Protein p130Cas constitutes an adaptor protein mainly involved in integrin signaling downstream of Src kinase. Owing to its modular structure, p130Cas acts as a general regulator of cancer cell growth and invasiveness induced by different oncogenes. However, other mechanisms of p130Cas signaling leading to malignant progression are poorly understood. Here, we show a novel interaction of p130Cas with Ser/Thr kinase PKN3, which is implicated in prostate and breast cancer growth downstream of phosphoinositide 3-kinase. This direct interaction is mediated by the p130Cas SH3 domain and the centrally located PKN3 polyproline sequence. PKN3 is the first identified Ser/Thr kinase to bind and phosphorylate p130Cas and to colocalize with p130Cas in cell structures that have a pro-invasive function. Moreover, the PKN3-p130Cas interaction is important for mouse embryonic fibroblast growth and invasiveness independent of Src transformation, indicating a mechanism distinct from that previously characterized for p130Cas. Together, our results suggest that the PKN3-p130Cas complex represents an attractive therapeutic target in late-stage malignancies.
- MeSH
- fibroblasty metabolismus MeSH
- fosforylace MeSH
- fosfothreonin metabolismus MeSH
- invazivní růst nádoru MeSH
- kontraktilní svazky metabolismus MeSH
- lidé MeSH
- myši nahé MeSH
- nádory metabolismus patologie MeSH
- podozomy metabolismus MeSH
- pohyb buněk MeSH
- proliferace buněk MeSH
- proteinkinasa C metabolismus MeSH
- pseudopodia metabolismus MeSH
- skupina kinas odvozených od src-genu metabolismus MeSH
- substrátový protein asociovaný s Crk metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Arginine-rich cell-penetrating peptides do not enter cells by directly passing through a lipid membrane; they instead passively enter vesicles and live cells by inducing membrane multilamellarity and fusion. The molecular picture of this penetration mode, which differs qualitatively from the previously proposed direct mechanism, is provided by molecular dynamics simulations. The kinetics of vesicle agglomeration and fusion by an iconic cell-penetrating peptide-nonaarginine-are documented via real-time fluorescence techniques, while the induction of multilamellar phases in vesicles and live cells is demonstrated by a combination of electron and fluorescence microscopies. This concert of experiments and simulations reveals that the identified passive cell penetration mechanism bears analogy to vesicle fusion induced by calcium ions, indicating that the two processes may share a common mechanistic origin.
- MeSH
- arginin metabolismus fyziologie MeSH
- biologický transport MeSH
- buněčná membrána metabolismus MeSH
- fúze membrán účinky léků fyziologie MeSH
- kinetika MeSH
- lipidové dvojvrstvy chemie MeSH
- membrány metabolismus MeSH
- penetrační peptidy chemie metabolismus MeSH
- peptidy chemie fyziologie MeSH
- pseudopodia metabolismus fyziologie MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Observation and analysis of cancer cell behaviour in 3D environment is essential for full understanding of the mechanisms of cancer cell invasion. However, label-free imaging of live cells in 3D conditions is optically more challenging than in 2D. Quantitative phase imaging provided by coherence controlled holographic microscopy produces images with enhanced information compared to ordinary light microscopy and, due to inherent coherence gate effect, enables observation of live cancer cells' activity even in scattering milieu such as the 3D collagen matrix. Exploiting the dynamic phase differences method, we for the first time describe dynamics of differences in cell mass distribution in 3D migrating mesenchymal and amoeboid cancer cells, and also demonstrate that certain features are shared by both invasion modes. We found that amoeboid fibrosarcoma cells' membrane blebbing is enhanced upon constriction and is also occasionally present in mesenchymally invading cells around constricted nuclei. Further, we demonstrate that both leading protrusions and leading pseudopods of invading fibrosarcoma cells are defined by higher cell mass density. In addition, we directly document bundling of collagen fibres by protrusions of mesenchymal fibrosarcoma cells. Thus, such a non-invasive microscopy offers a novel insight into cellular events during 3D invasion.
- MeSH
- buněčná membrána metabolismus MeSH
- buněčné kultury metody MeSH
- fibrosarkom diagnostické zobrazování patologie MeSH
- holografie přístrojové vybavení metody MeSH
- intravitální mikroskopie přístrojové vybavení metody MeSH
- invazivní růst nádoru diagnostické zobrazování patologie MeSH
- kolagen metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- pohyb buněk * MeSH
- pseudopodia metabolismus MeSH
- zobrazování trojrozměrné přístrojové vybavení metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.
- MeSH
- aktiny účinky léků metabolismus MeSH
- buněčná adheze MeSH
- CD antigeny účinky léků metabolismus MeSH
- cévní endotel MeSH
- endoteliální buňky pupečníkové žíly (lidské) MeSH
- endoteliální buňky účinky léků metabolismus fyziologie MeSH
- fyziologická neovaskularizace účinky léků fyziologie MeSH
- kadheriny účinky léků metabolismus MeSH
- komplex proteinů 2-3 souvisejících s aktinem metabolismus MeSH
- lehké řetězce myosinu metabolismus MeSH
- lidé MeSH
- mezibuněčné spoje účinky léků metabolismus MeSH
- mikrotubuly účinky léků metabolismus MeSH
- modely kardiovaskulární MeSH
- pohyb buněk účinky léků fyziologie MeSH
- polarita buněk účinky léků fyziologie MeSH
- protein 2 související s aktinem metabolismus MeSH
- protein 3 související s aktinem metabolismus MeSH
- protein Wiskottova-Aldrichova syndromu metabolismus MeSH
- pseudopodia účinky léků metabolismus fyziologie MeSH
- rac proteiny vázající GTP metabolismus MeSH
- receptor 2 pro vaskulární endoteliální růstový faktor metabolismus MeSH
- remodelace cév MeSH
- rodina proteinů Wiskottova-Aldrichova syndromu metabolismus MeSH
- signální transdukce MeSH
- srdeční myosiny metabolismus MeSH
- vaskulární endoteliální růstový faktor A metabolismus farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transmembrane adaptor proteins are membrane-anchored proteins consisting of a short extracellular part, a transmembrane domain, and a cytoplasmic part with various protein-protein interaction motifs but lacking any enzymatic activity. They participate in the regulation of various signaling pathways by recruiting other proteins to the proximity of cellular membranes where the signaling is often initiated and propagated. In this work, we show that LST1/A, an incompletely characterized protein encoded by MHCIII locus, is a palmitoylated transmembrane adaptor protein. It is expressed specifically in leukocytes of the myeloid lineage, where it localizes to the tetraspanin-enriched microdomains. In addition, it binds SHP-1 and SHP-2 phosphatases in a phosphotyrosine-dependent manner, facilitating their recruitment to the plasma membrane. These data suggest a role for LST1/A in negative regulation of signal propagation.
- MeSH
- buněčná membrána metabolismus MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- hlavní histokompatibilní komplex fyziologie MeSH
- Jurkat buňky MeSH
- lidé MeSH
- membránové proteiny chemie genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- myeloidní buňky cytologie metabolismus MeSH
- plakiny metabolismus MeSH
- primární buněčná kultura MeSH
- pseudopodia metabolismus MeSH
- sekvence aminokyselin MeSH
- signální transdukce fyziologie MeSH
- terciární struktura proteinů fyziologie MeSH
- transport proteinů fyziologie MeSH
- tyrosinfosfatasa nereceptorového typu 11 metabolismus MeSH
- tyrosinfosfatasa nereceptorového typu 6 metabolismus MeSH
- U937 buňky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
AIM: In this study, we analysed the post-translational modification of receptor tyrosine kinase-like orphan receptor (Ror1). Ror1 is highly upregulated in B cells of patients with chronic lymphocytic leukaemia (CLL). Molecularly, Ror1 acts as the Wnt receptor in the non-canonical Wnt pathway. METHODS: The level of Ror1 glycosylation in HEK293 cells and in primary human CLL cells was analysed by treatment of inhibitors interfering with different steps of glycosylation process and by direct treatment of cell lysates with N-glycosidase. Ror1 ubiquitination was determined by ubiquitination assay. Functional consequences of post-translational modifications were analysed by immunohistochemistry and by analysis of cell surface proteins. Differences in Ror1 glycosylation were confirmed by analysis of 14 samples of B cells from CLL patients. RESULTS: We demonstrate that Ror1 is extensively modified by N-linked glycosylation. Glycosylation produces several variants of Ror1 with electrophoretic migration of approx. 100, 115 and 130 kDa. Inhibition of glycosylation interferes with cell surface localization of the 130-kDa variant of Ror1 and prevents Ror1-induced formation of filopodia. Moreover, we show that 130-kDa Ror1 is mono-ubiquitinated. Furthermore, individual CLL patients show striking differences in the electrophoretic migration of Ror1, which correspond to the level of glycosylation. CONCLUSION: Our data show that Ror1 undergoes complex post-translational modifications by glycosylation and mono-ubiquitination. These modifications regulate Ror1 localization and signalling, and are highly variable among individual CLL patients. These may suggest that Ror1 signals only in a subset of CLL patients despite Ror1 levels are ubiquitously high in all CLL patients.
- MeSH
- B-lymfocyty metabolismus MeSH
- CHO buňky MeSH
- chronická lymfatická leukemie metabolismus MeSH
- Cricetulus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- glykosylace MeSH
- HEK293 buňky MeSH
- imunohistochemie MeSH
- konfokální mikroskopie MeSH
- křečci praví MeSH
- lidé MeSH
- molekulová hmotnost MeSH
- posttranslační úpravy proteinů účinky léků MeSH
- průtoková cytometrie MeSH
- pseudopodia metabolismus MeSH
- signální transdukce účinky léků MeSH
- sirotčí receptory podobné receptoru tyrosinkinasy chemie genetika metabolismus MeSH
- transfekce MeSH
- transport proteinů MeSH
- ubikvitinace MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The newly established breast cancer cell line G3S1, derived from EM-G3 breast cancer progenitors, was analyzed for functional changes related to neoplastic progression manifested by elevated invasiveness and enhanced capability to degrade gelatin. Degradation of gelatin and invasiveness of G3S1 cells was found to be dependent on the activity of matrix proteinases and actin cytoskeletal dynamics. Therefore, the expression and activity of these proteases was compared in G3S1 and EM-G3 cells. Despite enhanced capability of G3S1 cells to degrade gelatin, these cells exhibited lower levels of secreted extracellular matrix degrading proteases than parental EM-G3 cells. However, the expression of membrane-bound MT1-MMP was strongly elevated in G3S1 cells. While the degradation of gelatin was associated with invadopodia-like structures in both EM-G3 and G3S1 cells, the cytoskeletal remodeling dynamics was greatly elevated in G3S1 cells, suggesting that upregulation of MT1-MMP, together with elevation of cytoskeletal remodeling dynamics can effectively cause elevated invasiveness and enhanced matrix degrading capability in G3S1 cells.
- MeSH
- aktiny metabolismus MeSH
- aprotinin farmakologie MeSH
- cytoskelet účinky léků enzymologie patologie MeSH
- dipeptidy farmakologie MeSH
- inhibitory matrixových metaloproteinas MeSH
- inhibitory proteas farmakologie MeSH
- invazivní růst nádoru MeSH
- leucin analogy a deriváty farmakologie MeSH
- lidé MeSH
- matrixová metaloproteinasa 14 metabolismus MeSH
- metaloproteinasy secernované do matrix metabolismus MeSH
- mořské toxiny farmakologie MeSH
- nádorové buněčné linie MeSH
- nádorové kmenové buňky enzymologie patologie MeSH
- nádory prsu enzymologie patologie MeSH
- pohyb buněk účinky léků MeSH
- progrese nemoci MeSH
- pseudopodia enzymologie MeSH
- upregulace MeSH
- želatina metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
