Kees Jalink - Academia.edu (original) (raw)

Papers by Kees Jalink

Sensors, 2015

Optical (molecular) imaging can benefit from a combination of the high signal-to-background ratio... more Optical (molecular) imaging can benefit from a combination of the high signal-to-background ratio of activatable fluorescence imaging with the high specificity of luminescence lifetime imaging. To allow for this combination, both imaging techniques were integrated in a single imaging agent, a so-called activatable lifetime imaging agent. Important in the design of this imaging agent is the use of two luminophores that are tethered by a specific peptide with a hairpin-motive that ensured close proximity of the two while also having a specific amino acid sequence available for enzymatic cleavage by tumor-related MMP-2/9. Ir(ppy)3 and Cy5 were used because in close proximity the emission intensities of both luminophores were quenched and the influence of Cy5 shortens the Ir(ppy)3 luminescence lifetime from 98 ns to 30 ns. Upon cleavage in vitro, both effects are undone, yielding an increase in Ir(ppy)3 and Cy5 luminescence and a restoration of Ir(ppy)3 luminescence lifetime to 94 ns. As a reference for the luminescence activation, a similar imaging agent with the more common Cy3-Cy5 fluorophore pair was used. Our findings underline that the combination of enzymatic signal activation with lifetime imaging is possible and that it provides a promising method in the design of future disease specific imaging agents. OPEN ACCESS Sensors 2015, 15 11077

Journal of cell science, Jan 15, 2014

Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated f... more Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated forms, and is implicated in diverse cellular processes, ranging from ion channel formation to intracellular membrane remodeling. CLIC4 is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and serum, suggesting a possible role for CLIC4 in exocytic-endocytic trafficking. However, the function and subcellular target(s) of CLIC4 remain elusive. Here, we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell-matrix adhesion, cell spreading and integrin signaling, whereas it increases cell motility. LPA stimulates the recruitment of CLIC4 to β1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both the internalization and the serum- or LPA-induced recycling of β1 integrin, but not for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of β1 inte...

Nature, Jan 23, 2015

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR)... more Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pat...

Gap junctions (GJ) are clusters of transmembrane channels that allow direct cell-to-cell transfer... more Gap junctions (GJ) are clusters of transmembrane channels that allow direct cell-to-cell transfer of ions and small molecules. The GJ-permeant signaling molecule cAMP is of particular interest because of its numerous cellular effects. However, to assess the biological relevance of GJ-mediated cAMP transfer, quantitative aspects must be determined. Here we employed cAMP indicators based on fluorescence resonance energy transfer (FRET) to study propagation of cAMP signals to neighbor cells through connexin43 (Cx43)-based gap junctions in Rat-1 cells quantitatively. Intracellular cAMP levels were selectively raised in single cells by either photorelease of caged cAMP or stimulation of G(s)-coupled receptors.

Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAM... more Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP-Epac-YFP fusion construct. When expressed in mammalian cells, CFP-Epac-YFP shows significant fluorescence resonance energy transfer (FRET). FRET rapidly decreases in response to the cAMP-raising agents, whereas it fully recovers after addition of cAMP-lowering agonists. Thus, by undergoing a cAMP-induced conformational change, CFP-Epac-YFP serves as a highly sensitive cAMP indicator in vivo. When compared with a protein kinase A (PKA)-based sensor, Epacbased cAMP probes show an extended dynamic range and a better signal-to-noise ratio; furthermore, as a single polypeptide, CFP-Epac-YFP does not suffer from the technical problems encountered with multisubunit PKA-based sensors. These properties make Epac-based FRET probes the preferred indicators for monitoring cAMP levels in vivo.

Microdomains such as rafts are considered as scaffolds for phosphatidylinositol (4,5) bisphosphat... more Microdomains such as rafts are considered as scaffolds for phosphatidylinositol (4,5) bisphosphate (PIP 2 ) signaling, enabling PIP 2 to selectively regulate different processes in the cell. Enrichment of PIP 2 in microdomains was based on cholesterol-depletion and detergent-extraction studies.

Scientific Reports, 2014

The quality of super resolution images obtained by stochastic single-molecule microscopy critical... more The quality of super resolution images obtained by stochastic single-molecule microscopy critically depends on image analysis algorithms. We find that the choice of background estimator is often the most important determinant of reconstruction quality. A variety of techniques have found use, but many have a very narrow range of applicability depending upon the characteristics of the raw data. Importantly, we observe that when using otherwise accurate algorithms, unaccounted background components can give rise to biases on scales defeating the purpose of super-resolution microscopy. We find that a temporal median filter in particular provides a simple yet effective solution to the problem of background estimation, which we demonstrate over a range of imaging modalities and different reconstruction methods.

The mechanism by which CAMP inhibits growth factor-induced DNA synthesis in fibroblasts is not un... more The mechanism by which CAMP inhibits growth factor-induced DNA synthesis in fibroblasts is not understood. Here we show that in Rat-1 fibroblasts, C A M Praising agents inhibit p21"mediated mitogenactivated protein ( M A P ) kinase activation induced by either epidermal growth factor or lysophosphatidic acid. Under the same conditions, however, epidermal growth factoror lysophosphatidic acid-induced protein tyrosine phosphorylation, Ca2+ mobilization, and activation of Na+M+ exchange are not attenuated. In ras-transformed Rat-1 cells, 8-bromo-CAMP rapidly deactivates constitutively active MAP kinase without reducing p21".GTP levels; long term 8-bromo-CAMP treatment of these cells leads to growth arrest and reversion of the transformed phenotype. These results show that elevation of intracellular CAMP levels abrogates the p21-MAP kinase pathway at a step downstream of p21" activation. This finding provides a molecular basis for the growth-inhibitory action of CAMP in normal and transformed fibroblasts.

Reviews of Physiology, Biochemistry and Pharmacology, 119, 1992

Trends in Pharmacological Sciences, 2005

Although an established regulator of many cellular functions, the phosphoinositide phosphatidylin... more Although an established regulator of many cellular functions, the phosphoinositide phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ] appears to have evaded the attention of drug-discovery companies. An increasing number of reports have identified potential links between PtdIns(4,5)P 2 -mediated signalling pathways and the aetiology of many human diseases. Here, we review current knowledge of the regulation and function of PtdIns(4,5)P 2 and discuss how aberrant PtdIns(4,5)P 2 -mediated signalling might contribute to human pathologies such as cardiac failure, bipolar disorder, channelopathies and the genetic disorder Lowe syndrome.

The Journal of Cell Biology, 1994

Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activat... more Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA-and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.

The Journal of Cell Biology, 1992

The protease thrombin is a potent activator of various cell types. Thrombin cleaves and thereby a... more The protease thrombin is a potent activator of various cell types. Thrombin cleaves and thereby activates its own seven-transmembrane-domain receptor which couples to G proteins. Thrombin also can inhibit neuronal differentiation, supposedly by degrading components of the extracellular matrix. Here we report that active thrombin induces immediate cell rounding and neurite retraction in differentiating N1E-115 and NG108-15 neural cells in serum-free culture. Serum (0.5-5% vol/vol) evokes similar responses, but the cell-rounding and neurite-retracting activity of serum is not attributable to thrombin. Neural cell rounding is transient, subsiding after 10-15 min, and subject to homologous desensitization, whereas retracted neurites rapidly degenerate. Thrombin action is inhibited by cytochalasin, but not colchicine. A novel peptide, LLRNPNDKYEPF (500 #M), had no effect. Traces show transition from a flattened state (i.e., phase-dark cells) to a fully rounded state (phase-bright cells), a-c show cellular morphology corresponding to the indicated intensity of emitted light (/). It should be noted that there is no simple relationship between phasebrightness and cell thickness.

The Journal of Cell Biology, 1998

Gap junctions mediate cell-cell communication in almost all tissues, but little is known about th... more Gap junctions mediate cell-cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell-cell communication is rapidly disrupted by G protein-coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1-2 h of receptor stimulation. In contrast, a desensitization-defective G protein-coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is con-trolled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca 2+ , protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein-coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell-cell communication.

The Journal of Cell Biology, 2005

During wound healing, angiogenesis, and tumor invasion, cells often change their expression profi... more During wound healing, angiogenesis, and tumor invasion, cells often change their expression profiles of fibronectin-binding integrins. Here, we show that beta1 integrins promote random migration, whereas beta3 integrins promote persistent migration in the same epithelial cell background. Adhesion to fibronectin by alpha(v)beta3 supports extensive actin cytoskeletal reorganization through the actin-severing protein cofilin, resulting in a single broad lamellipod with static cell-matrix adhesions at the leading edge. Adhesion by alpha5beta1 instead leads to the phosphorylation/inactivation of cofilin, and these cells fail to polarize their cytoskeleton but extend thin protrusions containing highly dynamic cell-matrix adhesions in multiple directions. The activity of the small GTPase RhoA is particularly high in cells adhering by alpha5beta1, and inhibition of Rho signaling causes a switch from a beta1- to a beta3-associated mode of migration, whereas increased Rho activity has the opposite effect. Thus, alterations in integrin expression profiles allow cells to modulate several critical aspects of the motile machinery through Rho GTPases.

The Journal of Cell Biology, 2007

The EMBO Journal, 2006

Actomyosin contractility regulates various cell biological processes including cytokinesis, adhes... more Actomyosin contractility regulates various cell biological processes including cytokinesis, adhesion and migration. While in lower eukaryotes, alpha-kinases control actomyosin relaxation, a similar role for mammalian alpha-kinases has yet to be established. Here, we examined whether TRPM7, a cation channel fused to an alpha-kinase, can affect actomyosin function. We demonstrate that activation of TRPM7 by bradykinin leads to a Ca(2+)- and kinase-dependent interaction with the actomyosin cytoskeleton. Moreover, TRPM7 phosphorylates the myosin IIA heavy chain. Accordingly, low overexpression of TRPM7 increases intracellular Ca2+ levels accompanied by cell spreading, adhesion and the formation of focal adhesions. Activation of TRPM7 induces the transformation of these focal adhesions into podosomes by a kinase-dependent mechanism, an effect that can be mimicked by pharmacological inhibition of myosin II. Collectively, our results demonstrate that regulation of cell adhesion by TRPM7 is the combined effect of kinase-dependent and -independent pathways on actomyosin contractility.

The EMBO Journal, 2007

Resistance to tamoxifen is observed in half of the recurrences in breast cancer, where the anti-e... more Resistance to tamoxifen is observed in half of the recurrences in breast cancer, where the anti-estrogen tamoxifen acquires agonistic properties for transactivating estrogen receptor a (ERa). In a previous study, we showed that protein kinase A (PKA)-mediated phosphorylation of serine 305 (S305) of ERa results in resistance to tamoxifen. Now, we demonstrate that phosphorylation of S305 in ERa by PKA leads to an altered orientation between ERa and its coactivator SRC-1, which renders the transcription complex active in the presence of tamoxifen. This altered orientation involves the C-termini of ERa and SRC-1, which required a prolonged AF-1-mediated interaction. This intermolecular reorientation as a result of PKAmediated phosphorylation of ERa-S305 and tamoxifen binding provides a unique model for resistance to the anticancer drug tamoxifen.

Proceedings of the National Academy of Sciences, 1993

The naturally occurring phospholipid lysophosphatidic acid (LPA) can induce a number of physiolog... more The naturally occurring phospholipid lysophosphatidic acid (LPA) can induce a number of physiological responses in vertebrate cells, including platelet aggrgation, smooth musde contraction, and fibroblast proliferation. LPA is thought to activate a specfic G-protein-coupled receptor, thereby triggering classic second messenger pathways such as stimulation of phospholipase C and inhibition of adenylate cyclase. Here we report that 1-oleoyl-LPA, at submicromolar concentrations, evokes a chemotactic response in amoebae of the cellular slime mold Dicayostelium discoideum.

PLoS ONE, 2010

Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescen... more Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions.

Molecular Biology of the Cell, 2002

Phosphatidylinositol 4, 5-bisphosphate (PIP 2 ) at the inner leaflet of the plasma membrane has b... more Phosphatidylinositol 4, 5-bisphosphate (PIP 2 ) at the inner leaflet of the plasma membrane has been proposed to locally regulate the actin cytoskeleton. Indeed, recent studies that use GFP-tagged pleckstrin homology domains (GFP-PH) as fluorescent PIP 2 sensors suggest that this lipid is enriched in membrane microdomains. Here we report that this concept needs revision. Using three distinct fluorescent GFP-tagged pleckstrin homology domains, we show that highly mobile GFP-PH patches colocalize perfectly with various lipophilic membrane dyes and, hence, represent increased lipid content rather than PIP 2 -enriched microdomains. We show that bright patches are caused by submicroscopical folds and ruffles in the membrane that can be directly visualized at ϳ15 nm axial resolution with a novel numerically enhanced imaging method. F-actin motility is inhibited significantly by agonist-induced PIP 2 breakdown, and it resumes as soon as PIP 2 levels are back to normal. Thus, our data support a role for PIP 2 in the regulation of cortical actin, but they challenge a model in which spatial differences in PIP 2 regulation of the cytoskeleton exist at a micrometer scale.