Endothelium and subendothelial matrix mechanics modulate cancer cell transendothelial migration

Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the invasion into the subendothelial extracellular matrix (ECM) of distant tissues. While cancer research has mostly focused on the biomechanical interactions between tumor cells (TCs) and ECM, particularly at the primary tumor site, very little is known about the mechanical properties of endothelial cells and the subendothelial ECM and how they contribute to the extravasation process. Here, an integrated experimental and theoretical framework is developed to investigate the mechanical crosstalk between TCs, endothelium and subendothelial ECM during in vitro cancer cell extravasation. It is found that cancer cell actin-rich protrusions generate complex push–pull forces to initiate and drive TEM, while transmigration success also relies on the forces generated by the endothelium. Consequently, mechanical properties of the subendothelial ECM and endothelial actomyosin contractility that mediate the endothelial forces also impact the endothelium's resistance to cancer cell transmigration. These results indicate that mechanical features of distant tissues, including force interactions between the endothelium and the subendothelial ECM, are key determinants of metastatic organotropism.Peer ReviewedPostprint (published version

Triazolopyrimidines stabilize microtubules by binding to the vinca inhibitor site of tubulin

53 p.-7 fig.+ 7 p.-1 tab.supl.-6 fig.supl. Saez Calvo, Gonzalo et al.Microtubule-targeting agents (MTAs) are some of the clinically most successful anti-cancer drugs. Unfortunately, instances of multidrug resistances to MTA have been reported, which highlights the need for developing MTAs with different mechanistic properties. One less explored class of MTAs are [1,2,4]triazolo[1,5-a]pyrimidines (TPs). These cytotoxic compounds are microtubule-stabilizing agents that inexplicably bind to vinblastine binding site on tubulin, which is typically targeted by microtubule-destabilizing agents. Here we used cellular, biochemical, and structural biology approaches to address this apparent discrepancy. Our results establish TPs as vinca-site microtubule-stabilizing agents that promote longitudinal tubulin contacts in microtubules, in contrast to classical microtubule-stabilizing agents that primarily promote lateral contacts. Additionally we observe that TPs studied here are not affected by p-glycoprotein overexpression, and suggest that TPs are promising ligands against multidrug-resistant cancer cells.This work was supported in part by grants BFU2016‐75319‐R (AEI/FEDER, UE) (JFD), BFU2014‐51823‐R (to JMA) from Ministerio de Economia y Competitividad and 31003A_166608 from the Swiss National Science Foundation (M.O.S.). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature ‐ from natural products chemistry to drug discovery”. The SAXS experiments were performed at BL11‐NCD beamline at ALBA Synchrotron with the collaboration of ALBA staff. X‐ray data were collected at beamline X06DA of the Swiss Light Source (Paul Scherrer Institut, Villigen, Switzerland).Peer reviewe

Zampanolide, a potent new microtubule stabilizing agent, covalently reacts with the taxane luminal site in both tubulin alpha-beta-heterodimers and microtubules

13 páginas, 3 figuras, 1 tabla -- PAGS nros. 686-698Zampanolide and its less active analog dactylolide compete with paclitaxel for binding to microtubules and represent a new class of microtubule-stabilizing agent (MSA). Mass spectrometry demonstrated that the mechanism of action of both compounds involved covalent binding to β-tubulin at residues N228 and H229 in the taxane site of the microtubule. Alkylation of N228 and H229 was also detected in α,β-tubulin dimers. However, unlike cyclostreptin, the other known MSA that alkylates β-tubulin, zampanolide was a strong MSA. Modeling the structure of the adducts, using the NMR-derived dactylolide conformation, indicated that the stabilizing activity of zampanolide is likely due to interactions with the M-loop. Our results strongly support the existence of the luminal taxane site of microtubules in tubulin dimers and suggest that microtubule nucleation induction by MSAs may proceed through an allosteric mechanismThis work was supported in part by grants BIO2010-16351 and CTQ2009-08536 from Ministerio de Economia y Competitividad to J.F.D. and J.J.B., respectively, and grant S2010/BMD-2457 BIPEDD2 from Comunidad Autónoma de Madrid to J.F.D., the Cancer Society of New Zealand, and the Wellington Medical Research Foundation (J.M.). The CNIC is supported by the Ministerio de Economía y Competitividad and the Fundación Pro CNICPeer reviewe

Ruralidad y campesinado

¿Existe aún el campo? ¿Desapareció el campesinado? ¿Triunfó la expansión del modo de vida urbano y de los urbanistas? ¿Subsiste aún un modo de vida rural? ¿Existe aún una cultura campesina? ¿Cuáles son las tensiones y principales amenazas que recorren el mundo rural latinoamericano? ¿Cuáles son las formas de resistencia desde la identidad campesina e indígena a la marea globalizante? ¿Qué experiencias promisorias y/ o exitosas se están desplegando en el mundo rural? ¿Cómo se ha transformado la ruralidad en las últimas décadas? ¿Cómo se enlaza la producción agraria con la emergencia de territorios intermedios? son algunas de las muchas preguntas que abordamos en este número de Polis

Molecular Recognition of Epothilones by Microtubules and Tubulin Dimers Revealed by Biochemical and NMR Approaches

The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are 4 orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained, supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogues has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.</p