| 1. |
- Aguilar, Ximena, 1978-, et al.
(författare)
-
Interaction Studies of the Human and Arabidopsis thaliana Med25-ACID Proteins with the Herpes Simplex Virus VP16-and Plant-Specific Dreb2a Transcription Factors
- 2014
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:5, s. e98575-
-
Tidskriftsartikel (refereegranskat)abstract
- Mediator is an evolutionary conserved multi-protein complex present in all eukaryotes. It functions as a transcriptional coregulator by conveying signals from activators and repressors to the RNA polymerase II transcription machinery. The Arabidopsis thaliana Med25 (aMed25) ACtivation Interaction Domain (ACID) interacts with the Dreb2a activator which is involved in plant stress response pathways, while Human Med25-ACID (hMed25) interacts with the herpes simplex virus VP16 activator. Despite low sequence similarity, hMed25-ACID also interacts with the plant-specific Dreb2a transcriptional activator protein. We have used GST pull-down-, surface plasmon resonance-, isothermal titration calorimetry and NMR chemical shift experiments to characterize interactions between Dreb2a and VP16, with the hMed25 and aMed25-ACIDs. We found that VP16 interacts with aMed25-ACID with similar affinity as with hMed25-ACID and that the binding surface on aMed25-ACID overlaps with the binding site for Dreb2a. We also show that the Dreb2a interaction region in hMed25-ACID overlaps with the earlier reported VP16 binding site. In addition, we show that hMed25-ACID/Dreb2a and aMed25-ACID/Dreb2a display similar binding affinities but different binding energetics. Our results therefore indicate that interaction between transcriptional regulators and their target proteins in Mediator are less dependent on the primary sequences in the interaction domains but that these domains fold into similar structures upon interaction.
|
|
| 2. |
|
|
| 3. |
- Blomberg, Jeanette, et al.
(författare)
-
An Ethnographic Approach to Design
- 2002
-
Ingår i: Handbook of Human-Computer Interaction in Interactive Systems. - New Jersey : Lawrence Erlbaum Associates, Inc..
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)
|
|
| 4. |
|
|
| 5. |
- Blomberg, Jeanette, et al.
(författare)
-
Ethnography : positioning ethnography within participatory design
- 2012
-
Ingår i: Routledge International Handbook of Participatory Design. - : Routledge Mental Health. - 9780415694407 - 9780203108543 ; , s. 86-116
-
Bokkapitel (refereegranskat)abstract
- This chapter explores the history of ethnography in Participatory Design, the varied approaches that have been developed to connect ethnography and cooperative design, and the association this particular history has with the more general question of the relation between the sensibilities, commitments and requirements of design and of ethnography. As part of this exploration we discuss the practical limits and philosophical synergies between ethnography and design with attention to the similarities and incongruities between Participatory Design and ethnography. We conclude by reimagining ethnography within Participatory Design, pointing to the opportunities and challenges for a new generation of participatory designers. The commitments and guiding principles of ethnography and Participatory Design are examined, followed by a presentation of the foundational research on the application of ethnography to Participatory Design. Then the (re)positioning of ethnography and design are explored, including discussion of the role of reflexivity in Participatory Design. Finally, new opportunities and challenges are presented.This chapter re-examines the relationship between ethnography and Participatory Design, suggesting that researchers continue to interrogate and reflect on the role of ethnography in Participatory Design. It notes that a dichotomy has emerged in how ethnography is positioned in relation to Participatory Design; on the one hand normalised, accepted as part of Participatory Design practice, and on the other ‘backgrounded’, secondary to those activities that directly engage participants in design. It argues that the analytic purchase of ethnography as more than method is just as important today as it was earlier and that Participatory Design could learn from ethnography’s reflexivity.Positioning ethnography within Participatory Design is taking us beyond familiar terrains, con- fronting the challenges of designing for large-scale systems and the socio-technical infrastructures that enable global connections, and doing so with the participation of people from different knowledge traditions and socio-economic circumstances. Given these challenges, the sensibilities of ethnography continue to provide an important guide to Participatory Design.
|
|
| 6. |
- Blomberg, Jeanette, et al.
(författare)
-
Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α in a resistant variant of the human U937 cell line
- 2011
-
Ingår i: Apoptosis (London). - : Springer Science and Business Media LLC. - 1360-8185 .- 1573-675X. ; 16:8, s. 783-794
-
Tidskriftsartikel (refereegranskat)abstract
- Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.
|
|
| 7. |
- Blomberg, Jeanette, et al.
(författare)
-
Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes
- 2012
-
Ingår i: Nucleic Acids Research. - Oxford : Oxford University Press. - 0305-1048 .- 1362-4962. ; 40:13, s. 5938-5950
-
Tidskriftsartikel (refereegranskat)abstract
- Mediator is a multiprotein coregulatory complex that conveys signals from DNA-bound transcriptional regulators to the RNA polymerase II transcription machinery in eukaryotes. The molecular mechanisms for how these signals are transmitted are still elusive. By using purified transcription factor Dreb2a, mediator subunit Med25 from Arabidopsis thaliana, and a combination of biochemical and biophysical methods, we show that binding of Dreb2a to its canonical DNA sequence leads to an increase in secondary structure of the transcription factor. Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes. However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25. We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.
|
|
| 8. |
- Blomberg, Jeanette, et al.
(författare)
-
Participatory Design : Issues and Concerns
- 1998
-
Ingår i: Computer Supported Cooperative Work. - : Springer. - 0925-9724 .- 1573-7551. ; 7:3-4, s. 167-185
-
Tidskriftsartikel (refereegranskat)
|
|
| 9. |
- Blomberg, Jeanette, et al.
(författare)
-
Pseudomonas syringae infectivity correlates to altered transcript and metabolite levels of Arabidopsis mediator mutants
- 2024
-
Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 14:1
-
Tidskriftsartikel (refereegranskat)abstract
- Rapid metabolic responses to pathogens are essential for plant survival and depend on numerous transcription factors. Mediator is the major transcriptional co-regulator for integration and transmission of signals from transcriptional regulators to RNA polymerase II. Using four Arabidopsis Mediator mutants, med16, med18, med25 and cdk8, we studied how differences in regulation of their transcript and metabolite levels correlate to their responses to Pseudomonas syringae infection. We found that med16 and cdk8 were susceptible, while med25 showed increased resistance. Glucosinolate, phytoalexin and carbohydrate levels were reduced already before infection in med16 and cdk8, but increased in med25, which also displayed increased benzenoids levels. Early after infection, wild type plants showed reduced glucosinolate and nucleoside levels, but increases in amino acids, benzenoids, oxylipins and the phytoalexin camalexin. The Mediator mutants showed altered levels of these metabolites and in regulation of genes encoding key enzymes for their metabolism. At later stage, mutants displayed defective levels of specific amino acids, carbohydrates, lipids and jasmonates which correlated to their infection response phenotypes. Our results reveal that MED16, MED25 and CDK8 are required for a proper, coordinated transcriptional response of genes which encode enzymes involved in important metabolic pathways for Arabidopsis responses to Pseudomonas syringae infections.
|
|
| 10. |
- Blomberg, Jeanette, 1977-, et al.
(författare)
-
Reduced FAS transcription in clones of U937 cells that have acquired resistance to Fas-induced apoptosis
- 2009
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:2, s. 497-508
-
Tidskriftsartikel (refereegranskat)abstract
- Susceptibility to cell death is a prerequisite for the elimination of tumour cells by cytotoxic immune cells, chemotherapy or irradiation. Activation of the death receptor Fas is critical for the regulation of immune cell homeostasis and efficient killing of tumour cells by apoptosis. To define the molecular changes that occur during selection for insensitivity to Fas-induced apoptosis, a resistant variant of the U937 cell line was established. Individual resistant clones were isolated and characterized. The most frequently observed defect in the resistant cells was reduced Fas expression, which correlated with decreased FAS transcription. Clones with such reduced Fas expression also displayed partial cross-resistance to tumour necrosis factor-alpha stimulation, but the mRNA expression of tumour necrosis factor receptors was not decreased. Reintroduction of Fas conferred susceptibility to Fas but not to tumour necrosis factor-alpha stimulation, suggesting that several alterations could be present in the clones. The reduced Fas expression could not be explained by mutations in the FAS coding sequence or promoter region, or by silencing through methylations. Protein kinase B and extracellular signal-regulated kinase, components of signalling pathways downstream of Ras, were shown to be activated in some of the resistant clones, but none of the three RAS genes was mutated, and experiments using chemical inhibitors could not establish that the activation of these proteins was the cause of Fas resistance as described in other systems. Taken together, the data illustrate that Fas resistance can be caused by reduced Fas expression, which is a result of an unidentified mode of regulation.
|
|
| 11. |
- Blomberg, Jeanette, et al.
(författare)
-
Reflections on 25 Years of Ethnography in CSCW
- 2013
-
Ingår i: Computer Supported Cooperative Work. - : Springer Science and Business Media LLC. - 0925-9724 .- 1573-7551. ; 22:4-6, s. 373-423
-
Tidskriftsartikel (refereegranskat)abstract
- In this article we focus attention on ethnography's place in CSCW by reflecting on how ethnography in the context of CSCW has contributed to our understanding of the sociality and materiality of work and by exploring how the notion of the 'field site' as a construct in ethnography provides new ways of conceptualizing 'work' that extends beyond the workplace. We argue that the well known challenges of drawing design implications from ethnographic research have led to useful strategies for tightly coupling ethnography and design. We also offer some thoughts on recent controversies over what constitutes useful and proper ethnographic research in the context of CSCW. Finally, we argue that as the temporal and spatial horizons of inquiry have expanded, along with new domains of collaborative activity, ethnography continues to provide invaluable perspectives.
|
|
| 12. |
- Blomberg, Jeanette, 1977-
(författare)
-
Regulation of apoptosis during treatment and resistance development in tumour cells
- 2008
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Induction of apoptosis is the most studied cell death process and it is a tightly regulated physiological event that enables elimination of damaged and unwanted cells. Apoptosis can be induced via activation of either the intrinsic or the extrinsic signalling pathway. The intrinsic pathway involves activation of the mitochondria by stress stimuli, whereas the extrinsic pathway is triggered by ligand induced activation of death receptors such as Fas. Apoptosis induction via Fas activation plays an important role in the function of cytotoxic T lymphocytes and in the control of immune cell homeostasis. Several studies have shown that anticancer therapies require functional cell death signalling pathways. Irradiation based therapy has been successful in treatment of several malignancies but the usage of high doses has been associated with side effects. Therefore, low dose therapies, that either is optimized for specific delivery or administrated in combination with other treatments, are promising modalities. However, in order to achieve high-quality effects of such treatments, the death effector mechanisms involved in tumour eradication needs to be further explored. Importantly, tumour cells frequently acquire resistance to apoptosis, which consequently allows tumour cells to escape from elimination by the immune system and/or treatment. Interferons constitute a large family of pleotrophic cytokines that are important for the immune response against viruses and other microorganisms. The interferon signalling pathway mediates transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. Interferon has successfully been used in therapy against some tumours. However, several drawbacks have been reported, such as reduced sensitivity to interferon during treatment. The aim of this thesis was to elucidate mechanisms that mediate resistance to death receptor or interferon induced apoptosis in human tumour cell models, as well as investigate what molecular events that underlie cell death following radiation therapy of tumour cells. In order to elucidate mechanisms involved in acquired resistance to Fas- or interferon-induced apoptosis, a Fas- and interferon-sensitive human cell line, U937, was subjected to conditions where resistance to either Fas- or interferon induced apoptosis was acquired. Characterization of the Fas resistant cells showed that multiple resistant mechanisms had been acquired. Reduced Fas expression and increased cFLIP expression, which is an inhibitor of death receptor signalling, were two important changes found. To further examine the importance of these two alterations, clones from the Fas resistant population were established. The reduced Fas expression was determined to account for the resistant phenotype in approximately 70% of the clones. In the Fas resistant clones with normal Fas expression, the importance of an increased amount of the cFLIP protein was confirmed with shRNA interference. A cross-resistance to death receptor induced apoptosis was detected in the interferon resistant variant, which illustrates that a connection between death receptor and interferon induced apoptosis exists. Notably, interferon resistant cells also contained increased cFLIP expression, which were determined to mediate resistance to both interferon and death receptor mediated apoptosis. Finally, when cell death induced by irradiation treatment was investigated in HeLa Hep2 cells we could demonstrate that cell death was mediated by centrosome hyperamplification and mitotic aberrations, which forced the cells into mitotic catastrophes and delayed apoptosis. In conclusion, we have described model systems where selection for resistance to Fas or interferon induced apoptosis generated a heterogeneous population, where several signalling molecules were altered. Furthermore, we have shown that a complex cell death network was activated by irradiation based therapy.
|
|
| 13. |
|
|
| 14. |
- Chereji, Razvan V., et al.
(författare)
-
Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly
- 2017
-
Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 45:15, s. 8806-8821
-
Tidskriftsartikel (refereegranskat)abstract
- Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Sac-charomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization.
|
|
| 15. |
- Crawford, Tim, et al.
(författare)
-
Specific functions for Mediator complex subunits from different modules in the transcriptional response of Arabidopsis thaliana to abiotic stress
- 2020
-
Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- Adverse environmental conditions are detrimental to plant growth and development. Acclimation to abiotic stress conditions involves activation of signaling pathways which often results in changes in gene expression via networks of transcription factors (TFs). Mediator is a highly conserved co-regulator complex and an essential component of the transcriptional machinery in eukaryotes. Some Mediator subunits have been implicated in stress-responsive signaling pathways; however, much remains unknown regarding the role of plant Mediator in abiotic stress responses. Here, we use RNA-seq to analyze the transcriptional response of Arabidopsis thaliana to heat, cold and salt stress conditions. We identify a set of common abiotic stress regulons and describe the sequential and combinatorial nature of TFs involved in their transcriptional regulation. Furthermore, we identify stress-specific roles for the Mediator subunits MED9, MED16, MED18 and CDK8, and putative TFs connecting them to different stress signaling pathways. Our data also indicate different modes of action for subunits or modules of Mediator at the same gene loci, including a co-repressor function for MED16 prior to stress. These results illuminate a poorly understood but important player in the transcriptional response of plants to abiotic stress and identify target genes and mechanisms as a prelude to further biochemical characterization.
|
|
| 16. |
- Davoine, Celine, et al.
(författare)
-
Functional metabolomics as a tool to analyze Mediator function and structure in plants
- 2017
-
Ingår i: PLOS ONE. - : Public Library Science. - 1932-6203. ; 12:6
-
Tidskriftsartikel (refereegranskat)abstract
- Mediator is a multiprotein transcriptional co-regulator complex composed of four modules; Head, Middle, Tail, and Kinase. It conveys signals from promoter-bound transcriptional regulators to RNA polymerase II and thus plays an essential role in eukaryotic gene regulation. We describe subunit localization and activities of Mediator in Arabidopsis through metabolome and transcriptome analyses from a set of Mediator mutants. Functional metabolomic analysis based on the metabolite profiles of Mediator mutants using multivariate statistical analysis and heat-map visualization shows that different subunit mutants display distinct metabolite profiles, which cluster according to the reported localization of the corresponding subunits in yeast. Based on these results, we suggest localization of previously unassigned plant Mediator subunits to specific modules. We also describe novel roles for individual subunits in development, and demonstrate changes in gene expression patterns and specific metabolite levels in med18 and med25, which can explain their phenotypes. We find that med18 displays levels of phytoalexins normally found in wild type plants only after exposure to pathogens. Our results indicate that different Mediator subunits are involved in specific signaling pathways that control developmental processes and tolerance to pathogen infections.
|
|
| 17. |
- Elfving, Nils, et al.
(författare)
-
The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development
- 2011
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:20, s. 8245-8250
-
Tidskriftsartikel (refereegranskat)abstract
- Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-amino-acids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.
|
|
| 18. |
- Eriksson, David, et al.
(författare)
-
Iodine-131 induces mitotic catastrophes and activates apoptotic pathways in HeLa Hep2 cells
- 2008
-
Ingår i: Cancer Biotherapy and Radiopharmaceuticals. - : Mary Ann Liebert Inc. - 1084-9785 .- 1557-8852. ; 23:5, s. 541-549
-
Tidskriftsartikel (refereegranskat)abstract
- Iodine-131 (131I) has been used both in unconjugated form and conjugated to antibody derivates (i.e., radioimmunotherapy; RIT) to treat malignant diseases. The mechanisms by which 131I-irradiation causes growth retardation are, however, inadequately understood. The aim of this study was to elucidate the sequential molecular and cellular events that initiate cell death in HeLa Hep2 cells exposed to 131I. In this paper, HeLa Hep2 cells were found to display a transient G2-M arrest following irradiation, but then reentered the cell cycle still containing unrepaired cellular damage. An increase of multipolar mitotic spindles, as well as a significant increase in centrosome numbers from 8.8% +/- 1.9% in controls to 54.7% +/- 2.2% in irradiated cells, was observed (p < 0.0001). A subsequent failure of cytokinesis caused the cells to progress into mitotic catastrophe. This was accompanied by the formation of giant cells with multiple nuclei, multilobulated nuclei, and an increased frequency of polyploidy cells. A fraction of the cells also displayed apoptotic features, including the activation of initiator caspases-2, -8, -9, and effector caspase-3, as well as cleavage of poly(ADP-ribose) polymerase, a cell-death substrate for active caspase-3. These findings demonstrate that mitotic catastrophes and the activation of a delayed type of apoptosis might be important mechanisms involved in cell death following the RIT of solid tumors with -emitting radionuclides, such as 131I.
|
|
| 19. |
- Finley, Jeanne, et al.
(författare)
-
O Night Without Objects
- 1999
-
Ingår i: Arti and Innovation. - Cambridge, MA : MIT Press.
-
Bokkapitel (övrigt vetenskapligt/konstnärligt)
|
|
| 20. |
|
|
| 21. |
- Groth, Kristina, 1963-
(författare)
-
On knowing who knows : An alternative approach to knowledge management
- 2004
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The topic of this thesis is how computer applications can support knowledge sharing between individuals in an organization. The thesis particularly focuses on solutions that facilitate for people to find other persons to share knowledge with, rather than solutions where information is stored in some kind of database for the purpose of being reused by other persons. The thesis describes one shorter and one longer ethnographic study about information and knowledge sharing in two different settings. The studies have shown that what actions people take when they search for information and knowledge depends on the problem itself, and on the situation in which the problem occurs. The results from the studies indicate that supporting people in knowing about others’ activities and availability would be more important when supporting knowledge sharing, than a specific knowledge system with the purpose of storing information to be reused as knowledge. This awareness can be supported in a number of different ways, some based on social activities, and some based on technical solutions. Social activities involve supporting the development of social networks, communities of practice, and other kinds of social activities that facilitate for people to get to know each other and get an opportunity to talk to each other. There exists many technologies that can support people’s knowledge about others’ activities and availability. Awareness systems focus on collecting and presenting information about, for example, where a person is located and how busy a person is. Some awareness systems collect such information automatically using, for example, sensor technology or electronic calendars, while others require the user to enter the information by him- or herself. It is more difficult to get the second kind of systems to work in practice because it requires that the time a user spends on supporting the system is also returning a benefit in the end. Ordinary information systems may also contribute to supporting people’s knowledge about others’ activities and availability, but they need to be structured and searchable in a way that fulfils this purpose. Also, there usually exist more than one documentation repository in an organization among which some may be structured and some not. Based on the studies that have been conducted a number of prototypes supporting knowledge sharing have been developed and evaluated. The technologies focused on are notification systems including mobile solutions to communicate with others, awareness systems focusing on activities and availability, and information management to make already existing written documentation structured and searchable. These prototypes have been evaluated using video recorded scenarios, based on the studies conducted, and focus groups in a medium sized consultancy organization. The results from the evaluation show that the suggested prototypes in the large fulfil the purpose of supporting knowledge sharing in an organization. Based on the three field studies conducted within the work of this thesis, a framework for supporting knowledge sharing through computer support is suggested. The framework focuses on issues such as annoying interruptions, platform independent communication, privacy aspects, and how the information can be presented.
|
|
| 22. |
- Hoernke, Maria, et al.
(författare)
-
EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation
- 2017
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:22
-
Tidskriftsartikel (refereegranskat)abstract
- The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.
|
|
| 23. |
|
|
| 24. |
- Holst, Mikkel Roland, et al.
(författare)
-
Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion
- 2017
-
Ingår i: Cell Reports. - : Cell Press. - 2211-1247. ; 20:8, s. 1893-1905
-
Tidskriftsartikel (refereegranskat)abstract
- Cellular blebbing, caused by local alterations in cellsurface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrinindependent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension- mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol- interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor.
|
|
| 25. |
- Kumar, Koppolu Raja Rajesh, et al.
(författare)
-
The MED7 subunit paralogs of Mediator function redundantly in development of etiolated seedlings in Arabidopsis
- 2018
-
Ingår i: The Plant Journal. - : John Wiley & Sons. - 0960-7412 .- 1365-313X. ; 96:3, s. 578-594
-
Tidskriftsartikel (refereegranskat)abstract
- MED7 is a subunit of the Mediator middle module and is encoded by two paralogs in Arabidopsis. We generated MED7 silenced lines using RNAi to study its impact on Arabidopsis growth and development. Compared with wild type, etiolated seedlings of the MED7 silenced lines exhibited reduced hypocotyl length caused by reduced cell elongation when grown in the dark. The hypocotyl length phenotype was rescued by exogenously supplied brassinosteroid. In addition, MED7 silenced seedlings exhibited defective hook opening in the dark as well as defective cotyledon expansion in the presence of the brassinosteroid inhibitor brassinazole. Whole transcriptome analysis on etiolated seedlings using RNA sequencing revealed several genes known to be regulated by auxin and brassinosteroids, and a broad range of cell wall-related genes that were differentially expressed in the MED7 silenced lines. This was especially evident for genes involved in cell wall extension and remodeling, such as EXPANSINs and XTHs. Conditional complementation with each MED7 paralog individually restored the hypocotyl phenotype as well as the gene expression defects. Additionally, conditional expression of MED7 had no effects that were independent of the Mediator complex on the observed phenotypes. We concluded that the MED7 paralogs function redundantly in regulating genes required for the normal development of etiolated Arabidopsis seedlings.
|
|
