The role of microtubules in electrotaxis of rat Walker carcinosarcoma WC256 cells

The endogenous electric field may provide an important signal for directional cell migration during cancer metastasis but the mechanism of cell electrotaxis is poorly understood. It was postulated that microtubules play a central role in the polarization and directional migration of several types of cells. In this paper we investigated the role of microtubules in electrotaxis of rat Walker carcinosarcoma WC256 cells. We found that colchicine-stimulated disassembly of microtubules caused the formation of blebs instead of lamellipodia at the front of about 45% of cells. Most of the remaining cells contracted and became rounded or transformed into non-polar cells. Depolymerization of microtubules in both subpopulations of cells reduced the directionality of cell migration to about 50% of the control, but bleb- forming cells migrated much more efficiently than lamellipodia-forming cells. The analysis of microtubules architecture in the presence of an endogenous electric field showed that there is no relationship between the direction of migration and the polarization of microtubules. These results suggest that microtubules are not indispensable for electrotaxis of WC256 cells, however they may improve the directionality of cell migration

The effect of applied direct current electric fields (dcEF) on the migratory strategy - dependent directional migration of Walker 256 rat carcinosarcoma cells.

Zjawisko powstawania endogennego pola elektrycznego (eEF) związanego z ak-tywnością różnych tkanek tj. mięśnie, tkanka nerwowa oraz w trakcie procesu gojenia się ran, znane jest już od wielu lat. Wraz z rozwojem zaawansowanych technik pomia-rowych i badawczych możliwe było wnikliwe zbadanie oddziaływania eEF na komórki. Wiele typów komórek prawidłowych i nowotworowych np. komórki endotelialne, fi-broblasty, mezenchymalne komórki macierzyste szpiku ludzkiego, komórki raka piersi lub płuc, reaguje kierunkową migracją w polu elektrycznym. Zjawisko to nazwane zo-stało elektrotaksją lub galwanotaksją. Kierunkowa migracja komórek ma duże znaczenie w procesach fizjologicznych oraz patologicznych do których należą: proces gojenia się ran, reakcja odpornościowa organizmu, rozwój embrionalny i tworzenie przerzutów przez komórki nowotworowe o charakterze metastatycznym. Różne strategie migracji komórek są wypadkową wielu czynników m.in. oddziaływań ze środowiskiem w jakim się znajdują, ich pochodzenia oraz pełnionej funkcji. Mechanizm kierunkowej migracji nie został do końca wyjaśniony. Rozpatruje się wiele czynników mogących pośrednio lub bezpośrednio wpływać na kierunkową migrację komórek. Zaliczane są do nich m.in. wewnątrzkomórkowe jony Ca2+, reorganizacja cytoszkieletu mikrotubularnego (MT), depolaryzacja i hyperpolary-zacja błon komórkowych oraz organelli wewnątrzkomórkowych, aktywacja ścieżek sygnalizacyjnych z udziałem białek z rodziny Rho, cAMP, kinaz MAP oraz ROCK. W niniejszej pracy zbadano wpływ pola elektrycznego na migrację dwóch subli-nii komórek mięsakoraka Walkera 256. Pierwsza Sublinia charakteryzowała się silną adhezją do podłoża oraz migracją o charakterze mezenchymalnym, natomiast komórki drugiej sublinii były słabiej zaadherowane i poruszały się przy użyciu spontanicznie wytwarzanych pęcherzykowatych wypustek.Zaobserwowano, że w polu elektrycznym o natężeniu 0 – 3 V/cm komórki obu sublinii poruszają się w stronę katody a ich kierunkowa reakcja zależna jest od jego natężenia. Następnie wykazano, że rozbicie MT, pod wpływem 1 µM kolchicyny, w komórkach sublinii adherentnej spowodowało zmniejszenie kierunkowości migracji w polu elektrycznym o natężeniu 3 V/cm oraz zainicjowało wytwarzanie pęcherzyko-watych wypustek. Analiza architektury cytoszkieletu mikrotubularnego tych komórek wykazała brak zależności pomiędzy kierunkową migracja a spolaryzowanym ułożeniem włókien mi-krotubul, co sugeruje, że mikrotubule nie są istotnym czynnikiem w kierunkowej mi-gracji komórek WC 256 w EF. Zaobserwowano również, że dodanie do komórek sub-linii adherentnej WC256 1 µM kolchicyny razem z 10 µM Y-27632 inhibitorem kinazy ROCK zahamowało wytwarzanie pęcherzykowatych wypustek oraz częściowo przy-wróciło kierunkowość migracji. Komórki po inkubacji z samym Y-27632 charaktery-zowały się wydłużonym kształtem. Analiza aktywności ruchowej komórek sublinii słabo adherentnej WC 256 w obecności 1 µM kolchicyny wykazały jej hamujący wpływ na kierunkowość migracji w polu elektrycznym o natężeniu 3 V/cm, co sugeruje że wytwarzanie pęcherzyków nie ma decydującego udziału w kierunkowej migracji. Otrzymane wyniki świadczą o braku udziału w kierunkowej migracji komórek WC 256 zarówno spolaryzowanych mikrotubul jak i pęcherzykowatych wypustek. Efekt zahamowania kierunkowej migracji komórek WC 256 w polu elektrycz-nym, w wyniku potraktowania kolchicyną, może zachodzić poprzez wpływ tego związ-ku na inne procesy. Nie można wykluczyć udziału kolchicyny w regulacji takich proce-sów jak płynność i integralność błon, adhezję komórek do podłoża lub kanały jonowe, skutkujące dezorganizacją funkcji komórki.Many types of cells, including normal and cancer cells, migrate directionally in direct current electric field (dcEF). This phenomenon is called electrotaxis or galvanotaxis if cells migrate to the cathode. Directional cell migration is important in many physiological and pathological processes, which include: the process of wound healing, the body's immune responses, embryonic development and metastasis for-mation. Probably many factors participate in the mechanism of directional migration. Examples include the intracellular Ca2+ ions, the reorganization of microtubules cyto-skeleton and hyper-polarization and depolarization of cell membranes or organelles, activation of signaling pathways involving the Rho family protein, cAMP, MAP kinase, and ROCK. In the present study the effect of electric field on migration of two cells sublines carcinosarcoma Walker 256 was investigated. The first (subline 2) is characterized by strong adhesion to the substrate and less efficient locomotion, while the second (subline 3) was less adherent and migrates in non-adhesive mode using spontaneously generated blebs. Cells exposed to the electric current field between of 0 - 3 V/cm showed elctrotaxis toward the cathode and the directional response depended on the current intensity. Furthermore, colchicines-stimulated depolymerization of MTs reduced the directionality of migration in the electric field (3V/cm), and initiated the production of membrane blebs. Analysis of microtubules cytoskeleton architecture during direc-tional migration showed that there is no relationship between the direction of migration and polarization of MTs. Preincubation of the adhesive subline Walker 256 cells in the presence of 1 µM colchicine together with 10 µM Y-27632 inhibited the production of blebs and partially restored the directionality of migration. Moreover, it was found that the addition of 1 µM colchicine reduces directional migration in the electric field inten-sity of 3 V / cm in less adherent subline WC 256, which suggests that the production of blebs doesn’t affect the direction of migration. In summary, the results suggest that microtubule polarity and the production of blebs are not determining factors in the regulation of directional migration of WC 256 in the direct currents electric fields. The inhibitory effect of colchicines on the electrotaxis of WC 256 cell may be mediated by different mechanism. We cannot exclude the direct effect of colchicines on membrane structure or its integrity, adhesion, ion channels, resulting in the disorganisation of function

HPat a Decapping Activator Interacting with the miRNA Effector Complex

<div><p>Animal miRNAs commonly mediate mRNA degradation and/or translational repression by binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). The CCR4-NOT1 complex required for the deadenylation of target mRNAs is directly recruited to the miRNA effector complex. However, it is unclear whether the following decapping step is only a consequence of deadenylation occurring independent of the miRNA effector complex or e.g. decapping activators can get recruited to the miRNA effector complex. In this study we performed split-affinity purifications in <i>Drosophila</i> cells and provide evidence for the interaction of the decapping activator HPat with the miRNA effector complex. Furthermore, in knockdown analysis of various mRNA degradation factors we demonstrate the importance of NOT1 for this interaction. This suggests that deadenylation and/or the recruitment of NOT1 protein precedes the association of HPat with the miRNA effector complex. Since HPat couples deadenylation and decapping, the recruitment of HPat to the miRNA effector complex provides a mechanism to commit the mRNA target for degradation.</p></div

The interaction of HPat and GW182 protein.

<p>Immunoprecipitation analysis of <i>Drosophila</i> S2 cell lysates using anti-HPat (<b>A</b>) or anti-GW182 (<b>B, C</b>) antibodies or preimmune sera. Input (lane 1) and immunoprecipitates (lanes 2, 3) were separated on SDS-PAGE and analyzed by Western blot analysis using anti-HPat, anti-GW182 or anti-AGO1 antibody. In A) and B) 1.5% of the input (total clarified cell lysate) and 40% of the immunoprecipitate were separated on a SDS-PAGE, while in C) 2.5% of the input and only 10% of the immunoprecipitate were separated. The asterix indicates cross-reactivity of the secondary antibody with the immunoglobulin heavy chain of the antibody used for immunoprecipitation.</p

Co-purification of HPat (A) or AGO1 (B) with GW182 in NOT1 knockdown cells.

<p><b>A, B:</b> Protein complexes were immunoprecipiated using monoclonal anti-HA antibody from cell lysates. Cells stable expressing HA-GW182 and Myc-HPat were treated with dsRNA against YFP (control KD) or NOT1 (NOT1 KD). Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Input in A: lanes 1–5 and 14–18, in B: lanes 1–6 and 15–21. IPs in A lanes 6 and 19, in B lanes 7 and 22), anti-c-myc (Input in A: lanes 7–12 and 20–23. IPs in A lanes 13 and 24) or anti-AGO1 antibody (Input in B: lanes 8–13 and 23–29. IPs in B lanes 14 and 30). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. <b>C, D:</b> The amount of Myc-HPat/HA-GW182 (<b>C</b>) or AGO1/HA-GW182 (<b>D</b>) in immunoprecipitates (IP) from lysates of control and NOT1 knockdown cells. The IP was normalized (Supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071860#pone.0071860.s004" target="_blank">Figure S4</a>) and the value of the control IP set to 1. <b>E:</b> Analysis of <i>NOT1</i> mRNA levels in knockdown cells compared to control cells treated with dsYFP RNA. The levels of <i>NOT1</i> mRNA in total RNA of input samples were analyzed by RT-qPCR and normalized to <i>rp49</i> mRNA levels. The values of dsYFP treated cells were set to 1. <b>F:</b> Upregulation of endogenous miRNA targets in knockdown cells. Total RNA of input samples were analyzed by RT-qPCR for changes of <i>CG5123</i> mRNA levels in NOT1 knockdown cells. mRNA levels were normalized to <i>rp49</i> mRNA levels. The values of dsYFP treated cells were set to 1. Statistical analysis was performed using the Student’s <i>t</i> test and significance values are as follows: ns, not significant; *, p<0.005; **, p<0.001.</p

Co-purification of HPat with GW182 in EDC4 and Dcp1 (A), or XRN1 (B) knockdown cells.

<p><b>A, B:</b> Protein complexes were immunoprecipiated using monoclonal anti-HA antibody from cell lysates. Cells stable expressing HA-GW182 and Myc-HPat were treated with dsRNA against YFP (control KD), EDC4 and Dcp1 (EDC4/Dcp1 KD, <b>A</b>) or XRN1 (XRN1 KD, <b>B</b>). Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Input in A: lanes 1–3 and 11–14, in B: lanes 1–5 and 14–18. IPs in A: lanes 4 and 16, in B: lanes 6 and 19) or anti-c-myc antibody (Input in A: lanes 5–9 and 17–21, in B: lanes 7–12 and 20–25. IPs in A: lanes 10 and 22, in B: lanes 13 and 26). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. <b>C:</b> The amount of Myc-HPat/HA-GW182 in immunoprecipitates (IP) from lysates of control, EDC4 and Dcp1, or XRN1 knockdown cells. The IP was normalized (Supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071860#pone.0071860.s005" target="_blank">Figure S5</a>) and the value of the control IP set to 1. <b>D:</b> Analysis of <i>EDC4, Dcp1,</i> and <i>XRN1</i> mRNA levels. The levels of <i>EDC4, Dcp1,</i> and <i>XRN1</i> mRNA in total RNA of input samples were analyzed by RT-qPCR and normalized to <i>rp49</i> mRNA levels. The values of dsYFP treated cells were set to 1. <b>E:</b> Upregulation of endogenous miRNA targets in knockdown cells. <i>CG6770</i> mRNA levels in total RNA of EDC4/Dcp1, XRN1, and YFP knockdown cells were analyzed by RT-qPCR. mRNA levels were normalized to <i>rp49</i> mRNA levels. The values of dsYFP treated cells were set to 1. Statistical analysis was performed using the Student’s <i>t</i> test and significance values are as follows: ns, not significant; *, p<0.02; **, p<0.001; ***, p<0.0001.</p