Bivalent Ligands with Rigid Double-Stranded DNA Spacers Reveal Structural Constraints on Signaling by FcεRI

Paar, Jodi; Harris, Nancie T.; Holowka, David; Baird, Barbara

doi:10.4049/jimmunol.169.2.856

Cited by 66 publications

(82 citation statements)

References 37 publications

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“…The oligonucleotide 5Ј-ATC TCG GTG CGT AAT ACT CAC GGG CAG GAC-3Ј (ref. 29 and D. Holowka, personal communication) and its complementary sequence (both labeled with Cy5 at the 5Ј end) were purchased from Synthegen (Houston). The oligonucleotides were annealed in 200 mM Tris⅐HCl, 10 mM MgCl 2 , pH 8.0 and stored at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Beyond Rayleigh's criterion: A resolution measure with application to single-molecule microscopy

Ram

Ward

Ober

2006

Proc. Natl. Acad. Sci. U.S.A.

245

285

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Rayleigh's criterion is extensively used in optical microscopy for determining the resolution of microscopes. This criterion imposes a resolution limit that has long been held as an impediment for studying nanoscale biological phenomenon through an optical microscope. However, it is well known that Rayleigh's criterion is based on intuitive notions. For example, Rayleigh's criterion is formulated in a deterministic setting that neglects the photon statistics of the acquired data. Hence it does not take into account the number of detected photons, which, in turn, raises concern over the use of Rayleigh's criterion in photon-counting techniques such as single-molecule microscopy. Here, we re-examine the resolution problem by adopting a stochastic framework and present a resolution measure that overcomes the limitations of Rayleigh's criterion. This resolution measure predicts that the resolution of optical microscopes is not limited and that it can be improved by increasing the number of detected photons. Experimental verification of the resolution measure is carried out by imaging single-molecule pairs with different distances of separation. The resolution measure provides a quantitative tool for designing and evaluating single-molecule experiments that probe biomolecular interactions.Cramer-Rao lower bound ͉ photon statistics ͉ Fisher information matrix ͉ fluorescence microscopy A ccording to Rayleigh's criterion, the resolution of an optical microscope is defined as the minimum distance between two point sources such that their presence can be distinguished in the image (1). It is widely believed that the resolution limit imposed by this criterion precludes the single-molecule study of molecular interactions at distances of Ͻ200 nm. Despite the widespread use of Rayleigh's criterion, it is well known that this criterion is based on heuristic notions (2). Formulated within a deterministic framework, Rayleigh's criterion neglects the stochastic nature of the photon-detection process and hence does not consider the total photon count in the acquired data. This formulation is not surprising, because Rayleigh's criterion was developed at a time when the unaided human eye was used as the detector. Therefore, Rayleigh's criterion is not well adapted to current microscope setups, in which highly sensitive photon-counting cameras are used. Recent singlemolecule experiments have shown that Rayleigh's criterion can, in fact, be surpassed in an optical microscope setup (3-5). Thus Rayleigh's resolution limit is an inadequate performance criterion for current quantitative imaging techniques.By adopting a stochastic framework, we propose a resolution measure that overcomes the shortcomings of Rayleigh's criterion and provides a quantitative measure of a microscope's ability to determine the distance between two point sources. Unlike Rayleigh's criterion our resolution measure predicts that the resolution of a microscope can be improved by increasing the number of photons collected from the point sources. The resolution measu...

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Section: Methodsmentioning

confidence: 99%

Beyond Rayleigh's criterion: A resolution measure with application to single-molecule microscopy

Ram

Ward

Ober

2006

Proc. Natl. Acad. Sci. U.S.A.

245

285

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“…4B. More striking distinctions in Ca 2ϩ responses among constructs were observed compared with degranulation responses, in part because stimulated degranulation requires only very small amounts of Ca 2ϩ mobilization (32,33). In the averaged Ca 2ϩ responses, ␣ and ␣F are similar in magnitude as for degranulation, ␣DA is about half that of its parent, and ␣T is even smaller than this, with an average value that is 14% of the response via endogenous Fc⑀RI.…”

Section: Degranulation and Calcium Responses Via Chimeric Receptors Amentioning

confidence: 97%

Transmembrane Sequences Are Determinants of Immunoreceptor Signaling

Gosse

Wagenknecht-Wiesner

Holowka

et al. 2005

The Journal of Immunology

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To investigate structural features critical for signal initiation by Ag-stimulated immunoreceptors, we constructed a series of single-chain chimeric receptors that incorporate extracellular human FcεRIα for IgE binding, a variable transmembrane (TM) segment, and the ITAM-containing cytoplasmic tail of the TCR ζ-chain. We find that functional responses mediated by these receptors are strongly dependent on their TM sequences, and these responses are highly correlated to cross-link-dependent association with detergent-resistant lipid rafts. For one chimera designated αFζ, mutation of a TM cysteine abolishes robust signaling and lipid raft association. In addition, TM disulfide-mediated oligomerization of another chimeric receptor, αζζ, enhances signaling. These results demonstrate an important role for TM segments in immunoreceptor signaling and a strong correspondence between strength of signaling and cross-link-dependent partitioning into ordered membrane domains.

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“…The internal diameter of the ␤-barrel is estimated to be 8 Å (20), and the external one is ϳ20 Å (our estimation). Because of the flexible hinge present in the antibodies, these molecules bivalently bind epitopes separated by 40 -100 Å (36,37). Using the crystal structure of the Fab antibody fragment with six histidines bound, we estimated the minimal distance for bivalent binding to be ϳ37 Å.…”

Section: Discussionmentioning

confidence: 99%

C Termini of the Escherichia coli Mechanosensitive Ion Channel (MscS) Move Apart upon the Channel Opening

Koprowski¹,

Kubalski

2003

Journal of Biological Chemistry

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Heptameric YggB is a mechanosensitive ion channel (MscS) from the inner membrane of Escherichia coli. We demonstrate, using the patch clamp technique, that cross-linking of the YggB C termini led to irreversible inhibition of the channel activities. Application of Ni 2؉ to the YggB-His 6 channels with the hexahistidine tags added to the ends of their C termini also resulted in a marked but reversible decrease of activities. Western blot revealed that YggB-His 6 oligomers are more stable in the presence of Ni 2؉ , providing evidence that Ni 2؉ is coordinated between C termini from different subunits of the channel. Intersubunit coordination of Ni 2؉ affecting channel activities occurred in the channel closed conformation and not in the open state. This may suggest that the C termini move apart upon channel opening and are involved in the channel activation. We propose that the as yet undefined C-terminal region may form a cytoplasmic gate of the channel. The results are discussed and interpreted based on the recently released quaternary structure of the channel. Mechanosensitive (MS)1 ion channels open upon membrane tension, and therefore they represent the simplest mechanosensors. MS channels have been implicated in many physiological processes from growth and cell volume regulation to hearing, blood pressure regulation, and pain sensation (reviewed in Ref. 1). Bacterial MS channels protect these cells against hypoosmotic shock. Two types of MS channels from the cytoplasmic membrane of Escherichia coli, MscL and MscS, play an essential role in the physiology of this bacterium, allowing the efflux of solutes from the cytoplasm when osmolarity of the external medium decreases (2-4). MscL, the large conductance MS channel, has been cloned (5), and a quaternary structure of its closed conformation has been determined (6). Based on this structure and the analysis of the channel gating, the open conformation has been predicted (7, 8) and experimentally confirmed (9, 10). Functional homologues of this channel have been found in other bacteria (11) and Archea (12), and structurally related protein from Neurospora has been also reported (13). The functional channel is a pentamer, and each subunit consists of two ␣-helical membrane-spanning domains TM1 and TM2 with both the C and N termini located in the cytoplasm (6). TM1s line the pore, and their hydrophobic residues form the primary, transmembrane gate (6, 14). It is postulated that there are two gates involved in the opening of the channel: the transmembrane and the cytoplasmic gates (7, 8) acting in accordance (15). The transmembrane gate is proposed to act as a pressure sensor, and upon application of pressure, this gate permits initial expansion of the channel without its full opening (7,8,10). It is proposed that the other, cytoplasmic gate, which allows full activation of the channel, is composed of five ␣-helical S1 segments of the cytoplasmic N termini being connected with TM1s via flexible linkers. According to the model, the applied pressure is transmitted to...

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Bivalent Ligands with Rigid Double-Stranded DNA Spacers Reveal Structural Constraints on Signaling by FcεRI

Cited by 66 publications

References 37 publications

Beyond Rayleigh's criterion: A resolution measure with application to single-molecule microscopy

Beyond Rayleigh's criterion: A resolution measure with application to single-molecule microscopy

Transmembrane Sequences Are Determinants of Immunoreceptor Signaling

C Termini of the Escherichia coli Mechanosensitive Ion Channel (MscS) Move Apart upon the Channel Opening

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