Una Red de Reguladores de Actomiosin Controla la Maduración Apical en Epitelios

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-06-2018Esta tesis tiene embargado el acceso al texto completo hasta el 15-12-201

Endocytic turnover of Rab8 controls cell polarization

Adaptation of cell shape and polarization through the formation and retraction of cellular protrusions requires balancing of endocytosis and exocytosis combined with fine-tuning of the local activity of small GTPases like Rab8. Here, we show that endocytic turnover of the plasma membrane at protrusions is directly coupled to surface removal and inactivation of Rab8. Removal is induced by reduced membrane tension and mediated by the GTPase regulator associated with focal adhesion kinase-1 (GRAF1, also known as ARHGAP26), a regulator of clathrin-independent endocytosis. GRAF1-depleted cells were deficient in multi-directional spreading and displayed elevated levels of GTP-loaded Rab8, which was accumulated at the tips of static protrusions. Furthermore, GRAF1 depletion impaired lumen formation and spindle orientation in a 3D cell culture system, indicating that GRAF1 activity regulates polarity establishment. Our data suggest that GRAF1-mediated removal of Rab8 from the cell surface restricts its activity during protrusion formation, thereby facilitating dynamic adjustment of the polarity axis.Peer reviewe

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis

The actin cortex is involved in many biological processes and needs to be significantly remodeled during cell differentiation. Developing epithelial cells construct a dense apical actin cortex to carry out their barrier and exchange functions. The apical cortex assembles in response to three-dimensional (3D) extracellular cues, but the regulation of this process during epithelial morphogenesis remains unknown. Here, we describe Smoothelin-like 2 (SMTNL2) function, a member of the smooth-muscle related Smoothelin protein family, in apical cortex maturation. SMTNL2 is induced during the development of multiple epithelial tissues and localizes to the apical and junctional actin cortex in intestinal and kidney epithelial cells. SMTNL2 deficiency leads to membrane herniations in the apical domain of epithelial cells, indicative of cortex abnormalities. We find that SMTNL2 binds to actin filaments and is required to slow down the turnover of apical actin. We also characterize the SMTNL2 proximal interactome and find that SMTNL2 executes its functions partly through inhibition of Coronin-1B. While Coronin-1B-mediated actin dynamics are required for early morphogenesis, its sustained activity is detrimental for the mature apical shape. SMTNL2 binds to Coronin-1B through its N-terminal coiled-coil region and negates its function to stabilize the apical cortex. In sum, our results unveil a mechanism for regulating actin dynamics during epithelial morphogenesis, providing critical insights on the developmental control of the cellular corte

A humanized mouse model of HPV-associated pathology driven by E7 expression

Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPV-associated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPV-associated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies.This work was supported by Comunidad Autonoma de Madrid (Oncocycle S2006/BIO-0232), by Ministerio de Ciencia e Innovacion (ISCIII-RETIC RD06/0020 and SAF2008-00121), and by Fundación Sandra Ibarra. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Sfrp3 modulates stromal–epithelial crosstalk during mammary gland development by regulating Wnt levels

Mammary stroma is essential for epithelial morphogenesis and development. Indeed, postnatal mammary gland (MG) development is controlled locally by the repetitive and bi-directional cross-talk between the epithelial and the stromal compartment. However, the signalling pathways involved in stromal–epithelial communication are not entirely understood. Here, we identify Sfrp3 as a mediator of the stromal–epithelial communication that is required for normal mouse MG development. Using Drosophila wing imaginal disc, we demonstrate that Sfrp3 functions as an extracellular transporter of Wnts that facilitates their diffusion, and thus, their levels in the boundaries of different compartments. Indeed, loss of Sfrp3 in mice leads to an increase of ductal invasion and branching mirroring an early pregnancy state. Finally, we observe that loss of Sfrp3 predisposes for invasive breast cancer. Altogether, our study shows that Sfrp3 controls MG morphogenesis by modulating the stromal-epithelial cross-talk during pubertal development.MINECO (BFU2015-71244-ERC; BFU2014-52125-REDT; BFU2014-57831), and Fundacion Ramón Areces to F.M-B. M.D.B. was supported by a MINECOFPI 2015 PhD fellowship. M.H. was supported by a MINECO-FPI 2012 PhD fellowship. M.B.F. was supported by a La-Caix

Micropattern-based platform as a physiologically relevant model to study epithelial morphogenesis and nephrotoxicity

International audienc

Endocytic turnover of Rab8 controls cell polarization

Adaptation of cell shape and polarization through the formation and retraction of cellular protrusions requires balancing of endocytosis and exocytosis combined with fine-tuning of the local activity of small GTPases like Rab8. Here, we show that endocytic turnover of the plasma membrane at protrusions is directly coupled to surface removal and inactivation of Rab8. Removal is induced by reduced membrane tension and mediated by the GTPase regulator associated with focal adhesion kinase-1 (GRAF1, also known as ARHGAP26), a regulator of clathrin-independent endocytosis. GRAF1-depleted cells were deficient in multi-directional spreading and displayed elevated levels of GTP-loaded Rab8, which was accumulated at the tips of static protrusions. Furthermore, GRAF1 depletion impaired lumen formation and spindle orientation in a 3D cell culture system, indicating that GRAF1 activity regulates polarity establishment. Our data suggest that GRAF1-mediated removal of Rab8 from the cell surface restricts its activity during protrusion formation, thereby facilitating dynamic adjustment of the polarity axis.Vetenskapsrådet (Swedish Research Council) (dnr 811-2014-59), Stiftelsen fö r Strategisk Forskning (Swedish Foundation for Strategic Research) (ref no. FFL09-0181), the Kempestiftelserna (Kempe Foundation) (SMK-1348) and the Laboratory for Molecular Infection Medicine Sweden (MIMS) (ref no.316-706-10).Peer Reviewe

HPV surrogate marker MCM7 is ectopically expressed in E7-transplants.

<p>Sections of paraffin-embedded grafts were processed for immunofluorescence staining with antibodies to MCM7. MCM7 is normally confined to the basal layer as in control vector transplants (<b>A</b>). Suprabasal expression is eventually observed in HPV5 E7-samples (<b>B</b>). Importantly, MCM7 is expressed in HPV16 E7-grafts in most suprabasal cells, reaching the uppermost spinous cells. Representative images of different 16E7-grafts are shown (<b>C, C’, C’’, C’’’</b>).</p

E7-grafts showed no significant apoptosis.

<p>Sections of paraffin-embedded grafts were processed for immunofluorescence staining with antibodies to the apoptosis markers caspase-3 active and p53. Caspase-3 active is not expressed in control vector samples (<b>A</b>), but some scattered positive cells are observed in HPV5 E7 (<b>B</b>) and HPV16 E7 grafts (<b>C</b> and <b>D</b>). No p53 staining was observed in control and HPV5 E7 samples (not shown), but eventual, p53-positive cells were observed in the HPV16 E7-transplants (<b>D</b>). Dotted line indicates the location of the basal membrane. Inserts in panels <b>B</b>–<b>D</b> show magnified areas of staining. DAPI was used to visualize cell nuclei.</p

Pocket proteins expression in skin grafts.

<p>(<b>A</b>) Pocket protein and eGFP expression was analyzed by immunoblotting with specific antibodies using graft protein lysates from control vector, 5E7 and 16E7 samples. (<b>B</b>) Pocket protein and eGFP protein expression bands were quantified and relative (pocket protein/eGFP) values plotted. Horizontal green lines represents mean relative values. According to Student’s t-test, significant differences were only detected in pRb/eGFP expression values between vector and 16E7 samples (threshold p-val<0.05). *: p-val<0.05.</p