Ekaterina Papusheva | Novosibirsk State University (original) (raw)

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Papers by Ekaterina Papusheva

Journal of Cell Science, 2009

Embo Journal, 2010

Integrin-and cadherin-mediated adhesion is central for cell and tissue morphogenesis, allowing ce... more Integrin-and cadherin-mediated adhesion is central for cell and tissue morphogenesis, allowing cells and tissues to change shape without loosing integrity. Studies predominantly in cell culture showed that mechanosensation through adhesion structures is achieved by force-mediated modulation of their molecular composition. The specific molecular composition of adhesion sites in turn determines their signalling activity and dynamic reorganization. Here, we will review how adhesion sites respond to mecanical stimuli, and how spatially and temporally regulated signalling from different adhesion sites controls cell migration and tissue morphogenesis.

Nature Cell Biology, 2009

protrusions to bring about movement in the context of the three-dimensional cellular environment ... more protrusions to bring about movement in the context of the three-dimensional cellular environment 5,6 . Here we show that the motility of chemokine-guided germ cells within the zebrafish embryo requires the function of the small Rho GTPases Rac1 and RhoA, as well as E-cadherin-mediated cellcell adhesion. Using fluorescence resonance energy transfer we demonstrate that Rac1 and RhoA are activated in the cell front. At this location, Rac1 is responsible for the formation of actinrich structures, and RhoA promotes retrograde actin flow. We propose that these actin-rich structures undergoing retrograde flow are essential for the generation of E-cadherin-mediated traction forces between the germ cells and the surrounding tissue and are therefore crucial for cell motility in vivo.

Journal of Molecular Evolution, 2004

Genomic DNA amplification from 51 species of the family Chironomidae shows that most contain rela... more Genomic DNA amplification from 51 species of the family Chironomidae shows that most contain relatives of NLRCth1 LINE and CTRT1 SINE retrotransposons first found in Chironomus thummi. More than 300 cloned PCR products were sequenced. The amplified region of the reverse transcriptase gene in the LINEs is intact and highly conserved, suggesting active elements. The SINEs are less conserved, consistent with minimal/no selection after transposition. A mitochondrial gene phylogeny resolves the Chironomus genus into six lineages (Guryev et al. 2001). LINE and SINE phylogenies resolve five of these lineages, indicating their monophyletic origin and vertical inheritance. However, both the LINE and the SINE tree topologies differ from the species phylogeny, resolving the elements into “clusters I–IV” and “cluster V” families. The data suggest a descent of all LINE and SINE subfamilies from two major families. Based on the species phylogeny, a few LINEs and a larger number of SINEs are cladisitically misplaced. Most misbranch with LINEs or SINEs from species with the same families of elements. From sequence comparisons, cladistically misplaced LINEs and several misplaced SINEs arose by convergent base substitutions. More diverged SINEs result from early transposition and some are derived from multiple source SINEs in the same species. SINEs from two species (C. dorsalis, C. pallidivittatus), expected to belong to the clusters I–IV family, branch instead with cluster V family SINEs; apparently both families predate separation of cluster V from clusters I–IV species. Correlation of the distribution of active SINEs and LINEs, as well as similar 3′ sequence motifs in CTRT1 and NLRCth1, suggests coevolving retrotransposon pairs in which CTRT1 transposition depends on enzymes active during NLRCth1 LINE mobility.

Russian Journal of Genetics, 2011

Non-Long Terminal Repeats (non-LTR or LINE) retrotransposons belong to the class of mobile geneti... more Non-Long Terminal Repeats (non-LTR or LINE) retrotransposons belong to the class of mobile genetic elements that are transposed into the host genome by reverse transcription of the RNA intermediate. Most of non-LTR retrotransposons contain two open reading frames (ORFs). The ORF1 codes for a gag-like protein, while the ORF2 codes for a reverse transcriptase (RT). We cloned two constructs based on Jockey-like non-LTR retrotransposon from genome Chironomus thummi (NLR1Cth). The retroposition assay performed in Chinese hamster ovary (CHO) cells demonstrated genome integrations of both constructs. The finding that the insect mobile element NLR1Cth is functional in mammalian cells demonstrates that this element possess universal enzymatic machinery allowing for active propagation in the genome of distant taxa. This suggests that the NLR1Cth transposon system may represent a useful tool for genetic analysis and manipulation in vertebrate cells.

Biochimica Et Biophysica Acta-molecular Cell Research, 2008

In the present study we analyzed the oligomerization state of the serotonin 5-HT1A receptor and s... more In the present study we analyzed the oligomerization state of the serotonin 5-HT1A receptor and studied oligomerization dynamics in living cells. We also investigated the role of receptor palmitoylation in this process. Biochemical analysis performed in neuroblastoma N1E-115 cells demonstrated that both palmitoylated and non-palmitoylated 5-HT1A receptors form homo-oligomers and that the prevalent receptor species at the plasma membrane are dimers. A combination of an acceptor-photobleaching FRET approach with fluorescence lifetime measurements verified the interaction of CFP-and YFP-labeled wild-type as well as acylation-deficient 5-HT1A receptors at the plasma membrane of living cells. Using a novel FRET technique based on the spectral analysis we also confirmed the specific nature of receptor oligomerization. The analysis of oligomerization dynamics revealed that apparent FRET efficiency measured for wild-type oligomers significantly decreased in response to agonist stimulation, and our combined results suggest that this decrease was mediated by accumulation of FRET-negative complexes rather than by dissociation of oligomers to monomers. In contrast, the agonist-mediated decrease of FRET signal was completely abolished in oligomers composed by non-palmitoylated receptor mutants, demonstrating the importance of palmitoylation in modulation of the structure of oligomers.

Molecular Pharmacology, 2007

In the present study, we have used wild-type and palmitoylation-deficient mouse 5-hydroxytryptami... more In the present study, we have used wild-type and palmitoylation-deficient mouse 5-hydroxytryptamine 1A receptor (5-HT1A) receptors fused to the yellow fluorescent protein-and the cyan fluorescent protein (CFP)-tagged ␣ i3 subunit of heterotrimeric G-protein to study spatiotemporal distribution of the 5-HT1Amediated signaling in living cells. We also addressed the question on the molecular mechanisms by which receptor palmitoylation may regulate communication between receptors and G i -proteins. Our data demonstrate that activation of the 5-HT1A receptor caused a partial release of G␣ i protein into the cytoplasm and that this translocation is accompanied by a significant increase of the intracellular Ca 2ϩ con-centration. In contrast, acylation-deficient 5-HT1A mutants failed to reproduce both G␣ i3 -CFP relocation and changes in [Ca 2ϩ ] i upon agonist stimulation. By using gradient centrifugation and copatching assays, we also demonstrate that a significant fraction of the 5-HT1A receptor resides in membrane rafts, whereas the yield of the palmitoylation-deficient receptor in these membrane microdomains is reduced considerably. Our results suggest that receptor palmitoylation serves as a targeting signal responsible for the retention of the 5-HT1A receptor in membrane rafts. More importantly, the raft localization of the 5-HT1A receptor seems to be involved in receptor-mediated signaling.

Journal of Cell Science, 2009

Embo Journal, 2010

Integrin-and cadherin-mediated adhesion is central for cell and tissue morphogenesis, allowing ce... more Integrin-and cadherin-mediated adhesion is central for cell and tissue morphogenesis, allowing cells and tissues to change shape without loosing integrity. Studies predominantly in cell culture showed that mechanosensation through adhesion structures is achieved by force-mediated modulation of their molecular composition. The specific molecular composition of adhesion sites in turn determines their signalling activity and dynamic reorganization. Here, we will review how adhesion sites respond to mecanical stimuli, and how spatially and temporally regulated signalling from different adhesion sites controls cell migration and tissue morphogenesis.

Nature Cell Biology, 2009

protrusions to bring about movement in the context of the three-dimensional cellular environment ... more protrusions to bring about movement in the context of the three-dimensional cellular environment 5,6 . Here we show that the motility of chemokine-guided germ cells within the zebrafish embryo requires the function of the small Rho GTPases Rac1 and RhoA, as well as E-cadherin-mediated cellcell adhesion. Using fluorescence resonance energy transfer we demonstrate that Rac1 and RhoA are activated in the cell front. At this location, Rac1 is responsible for the formation of actinrich structures, and RhoA promotes retrograde actin flow. We propose that these actin-rich structures undergoing retrograde flow are essential for the generation of E-cadherin-mediated traction forces between the germ cells and the surrounding tissue and are therefore crucial for cell motility in vivo.

Journal of Molecular Evolution, 2004

Genomic DNA amplification from 51 species of the family Chironomidae shows that most contain rela... more Genomic DNA amplification from 51 species of the family Chironomidae shows that most contain relatives of NLRCth1 LINE and CTRT1 SINE retrotransposons first found in Chironomus thummi. More than 300 cloned PCR products were sequenced. The amplified region of the reverse transcriptase gene in the LINEs is intact and highly conserved, suggesting active elements. The SINEs are less conserved, consistent with minimal/no selection after transposition. A mitochondrial gene phylogeny resolves the Chironomus genus into six lineages (Guryev et al. 2001). LINE and SINE phylogenies resolve five of these lineages, indicating their monophyletic origin and vertical inheritance. However, both the LINE and the SINE tree topologies differ from the species phylogeny, resolving the elements into “clusters I–IV” and “cluster V” families. The data suggest a descent of all LINE and SINE subfamilies from two major families. Based on the species phylogeny, a few LINEs and a larger number of SINEs are cladisitically misplaced. Most misbranch with LINEs or SINEs from species with the same families of elements. From sequence comparisons, cladistically misplaced LINEs and several misplaced SINEs arose by convergent base substitutions. More diverged SINEs result from early transposition and some are derived from multiple source SINEs in the same species. SINEs from two species (C. dorsalis, C. pallidivittatus), expected to belong to the clusters I–IV family, branch instead with cluster V family SINEs; apparently both families predate separation of cluster V from clusters I–IV species. Correlation of the distribution of active SINEs and LINEs, as well as similar 3′ sequence motifs in CTRT1 and NLRCth1, suggests coevolving retrotransposon pairs in which CTRT1 transposition depends on enzymes active during NLRCth1 LINE mobility.

Russian Journal of Genetics, 2011

Non-Long Terminal Repeats (non-LTR or LINE) retrotransposons belong to the class of mobile geneti... more Non-Long Terminal Repeats (non-LTR or LINE) retrotransposons belong to the class of mobile genetic elements that are transposed into the host genome by reverse transcription of the RNA intermediate. Most of non-LTR retrotransposons contain two open reading frames (ORFs). The ORF1 codes for a gag-like protein, while the ORF2 codes for a reverse transcriptase (RT). We cloned two constructs based on Jockey-like non-LTR retrotransposon from genome Chironomus thummi (NLR1Cth). The retroposition assay performed in Chinese hamster ovary (CHO) cells demonstrated genome integrations of both constructs. The finding that the insect mobile element NLR1Cth is functional in mammalian cells demonstrates that this element possess universal enzymatic machinery allowing for active propagation in the genome of distant taxa. This suggests that the NLR1Cth transposon system may represent a useful tool for genetic analysis and manipulation in vertebrate cells.

Biochimica Et Biophysica Acta-molecular Cell Research, 2008

In the present study we analyzed the oligomerization state of the serotonin 5-HT1A receptor and s... more In the present study we analyzed the oligomerization state of the serotonin 5-HT1A receptor and studied oligomerization dynamics in living cells. We also investigated the role of receptor palmitoylation in this process. Biochemical analysis performed in neuroblastoma N1E-115 cells demonstrated that both palmitoylated and non-palmitoylated 5-HT1A receptors form homo-oligomers and that the prevalent receptor species at the plasma membrane are dimers. A combination of an acceptor-photobleaching FRET approach with fluorescence lifetime measurements verified the interaction of CFP-and YFP-labeled wild-type as well as acylation-deficient 5-HT1A receptors at the plasma membrane of living cells. Using a novel FRET technique based on the spectral analysis we also confirmed the specific nature of receptor oligomerization. The analysis of oligomerization dynamics revealed that apparent FRET efficiency measured for wild-type oligomers significantly decreased in response to agonist stimulation, and our combined results suggest that this decrease was mediated by accumulation of FRET-negative complexes rather than by dissociation of oligomers to monomers. In contrast, the agonist-mediated decrease of FRET signal was completely abolished in oligomers composed by non-palmitoylated receptor mutants, demonstrating the importance of palmitoylation in modulation of the structure of oligomers.

Molecular Pharmacology, 2007

In the present study, we have used wild-type and palmitoylation-deficient mouse 5-hydroxytryptami... more In the present study, we have used wild-type and palmitoylation-deficient mouse 5-hydroxytryptamine 1A receptor (5-HT1A) receptors fused to the yellow fluorescent protein-and the cyan fluorescent protein (CFP)-tagged ␣ i3 subunit of heterotrimeric G-protein to study spatiotemporal distribution of the 5-HT1Amediated signaling in living cells. We also addressed the question on the molecular mechanisms by which receptor palmitoylation may regulate communication between receptors and G i -proteins. Our data demonstrate that activation of the 5-HT1A receptor caused a partial release of G␣ i protein into the cytoplasm and that this translocation is accompanied by a significant increase of the intracellular Ca 2ϩ con-centration. In contrast, acylation-deficient 5-HT1A mutants failed to reproduce both G␣ i3 -CFP relocation and changes in [Ca 2ϩ ] i upon agonist stimulation. By using gradient centrifugation and copatching assays, we also demonstrate that a significant fraction of the 5-HT1A receptor resides in membrane rafts, whereas the yield of the palmitoylation-deficient receptor in these membrane microdomains is reduced considerably. Our results suggest that receptor palmitoylation serves as a targeting signal responsible for the retention of the 5-HT1A receptor in membrane rafts. More importantly, the raft localization of the 5-HT1A receptor seems to be involved in receptor-mediated signaling.