In JoVE (1)

Other Publications (41)

Articles by Rajesh Menon in JoVE

Other articles by Rajesh Menon on PubMed

Specificity of Interactions of Galectin-3 with Chrp, a Cysteine- and Histidine-rich Cytoplasmic Protein

Biochimie. Jan-Feb, 2003  |  Pubmed ID: 12765788

Earlier work described the cloning of a gene from murine 3T3 cells encoding a cytoplasmic protein Chrp containing a cysteine- and histidine-rich motif characteristic of Zn-finger proteins. The interaction of Chrp with murine galectin-3 first became evident in a yeast two-hybrid screen, but it was also observed in co-precipitation experiments from 3T3 cell lysates. Here, the formation of equimolar complexes by murine Chrp and hamster galectin-3 is shown. Moreover, we found that Chrp binds to the carbohydrate-recognition domain (CRD) of hamster galectin-3 and not to the N-terminal domain carrying the proline- and glycine-rich repeats characteristic of galectin-3 and absent in other galectins. However, galectin-1 does not bind to Chrp, although its CRD is homologous to the galectin-3 CRD. Finally, we report that galectin-3, in a complex with Chrp, binds to laminin in surface plasmon resonance experiments with similar kinetics and affinity as it does in the free state. The formation of higher-order complexes containing these proteins and additional binding partners may be relevant to cytoplasmic functions involving galectin-3.

Domain Architecture of the Polyglutamine Protein Ataxin-3: a Globular Domain Followed by a Flexible Tail

FEBS Letters. Aug, 2003  |  Pubmed ID: 12914917

Anomalous expansion of a polyglutamine (polyQ) tract in the protein ataxin-3 causes spinocerebellar ataxia type 3, an autosomal dominant neurodegenerative disease. Very little is known about the structure and the function of ataxin-3, although this information would undoubtedly help to understand why the expanded protein forms insoluble nuclear aggregates and causes neuronal cell death. With the aim of establishing the domain architecture of ataxin-3 and the role of the polyQ tract within the protein context, we have studied the human and murine orthologues using a combination of techniques, which range from limited proteolysis to circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The two protein sequences share a highly conserved N-terminus and differ only in the length of the glutamine repeats and in the C-terminus. Our data conclusively indicate that ataxin-3 is composed by a structured N-terminal domain, followed by a flexible tail. Moreover, [(15)N]glutamine selectively labelled samples allowed us to have a direct insight by NMR into the structure of the polyQ region.

The C Terminus of Fragile X Mental Retardation Protein Interacts with the Multi-domain Ran-binding Protein in the Microtubule-organising Centre

Journal of Molecular Biology. Oct, 2004  |  Pubmed ID: 15381419

Absence of the fragile X mental retardation protein (FMRP) causes fragile X syndrome, the most common form of hereditary mental retardation. FMRP is a mainly cytoplasmic protein thought to be involved in repression of translation, through a complex network of protein-protein and protein-RNA interactions. Most of the currently known protein partners of FMRP recognise the conserved N terminus of the protein. No interaction has yet been mapped to the highly charged, poorly conserved C terminus, so far thought to be involved in RNA recognition through an RGG motif. In the present study, we show that a two-hybrid bait containing residues 419-632 of human FMRP fishes out a protein that spans the sequence of the Ran-binding protein in the microtubule-organising centre (RanBPM/RanBP9). Specific interaction of RanBPM with FMRP was confirmed by in vivo and in vitro assays. In brain tissue sections, RanBPM is highly expressed in the neurons of cerebral cortex and the cerebellar purkinje cells, in a pattern similar to that described for FMRP. Sequence analysis shows that RanBPM is a multi-domain protein. The interaction with FMRP was mapped in a newly identified CRA motif present in the RanBPM C terminus. Our results suggest that the functional role of RanBPM binding is modulation of the RNA-binding properties of FMRP.

Characterization of the Structure and the Amyloidogenic Properties of the Josephin Domain of the Polyglutamine-containing Protein Ataxin-3

Journal of Molecular Biology. Dec, 2004  |  Pubmed ID: 15544810

Expansion of the polyglutamine (polyQ) region in the protein ataxin-3 is associated with spinocerebellar ataxia type 3, an inherited neurodegenerative disorder that belongs to the family of polyQ diseases. Increasing evidence indicates that protein aggregation and fibre formation play an important role in these pathologies. In a previous study, we determined the domain architecture of ataxin-3, suggesting that it comprises a globular domain, named Josephin, and a more flexible C-terminal region, that includes the polyQ tract. Here, we have characterised for the first time the biophysical properties of the isolated Josephin motif, showing that it is an autonomously folded unit and that it has no significant interactions with the C-terminal region. Study of its thermodynamic stability indicates that Josephin has an intrinsic tendency to aggregate and forms temperature-induced fibrils similar to those described for expanded ataxin-3. We show that, under destabilising conditions, the behaviours of the isolated Josephin domain and ataxin-3 are extremely similar. Our data therefore strongly suggest that the stability and aggregation properties of non-expanded ataxin-3 are determined by those of the Josephin domain, which is sufficient to reproduce the behaviour of the full-length protein. Our data support a mechanism in which the thermodynamic stability of ataxin-3 is governed by the properties of the Josephin domain, but the presence of an expanded polyQ tract increases dramatically the protein's tendency to aggregate.

Assignment of the 1H, 13C, and 15N Resonances of the Josephin Domain of Human Ataxin-3

Journal of Biomolecular NMR. Dec, 2004  |  Pubmed ID: 15630566

Photon-sieve Lithography

Journal of the Optical Society of America. A, Optics, Image Science, and Vision. Feb, 2005  |  Pubmed ID: 15717565

We present the first lithography results that use high-numerical-aperture photon sieves as focusing elements in a scanning-optical-beam-lithography system [J. Vac. Sci. Technol. B 21, 2810 (2003)]. Photon sieves are novel optical elements that offer the advantages of higher resolution and improved image contrast compared with traditional diffractive optics such as zone plates [Nature 414, 184 (2001)]. We fabricated the highest-numerical-aperture photon sieves reported to date and experimentally verified their focusing characteristics. We propose two new designs of the photon sieve that have the potential to significantly increase focusing efficiency.

The AXH Domain Adopts Alternative Folds the Solution Structure of HBP1 AXH

Structure (London, England : 1993). May, 2005  |  Pubmed ID: 15893665

AXH is a protein module identified in two unrelated families that comprise the transcriptional repressor HBP1 and ataxin-1 (ATX1), the protein responsible for spinocerebellar ataxia type-1 (SCA1). SCA1 is a neurodegenerative disorder associated with protein misfolding and formation of toxic intranuclear aggregates. We have solved the structure in solution of monomeric AXH from HBP1. The domain adopts a nonclassical permutation of an OB fold and binds nucleic acids, a function previously unidentified for this region of HBP1. Comparison of HBP1 AXH with the crystal structure of dimeric ATX1 AXH indicates that, despite the significant sequence homology, the two proteins have different topologies, suggesting that AXH has chameleon properties. We further demonstrate that HBP1 AXH remains monomeric, whereas the ATX1 dimer spontaneously aggregates and forms fibers. Our results describe an entirely novel, to our knowledge, example of a chameleon fold and suggest a link between these properties and the SCA1 pathogenesis.

The Solution Structure of the Josephin Domain of Ataxin-3: Structural Determinants for Molecular Recognition

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2005  |  Pubmed ID: 16020535

The Josephin domain plays an important role in the cellular functions of ataxin-3, the protein responsible for the neurodegenerative Machado-Joseph disease. We have determined the solution structure of Josephin and shown that it belongs to the family of papain-like cysteine proteases, sharing the highest degree of structural similarity with bacterial staphopain. A currently unique structural feature of Josephin is a flexible helical hairpin formed by a 32-residue insertion, which could determine substrate specificity. By using the Josephin structure and the availability of NMR chemical shift assignments, we have mapped the enzyme active site by using the typical cysteine protease inhibitors, transepoxysuccinyl-L-eucylamido-4-guanidino-butane (E-64) and [L-3-trans-(propylcarbamyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline (CA-074). We also demonstrate that the specific interaction of Josephin with the ubiquitin-like domain of the ubiquitin- and proteasome-binding factor HHR23B involves complementary exposed hydrophobic surfaces. The structural similarity with other deubiquitinating enzymes suggests a model for the proteolytic enzymatic activity of ataxin-3.

Polyglutamine is Not All: the Functional Role of the AXH Domain in the Ataxin-1 Protein

Journal of Molecular Biology. Dec, 2005  |  Pubmed ID: 16277991

A family of neurodegenerative diseases is associated with anomalous expansion of a polyglutamine tract in the coding region of the corresponding proteins. The current working hypothesis is that polyglutamine diseases are caused by misfolding and aggregation of the proteins with a process dictated by the polyglutamine tracts, although increasing evidence suggests an involvement of the protein context in modulating these properties. Here, we show that the AXH domain of ataxin-1, the protein involved in spinocerebellar ataxia type-1, is the region responsible for the transcriptional repression activity of ataxin-1 and participates in protein aggregation. In vitro, the isolated domain undergoes a conformational transition towards a beta-enriched structure associated with aggregation and amyloid fibre formation spontaneously and without need for destabilizing conditions. Using a transfected cell line, we demonstrate that, while determined by polyglutamine expansion, ataxin-1 aggregation is noticeably reduced by deletion of AXH or by replacement with the homologous sequence from the transcription factor HBP1, which has no known tendency to aggregate. These results provide the first direct evidence of an involvement of a region other than the polyglutamine tract in polyglutamine pathologies.

Hammerhead Ribozyme-mediated Silencing of the Mutant Fibrillin-1 of Tight Skin Mouse: Insight into the Functional Role of Mutant Fibrillin-1

Experimental Cell Research. May, 2006  |  Pubmed ID: 16488411

The tight skin (Tsk/+) mouse is a model for fibrotic disorders. The genetic defect in the Tsk/+ is an in-frame duplication between exons 17 and 40 of the fibrillin-1 gene which gives rise to a large transcript and protein. Mice homozygous for the mutation die in utero, whereas heterozygotes survive and spontaneously develop connective tissue disease. In this study, we generated hammerhead ribozymes directed against the mutant fibrillin-1 transcript. A partially mispairing ribozyme was the most effective vehicle to cleave the mutant transcript without undesired cleavage of wild type transcripts, as shown by cell-free RNA cleavage and cleavage in cell lines harboring the ribozyme, by RT-PCR, Northern and Western Blotting. Global gene expression profiling using oligonucleotide microarrays showed the expected reduction in fibrillin-1 mRNA, and down-regulation of several gene cohorts in ribozyme harboring TskR1 cells compared to Tsk/+ cells. Two of the functional clusters included genes regulating extracellular matrix such as connective tissue growth factor, serpine-1 (plasminogen activator inhibitor-1) and TIMP-1 and TIMP-3, and those involved in cytoskeletal organization and myofibroblast formation including calponins and transgelin. Ribozyme-mediated inhibition was confirmed by Western Blot and functional analysis using cell-reporter systems and remodeling of three dimensional collagen gels. Our results underline the therapeutic potential of hammerhead ribozymes in dominant negative defects and suggest that changes in microfibril architecture brought about by fibrillin-1 mutation lead to a complex disease phenotype.

Experimental Characterization of Focusing by High-numerical-aperture Zone Plates

Journal of the Optical Society of America. A, Optics, Image Science, and Vision. Mar, 2006  |  Pubmed ID: 16539052

High-numerical-aperture zone plates have important applications in high-resolution optical maskless lithography as well as scanning confocal microscopy. We describe two methods to experimentally characterize the focusing properties, i.e., the point-spread function, of such diffractive lenses. The first method uses spot exposures in photoresist and the second uses a conventional knife-edge scan. The experimental results agree well with rigorous theoretical calculations.

Absorbance-modulation Optical Lithography

Journal of the Optical Society of America. A, Optics, Image Science, and Vision. Sep, 2006  |  Pubmed ID: 16912755

We describe a new mode of optical lithography called absorbance-modulation optical lithography (AMOL) in which a thin film of photochromic material is placed on top of a conventional photoresist and illuminated simultaneously by a focal spot of wavelength lambda1 and a ring-shaped illumination of wavelength lambda2. The lambda1 radiation converts the photochromic material from an opaque to a transparent configuration, thereby enabling exposure of the photoresist, while the lambda2 radiation reverses the transformation. As a result of these competing effects, the point-spread function that exposes the resist is strongly compressed, resulting in higher photolithographic resolution and information density. We show by modeling that the point-spread-function compression achieved via AMOL depends only on the absorbance distribution in the photostationary state. In this respect, absorbance modulation represents an optical nonlinearity that depends on the intensity ratio of lambda1 and lambda2 and not on the absolute intensity of either one alone. By inserting material parameters into the model, a lithographic resolution corresponding to lambda1/13 is predicted.

Far-field Generation of Localized Light Fields Using Absorbance Modulation

Physical Review Letters. Jan, 2007  |  Pubmed ID: 17358771

In this Letter, we report the confinement of a uniform beam of light (lambda(1) = 400 nm) at the nodes of a standing wave (lambda(2) = 532 nm) via absorbance modulation. In the present implementation of absorbance modulation, a thin polymer film containing a photochromic azobenzene side chain is exposed to a standing wave at lambda(2) and a uniform beam at lambda(1), resulting in alternate regions of high and low absorbance. Light at lambda(1) is localized around the low-absorbance regions. Using photoresist exposures, we mapped out the localized light intensity distribution, which agrees well with our theoretical model. Since the width of this distribution is primarily determined by the ratio of the intensities at the two wavelengths, this technique opens up the possibility of localizing light fields below the diffraction limit using far-field optics.

Down-regulation of the Dopamine Receptor D2 in Mice Lacking Ataxin 1

Human Molecular Genetics. Sep, 2007  |  Pubmed ID: 17599952

Ataxin 1 (Atxn1) is a protein of unknown function associated with spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease of late onset with variable degrees of cerebellar ataxia, ophthalmoplegia and neuropathy. SCA1 is caused by the toxic effects triggered by an expanded polyglutamine (polyQ) within Atxn1 resulting in neurodegeneration in the cerebellum, brain stem and spinocerebellar tracts. To gain insights into Atxn1 function, we have analysed the cerebellar gene expression profiles by microarray analysis in Atxn1-null mice, and identified alterations in expression of genes regulated by Sp1-dependent transcription, including the dopamine receptor D2 (Drd2), retinoic acid/thyroid hormone and Wnt-signalling. Interestingly, Drd2 expression levels are reduced in both Atxn1-null and transgenic mice expressing a pathogenic human Atxn1 with an expanded polyglutamine in cerebellar Purkinje cells. Our co-transfection experiments in human neuroblastoma SH-SY5Y cells and luciferase assays provide evidence for transcriptional regulation of Drd2 by Atxn1 and its AXH module. We show that Atxn1 occupies at the Drd2 promoter in vivo, and interacts and functions synergistically with the zinc-finger transcription factor Sp1 to co-regulate Drd2 expression. The interaction and transcriptional effects are mediated by the AXH domain within Atxn1 and are abrogated by the expanded polyQ within Atxn1. Therefore, this study identifies novel molecular targets that are regulated by Atxn1 which might contribute to the motor deficits in SCA1, and provides new insights into the mechanisms by which Atxn1 co-regulates transcription.

Structural Bases for Recognition of Anp32/LANP Proteins

The FEBS Journal. May, 2008  |  Pubmed ID: 18410380

The leucine-rich repeat acidic nuclear protein (Anp32a/LANP) belongs to a family of evolutionarily-conserved phosphoproteins involved in a complex network of protein-protein interactions. In an effort to understand the cellular role, we have investigated the mode of interaction of Anp32a with its partners. As a prerequisite, we solved the structure in solution of the evolutionarily conserved N-terminal leucine-rich repeat (LRR) domain and modeled its interactions with other proteins, taking PP2A as a paradigmatic example. The interaction between the Anp32a LRR domain and the AXH domain of ataxin-1 was probed experimentally. The two isolated and unmodified domains bind with very weak (millimolar) affinity, thus suggesting the necessity either for an additional partner (e.g. other regions of either or both proteins or a third molecule) or for a post-translational modification. Finally, we identified by two-hybrid screening a new partner of the LRR domain, i.e. the microtubule plus-end tracking protein Clip 170/Restin, known to regulate the dynamic properties of microtubules and to be associated with severe human pathologies.

Reduction of Focal-spot Size Using Dichromats in Absorbance Modulation

Optics Letters. Dec, 2008  |  Pubmed ID: 19079491

We experimentally verify the focusing characteristics of dichromats, a new class of circular-symmetric diffractive-optical lenses that generate, in the same focal plane, focal spots for one wavelength and ring-shaped spots with central nodes for another wavelength. Using a dichromat, we illuminate a thin photochromic layer and demonstrated point-spread-function compression of the transmitted focal spot.

Design of Diffractive Lenses That Generate Optical Nulls Without Phase Singularities

Journal of the Optical Society of America. A, Optics, Image Science, and Vision. Feb, 2009  |  Pubmed ID: 19183681

We exploit a technique, based on nonlinear optimization, to design diffractive lenses that focus optical nulls without any phase singularities. To ensure ease of fabrication, these lenses are composed of concentric circular zones. Furthermore, we show that this technique is readily extended to multiple wavelengths and can be used to improve tolerance to fabrication errors.

Confining Light to Deep Subwavelength Dimensions to Enable Optical Nanopatterning

Science (New York, N.Y.). May, 2009  |  Pubmed ID: 19359545

In the past, the formation of microscale patterns in the far field by light has been diffractively limited in resolution to roughly half the wavelength of the radiation used. Here, we demonstrate lines with an average width of 36 nanometers (nm), about one-tenth the illuminating wavelength lambda1 = 325 nm, made by applying a film of thermally stable photochromic molecules above the photoresist. Simultaneous irradiation of a second wavelength, lambda2 = 633 nm, renders the film opaque to the writing beam except at nodal sites, which let through a spatially constrained segment of incident lambda1 light, allowing subdiffractional patterning. The same experiment also demonstrates a patterning of periodic lines whose widths are about one-tenth their period, which is far smaller than what has been thought to be lithographically possible.

Josephin Domain of Ataxin-3 Contains Two Distinct Ubiquitin-binding Sites

Biopolymers. Dec, 2009  |  Pubmed ID: 19382171

Joseph-Machado is an incurable neurodegenerative disease caused by toxic aggregation of ataxin-3, a ubiquitin-specific cysteine protease, involved in the ubiquitin-proteasome pathway and known to bind poly-ubiquitin chains of four or more subunits. The enzymatic site resides in the N-terminal josephin domain of ataxin-3. We have characterized the ubiquitin-binding properties of josephin and showed that, unexpectedly, josephin contains two contiguous but distinct ubiquitin-binding sites. One is close to the enzymatic cleft and exploits an induced fit mechanism, which involves a flexible helical hairpin; the other overlaps with the site involved in recognition of HHR23B, a protein involved in delivering proteolytic substrates to the proteasome. To gain a structural description of the system, we had to overcome the nontrivial problem of dealing with a weak ternary complex. This was done by designing josephin mutants, which retain only one binding site and by characterizing the complexes with complementary computational and experimental techniques. The presence of two ubiquitin-binding sites explains how ataxin-3 binds poly-ubiquitin chains and provides new insights into the molecular mechanism of ubiquitin recognition.

Phosphorylation of S776 and 14-3-3 Binding Modulate Ataxin-1 Interaction with Splicing Factors

PloS One. 2009  |  Pubmed ID: 20037628

Ataxin-1 (Atx1), a member of the polyglutamine (polyQ) expanded protein family, is responsible for spinocerebellar ataxia type 1. Requirements for developing the disease are polyQ expansion, nuclear localization and phosphorylation of S776. Using a combination of bioinformatics, cell and structural biology approaches, we have identified a UHM ligand motif (ULM), present in proteins associated with splicing, in the C-terminus of Atx1 and shown that Atx1 interacts with and influences the function of the splicing factor U2AF65 via this motif. ULM comprises S776 of Atx1 and overlaps with a nuclear localization signal and a 14-3-3 binding motif. We demonstrate that phosphorylation of S776 provides the molecular switch which discriminates between 14-3-3 and components of the spliceosome. We also show that an S776D Atx1 mutant previously designed to mimic phosphorylation is unsuitable for this aim because of the different chemical properties of the two groups. Our results indicate that Atx1 is part of a complex network of interactions with splicing factors and suggest that development of the pathology is the consequence of a competition of aggregation with native interactions. Studies of the interactions formed by non-expanded Atx1 thus provide valuable hints for understanding both the function of the non-pathologic protein and the causes of the disease.

Parallel Scanning-optical Nanoscopy with Optically Confined Probes

Optics Express. Jul, 2010  |  Pubmed ID: 20720986

We report the imaging of sub-diffraction limited features using an optical probe generated by focusing a round spot at one wavelength, lambda(1) = 405 nm, and a ring-shaped spot at a second wavelength, lambda(2) = 532 nm, onto a thin photochromic layer that coats the nanostructures. Illumination at lambda(2) turns the photochromic layer opaque to lambda(1) everywhere except at the centre of the ring, where the illumination at lambda(1) penetrates and probes the underlying nanostructure. We confirm that this optically confined probe increases image contrast and is able to resolve features smaller than the far-field diffraction limit. Furthermore, by using an array of dual-wavelength diffractive microlenses, we demonstrate the feasibility of parallelizing this approach. Compared to previous approaches, our technique is not limited to fluorescence imaging.

Breaking the Far-field Diffraction Limit in Optical Nanopatterning Via Repeated Photochemical and Electrochemical Transitions in Photochromic Molecules

Physical Review Letters. Nov, 2011  |  Pubmed ID: 22181742

By saturating a photochromic transition with a nodal illumination (wavelength, λ), one isomeric form of a small molecule is spatially localized to a region smaller than the far-field diffraction limit. A selective oxidation step effectively locks this pattern allowing repeated patterning. Using this approach and a two-beam interferometer, we demonstrate isolated lines as narrow as λ/8 (78 nm) and spacing between features as narrow as λ/4 (153 nm). This is considerably smaller than the minimum far-field diffraction limit of λ/2. Most significantly, nanopatterning is achieved via single-photon reactions and at low light levels, which in turn allow for high throughput.

Simulation and Optimization of 1-D Periodic Dielectric Nanostructures for Light-trapping

Optics Express. Jan, 2012  |  Pubmed ID: 22274529

Light-trapping is essential to improve the performance of thin-film solar cells. In this paper, we perform a parametric optimization of 1-D square and sinusoidal grating structures that act as nanophotonic scatterers to increase light absorption in ultra-thin (10nm) solar cells. Our optimization reveals that the short-circuit current density in a device of active-layer thickness 10nm can be improved by a factor of ~5 in the presence of the scattering structure. More complex geometries allow for increased degrees of design freedom and potentially high enhancement of light absorption.

Design and Analysis of Multi-wavelength Diffractive Optics

Optics Express. Jan, 2012  |  Pubmed ID: 22330517

We present an extension of the direct-binary-search algorithm for designing high-efficiency multi-wavelength diffractive optics that reconstruct in the Fresnel domain. A fast computation method for solving the optimization problem is proposed. Examples of three-wavelength diffractive optics with over 90% diffraction efficiency are presented. These diffractive optical elements reconstruct three distinct image patterns when probed using the design wavelengths. Detailed parametric and sensitivity studies are conducted, which provide insight into the diffractive optic's performance when subject to different design conditions as well as common systematic and fabrication errors.

Simulation and Analysis of the Angular Response of 1D Dielectric Nanophotonic Light-trapping Structures in Thin-film Photovoltaics

Optics Express. Jul, 2012  |  Pubmed ID: 22828623

Nanophotonics can guide the design of novel structures for light-trapping in ultra-thin photovoltaic cells. Here, we report on the systematic study of the effect of the angle of incidence of sunlight on the performance of such structures. We also conduct a parametric study of a sinusoidal grating and demonstrate that light intensity in the active region averaged over a range of input angles from 0° to 80° can be enhanced by more than 3 times compared to the bare device. Such a broadband light-trapping nanostructure can increase the total daily energy production of a fixed (non-tracking) device by over 60%, compared to a reference device with an anti-reflection coating.

The Importance of Serine 776 in Ataxin-1 Partner Selection: a FRET Analysis

Scientific Reports. 2012  |  Pubmed ID: 23213356

Anomalous expansion of a polymorphic tract in Ataxin-1 causes the autosomal dominant spinocerebellar ataxia type 1. In addition to polyglutamine expansion, requirements for development of pathology are phosphorylation of serine 776 in Ataxin-1 and nuclear localization of the protein. The phosphorylation state of serine 776 is also crucial for selection of the Ataxin-1 multiple partners. Here, we have used FRET for an in cell study of the interaction of Ataxin-1 with the spliceosome-associated U2AF65 and the adaptor 14-3-3 proteins. Using wild-type Ataxin-1 and Ser776 mutants to a phosphomimetic aspartate and to alanine, we show that U2AF65 binds Ataxin-1 in a Ser776 phosphorylation independent manner whereas 14-3-3 interacts with phosphorylated wild-type Ataxin-1 but not with the mutants. These results indicate that Ser776 acts as the molecular switch that discriminates between normal and aberrant function and that phosphomimetics is not a generally valid approach whose applicability should be carefully validated.

Optical Patterning of Features with Spacing Below the Far-field Diffraction Limit Using Absorbance Modulation

Optics Express. Feb, 2013  |  Pubmed ID: 23482054

Absorbance modulation is an approach that enables the localization of light to deep sub-wavelength dimensions by the use of photochromic materials. In this article, we demonstrate the application of absorbance modulation on a transparent (quartz) substrate, which enables patterning of isolated lines of width 60 nm for an exposure wavelength of 325 nm. Furthermore, by moving the optical pattern relative to the sample, we demonstrate patterning of closely spaced lines, whose spacing is as small as 119 nm.

Optimization of Periodic Nanostructures for Enhanced Light-trapping in Ultra-thin Photovoltaics

Optics Express. Mar, 2013  |  Pubmed ID: 23482196

Nanophotonic light trapping offers a promising approach to increased efficiency in thin-film organic photovoltaics. In this paper, an extension of the direct-binary-search algorithm was adopted to optimize dielectric nanophotonic structures for increasing power output of ultra-thin organic solar cells. The optimized devices were comprised of an absorber layer sandwiched between two patterned, transparent, conducting cladding layers. Light trapping in such devices with an absorber thickness of only 10nm increases power output by a factor of 16 when compared to a flat reference device. We further show that even under oblique illumination with angles ranging from 0 to 60 degrees, such a device could produce over 7 times more power compared to a flat reference device. Finally, we also performed a spectral and parametric analysis of the optimized design, and show that the increase is primarily due to guided-mode resonances. Our simulations indicate that this new design approach has the potential to significantly increase the performance of ultra-thin solar cells in realistic scenarios.

The Role of Interruptions in PolyQ in the Pathology of SCA1

PLoS Genetics. 2013  |  Pubmed ID: 23935513

At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission.

Modeling Absorbance-modulation Optical Lithography in Photochromic Films

Optics Letters. Aug, 2013  |  Pubmed ID: 24104638

A kinetic model describing the conversion of a photochromic layer under complex illumination conditions is applied to absorbance-modulation optical lithography to determine the influence of the material characteristics on the confinement to subdiffraction dimensions of the transmitted dose. We show that the most important parameters are the intensity ratio between the confining and writing beams, the overall absorption at the writing wavelength, the relative absorption coefficients, and the photoreaction quantum yields at the two wavelengths. As the confining beam ultimately determines the transferred dose pattern, we conclude that the modulation of the writing beam is not strictly necessary to produce subwavelength apertures.

Protein-protein Interactions As a Strategy Towards Protein-specific Drug Design: the Example of Ataxin-1

PloS One. 2013  |  Pubmed ID: 24155902

A main challenge for structural biologists is to understand the mechanisms that discriminate between molecular interactions and determine function. Here, we show how partner recognition of the AXH domain of the transcriptional co-regulator ataxin-1 is fine-tuned by a subtle balance between self- and hetero-associations. Ataxin-1 is the protein responsible for the hereditary spinocerebellar ataxia type 1, a disease linked to protein aggregation and transcriptional dysregulation. Expansion of a polyglutamine tract is essential for ataxin-1 aggregation, but the sequence-wise distant AXH domain plays an important aggravating role in the process. The AXH domain is also a key element for non-aberrant function as it intervenes in interactions with multiple protein partners. Previous data have shown that AXH is dimeric in solution and forms a dimer of dimers when crystallized. By solving the structure of a complex of AXH with a peptide from the interacting transcriptional repressor CIC, we show that the dimer interface of AXH is displaced by the new interaction and that, when blocked by the CIC peptide AXH aggregation and misfolding are impaired. This is a unique example in which palindromic self- and hetero-interactions within a sequence with chameleon properties discriminate the partner. We propose a drug design strategy for the treatment of SCA1 that is based on the information gained from the AXH/CIC complex.

Enzymatic Production of Mono-ubiquitinated Proteins for Structural Studies: The Example of the Josephin Domain of Ataxin-3

FEBS Open Bio. 2013  |  Pubmed ID: 24251111

Protein ubiquitination occurs through formation of an isopeptide bond between the C-terminal glycine of ubiquitin (Ub) and the ɛ-amino group of a substrate lysine residue. This post-translational modification, which occurs through the attachment of single and/or multiple copies of mono-ubiquitin and poly-ubiquitin chains, is involved in crucial cellular events such as protein degradation, cell-cycle regulation and DNA repair. The abnormal functioning of ubiquitin pathways is also implicated in the pathogenesis of several human diseases ranging from cancer to neurodegeneration. However, despite the undoubted biological importance, understanding the molecular basis of how ubiquitination regulates different pathways has up to now been strongly limited by the difficulty of producing the amounts of highly homogeneous samples that are needed for a structural characterization by X-ray crystallography and/or NMR. Here, we report on the production of milligrams of highly pure Josephin mono-ubiquitinated on lysine 117 through large scale in vitro enzymatic ubiquitination. Josephin is the catalytic domain of ataxin-3, a protein responsible for spinocerebellar ataxia type 3. Ataxin-3 is the first deubiquitinating enzyme (DUB) reported to be activated by mono-ubiquitination. We demonstrate that the samples produced with the described method are correctly folded and suitable for structural studies. The protocol allows facile selective labelling of the components. Our results provide an important proof-of-concept that may pave the way to new approaches to the in vitro study of ubiquitinated proteins.

Improved Localization Accuracy in Stochastic Super-resolution Fluorescence Microscopy by K-factor Image Deshadowing

Biomedical Optics Express. Dec, 2013  |  Pubmed ID: 24466491

Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution.

Increased Photovoltaic Power Output Via Diffractive Spectrum Separation

Physical Review Letters. Mar, 2013  |  Pubmed ID: 25166805

In this Letter, we report the preliminary demonstration of a new paradigm for photovoltaic power generation that utilizes a broadband diffractive-optical element (BDOE) to efficiently separate sunlight into laterally spaced spectral bands. These bands are then absorbed by single-junction photovoltaic cells, whose band gaps correspond to the incident spectral bands. We designed such BDOEs by utilizing a modified version of the direct-binary-search algorithm. Gray scale lithography was used to fabricate these multilevel optics. They were experimentally characterized with an overall optical efficiency of 70% over a wavelength range of 350-1100 nm, which was in excellent agreement with simulation predictions. Finally, two prototype devices were assembled: one with a pair of copper indium gallium selenide based photovoltaic devices, and another with GaAs and c-Si photovoltaic devices. These devices demonstrated an increase in output peak electrical power of ∼ 42% and ∼ 22%, respectively, under white-light illumination. Because of the optical versatility and manufacturability of the proposed BDOEs, the reported spectrum-splitting approach provides a new approach toward low-cost solar power.

Chemical Shift Assignment of the Ataxin-1 AXH Domain in Complex with a CIC Ligand Peptide

Biomolecular NMR Assignments. Oct, 2014  |  Pubmed ID: 23853075

Ataxin-1 is the protein responsible for the genetically-inherited neurodegenerative disease spinocerebellar ataxia type-1 linked to the expansion of a polyglutamine tract within the protein sequence. The AXH domain of ataxin-1 is essential for the protein to function as a transcriptional co-repressor and mediates the majority of the interactions of ataxin-1 with cellular partners, mainly transcriptional regulators. One of the best characterized ataxin-1 functional partners is Capicua (CIC), a transcriptional repressor involved in signalling pathways that regulate mammalian development, tumorigenesis and, through the interaction with ataxin-1, also neurodegeneration. Complex formation of ataxin-1 with CIC is important both for the function of the wild-type protein and for pathogenesis as transcriptional disregulation is observed since the early stages of the development of the disease. Here we report the (1)H, (13)C and (15)N backbone and side-chain chemical shift assignments of the human ataxin-1 AXH domain in complex with a CIC ligand-peptide.

Mapping the Self-association Domains of Ataxin-1: Identification of Novel Non Overlapping Motifs

PeerJ. 2014  |  Pubmed ID: 24711972

The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is caused by aggregation and misfolding of the ataxin-1 protein. While the pathology correlates with mutations that lead to expansion of a polyglutamine tract in the protein, other regions contribute to the aggregation process as also non-expanded ataxin-1 is intrinsically aggregation-prone and forms nuclear foci in cell. Here, we have used a combined approach based on FRET analysis, confocal microscopy and in vitro techniques to map aggregation-prone regions other than polyglutamine and to establish the importance of dimerization in self-association/foci formation. Identification of aggregation-prone regions other than polyglutamine could greatly help the development of SCA1 treatment more specific than that based on targeting the low complexity polyglutamine region.

Optimization of Generalized Dielectric Nanostructures for Enhanced Light Trapping in Thin-film Photovoltaics Via Boosting the Local Density of Optical States

Optics Express. Jan, 2014  |  Pubmed ID: 24922004

Recent work has shown that using a high-index cladding atop a lower-index photovoltaic absorber enables absorption of light beyond the ergodic (4n2) limit. In this paper, we propose a generalized optimization method for deriving optimal geometries that allow for such enhancement. Specifically, we adapted the direct-binary-search algorithm to optimize a complex 2-D multi-layer structure with the explicit goal of increasing photocurrent. We show that such an optimization results in enhancing the local density of optical states in an ultra-thin absorber, which forms a slot-waveguide geometry in the presence of a higher-index overcladding. Numerical simulations confirmed optical absorption approaching 100% and absorption-enhancement beyond the ergodic (4n2) limit for specific spectral bands of interest. Our method provides a direct, intuitive and computationally scalable approach for designing light-trapping nanostructures.

Optimization and Analysis of 3D Nanostructures for Power-density Enhancement in Ultra-thin Photovoltaics Under Oblique Illumination

Optics Express. Mar, 2014  |  Pubmed ID: 24922240

Nanostructures have the potential to significantly increase the output power-density of ultra-thin photovoltaic devices by scattering incident sunlight into resonant guided modes. We applied a modified version of the direct-binary-search algorithm to design such nanostructures in order to maximize the output power-density under oblique-illumination conditions. We show that with appropriate design of nanostructured cladding layers, it is possible for a 10nm-thick organic absorber to produce an average peak power-density of 4 mW/cm² with incident polar angle ranging from -90° to 90° and incident azimuthal angle ranging from -23.5° to 23.5°. Using careful modal and spectral analysis, we further show that an optimal trade-off of absorption at λ~510 nm among various angles of incidence is essential to excellent performance under oblique illumination. Finally, we show that the optimized device with no sun tracking can produce on an average 7.23 times more energy per year than that produced by a comparable unpatterned device with an optimal anti-reflection coating.

Computational Spectrometer Based on a Broadband Diffractive Optic

Optics Express. Jun, 2014  |  Pubmed ID: 24977553

We describe a simple, compact, low-cost spectrometer comprised of a broadband diffractive optic and a sensor array. The diffractive optic is designed to disperse incident collimated light onto the sensor array in a prescribed manner defined by its spatial-spectral point-spread function. By applying a novel nonlinear optimization method, we show that it is possible to reconstruct the unknown spectrum from the measured image on the sensor array. We experimentally reconstructed numerous spectra with resolution as small as ~1 nm and bandwidths as large as 450 nm. Furthermore, we readily resolved two spatially overlapping but spectrally distinct objects. The spectral resolution is determined by dispersion of the diffractive optic via a spectral correlation function, while the bandwidth is limited primarily by the quantum efficiency of the sensor array. Using simulations, we present a spectral extraction of solar radiation from 300 nm to 2,500 nm with a resolution of ~0.11 nm. Moreover, our technique utilizes almost all the incident photons owing to the high transmission efficiency of the broadband diffractive optic, which allows for fast spectroscopy with dim illumination. Due to its simple construction with no moving parts, our technique could have important applications in portable, low-cost spectroscopy.

Computational Spectroscopy Via Singular-value Decomposition and Regularization

Optics Express. Sep, 2014  |  Pubmed ID: 25321533

Computational spectrometer based on a broadband diffractive optic was demonstrated with high spectral resolution over large bandwidth and high photon utilization efficiency. In this paper, we analyze such a spectrometer using singular value decomposition and propose a faster spectrum reconstruction algorithm with excellent accuracy by regularization. A new definition of spectral resolution based upon the Fourier analysis of singular vectors is described as well.

Integrated Metamaterials for Efficient and Compact Free-space-to-waveguide Coupling

Optics Express. Nov, 2014  |  Pubmed ID: 25401868

We applied nonlinear optimization to design nanophotonics-based metamaterials for efficient free-space-to-waveguide coupling. Three devices were designed, fabricated and characterized. The first device couples incident light into a multi-mode waveguide, the second device couples incident light into a single-mode waveguide directly, and the third device couples and separates two orthogonal polarizations into two multi-mode waveguides. All devices offer comparable or higher coupling efficiencies, are easier to fabricate, and demonstrate higher bandwidth when compared to conventional devices. Furthermore, each device is at least an order of magnitude smaller in area than previously reported devices. The highly efficient single-mode waveguide-coupler is a unique device that has not been experimentally demonstrated before. We further performed careful simulations to underscore the tolerance of these devices to fabrication errors. Their robustness is primarily a result of the large number of coupled guided-mode resonances that are responsible for each device performance.

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