Biological applications of an LCoS-based programmable array microscope (PAM)

Hagen, Guy M.; Caarls, Wouter; Thomas, Mandy; Hill, Andrew H.; Lidke, Keith A.; Rieger, Bernd; Fritsch, Cornelia; Geest, Bert van; Jovin, Thomas M.; Arndt‐Jovin, Donna J.

doi:10.1117/12.710995

Cited by 22 publications

(19 citation statements)

References 29 publications

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“…We have generated tagged insulin and insulin receptors and used advanced imaging tools to study insulin binding and receptor internalization with a focus on the comparative properties of the two IR isoforms. Highspeed optical sectioning confocal and structured illumination microscopy with a programmable array microscope (PAM) (Heintzmann et al, 2001;Hagen et al, 2007) revealed the early steps in ligand-dependent receptor activation and endocytosis. We demonstrated a higher initial rate of internalization of IR-A than IR-B and explored the potential influences on the signaling cascades linked to the IR.…”

Section: Discussionmentioning

confidence: 99%

Differential endocytosis and signaling dynamics of insulin receptor variants IR-A and IR-B

Giudice

Leskow

Arndt‐Jovin

et al. 2011

Journal of Cell Science

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Insulin signaling comprises a complex cascade of events, playing a key role in the regulation of glucose metabolism and cellular growth. Impaired response to insulin is the hallmark of diabetes, whereas upregulated insulin activity occurs in many cancers. Two splice variants of the insulin receptor (IR) exist in mammals: IR-A, lacking exon 11, and full-length IR-B. Although considerable biochemical data exist on insulin binding and downstream signaling, little is known about the dynamics of the IR itself. We created functional IR transgenes fused with visible fluorescent proteins for use in combination with biotinamido-caproyl insulin and streptavidin quantum dots. Using confocal and structured illumination microscopy, we visualized the endocytosis of both isoforms in living and fixed cells and demonstrated a higher rate of endocytosis of IR-A than IR-B. These differences correlated with higher and sustained activation of IR-A in response to insulin and with distinctive ERK1/2 activation profiles and gene transcription regulation. In addition, cells expressing IR-B showed higher AKT phosphorylation after insulin stimulation than cells expressing IR-A. Taken together, these results suggest that IR signaling is dependent on localization; internalized IRs regulate mitogenic activity, whereas metabolic balance signaling occurs at the cell membrane.

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Section: Discussionmentioning

confidence: 99%

Differential endocytosis and signaling dynamics of insulin receptor variants IR-A and IR-B

Giudice

Leskow

Arndt‐Jovin

et al. 2011

Journal of Cell Science

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“…Eightphase step images were acquired in random order. Confocal microscopy was performed at room temperature on a Zeiss LSM-510 Meta instrument using laser excitation at 488 or 633 nm and a 63ϫ water immersion objective or on a programmable array microscope fitted on an Olympus IX71 microscope using laser excitation at 488 nm and a 150ϫ oil immersion objective (48). Quantum dot tracking was performed using the View5D plugin (Rainer Heintzmann, King's College, London) for ImageJ (National Institutes of Health), and diffusion coefficients were calculated from the exported coordinates as described before (49).…”

Section: Methodsmentioning

confidence: 99%

Neuroprotective Secreted Amyloid Precursor Protein Acts by Disrupting Amyloid Precursor Protein Dimers

Gralle

Botelho

Wouters

2009

Journal of Biological Chemistry

121

103

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The amyloid precursor protein (APP) is implied both in cell growth and differentiation and in neurodegenerative processes in Alzheimer disease. Regulated proteolysis of APP generates biologically active fragments such as the neuroprotective secreted ectodomain sAPP␣ and the neurotoxic ␤-amyloid peptide. Furthermore, it has been suggested that the intact transmembrane APP plays a signaling role, which might be important for both normal synaptic plasticity and neuronal dysfunction in dementia. To understand APP signaling, we tracked single molecules of APP using quantum dots and quantitated APP homodimerization using fluorescence lifetime imaging microscopy for the detection of För-ster resonance energy transfer in living neuroblastoma cells. Using selective labeling with synthetic fluorophores, we show that the dimerization of APP is considerably higher at the plasma membrane than in intracellular membranes. Heparan sulfate significantly contributes to the almost complete dimerization of APP at the plasma membrane. Importantly, this technique for the first time structurally defines the initiation of APP signaling by binding of a relevant physiological extracellular ligand; our results indicate APP as receptor for neuroprotective sAPP␣, as sAPP␣ binding disrupts APP dimers, and this disruption of APP dimers by sAPP␣ is necessary for the protection of neuroblastoma cells against starvation-induced cell death. Only cells expressing reversibly dimerized wild-type, but not covalently dimerized mutant APP are protected by sAPP␣. These findings suggest a potentially beneficial effect of increasing sAPP␣ production or disrupting APP dimers for neuronal survival. The amyloid precursor protein (APP)4 is known both for its important role in the development and plasticity of the nervous system (1-6) and for its involvement in Alzheimer disease (AD) (7,8). Despite intensive research efforts, the initial events that lead to the prevalent sporadic, i.e. non-familial, forms of AD are still unclear. Furthermore, although a higher gene dose of APP (9) or the presence of pathological APP mutations is sufficient to induce familial AD (for review, see Ref. 10), the exact pathological mechanism that is triggered by APP is still under debate.Some fragments of APP, such as the ␤-amyloid peptide (A␤), are thought to contribute to synaptic dysfunction and neurotoxicity (11,12). On the other hand, the ␣-secretasederived extracellular fragment of APP (sAPP␣), which is present at lower levels in AD patients than in controls (13), has been shown to be beneficial for memory function, to possess neuroprotective properties, and to counteract the effects of A␤ (14 -18).Signaling by transmembrane APP may directly contribute to neurodegeneration in AD (19 -24); however, the signal transduction pathway for transmembrane APP remains unknown, although several potential regulatory proteins, glycosaminoglycans, and metal ions are known to bind with high affinity to APP and sAPP␣ (25,26). The most common form of signal transduction for single-pass transm...

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“…The Gen-2 PAM is capable of video rate, real-time sectioned imaging (Hagen et al, 2007). Its inherently versatile nature provides for a variety of imaging modalities besides mere intensity: hyperspectral imaging using Hadamard encoding masks (Hanley et al, 2000; Hanley et al, 1999b; Harwit and Sloane, 1979); frequency domain fluorescence lifetime imaging (FLIM) (Hagen et al, 2007; Hanley et al, 2005); and polarization (anisotropy). For a brief review of PAMs and their applications see Hagen et al (2007).…”

Section: Introductionmentioning

confidence: 99%

“…fluorescence recovery after photobleaching (FRAP) constitute important tools in cell biology for studying the kinetics of macromolecular motion, interactions and activity (for reviews, see McNally (2008); Rabut and Ellenberg (2005). We have reported previously experiments with the PAM involving photoconversion (Fulwyler et al, 2005; Hagen et al, 2007). One application explored with the newer LCoS-based PAM is the measurement of lateral diffusion of fluorescent protein-membrane receptor fusion proteins by FRAP.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence recovery after photobleaching and photoconversion in multiple arbitrary regions of interest using a programmable array microscope

Hagen

Caarls

Lidke

et al. 2009

Microscopy Res & Technique

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Photomanipulation (photobleaching, photoactivation, or photoconversion) is an essential tool in fluorescence microscopy. Fluorescence recovery after photobleaching (FRAP) is commonly used for the determination of lateral diffusion constants of membrane proteins, and can be conveniently implemented in confocal laser scanning microscopy (CLSM). Such determinations provide important information on molecular dynamics in live cells. However, the CLSM platform is inherently limited for FRAP because of its inflexible raster (spot) scanning format. We have implemented FRAP and photoactivation protocols using structured illumination and detection in a programmable array microscope (PAM). The patterns are arbitrary in number and shape, dynamic and adjustable to and by the sample characteristics. We have used multi-spot PAM-FRAP to measure the lateral diffusion of the erbB3 (HER3) receptor tyrosine kinase labeled by fusion with mCitrine on untreated cells and after treatment with reagents that perturb the cytoskeleton or plasma membrane or activate co-expressed erbB1 (HER1, the EGF receptor EGFR). We also show the versatility of the PAM for photoactivation in arbitrary regions of interest, in cells expressing erbB3 fused with the photoconvertible fluorescent protein dronpa.

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Biological applications of an LCoS-based programmable array microscope (PAM)

Cited by 22 publications

References 29 publications

Differential endocytosis and signaling dynamics of insulin receptor variants IR-A and IR-B

Differential endocytosis and signaling dynamics of insulin receptor variants IR-A and IR-B

Neuroprotective Secreted Amyloid Precursor Protein Acts by Disrupting Amyloid Precursor Protein Dimers

Fluorescence recovery after photobleaching and photoconversion in multiple arbitrary regions of interest using a programmable array microscope

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