Preparation of Xenopus laevis retinal cryosections for electron microscopy

Tam, Beatrice M.; Yang, Lee Ling; Bogéa, Tami; Ross, Bradford; Martens, Garnet; Moritz, Orson L.

doi:10.1016/j.exer.2015.05.014

Cited by 9 publications

(8 citation statements)

References 19 publications

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“…The eyecups were then immediately immersed in a fixative solution containing 4% paraformaldehyde (p-FA) and 1% glutaraldehyde (GA) buffered with 0.1 M sodium phosphate, pH 7.3. 24 All eyecups were fixated for at least 48 hours before cryo-sectioning. For the dark-adapted group, all the procedures were performed in a dark room under dim red light.…”

Section: Methodsmentioning

confidence: 99%

“…The thawing and washing process had been demonstrated to have negligible effect upon photoreceptor ultrastructure, and the acquired images were comparable in quality and the measurements were consistent with other published results. 24,26–29 After being thawed and washed, the retinal samples underwent a secondary fixation (osmication) as the lipid-rich structures (including membranes) were not well preserved by aldehydes. 30 This secondary fixation was performed using osmium tetraoxide (OsO 4 ), which also helped to stabilize proteins.…”

Section: Methodsmentioning

confidence: 99%

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Light-Induced Length Shrinkage of Rod Photoreceptor Outer Segments

Benedetti

Yao

2018

Trans. Vis. Sci. Tech.

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PurposeThis study was designed to verify light-induced outer segment (OS) length shrinkage of rod photoreceptors and to characterize its anatomic source at disc-level resolution.MethodsFrog (Rana pipiens) retinas were used for this study. Time-lapse light microscopy of freshly isolated OSs was employed to test transient rod OS changes at 10 ms temporal resolution. Histological light microscopy of dark- and light-adapted retinas was used to confirm light-induced rod OS length changes; and transmission electron microscopy (TEM) was used to quantify light-driven structural perturbation of rod OSs at disc level resolution.ResultsTime-lapse light microscopy images verified transient length shrinking responses in freshly isolated rod OSs. Histological light microscopy images confirmed reduced rod OS lengths in light-adapted retinas, compared to that of dark-adapted retinas. TEM images disclosed shortened inter-disc distances in light-adapted retinas compared to dark-adapted retinas.ConclusionsLight-induced rod OS length shrinkage was confirmed using time-lapse light microscopy of isolated rod OSs and histological light microscopy of dark- and light-adapted retinas. TEM revealed that the rod OS length shrinkage was correlated to the light-driven decrease of the space between individual discs, not the disc thickness itself.Translational RelevanceLight-induced transient rod response promises a noninvasive biomarker for early diagnosis of age-related macular degeneration and retinitis pigmentosa, in which the rod photoreceptors are known to be more vulnerable than cone photoreceptors.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Light-Induced Length Shrinkage of Rod Photoreceptor Outer Segments

Benedetti

Yao

2018

Trans. Vis. Sci. Tech.

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“…Animals were fixed in 1% glutaraldehyde, 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4. Cryosections were obtained, embedded in Eponate12 resin, and processed for electron microscopy using procedures identical to those previously described 38 .…”

Section: Methodsmentioning

confidence: 99%

Modeling Dominant and Recessive Forms of Retinitis Pigmentosa by Editing Three Rhodopsin-Encoding Genes in Xenopus Laevis Using Crispr/Cas9

Feehan

Chiu

Stanar

et al. 2017

Sci Rep

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The utility of Xenopus laevis, a common research subject for developmental biology, retinal physiology, cell biology, and other investigations, has been limited by lack of a robust gene knockout or knock-down technology. Here we describe manipulation of the X. laevis genome using CRISPR/Cas9 to model the human disorder retinitis pigmentosa, and to introduce point mutations or exogenous DNA sequences. We introduced and characterized in-frame and out-of-frame insertions and deletions in three genes encoding rhodopsin by co-injection of Cas9 mRNA, eGFP mRNA, and single guide RNAs into fertilized eggs. Deletions were characterized by direct sequencing and cloning; phenotypes were assessed by assays of rod opsin in retinal extracts, and confocal microscopy of cryosectioned and immunolabeled contralateral eyes. We obtained germline transmission of editing to F1 offspring. In-frame deletions frequently caused dominant retinal degeneration associated with rhodopsin biosynthesis defects, while frameshift phenotypes were consistent with knockout. We inserted eGFP or point mutations into rhodopsin genes by co-injection of repair fragments with homology to the Cas9 target sites. Our techniques can produce high frequency gene editing in X. laevis, permitting analysis in the F0 generation, and advancing the utility of X. laevis as a subject for biological research and disease modeling.

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“…To define the origin of the GFP-VAMP7-R150E-containing membranes, we examined transgenic Xenopus expressing this mutant by correlative light electron microscopy (CLEM). Transgenic retinas were first examined by confocal microscopy and then processed for EM analysis, as described previously (Tam et al, 2015). Images were superimposed and six cells within the boxed area of the fluorescent micrograph (Fig.…”

Section: Aberrant Vesicular Structures Containing Gfp-vamp7-r150e Arementioning

confidence: 99%

“…Nuclei were counterstained for 10 min at RT with TO-PRO-3 (1:500; Molecular Probes) and sections were mounted on slides with Vectashield (Vector Laboratories). CLEM of transgenic retinas expressing GFP-VAMP7-R150E was performed as previously described (Tam et al, 2015).…”

Section: Confocal Microscopy Clem and Plamentioning

confidence: 99%

An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex

Kandachar

Tam

Moritz

et al. 2018

Journal of Cell Science

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The Arf4-rhodopsin complex (mediated by the VxPx motif in rhodopsin) initiates expansion of vertebrate rod photoreceptor ciliaderived light-sensing organelles through stepwise assembly of a conserved trafficking network. Here, we examine its role in the sorting of VAMP7 (also known as TI-VAMP)an R-SNARE possessing a regulatory longin domain (LD)into rhodopsin transport carriers (RTCs). During RTC formation and trafficking, VAMP7 colocalizes with the ciliary cargo rhodopsin and interacts with the Rab11-Rabin8-Rab8 trafficking module. Rab11 and Rab8 bind the VAMP7 LD, whereas Rabin8 (also known as RAB3IP) interacts with the SNARE domain. The Arf/Rab11 effector FIP3 (also known as RAB11FIP3) regulates VAMP7 access to Rab11. At the ciliary base, VAMP7 forms a complex with the cognate SNAREs syntaxin 3 and SNAP-25. When expressed in transgenic animals, a GFP-VAMP7ΔLD fusion protein and a Y45E phosphomimetic mutant colocalize with endogenous VAMP7. The GFP-VAMP7-R150E mutant displays considerable localization defects that imply an important role of the R-SNARE motif in intracellular trafficking, rather than cognate SNARE pairing. Our study defines the link between VAMP7 and the ciliary targeting nexus that is conserved across diverse cell types, and contributes to general understanding of how functional Arf and Rab networks assemble SNAREs in membrane trafficking.

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Preparation of Xenopus laevis retinal cryosections for electron microscopy

Cited by 9 publications

References 19 publications

Light-Induced Length Shrinkage of Rod Photoreceptor Outer Segments

Light-Induced Length Shrinkage of Rod Photoreceptor Outer Segments

Modeling Dominant and Recessive Forms of Retinitis Pigmentosa by Editing Three Rhodopsin-Encoding Genes in Xenopus Laevis Using Crispr/Cas9

An interaction network between the SNARE VAMP7 and Rab GTPases within a ciliary membrane-targeting complex

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