| 1. |
- Gad, Annica K B, et al.
(författare)
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RhoD regulates cytoskeletal dynamics via the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules
- 2012
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Ingår i: Molecular Biology of the Cell. - Stockholm : Karolinska Institutet, Dept of Microbiology, Tumor and Cell Biology. - 1939-4586 .- 1059-1524.
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Tidskriftsartikel (refereegranskat)abstract
- The Rho GTPases have mainly been studied in association with their roles in the regulation of actin filament organization. These studies have shown that the Rho GTPases are essential for basic cellular processes, such as cell migration, contraction, and division. In this paper, we report that RhoD has a role in the organization of actin dynamics that is distinct from the roles of the better-studied Rho members Cdc42, RhoA, and Rac1. We found that RhoD binds the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules (WHAMM), as well as the related filamin A–binding protein FILIP1. Of these two RhoD-binding proteins, WHAMM was found to bind to the Arp2/3 complex, while FILIP1 bound filamin A. WHAMM was found to act downstream of RhoD in regulating cytoskeletal dynamics. In addition, cells treated with small interfering RNAs for RhoD and WHAMM showed increased cell attachment and decreased cell migration. These major effects on cytoskeletal dynamics indicate that RhoD and its effectors control vital cytoskeleton-driven cellular processes. In agreement with this notion, our data suggest that RhoD coordinates Arp2/3-dependent and FLNa-dependent mechanisms to control the actin filament system, cell adhesion, and cell migration.
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| 2. |
- Nehru, Vishal, et al.
(författare)
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RhoD Binds the Rab5 Effector Rabankyrin-5 and has a Role in Trafficking of the Platelet-derived Growth Factor Receptor
- 2013
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Ingår i: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 14:12, s. 1242-1254
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Tidskriftsartikel (refereegranskat)abstract
- RhoD is a member of the classical Rho GTPases and it has essential roles in the regulation of actin dynamics. RhoD localizes to early endosomes and recycling endosomes, which indicates its important role in the regulation of endosome trafficking. Here, we show that RhoD binds to the Rab5 effector Rabankyrin-5, and RhoD and Rabankyrin-5 colocalize to Rab5-positive endosomes, which suggests a role for Rabankyrin-5 in the coordination of RhoD and Rab5 in endosomal trafficking. Interestingly, depletion of RhoD using siRNA techniques interfered with the internalization of the PDGF receptor and the subsequent activation of the downstream signaling cascades. Our data suggest that RhoD and Rabankyrin-5 have important roles in coordinating RhoD and Rab activities during internalization and trafficking of activated tyrosine kinase receptors.
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| 3. |
- Nehru, Vishal
(författare)
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The role of RhoD in actin dynamics, receptor trafficking and cancer cell migration
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Ras subfamily of Rho GTPases plays an important role in myriads of cell processes, such as actin cytoskeleton dynamics, membrane trafficking and cell migration. Additionally, they also participate in diverse signal transduction pathways that regulate gene transcription, cell survival and cell growth. The ability of these proteins to function as a signaling molecule depends on their capacity to cycle between an active GTP-bound conformation and an inactive GDP-bound conformation. Till date, three Rho family members namely, RhoA, Rac1 and Cdc42 have been well studied as compared to other Rho family members. This thesis highlights a less studied Rho GTPase, RhoD, which together with Rif constitutes a separate subgroup of the Rho GTPases. Ectopic expression of RhoD and Rif has a dramatic effect on the organization of the actin filament system observed as long flexible filopodia protrusions and the formation of short bundles of actin filaments. Moreover, RhoD has a role in regulating endosome dynamics and is a negative regulator of cell motility. This motivated us to find out more about the signaling pathways downstream of RhoD. We initiated our studies by identifying a number of novel binding partners for RhoD for instance, FILIP1, WHAMM, Rabankyrin-5 and the ZIP kinase. We observed that RhoD together with FILIP1 and WHAMM had a distinct role in actin dynamics, cell adhesion and cell migration as compared to the better studied members of the Rho subfamily. Furthermore, with the same RhoD binding partners, we elucidated another regulatory role of RhoD in Golgi homeostasis and ER-to-Golgi transport measured by VSV-G protein transport assay. Previous studied have identified Rabankyrin-5 as a Rab5 effector. Interestingly, these studies also reported that Rabankyrin-5 localizes to early endosomes and to macropinosomes of epithelial cells. We found that Rabankyrin-5 participates in coordinating Rab5 and RhoD in endosome trafficking. We describe a novel mechanism by which RhoD, Rab5 and Rabankyrin-5 coordinate membrane trafficking events and endocytosis, for instance during the internalization of activated tyrosine kinase receptor, such as the PDGFRβ. Finally, we found that RhoD modulates focal adhesion dynamics and actin filament assembly through a novel effector, ZIP kinase. In summary, this thesis sheds light on the less studied Rho GTPase RhoD and provides novel insight into the mechanisms underlying its diverse cellular effects.
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| 4. |
- Nilsang, Suthasinee, et al.
(författare)
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Three-dimensional culture for monoclonal antibody production by hybridoma cells immobilized in macroporous gel particles
- 2008
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Ingår i: Biotechnology progress (Print). - Malden, MA : Wiley-Blackwell Publishing Inc.. - 8756-7938 .- 1520-6033. ; 24:5, s. 1122-1131
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Tidskriftsartikel (refereegranskat)abstract
- Cell proliferation and long-term production of monoclonal antibody IgG(2b) by M2139 hybridoma cells immobilized in macroporous gel particles (MGPs) in packed-bed reactor were studied for a period of 60 days. The MGPs were made of supermacroporous gels produced in frozen conditions from crosslinked polyacrylamide and modified with gelatin which were housed in special plastic carriers (7 x 9 mm(2)). Cells were trapped in the interior part of MGPs by attaching to the void space of the gel matrix as three-dimensional (3D) cultivation using gelatin as a substrate layer. Optimizing productivity by hybridoma cell relies on understanding regulation of antibody production. In this study, the behavior of M2139 cells in two-dimensional cultures on multiwell plate surfaces was also investigated. The effect of three different medium such as basal medium Dulbecco's modified Eagle's medium (D-MEM) containing L-glutamine or L-glutamine + 2 mM alpha-ketoglutarate or L-alanyl-glutamine (GlutaMAXtrade mark) was studied prior to its use in 3D cultivation. The kinetics of cell growth in basal medium containing L-glutamine + alpha-ketoglutarate was similar to cells grown on GlutaMAX containing medium, whereas D-MEM containing L-glutamine showed lower productivity. With the maximal viable cell density (6.85 x 10(6) cells mL(-1)) and highest specific mAb production rate (3.9 mug mL(-1) 10(-4) viable cell day(-1)), D-MEM-GlutaMAX was further selected for 3D cultivation. Cells in MGPs were able to grow and secrete antibody for 30 days in packed-bed batch reactor, before a fresh medium reservoir was replaced. After being supplied with fresh medium, cells again showed continuous growth for another 30 days with mAb production efficiency of 50%. These results demonstrate that MGPs can be used efficiently as supporting carrier for long-term monoclonal antibody production. © 2008 American Institute of Chemical Engineers.
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