Papers by Donna Arndt-jovin
Journal of Cell Biology, Apr 21, 1997
Journal of Cell Biology, Aug 15, 2005
rbB1 receptors situated on cellular filopodia undergo systematic retrograde transport after bindi... more rbB1 receptors situated on cellular filopodia undergo systematic retrograde transport after binding of the epidermal growth factor (EGF) and activation of the receptor tyrosine kinase. Specific inhibitors of the erbB1 receptor tyrosine kinase as well as cytochalasin D, a disruptor of the actin cytoskeleton, abolish transport but not free diffusion of the receptor-ligand complex. Diffusion constants and transport rates were determined with single molecule sensitivity by tracking receptors labeled with EGF conjugated to fluorescent quantum dots. Retro-E grade transport precedes receptor endocytosis, which occurs at the base of the filopodia. Initiation of transport requires the interaction and concerted activation of at least two liganded receptors and proceeds at a constant rate mediated by association with actin. These findings suggest a mechanism by which filopodia detect the presence and concentration of effector molecules far from the cell body and mediate cellular responses via directed transport of activated receptors.
Proceedings of SPIE, Mar 10, 2015
We report on the current version of the optical sectioning programmable array microscope (PAM) im... more We report on the current version of the optical sectioning programmable array microscope (PAM) implemented with a single digital micro-mirror device (DMD) spatial light modulator utilized as a mask in both the fluorescence excitation and emission paths. The PAM incorporates structured illumination and structured detection operating in synchrony. A sequence of binary patterns of excitation light in high definition format (1920×1080 elements) is projected into the focal plane of the microscope at the 18 kHz binary frame rate of the Texas Instruments 1080p DMD. The resulting fluorescent emission is captured as two distinct signals: conjugate (c, ca. “on-focus”) consisting of light impinging on and deviated from the “on” elements of the DMD, and the non-conjugate (nc, ca. “out-of-focus”) light falling on and deviated from the “off” elements. The two distinct, deflected beams are optically filtered and detected either by two individual cameras or captured as adjacent images on a single camera after traversing an image combiner. The sectioned image is gained from a subtraction of the nc image from the c image, weighted in accordance with the pattern(s) used for illumination and detection and the relative exposure times of the cameras. The widefield image is given by the sum of the c and nc images. This procedure allows a high duty cycle (typically 25-50%) of on-elements in the excitation patterns and thus functions with low light intensities, preventing saturation and minimizing photobleaching of sensitive fluorophores. The corresponding acquisition speed is also very high, limited only by the bandwidth of the camera(s) (100 fps full frame with the sCMOS camera in current use) and the optical power of the light source (lasers, large area LEDs). In contrast to the static patterns typical of SIM systems, the programmable array allows optimization of the patterns (duty cycle, feature size and distribution), thus enabling a wide range of applications, ranging from patterned photobleaching, (e.g., FRAP, FLIP) and photoactivation, spatial superresolution, automated adaptive tracking and minimization of light exposure (MLE), and photolithography.
Microscopy Today, Mar 1, 2001
Biophysical Journal, Feb 1, 2009
Journal of Fluorescence, Dec 1, 1994
The physical interaction between plasma-membrane lipids and the epidermal growth factor (EGF)rece... more The physical interaction between plasma-membrane lipids and the epidermal growth factor (EGF)receptor was investigated on single A431 human epidermoid carcinoma cells by monitoring fluorescence resonance energy transfer (FRET) between exogeneously added fluorescein-EGF (donor) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoybsnglycero-3-phosphocholine ('Bodipy-PC, aceeptor) using donor-photobleaching FRET-microscopy. The measured mean FRET-efficiency of 13% is indicative of such a physical interaction and exemplifies the great potential and sensitivity of time-resolved imaging fluorescence microscopy techniques for the study of lipid-receptor interactions on single ceUs.
Humana Press eBooks, Jan 20, 2007
The unique fluorescence properties of quantum dots (QDs), particulary their large extinction coef... more The unique fluorescence properties of quantum dots (QDs), particulary their large extinction coefficients and photostability, make them ideal probes for tracking proteins in live cells using real-time visualization. We have shown that QDs conjugated to epidermal growth factor act as functional ligands for their receptor, erbB1. Here, we describe protocols for (1) conjugation of streptavidin-QDs to biotinylated ligand, (2) formation of ligand-QD-receptor complexes, and (3) quantification of binding and internalization of receptor complex using both high-resolution fluorescence microscopy and flow cytometry.
Nucleic Acids Research, 1991
Biochemistry, May 11, 1993
Applied Spectroscopy, Feb 1, 2002
Proceedings of the National Academy of Sciences of the United States of America, Nov 1, 1985
An early step in the transposition of bacteriophage Mu DNA in vitro is a DNA strand-transfer reac... more An early step in the transposition of bacteriophage Mu DNA in vitro is a DNA strand-transfer reaction that generates an intermediate DNA structure in which the Mu donor DNA and the target DNA are covalently joined. DNA replication, initiated at the DNA forks in this intermediate, generates a cointegrate product; simple insert products can also be formed from the same intermediate by degradation of a specific segment of the structure, followed by gap repair. This DNA strand-transfer reaction requires ATP, magnesium, the Mu A and Mu B proteins, and a factor supplied by an Escherichia coli cell extract. We have now shown that the host protein factor requirement can be satisfied by purified protein HU. The defined system has been used to determine the DNA substrate requirements for the reaction. The reaction requires the two Mu ends, located on the same DNA molecule, in the same relative orientation to one another as in the phage Mu genome. To participate in the strand-transfer reaction efficiently the mini-Mu plasmid, used as the transposon donor, must be supercoiled; the target DNA molecule may be supercoiled, relaxed circular, or linear.
FEBS Journal, Jul 1, 2005
In thermophilic bacteria of Thermus genus there are two GreAlike transcription factors, GreA1 and... more In thermophilic bacteria of Thermus genus there are two GreAlike transcription factors, GreA1 and GreA2 (Gfh) that display opposite functional properties. GreA1, which is a functional analog of E. coli GreA, possesses nucleolytic activity and stimulates transcription elongation, whereas Gfh does not display any nucleolytic activity, but inhibits the activity of GreA1. We demonstrated that Gfh functions as a general transcription repressor that inhibits all enzymatic activities of RNA polymerase (RNAP) including RNA synthesis, pyrophosphorolysis and hydrolysis. During transcription initiation, Gfh decreases the rates of abortive synthesis, affecting both the Vmax and Km for NTPs. During elongation, Gfh increases the Km, and its inhibitory activity is stimulated by Mg-ions. Gfh binds to RNAP through its C-terminal domain (CTD), whereas Gfh's inhibitory activity resides in the lower portion of the N-terminal domain (NTD) containing the ''tip'' region with four essential Asp residues. In Gfh-RNAP complexes, the ''tip'' of NTD reaches the RNAP catalytic center. Gfh and GreA1 bind competitively to RNAP with apparent Kd values of 1.0 and 0.2 lm respectively. We determined the crystal structure of Gfh at 2.6 Å resolution. The overall structure is similar to that of E. coli GreA, however, the CTD of Gfh is flipped and rotated around the linker loop by 120°relative to NTD. Conclusion: Based on our structural, biochemical and genetic data, we proposed a model in which Gfh could assume two conformations in the cell. In the ''flipped'' conformation, Gfh is unable to bind to RNAP and is functionally inactive. In the alternate, GreA-like conformation, Gfh binds to RNAP and inhibits its catalytic activity. The Gfh action entails a partial occlusion of NTPs to the active site and an alteration of the geometry of Mg-ion coordination in the catalytic center.
Springer eBooks, Jan 14, 2006
Semiconductor nanocrystals, or quantum dots (QDs), are exciting new fluorescent probes useful in ... more Semiconductor nanocrystals, or quantum dots (QDs), are exciting new fluorescent probes useful in imaging at the single molecule to the whole animal level. Some recent applications of QDs are reviewed here and the reader is directed to the original literature for further details.
Cytometry Part A, Nov 1, 2016
After receiving his BA degree, in physics from New York University in 1946, Bernie worked for the... more After receiving his BA degree, in physics from New York University in 1946, Bernie worked for the Army Signal Corps and Sperry Gyroscope Co. In 1966 he began working for Endevco Corp which was eventually bought by Becton Dickinson (BD). In 1970 he became manager of BD's Mountain View Lab in CA. It was, in large part, Shoor's vision of the future that led BD to venture into the field of flow cytometry and monoclonal antibodies. In 1973, BD built its first two fluorescence-activated cell sorters, one for Stanford and one for NIH. In 1977 he was named VP of research and design at BD corporate headquarters in New Jersey. He returned to California in 1981 to establish the BD Monoclonal Center. After retiring in 1984, he continued to consult with BD for many years. Bernie was honored with a Distinguished Service Award by ISAC in 1993. In 2005 the first BD Lifetime Achievement Award in the Company's history was presented to Bernie for his development of the BD FACS fluorescence-activated cell sorter and the BD Monoclonal Center in Mountain View. REMEMBERING BERNIE SHOOR How does Bernie Shoor fit into the legacy of ISAC? He was an active promoter and participant of the early Engineering Foundation Conferences on automated cytology as well as of ISAC, which arose after SAC was constituted at Schloss Elmau in Germany in 1978. What made Bernie, an executive of a large health-care company, unique in his relation to ISAC and Cytometry? We can think of the following: first, his ability to recognize a technology (fluorescence flow sorting) in its early stage of development as a revolution in diagnostics as well as basic research; second, his understanding of what other tools (commercial monoclonal antibodies) were needed to bring that technology to the clinic; and finally, his respect for other
Proceedings of SPIE, Jun 1, 1991
ABSTRACT
Biophysical Journal, 2014
Biological processes are carried out through conformational transitions, ranging from the structu... more Biological processes are carried out through conformational transitions, ranging from the structural changes within biomolecules to the formation of macromolecular complexes and the associations between the complexes themselves. These transitions cover a vast range of timescales and are governed by a tangled network of molecular interactions. The resulting hierarchy of interactions, in turn, becomes encoded in the experimentally measurable "mechanical fingerprints" of the biomolecules, their force-extension curves. How can we decode these fingerprints so that they reveal the kinetic barriers and the associated timescales of a biological process? Here, we show [1] that this can be accomplished with a simple, model-free transformation that is general enough to be applicable to molecular interactions involving an arbitrarily large number of barriers. Specifically, the transformation converts the mechanical fingerprints of the system directly into a map of force-dependent rate constants. This map reveals the kinetics of the multitude of rate processes beyond what is typically accessible to direct measurements. With the contributions from individual barriers to the interaction network now "untangled", the map is straightforward to analyze in terms of the barriers and timescales. [1] Y.
International Journal of Molecular Sciences, Jun 27, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
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Papers by Donna Arndt-jovin