Cilia Independent Role of Ift88/Polaris during Cell Migration

<div><p>Ift88 is a central component of the intraflagellar transport (Ift) complex B, essential for the building of cilia and flagella from single cell organisms to mammals. Loss of Ift88 results in the absence of cilia and causes left-right asymmetry defects, disordered Hedgehog signaling, and polycystic kidney disease, all of which are explained by aberrant ciliary function. In addition, a number of extraciliary functions of Ift88 have been described that affect the cell-cycle, mitosis, and targeting of the T-cell receptor to the immunological synapse. Similarly, another essential ciliary molecule, the kinesin-2 subunit Kif3a, which transports Ift-B in the cilium, affects microtubule (MT) dynamics at the leading edge of migrating cells independently of cilia. We now show that loss of Ift88 impairs cell migration irrespective of cilia. Ift88 is required for the polarization of migrating MDCK cells, and Ift88 depleted cells have fewer MTs at the leading edge. Neither MT dynamics nor MT nucleation are dependent on Ift88. Our findings dissociate the function of Ift88 from Kif3a outside the cilium and suggest a novel extraciliary function for Ift88. Future studies need to address what unifying mechanism underlies the different extraciliary functions of Ift88.</p></div

MDCK cells lose cilia during sheet migration.

<p><b>(A)</b> Confluent layers of ciliated MDCK cells (7 days after seeding) were scratched, fixed immediately (0h) or 6h after wounding, then stained with antibodies against acetylated Tubulin for cilia (magenta), Cep164 for the basal body (green) and Hoechst for nuclei (blue). Cilia occur at different lengths, basal bodies devoid of magenta are unciliated (squared). White lines indicate the leading edge. Scale bars: 10μm. <b>(B)</b> Quantification of ciliated cells at various time points in areas away from the leading edge and at the leading edge. 0h: 56.7%±3.3% vs. 31.0%±4.5%, 2h: 58.1%±1.7% vs. 20.2%±4.7%, 4h: 53.3%±2.1% vs. 15.0%±3.0%, 6h: 54.6%±3.1% vs. 7.5%±2.7% (asterisk: p < 0.01). n = 10 fields of view in two independent experiments. Note that the number of cilia at time point 0h at the leading edge is decreased compared to distant cells due to mechanical injury from wounding. <b>(C)</b> Analysis of proliferation in cells after wounding. MDCK cells stably expressing Fucci cell-cycle indicators were monitored after 7 days post seeding after scratch wounding. Nuclei of non-proliferating cells in G1 express RFP (red), nuclei of proliferating cells (S/G2/M) express GFP (green). Scale bars: 100μm. <b>(D)</b> Quantification of proliferating cells at the leading edge and cells away from the wound over 21 hours after scratch. n = 10 fields of view from two independent experiments.</p

The number of ciliated MDCK cells after seeding increases over time.

<p><b>(A)</b> Cells were fixed at respective time points and stained with antibodies against acetylatedTubulin for cilia (magenta), Cep164 for the mother centriole (green) and Hoechst for nuclei (blue). Arrows point to mother centrioles which do not have cilia. <b>(B)</b> The number of ciliated cells was quantified over time: 6h: 0.0% ±0.0%, 3420 cells; 1d: 0.0% ±0.0%, 9347 cells; 2d: 0.1% ±0.1%, 18600 cells; 3d: 3.5% ±1.7%, 18309 cells; 7d: 43.2% ±4.7%, 17418 cells and 10d: 51.9% ±3.6%, 17475 cells. n = 3 (32 fields of view per N).</p

Ift88 expedites cell migration in previously unciliated MDCK cells.

<p><b>(A)</b> Ift88-i1 cells were grown to confluency for 2 days (unciliated stage, compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140378#pone.0140378.g003" target="_blank">Fig 3</a>) and subjected to wounding. Cells depleted of Ift88 by inducible shRNA (+Tet) migrate more slowly compared with non-induced control cells (-Tet). The leading edge is shown after 2h and 6h. Scale bars: 100μm. <b>(B)</b> Quantification reveals no significant reduction of migration speed for a control cell line: -Tet: 157.1 ±12.8 μm²/minute vs. +Tet: 144.8 ±10.6 μm²/minute (n.s.; p = 0.5). Migration speed is reduced in two independent cell lines after induced depletion of Ift88. Ift88-i1: -Tet: 133.9 ±7.0 μm²/minute vs. +Tet: 50.4 ±2.5 μm²/minute, p<0.01, Ift88-i2: -Tet: 193.0 ±7.4 μm²/minute vs. + Tet: 86.0 ±2.9 μm²/minute, p<0.01. Migration speed is restored in Ift88-i1 cells expressing non-degradable Ift88 mRNA (Ift88-i1.rescue): -Tet: 162.0 ±9.8 μm²/minute vs. +Tet: 154.6 ±10.0 μm²/minute, p = 0.63. All n = 3. <b>(C)</b> Sparsely seeded Ift88-i1 cells were stimulated with HGF and the morphology was analyzed. Ift88 depleted cells (+Tet) frequently exhibit an unpolarized pancake shape, while non-induced controls (-Tet) more often show a leading and a trailing edge (A representative cell stained for α-Tubulin and a Golgi marker is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140378#pone.0140378.s001" target="_blank">S1E Fig</a>). Phase contrast images. Scale bars: 10μm. <b>(D)</b> Quantification of the proportion of pancake-shaped cells. No significant increase is seen after exposure to tetracycline in a control cell line: -Tet: 18.3 ±1.9% vs. +Tet: 17.8 ±0.7%, p = 0.87, n = 4, a total of 269 and 392 cells counted. Depletion of Ift88 increases the number of unpolarized cells: -Tet: 19.9 ±2.4% vs. +Tet: 50.2 ±2.9%, p<0.01, n = 4, a total of 454 and 362 cells counted. <b>(E)</b> Representative trajectories of single cell migration over 5 hours after stimulation of sparsely seeded cells with HGF (10ng/ml). Scale Bar: 300μm. <b>(F)</b> Quantification of track lengths over 5h. In control cells no significant reduction of track lengths is observed after incubation with tetracycline: -Tet: 360.4 ±27.0 μm vs. +Tet: 348.9 ±24.3 μm, p = 0.76, n = 40 tracks from four independent experiments. A reduction is observed in Ift88 depleted cells: -Tet: 426.7 ±28.5 μm vs. +Tet: 213.8 ±21.3 μm, p<0.05, n = 40 tracks from four independent experiments.</p

Ift88 expedites cell migration in ciliated MDCK cells.

<p><b>(A)</b> Ift88-i1 cells were grown to confluency for 7–8 days (ciliated stage) and subjected to wounding. Cells depleted of Ift88 by inducible shRNA (+Tet) migrate slower compared with non-induced control cells (-Tet). The leading edge is shown after 2h and 6h. Scale bars: 100μm. <b>(B)</b> Quantification of migration speed in different cell lines expressing different tetracycline inducible shRNAs. No significant (n.s.) reduction in migration speed is observed in cells expressing unspecific shRNA (control; -Tet: 111.5 ±5.8 μm²/minute vs. 105.3 ±10.2 μm²/minute, p = 0.46, n = 4). Two independent cell lines expressing different shRNAs against Ift88 show significantly reduced migration speed: Ift88-i1: -Tet: 111.0 ±4.7 μm²/minute vs. +Tet: 64.6 ±10.3 μm²/minute, p<0.01, n = 4; Ift88-i2: -Tet: 124.7 ±1.2 μm²/minute vs. +Tet: 56.7 ±6.8 μm²/minute, p<0.01, n = 3. Migration speed is partially restored in Ift88-i1 cells expressing non-degradable Ift88 mRNA (Ift88-i1.rescue): -Tet: 116.5 ±5.5 μm²/minute vs. +Tet: 85.8 ±4.3 μm²/minute, p<0.01, n = 4.</p

Ift88 facilitates polarization in migrating cells.

<p><b>(A)</b> Ift88 depleted (+Tet) or control cells (-Tet) were subjected to wound healing as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140378#pone.0140378.g004" target="_blank">Fig 4A</a>. After 6 hours cells were fixed, stained with antibodies against Scrib (green) and Hoechst for nuclei (blue), and the leading edge was imaged. Scale bars: 10μm. White arrows point to the leading edge in absence of Scrib. <b>(B)</b> Score based quantification of Scrib localization (0 (not polarized), 1 (partially polarized) or 2 (fully polarized)). After tetracycline induced depletion of Ift88 fewer cells have a fully polarized score (23.5%) compared to the absence of tetracycline (49.2%), n = 3, -Tet 478 cells/ +Tet 468 cells. <b>(C)</b> Ift88-i1 cells were subjected to wound healing assay as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140378#pone.0140378.g004" target="_blank">Fig 4A</a>. After 6 hours cells were fixed, stained against Golgin-97 for the Golgi apparatus (magenta) and Hoechst for nuclei (blue), and the leading edge was imaged. Red arrows show the direction of polarization of the Golgi apparatus relative to the nucleus and the leading edge within ±60°; white arrows illustrate unpolarized Golgi (not within ±60°). Scale bars: 10μm. White line illustrates the leading edge. <b>(D)</b> Quantification of Golgi apparatus polarization (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140378#sec009" target="_blank">methods</a>) showed a significant decrease of polarized Golgi in Ift88-knockdown conditions (+Tet: 37.5 ±4.8%) compared to non-induced controls (-Tet: 53.6 ±3.3%), p<0.05 (Chi²-Test), n = 3, -Tet: 258 cells/ +Tet: 269 cells. <b>(E)</b> After 6 hours of migration Ift88-i1 cells were fixed, stained against α-Tubulin (white) and Hoechst for nuclei (blue) showing reduced numbers of MTs in the leading edge in Ift88-knockdown conditions (+Tet). Scale bars: 10μm. <b>(F)</b> Quantification of the MTs in the leading edge (conducted in Ift88-i1/ α-Tubulin-YFP cells, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140378#pone.0140378.g006" target="_blank">Fig 6A</a>) revealed significantly fewer MT in Ift88 deficient cells (-Tet: 23.7±3.4 vs. +Tet: 12.4±2.5, p<0.05, n = 10/11 cells in two independent experiments).</p

Insulin Modulates the Inflammatory Granulocyte Response to Streptococci via Phosphatidylinositol 3-Kinase

Group B streptococci (GBS; Streptococcus agalactiae) are a major cause of invasive infections in newborn infants and in patients with type 2 diabetes. Both patient groups exhibit peripheral insulin resistance and alterations in polymorphonuclear leukocyte (PML) function. In this investigation, we studied the PML response repertoire to GBS with a focus on TLR signaling and the modulation of this response by insulin in mice and humans. We found that GBS-induced, MyD88-dependent chemokine formation of PML was specifically downmodulated by insulin via insulin receptor-mediated induction of PI3K. PI3K inhibited transcription of chemokine genes on the level of NF-kappa B activation and binding. Insulin specifically modulated the chemokine response of PML to whole bacteria, but affected neither activation by purified TLR agonists nor antimicrobial properties, such as migration, phagocytosis, bacterial killing, and formation of reactive oxygen species. The targeted modulation of bacteria-induced chemokine formation by insulin via PI3K may form a basis for the development of novel targets of adjunctive sepsis therapy. The Journal of Immunology, 2012, 189: 4582-459