Disruption of the Cdc42/Par6/aPKC or Dlg/Scrib/Lgl polarity complex promotes epithelial proliferation via overlapping mechanisms

The establishment and maintenance of apical-basal polarity is a defining characteristic and essential feature of functioning epithelia. Apical-basal polarity (ABP) proteins are also tumor suppressors that are targeted for disruption by oncogenic viruses and are commonly mutated in human carcinomas. Disruption of these ABP proteins is an early event in cancer development that results in increased proliferation and epithelial disorganization through means not fully characterized. Using the proliferating Drosophila melanogaster wing disc epithelium, we demonstrate that disruption of the junctional vs. basal polarity complexes results in increased epithelial proliferation via distinct downstream signaling pathways. Disruption of the basal polarity complex results in JNK-dependent proliferation, while disruption of the junctional complex primarily results in p38-dependent proliferation. Surprisingly, the Rho-Rok-Myosin contractility apparatus appears to play opposite roles in the regulation of the proliferative phenotype based on which polarity complex is disrupted. In contrast, non-autonomous Tumor Necrosis Factor (TNF) signaling appears to suppress the proliferation that results from apical-basal polarity disruption, regardless of which complex is disrupted. Finally we demonstrate that disruption of the junctional polarity complex activates JNK via the Rho-Rok-Myosin contractility apparatus independent of the cortical actin regulator, Moesin

Cell density and actomyosin contractility control the organization of migrating collectives within an epithelium

The mechanisms underlying collective migration are important for understanding development, wound healing, and tumor invasion. Here we focus on cell density to determine its role in collective migration. Our findings show that increasing cell density, as might be seen in cancer, transforms groups from broad collectives to small, narrow streams. Conversely, diminishing cell density, as might occur at a wound front, leads to large, broad collectives with a distinct leader–follower structure. Simulations identify force-sensitive contractility as a mediator of how density affects collectives, and guided by this prediction, we find that the baseline state of contractility can enhance or reduce organization. Finally, we test predictions from these data in an in vivo epithelium by using genetic manipulations to drive collective motion between predicted migratory phases. This work demonstrates how commonly altered cellular properties can prime groups of cells to adopt migration patterns that may be harnessed in health or exploited in disease

Expression Levels of a Kinesin-13 Microtubule Depolymerase Modulates the Effectiveness of Anti-Microtubule Agents

Chemotherapeutic drugs often target the microtubule cytoskeleton as a means to disrupt cancer cell mitosis and proliferation. Anti-microtubule drugs inhibit microtubule dynamics, thereby triggering apoptosis when dividing cells activate the mitotic checkpoint. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs); however, we lack a comprehensive understanding about how anti-microtubule agents functionally interact with MAPs. In this report, we test the hypothesis that the cellular levels of microtubule depolymerases, in this case kinesin-13 s, modulate the effectiveness of the microtubule disrupting drug colchicine.We used a combination of RNA interference (RNAi), high-throughput microscopy, and time-lapse video microscopy in Drosophila S2 cells to identify a specific MAP, kinesin-like protein 10A (KLP10A), that contributes to the efficacy of the anti-microtubule drug colchicine. KLP10A is an essential microtubule depolymerase throughout the cell cycle. We find that depletion of KLP10A in S2 cells confers resistance to colchicine-induced microtubule depolymerization to a much greater extent than depletion of several other destabilizing MAPs. Using image-based assays, we determined that control cells retained 58% (+/-2%SEM) of microtubule polymer when after treatment with 2 microM colchicine for 1 hour, while cells depleted of KLP10A by RNAi retained 74% (+/-1%SEM). Likewise, overexpression of KLP10A-GFP results in increased susceptibility to microtubule depolymerization by colchicine.Our results demonstrate that the efficacy of microtubule destabilization by a pharmacological agent is dependent upon the cellular expression of a microtubule depolymerase. These findings suggest that expression levels of Kif2A, the human kinesin-13 family member, may be an attractive biomarker to assess the effectiveness of anti-microtubule chemotherapies. Knowledge of how MAP expression levels affect the action of anti-microtubule drugs may prove useful for evaluating possible modes of cancer treatment

The ERM protein Moesin regulates Rho-JNK independent of the junctional polarity complex.

<p>Confocal immunofluorescent localization of DE-cadherin, GFP, and MMP1 in wing discs expressing GFP with P35 alone (Aa and Ab), Cdc42-RNAi and P35 (Ba and Bb), Moe-RNAi and P35 (Ca and Cb), Cdc42-RNAi, Moe-RNAi, and P35 (Da and Db), Cdc42-RNAi, Moe, and P35 (Ea and Eb), via <i>ptc-GAL4</i>. (F) Quantification of GFP area/total wing disc area in conditions shown in A-D, n>12. Scale bars represent 100μm. AU—Arbitrary Units.</p

P38 MAPK is required for proliferation following junctional complex disruption, but not for proliferation following basolateral complex disruption.

<p>(A-D, F-H) Confocal immunofluorescent localization of DE-cadherin and GFP (Aa-Da), and MMP1 (Ab-Db) in wing discs expressing GFP with P35 only (Aa and Ab), Cdc42-RNAi and P35 (Ba and Bb), Cdc42-RNAi, p38a-RNAi, and P35 (Ca and Cb), Cdc42-RNAi, p38b-RNAi, and P35 (Da and Db), via <i>ptc-GAL4</i>. (E) Quantification of GFP area/total wing disc area in conditions shown in A-D, n>13. Statistical comparisons to “A” are shown in black, while statistical comparisons to “B” are shown in red. Confocal immunofluorescent localization of DE-cadherin and GFP (Fa-Ha), and MMP1 (Fb-Hb) in wing discs expressing GFP with Dlg-RNAi#1 and P35 (Fa and Fb), Dlg-RNAi#1, P35, and p38a-RNAi (Ga and Gb), Dlg-RNAi#1, P35, and p38b-RNAi (Ha and Hb), via <i>ptc-GAL4</i>. (I) Quantification of GFP area/total wing disc area in conditions shown in A and F-H, n>8. Scale bars represent 100μm. AU—Arbitrary Units.</p

Hyperproliferation following junctional complex disruption specifically requires the upstream JNK-KK, Mekk1; hyperproliferation following basolateral complex disruption is dependent on multiple upstream kinases.

<p>(A) Schematic of JNK MAPK signaling in <i>Drosophila melanogaster</i>. (B-H, J-O) Confocal immunofluorescent localization of DE-cadherin, GFP, and MMP1 in wing discs expressing P35 and GFP alone (Aa and Ab), Cdc42-RNAi, P35 (Ca and Cb), Cdc42-RNAi, Slpr-RNAi, and P35 (Da and Db), Cdc42-RNAi, Tak1-RNAi, and P35 (Ea and Eb), Cdc42-RNAi, Mekk1-RNAi, and P35 (Fa and Fb), Cdc42-RNAi, Wnd-RNAi, and P35 (Ga and Gb), Cdc42-RNAi, Ask1-RNAi, and P35 (Ha and Hb), via <i>ptc-GAL4</i>. (I) Quantification of GFP area/total wing disc area in conditions shown in B-H, n>13. (J-O) Confocal immunofluorescent localization of DE-cadherin and MMP1 in wing discs expressing Dlg-RNAi and P35 (Ja and Jb), Dlg-RNAi, Bsk-RNAi, and P35 (Ka and Kb), Dlg-RNAi, Mkk4-RNAi, and P35 (La and Lb), Dlg-RNAi, Tak1-RNAi, and P35 (Ma and Mb), Dlg-RNAi, Hep-RNAi, and P35 (Na and Nb), and Dlg-RNAi, Mekk1-RNAi, and P35 (Oa and Ob), via <i>ptc-GAL4</i>. (P) Quantification of GFP area/total wing disc area in conditions shown in B and J-O, n>13. Statistical comparisons to “B” are shown in black, while statistical comparisons to “J” are shown in red. Scale bars represent 100μm. AU—Arbitrary Units.</p

Junctional or basolateral polarity disruption in the presence of P35 leads to hyperproliferation, proliferation following basolateral complex disruption is dependent on JNK, proliferation following junctional complex disruption is largely JNK-independent.

<p>(A-H) Confocal immunofluorescent localization of DE-cadherin (DE-cad) (Aa-Ha), matrix metalloproteinase 1 (MMP1) (Ab-Bb, Db-Eb, Gb-Hb) and β-galactosidase (β-gal) (Cb and Fb) in larval wing discs expressing GFP with P35 alone (Aa and Ab), Dlg-RNAi and P35 (Ba and Bb), Dlg-RNAi and P35 in a heterozygous background of <i>pucE69</i> (<i>puc-LacZ</i>) (Ca and Cb), Dlg-RNAi, P35, and Bsk-RNAi (Da and Db), Cdc42-RNAi and P35 (Ea and Eb), Cdc42-RNAi and P35 in a <i>pucE69</i> heterozygous background (Fa and Fb), Cdc42-RNAi, P35, and Bsk-RNAi (Ga and Gb), Cdc42-RNAi and P35 in a <i>bsk1</i> heterozygous background, via <i>ptc-GAL4</i>. Quantification of GFP area/total wing disc area in tissues from conditions listed (I and J), n>14. Scale bars represent 100μm. Black statistical marks represent comparisons to the ptc>GFP control case, red statistical marks represent comparisons to the ptc>p35 case, unless otherwise indicated. AU—Arbitrary Units.</p