Chromophore-assisted laser inactivation of patched protein switches cell fate in the larval visual system of Drosophila.

Schmucker, Dietmar; Su, A L; Beermann, Anke; Jäckle, Herbert; Jay, Daniel G.

doi:10.1073/pnas.91.7.2664

Cited by 48 publications

(38 citation statements)

References 30 publications

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“…Cold Spring Harbor Laboratory Press on April 4, 2019 -Published by genesdev.cshlp.org Downloaded from survival of secondary precursors (Green et al 1993;Schmucker et al 1994;Suzuki and Saigo 2000).…”

Section: Genes and Development 2191mentioning

confidence: 99%

“…Development of larval PR precursor cells proceeds in a two-step process. First, primary precursors (also called BO founder cells) are specified; they express and require the proneural gene atonal (ato) and the retinal patterning genes sine oculis (so) and eyes absent (eya) as well as hedgehog (hh) signaling (Schmucker et al 1994;Suzuki and Saigo 2000). Then, primary precursors signal to the surrounding tissue by expressing the TGF␣ homolog spitz (spi), which activates EGF receptor (EGFR) and recruits adjacent cells to develop as secondary precursors (Daniel et al 1999;Suzuki and Saigo 2000).…”

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confidence: 99%

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Adult and larval photoreceptors use different mechanisms to specify the same Rhodopsin fates

Sprecher¹,

Pichaud²,

Desplan³

2007

Genes Dev.

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Although development of the adult Drosophila compound eye is very well understood, little is known about development of photoreceptors (PRs) in the simple larval eye. We show here that the larval eye is composed of 12 PRs, four of which express blue-sensitive rhodopsin5 (rh5) while the other eight contain green-sensitive rh6. This is similar to the 30:70 ratio of adult blue and green R8 cells. However, the stochastic choice of adult color PRs and the bistable loop of the warts and melted tumor suppressor genes that unambiguously specify rh5 and rh6 in R8 PRs are not involved in specification of larval PRs. Instead, primary PR precursors signal via EGFR to surrounding tissue to develop as secondary precursors, which will become Rh6-expressing PRs. EGFR signaling is required for the survival of the Rh6 subtype. Primary precursors give rise to the Rh5 subtype. Furthermore, the combinatorial action of the transcription factors Spalt, Seven-up, and Orthodenticle specifies the two PR subtypes. Therefore, even though the larval PRs and adult R8 PRs express the same rhodopsins (rh5 and rh6), they use very distinct mechanisms for their specification.[Keywords: Drosophila; visual system development; photoreceptor specification; transcription factor interaction; EGFR signaling] Supplemental material is available at http://www.genesdev.org. In spite of the morphological and developmental differences between vertebrate and invertebrate eyes, their basic function to translate light information from the environment to the brain is maintained. In Drosophila, the adult compound eye has been studied in great detail. It consists of ∼800 individual ommatidia. Each ommatidium contains eight photoreceptor cells (PRs): six outer PRs (R1-R6) and two inner PRs (R7 and R8). Different PRs are sensitive to different wavelengths of light, depending on the rhodopsin gene (rh) they express. Outer PRs are involved in motion detection and contain Rh1, a broad-spectrum photopigment. R7 and R8 each expresses a distinct rh with restricted absorption spectra-rh3, rh4, rh5, and rh6. The type of rh expressed in inner PRs defines two major types of ommatidia: The pale (p) ommatidia have R7 that contain UV-sensitive Rh3 with the corresponding R8 expressing blue Rh5, whereas in yellow (y) ommatidia, R7 expresses UV-sensitive Rh4 and R8 expresses green Rh6.Recently, substantial progress has been achieved in understanding the molecular basis of how different subtypes of PRs are specified (Wernet and Desplan 2004;Mikeladze-Dvali et al. 2005a). Initially, R7 and R8 express the transcription factor spalt (sal) that is required to specify them as inner PRs and distinguish them from outer PR identity (Mollereau et al. 2001). Then, the expression in R7 of the gene prospero (pros), which encodes a homeodomain transcription factor, further distinguishes R7 from R8 by repressing R8 rhs, rh5, and rh6 .The generation of the two types of ommatidia, yellow and pale, includes several steps. First, the stochastic expression of the transcription factor Spineless (Ss) in ...

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Section: Genes and Development 2191mentioning

confidence: 99%

mentioning

confidence: 99%

Adult and larval photoreceptors use different mechanisms to specify the same Rhodopsin fates

Sprecher¹,

Pichaud²,

Desplan³

2007

Genes Dev.

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“…Upon illumination, the dye generates free radicals that then destroy the labeled protein (5). So far, CALI has been successfully applied to the study of growth processes and guidance of neuronal growth cones (6)(7)(8)(9) and of cell fate switching during the development of the visual system (10). Nevertheless, some concerns about the technique remain that must be addressed before it can be used as routinely as genetic techniques are used today.…”

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confidence: 99%

Chromophore-assisted light inactivation and self-organization of microtubules and motors

Surrey

Elowitz

Wolf

et al. 1998

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

145

119

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Chromophore-assisted light inactivation (CALI) offers the only method capable of modulating specific protein activities in localized regions and at particular times. Here, we generalize CALI so that it can be applied to a wider range of tasks. Specifically, we show that CALI can work with a genetically inserted epitope tag; we investigate the effectiveness of alternative dyes, especially f luorescein, comparing them with the standard CALI dye, malachite green; and we study the relative efficiencies of pulsed and continuous-wave illumination. We then use f luorescein-labeled hemagglutinin antibody fragments, together with relatively low-power continuous-wave illumination to examine the effectiveness of CALI targeted to kinesin. We show that CALI can destroy kinesin activity in at least two ways: it can either result in the apparent loss of motor activity, or it can cause irreversible attachment of the kinesin enzyme to its microtubule substrate. Finally, we apply this implementation of CALI to an in vitro system of motor proteins and microtubules that is capable of self-organized aster formation. In this system, CALI can effectively perturb local structure formation by blocking or reducing the degree of aster formation in chosen regions of the sample, without inf luencing structure formation elsewhere.

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“…CALI has been used to show the in vivo roles of several proteins in insect neural development (3)(4)(5).…”

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confidence: 99%

Chromophore-assisted laser inactivation of proteins is mediated by the photogeneration of free radicals.

Liao

Roider

Jay

1994

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

Self Cite

181

135

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Chromophore-assisted laser inactivation (CALI) is a technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions and an understanding of its mechanism is required for its judicious application. We report here that CALI is not mediated by photoinduced thermal denaturation but by photogenerated free radicals. Thermal diffusion calculations suggest that the temperature changes resulting from CALI are too small to cause thermal denaturation, and Arrhenius plots of CALI are inconsistent with a photothermal mechanism. CALI shows an energy dose reciprocity above a threshold and can be inhibited by free-radical quenchers, thus demonstrating a photochemical mechanism of protein inactivation. The type of quenchers that are effective in inhibiting CALl indicates that the active species is a hydrogen abstractor which is not derived from molecular oxygen. We suggest that the active free-radical species is the hydroxyl radical and its very short lifetime explains the spatial specificity of CALI such that half-maximal damage is effected within 15 A from the dye moiety and no signicant damage occurs at 34 A. The data are consistent with free-radical formation resulting from a sequential two-photon process.Chromophore-assisted laser inactivation (CALI) of protein function (1) targets laser energy to damage single protein species by binding the protein with a specific antibody that has been labeled with the chromophore malachite green. The bound dye is excited by 620-nm light, a wavelength not significantly absorbed by most cellular components, and damage is localized to within 60 A of the chromophore (2).CALI has been used to show the in vivo roles of several proteins in insect neural development (3-5).Further characterization and understanding of CALI are required for its optimization and judicious application in biological research. A dependence of CALI on the distance from the dye, the laser power, the number of pulses, and the concentration of dye-labeled ligand has been shown (1, 2). Although thermal denaturation was postulated as the molecular mechanism of CALI, the precise nature of the lasermediated damage to the protein function has not been established. This study was undertaken to ascertain the mechanism of protein damage by CALI.The likely sources of photoinduced damage through the absorption of light energy by a chromophore at the radiant flux used for CALI are photothermal and photochemical (6). Photothermal damage would occur through highly localized denaturation of the antibody-antigen complex caused by the rapid laser excitation and the relaxation of the bound dye (5 ps) through vibrational modes to generate heat. For CALI to be based on a photothermal mechanism, the temperature increase at the site of the protein of interest after laser treatment must be sufficiently high to result in thermal denaturation. Photochemical damage would occur through the generation offree radicals by the relaxation ofthe...

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Chromophore-assisted laser inactivation of patched protein switches cell fate in the larval visual system of Drosophila.

Abstract: The

Cited by 48 publications

References 30 publications

Adult and larval photoreceptors use different mechanisms to specify the same Rhodopsin fates

Adult and larval photoreceptors use different mechanisms to specify the same Rhodopsin fates

Chromophore-assisted light inactivation and self-organization of microtubules and motors

Chromophore-assisted laser inactivation of proteins is mediated by the photogeneration of free radicals.

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