Nuclear/Cytoplasmic Fractionation of Proteins from Caenorhabditis elegans

Mata‐Cabana, Alejandro; Sin, Olga; Seinstra, Renée I.; Nollen, Ellen A. A.

doi:10.21769/bioprotoc.3053

Cited by 8 publications

(6 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Samples were prepared with Laemmli sample buffer with 0.1 mM DTT. Equal protein amounts within respective fractions were loaded 60 and assessed via SDS-PAGE on 9% gels or gradient gels. Proteins were transferred to PVDF membranes.…”

Section: Methodsmentioning

confidence: 99%

“…In this protocol, we loaded equal protein amounts for each respective fractions, and normalized the levels of the target proteins within respective fractions to a fraction marker (GAPDH and p84 for cytoplasmic and nuclear fractions, respectively), to cancel out the potential loading error [61][62][63] . This protocol is suited for comparing protein levels in the nuclear fraction before and after PDGF treatment but not designed to compare protein abundance between the cytoplasmic and nuclear fractions 60 .…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus

et al. 2020

View full text Add to dashboard Cite

The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth, nutrient and energy status cues to control cell growth and metabolism. While mTORC1 activation at the lysosome is well characterized, it is not clear how this complex is regulated at other subcellular locations. Here, we combine location-selective kinase inhibition, live-cell imaging and biochemical assays to probe the regulation of growth factor-induced mTORC1 activity in the nucleus. Using a nuclear targeted Akt Substrate-based Tandem Occupancy Peptide Sponge (Akt-STOPS) that we developed for specific inhibition of Akt, a critical upstream kinase, we show that growth factor-stimulated nuclear mTORC1 activity requires nuclear Akt activity. Further mechanistic dissection suggests that nuclear Akt activity mediates growth factor-induced nuclear translocation of Raptor, a regulatory scaffolding component in mTORC1, and localization of Raptor to the nucleus results in nuclear mTORC1 activity in the absence of growth factor stimulation. Taken together, these results reveal a mode of regulation of mTORC1 that is distinct from its lysosomal activation, which controls mTORC1 activity in the nuclear compartment.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Subcellular fractionation was performed as described in ref. 36 . Briefly, one-day-old animals grown at 20˚C were washed with M9 buffer until the supernatant was clear and then washed twice with 1 ml cold hypotonic buffer (15 mM HEPES KOH pH7.6, 10 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 350 mM sucrose).…”

Section: Cytoplasmic and Nuclear Fractionationmentioning

confidence: 99%

Reprogramming of transcriptome after hormetic heat stress by the endoribonuclease ENDU‑2 improves lifespan and stress resistance in C. elegans

Gromoff

Drepper

et al. 2022

Preprint

View full text Add to dashboard Cite

Any organism is constantly exposed to dangerous, potentially life-threatening environmental impacts that demand coordinated responses. Although acute stress responses as temporal reactions to unfavorable external or internal factors are well known, transient stress experiences may have long-term physiological consequences. The underlying mechanisms adjusting cellular activities across time scale is poorly understood. Here, we investigate the long-term alteration of C. elegans transcriptome after a short heat shock (HS) exposure and discovered that the canonical HS response is followed by a profound transcriptional reprogramming that affects many genes involved in innate immunity response. This transcriptional reprogramming depends on the function of the endoribonuclease ENDU-2 but not the heat shock factor 1 (HSF-1). ENDU-2 in this context co-localizes with chromatin and interacts with RNA polymerase Pol II to specifically regulate transcription in the post-HS period. Failure to activate this post-HS response does not impair survival of animals upon HS insult, but abolishes the hormetic HS mediated beneficial effects. In summary, our work discovers that the hormetic long-term effects of a transient HS are mediated by the RNA-binding protein ENDU-2 that determines aging and longevity.

show abstract

“…After homogenization, the cytoplasmic fraction was separated from the nuclei by centrifugation, and the nuclei were lysed in a hypotonic buffer to obtain nuclear extract. We subsequently checked the effectiveness of our subcellular fractionation through Western blot analyses of the cytoplasmic fraction and nuclear extract using αtubulin and histone H3 as marker proteins, respectively (17). Western results showed that α-tubulin mainly appeared in the cytoplasmic fraction and not in the nuclear fraction, whereas histone H3 was exclusively detected in the nuclear extract (Fig.…”

Section: Preparation Of C Elegans Nuclear Extractmentioning

confidence: 99%

A novelin vitro Caenorhabditis eleganstranscription system

Wibisono

Liu

Sun

2020

Preprint

View full text Add to dashboard Cite

11Caenorhabditis elegans is an excellent model organism for biological research, but its contributions to 12 biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest 13 obstacles for biochemical studies of C. elegans is the high difficulty of preparing functionally active nuclear 14 extract due to its thick surrounding cuticle. By employing Balch homogenization, we have achieved effective 15 disruption of larval and adult worms and have obtained functionally active nuclear extract through subcellular 16 fractionation. In vitro transcription reactions were successfully re-constituted using such nuclear extract. 17Furthermore, two non-radioactive detection methods, PCR and qRT-PCR, have been adapted into our system to 18 qualitatively and quantitatively detect transcription, respectively. Using this system to assess how pathogen 19 infection affects C. elegans transcription revealed that Pseudomonas aeruginosa infection increased 20 transcription activity. Our in vitro system is useful for biochemically studying C. elegans transcription 21 mechanisms and gene expression regulations. The effective preparation of functionally active nuclear extract in 22 our system fills a technical gap in biochemical studies of C. elegans and will expand the usefulness of this 23 model organism in addressing many biological questions beyond transcription. 24 25 2 Keywords 26 in vitro transcription, Caenorhabditis elegans, Balch homogenizer, subcellular fractionation, non-radioactive 27 detection 28 29 Background 30Caenorhabditis elegans is a free-living, 1-millimeter-long, nematode worm found in soil and decaying organic 31 matter. In 1963, Sydney Brenner proposed research into C. elegans, stating that "I would like to tame a small 32 metazoan organism to study development directly" (1). Since then, C. elegans has been used as a model 33 organism to address a wide range of biological questions such as those relating to development, metabolism, 34 neurobiology, and aging. The nematode has many characteristics that make it an excellent model system, 35 including, but not limited to, its rapid (3-day) life cycle, small size, ease of laboratory cultivation, genetic 36 tractability, invariant lineage, effectiveness of RNA interference, and transparent body that allows for 37 monitoring development or gene expression with single-cell resolution. Research involving the use of C. 38 elegans, including Brenner's work on organ development, was awarded with the Nobel Prize in 2002, 2006, and 39 2008.40 41Because of its genomic simplicity and physical characteristics, C. elegans offers a unique system to study 42 transcription mechanisms and regulation. For example, mutations in pre-initiation complex genes have been 43 recovered in genetic screens of C. elegans and have linked regulation that involves these factors to specific 44 biological processes (2); the nematode is transparent throughout its entire life cycle making it an ideal system to 45 use fluorescent protein reporter genes to monitor g...

show abstract

Nuclear/Cytoplasmic Fractionation of Proteins from Caenorhabditis elegans

Cited by 8 publications

References 5 publications

Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus

Location-specific inhibition of Akt reveals regulation of mTORC1 activity in the nucleus

Reprogramming of transcriptome after hormetic heat stress by the endoribonuclease ENDU‑2 improves lifespan and stress resistance in C. elegans

A novelin vitro Caenorhabditis eleganstranscription system

Contact Info

Product

Resources

About