Preparation of the planarian Schmidtea mediterranea for high-resolution histology and transmission electron microscopy

Brubacher, John L.; Vieira, Ana Paula; Newmark, Phillip A.

doi:10.1038/nprot.2014.041

Cited by 30 publications

(20 citation statements)

References 44 publications

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“…For TEM analysis, dissected ovaries were fixed with 2.5% paraformaldehyde/2% glutaraldehyde/PBS for overnight at 4°C. Then the tissues were processed as described with modifications [74–76]. Briefly, the tissues were washed with 0.1 M sodium cacodylate buffer (pH=6.8), intensified in 2% OsO4/0.1 M sodium cacodylate buffer (pH=6.8), stained with 2% uranium acetate en bloc , dehydrated with a graded ethanol series (30%, 50%, 70%, 95%, and two times 100%, 10 min each), equilibrated with two incubations (10 min) in propylene oxide, and incubated in 50% propylene oxide/50% Epon resin (EMS, Fort Washington, PA) mixture overnight.…”

Section: Methodsmentioning

confidence: 99%

Subcellular analyses of planarian meiosis implicates a novel, double-membraned vesiculation process in nuclear envelope breakdown

Guo

Zhang

et al. 2019

Preprint

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19The cell nuclei of Ophisthokonts, the eukaryotic supergroup defined by fungi and 20 metazoans, is remarkable in the constancy of both their double-membraned structure and 21 protein composition. Such remarkable structural conservation underscores common and 22 ancient evolutionary origins. Yet, the dynamics of disassembly and reassembly displayed 23 by Ophisthokont nuclei vary extensively. Besides closed mitosis in fungi and open mitosis 24 in some animals, little is known about the evolution of nuclear envelope break down 25 (NEBD) during cell division. Here, we uncovered a novel form of NEBD in primary 26 oocytes of the flatworm Schmidtea mediterranea. From zygotene to metaphase II, both 27 nuclear envelope (NE) and peripheral endoplasmic reticulum (ER) expand notably in size, 28 likely involving de novo membrane synthesis. 3-D electron microscopy reconstructions 29 demonstrated that the NE transforms itself into numerous double-membraned vesicles 30 similar in membrane architecture to NE doublets in mammalian oocytes after germinal 31 vesicle breakdown. The vesicles are devoid of nuclear pore complexes and DNA, yet are 32 loaded with nuclear proteins, including a planarian homologue of PIWI, a protein essential 33 for the maintenance of stem cells in this and other organisms. Our data contribute a new 34 model to the canonical view of NE dynamics and support that NEBD is an evolutionarily 35 adaptable trait in multicellular organisms. 36 37 209 2. Kutay, U. and M.W. Hetzer, Reorganization of the Nuclear Envelope during 210 Open Mitosis. Current opinion in cell biology, 2008. 20

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Section: Methodsmentioning

confidence: 99%

Subcellular analyses of planarian meiosis implicates a novel, double-membraned vesiculation process in nuclear envelope breakdown

Guo

Zhang

et al. 2019

Preprint

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“…This protocol allows rapid fixation and is well adapted for observing not only cilia and centrioles (Figure 1(G)e(K)), but also more dynamic cytoskeletal structures such as cytoplasmic microtubules . Protocols for high-pressure freezing and a standard protocol for chemical fixation have also been described (Brubacher, Vieira, & Newmark, 2014;Salvenmoser, Egger, Achatz, Ladurner, & Hess, 2010). …”

Section: Electron Microscopymentioning

confidence: 99%

The planarian Schmidtea mediterranea as a model for studying motile cilia and multiciliated cells

Basquin

Orfila

Azimzadeh

2015

Methods in Cell Biology

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“…Sexual and asexual planarians originating from the Newmark laboratory were fixed and processed for epoxy (Epon-Araldite) embedding as previously described (62). For light-microscopic histology, 0.5 µm sections were stained with 1% (w/v) toluidine blue O in 1% (w/v) borax for 30 s at 100 º C, and imaged on a Zeiss Axio Observer.…”

Section: Histology and Transmission Electron Microscopymentioning

confidence: 99%

A planarian nidovirus expands the limits of RNA genome size

Saberi

Gulyaeva

Brubacher

et al. 2018

Preprint

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RNA viruses are the only known RNA-protein (RNP) entities capable of autonomous replication (albeit within a permissive environment). A 33.5-kb nidovirus has been considered close to the upper size limit for such entities; conversely, the minimal cellular DNA genome is ~200 kb. This large difference presents a daunting gap for the transition from primordial RNP to contemporary DNA-RNP-based life. Whether or not RNA viruses represent transitional steps on the road to DNA-based life, studies of larger RNA viruses advance our understanding of size constraints on RNP entities. For example, emergence of the largest previously known RNA genomes (20-34 kb in positive-stranded nidoviruses, including coronaviruses) is associated with a proofreading exoribonuclease encoded in the nidoviral open reading frame 1b (ORF1b). However, apparent constraints on the size of ORF1b, which encodes this and other key replicative enzymes, have been hypothesized to limit further expansion of viral RNA genomes. Here, we characterize a novel nidovirus (planarian secretory cell nidovirus; PSCNV) whose disproportionately large ORF1b-like region, and overall 41.1 kb genome, substantially extend the presumed limits on RNA genome size. This genome encodes a predicted 13,556-aa polyprotein in an unconventional single ORF, yet retains canonical nidoviral genome organization and expression, and key replicative domains. Our evolutionary analysis suggests that PSCNV diverged early from multi-ORF nidoviruses, and subsequently acquired additional genes, including those typical of large DNA viruses or hosts. PSCNV's greatly expanded genome, proteomic complexity, and unique featuresimpressive in themselvesattest to the likelihood of still-larger RNA genomes awaiting discovery. author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/299776 doi: bioRxiv preprint Significance StatementRNA viruses are the only known RNA-protein (RNP) entities capable of autonomous replication. The upper genome size for such entities was assumed to be <35 kb; conversely, the minimal cellular DNA genome is ~200 kb. This large difference presents a daunting gap for the proposed evolution of contemporary DNA-RNP-based life from primordial RNP entities. Here, we describe a nidovirus from planarians, whose 41.1 kb genome is 23% larger than the largest known of RNA virus. The planarian secretory cell nidovirus has broken apparent constraints on the size of the genomic subregion that encodes core replication machinery, and has acquired genes not previously observed in RNA viruses. This virus challenges and advances our understanding of the limits to RNA genome size. author/funder. All rights reserved. No reuse allowed without permission.

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Preparation of the planarian Schmidtea mediterranea for high-resolution histology and transmission electron microscopy

Cited by 30 publications

References 44 publications

Subcellular analyses of planarian meiosis implicates a novel, double-membraned vesiculation process in nuclear envelope breakdown

Subcellular analyses of planarian meiosis implicates a novel, double-membraned vesiculation process in nuclear envelope breakdown

The planarian Schmidtea mediterranea as a model for studying motile cilia and multiciliated cells

A planarian nidovirus expands the limits of RNA genome size

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