Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and... Show moreFilopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins as filopodia elongate and subsequently retract. Although several methods exist that analyze changes in filopodial shape, a software solution to reliably correlate growth dynamics with spatially resolved protein concentration along the filopodium independent of bending, lateral shift, or tilting is missing. Here we introduce a novel approach based on the convex-hull algorithm for parallel analysis of growth dynamics and relative spatiotemporal protein concentration along flexible filopodial protrusions. Detailed in silico tests using various geometries confirm that our technique accurately tracks growth dynamics and relative protein concentration along the filopodial length for a broad range of signal distributions. To validate our technique in living cells, we measure filopodial dynamics and quantify spatiotemporal localization of filo-podia- associated proteins during the filopodial extension-retraction cycle in a variety of cell types in vitro and in vivo. Together these results show that the technique is suitable for simultaneous analysis of growth dynamics and spatiotemporal protein enrichment along filopodia. To allow readily application by other laboratories, we share source code and instructions for software handling. Show less
Epithelial cell differentiation and polarized migration associated with epithelial-to-mesenchymal transition (EMT) in cancer requires integration of gene expression with cytoskeletal dynamics. Here... Show moreEpithelial cell differentiation and polarized migration associated with epithelial-to-mesenchymal transition (EMT) in cancer requires integration of gene expression with cytoskeletal dynamics. Here we show that the PDZ-LIM domain protein PDLIM2 (Mystique/SLIM), a known cytoskeletal protein and promoter of nuclear nuclear factor κB (NFκB) and signal transducer and activator of transcription (STAT) degradation, regulates transcription factor activity and gene expression through the COP9 signalosome (CSN). Although repressed in certain cancers, PDLIM2 is highly expressed in invasive cancer cells. Here we show that PDLIM2 suppression causes loss of directional migration, inability to polarize the cytoskeleton, and reversal of the EMT phenotype. This is accompanied by altered activity of several transcription factor families, including β-catenin, Ap-1, NFκB, interferon regulatory factors, STATs, JUN, and p53. We also show that PDLIM2 associates with CSN5, and cells with suppressed PDLIM2 exhibit reduced nuclear accumulation and deneddylation activity of the CSN toward the cullin 1 and cullin 3 subunits of cullin-RING ubiquitin ligases. Thus PDLIM2 integrates cytoskeleton signaling with gene expression in epithelial differentiation by controlling the stability of key transcription factors and CSN activity. Show less
Kreuk, B.J. de; Nethe, M.; Fernandez-Borja, M.; Anthony, E.; Hensbergen, P.; Deelder, A.; ... ; Hordijk, P. 2011
Glycogen synthase kinase 3 beta (GSK3 beta) regulates diverse physiological processes, including metabolism, development, oncogenesis, and neuroprotection. GSK3 beta kinase activity has been... Show moreGlycogen synthase kinase 3 beta (GSK3 beta) regulates diverse physiological processes, including metabolism, development, oncogenesis, and neuroprotection. GSK3 beta kinase activity has been reported to be critical for various types of cancer cells, but the mechanism has remained elusive. In this study we examine the mechanism by which GSK3 beta regulates the survival of leukemia cells. We demonstrate that upon GSK3 beta kinase inhibition different types of leukemia cells show severe proliferation defects as a result of apoptosis. The transcription factor c-Myb is found to be the main target of GSK3 beta inhibition in cell survival. GSK3 beta inactivation reduces the expression of c-Myb by promoting its ubiquitination-mediated degradation, thereby inhibiting the expression of c-Myb-dependent antiapoptotic genes Bcl2 and survivin. Coimmunoprecipitation, reporter assays, chromatin immunoprecipitation, and knockdown studies show that c-Myb needs to interact and cooperate with transcription factor LEF-1 in the activation of Bcl2 and survivin and that both transcription factors are required for cell survival. These data reveal an as-yet-unknown mechanism by which GSK3 beta controls cell survival. Show less
Zhang, L.; Zhou, F.F.; Laar, T. van; Zhang, J.; Dam, H. van; Dijke, P. ten 2011
The canonical Wnt pathway plays an important role in the regulation of cell proliferation and differentiation. Activation of this signaling pathway causes disruption of the Axin/adenomatous... Show moreThe canonical Wnt pathway plays an important role in the regulation of cell proliferation and differentiation. Activation of this signaling pathway causes disruption of the Axin/adenomatous polyposis coli/glycogen synthase kinase 3 beta complex, resulting in stabilization of beta-catenin and its association with lymphoid enhancer factor/T-cell factor in the nucleus. Here, we identify Fas-associated factor 1 (FAF1) as a negative regulator of Wnt/beta-catenin signaling. We found overexpression of FAF1 to strongly inhibit Wnt-induced transcriptional reporter activity and to counteract Wnt-induced beta-catenin accumulation. Moreover, knockdown of FAF1 resulted in an increase in beta-catenin levels and in activation of Wnt/beta-catenin-induced transcription. FAF1 was found to interact with beta-catenin upon inhibition of proteasome. Ectopic expression of FAF1 promoted beta-catenin degradation by enhancing its polyubiquitination. Functional studies in C2C12 myoblasts and KS483 preosteoblastic cells showed that FAF1 depletion resulted in activation of endogenous Wnt-induced genes and enhanced osteoblast differentiation, whereas FAF1 overexpression had the opposite effect. These results identify FAF1 as a novel inhibitory factor of canonical Wnt signaling pathway. Show less
Koningsbruggen, S. van; Gierlinski, M.; Schofield, P.; Martin, D.; Barton, G.J.; Ariyurek, Y.; ... ; Lamond, A.I. 2010
The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of... Show moreThe nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope. Show less
Bernardi, K.M.; Williams, J.M.; Kikkert, M.; Voorden, S. van; Wiertz, E.J.; Ye, Y.H.; Tsai, B. 2010
To cause disease, cholera toxin (CT) is transported from the cell surface to the endoplasmic reticulum (ER) lumen where the catalytic CTA1 subunit retro-translocates to the cytosol to induce... Show moreTo cause disease, cholera toxin (CT) is transported from the cell surface to the endoplasmic reticulum (ER) lumen where the catalytic CTA1 subunit retro-translocates to the cytosol to induce pathological water secretion. Two retro-translocon components are the Derlins and ER-associated multi-spanning E3 ubiquitin ligases including Hrd1 and gp78. We demonstrated previously that Derlin-1 facilitates CTA1 retro-translocation. However, as CTA1 is neither ubiquitinated on lysines nor at its N-terminus, the role of E3 ligases in toxin retro-translocation is unclear. Here, we show that expression of mutant Hrd1 and gp78 and a mutant E2-conjugating enzyme dedicated to retro-translocation (Ube2g2) decrease CTA1 retro-translocation. Hrd1 knockdown also attenuated toxin retro-translocation. Binding studies demonstrate that Hrd1 and gp78 interact with CT and protein disulfide isomerase, an ER chaperone that unfolds CTA1 to initiate translocation. Moreover, we find that the toxin's association with Hrd1 and gp78 is blocked by dominant-negative Derlin-1, suggesting that CT is targeted initially to Derlin-1 and then transferred to Hrd1 and gp78. These data demonstrate a role of the E3 ubiquitin ligases in CTA1 retro-translocation, implicate a sequence of events experienced by the toxin on the ER membrane, and raise the possibility that ubiquitination is involved in the transport process. Show less
