U.S. Department of Health and Human Services

Laboratory of Biochemistry and Genetics

Reed B. Wickner, M.D., NIH Distinguished Investigator, Chief

​Research Images

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Centrosome amplification in a C. elegans embryo.

This video was recorded by Jyoti Iyer, a research fellow in the laboratory of Dr. Kevin O’Connell and shows the first few cell cycles of a C. elegans embryo that lacks the protein phosphatase regulator SDS-22.   The embryo expresses GFP::SPD-2, which marks centrosomes in green and mCherry::Histone, which marks chromatin in red. Centrosomes initially duplicate properly yielding 2 centrosomes per cell through the two cell stage.  Centrosomes eventually over-duplicate giving rise to more than two centrosomes per cell at the four cell stage. From Peel N., Iyer J., Naik A., Dougherty M. P., Decker M., O'Connell K. F., 2017 Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication. PLoS Genet. 13: e1006543.

Centrosome duplication in a C. elegans embryo.

This video was recorded by Nina Peel, a postdoctoral fellow in the laboratory of Dr. Kevin O’Connell and shows the first few cell cycles of a wild-type C. elegans embryo.   The embryo expresses GFP::SPD-2, which marks centrosomes in green and mCherry::Histone, which marks chromatin in red. Notice that at the end of the first cell cycle, each centrosome duplicates precisely and resolves into two daughter centrosomes. These daughter centrosomes migrate around the DNA to direct chromosome segregation during the next cell cycle.

Spindle assembly in a C. elegans embryo.

This time-lapse recording of a wild-type C. elegans embryo expressing GFP::Tubulin was produced in the laboratory of Kevin O’Connell.  The video begins with the maternal and paternal pronuclei uniting at the posterior of the embryo.  Two microtubule asters formed by centrosomes associated with the paternal pronucleus grow in size and set up the first mitotic spindle. At the end of the first cell cycle, the spindle disassembles as each centrosome duplicates giving rise to another round of bipolar spindle formation at the two-cell stage.

Centrosome amplification in a C. elegans embryo

A four-cell C. elegans sds-22 mutant embryo with extra centrosomes.  This is from our paper: Peel N., Iyer J., Naik A., Dougherty M. P., Decker M., O'Connell K. F., 2017 Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication. PLoS Genet. 13: e1006543.

A four-cell C. elegans sds-22 mutant embryo with extra centrosomes.Enlarge
Electron micrograph of a C. elegans mother centriole

An electron micrograph of a C. elegans embryonic centriole. From: O'Connell K. F., Caron C., Kopish K. R., Hurd D. D., Kemphues K. J., Li Y., White J. G., 2001 The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo. Cell 105: 547–558.

An electron micrograph of a C. elegans embryonic centriole.Enlarge
Bipolar and monopolar spindle assembly in wild-type and mutant C. elegans embryos

Images of one- and two-cell C. elegans embryos stained for microtubules (red) and DNA (blue). Embryos on left are wild type and possess bipolar spindles. Embryos on right lack the kinase ZYG-1 and have monopolar spindles.

Images of one- and two-cell C. elegans embryos stained for microtubules (red) and DNA (blue).Enlarge
Microtubule organization in early C. elegans embryos

C. elegans embryos stained for microtubules (green), SPD-2 (red/yellow) and DNA (blue). From: Kemp C. A., Kopish K. R., Zipperlen P., Ahringer J., O'Connell K. F., 2004 Centrosome maturation and duplication in C. elegans require the coiled-coil protein SPD-2. Dev. Cell 6: 511–523.

C. elegans embryos stained for microtubules (green), SPD-2 (red/yellow) and DNA (blue).Enlarge
Imaging mRNA in dividing live yeast cells.

Cells are expressing a reporter mRNA that encodes MS2 stem loops in its 3’UTR and a second mRNA encoding fluorescent mKATE linked to MS2 coat protein (mKATE-MCP). Bright dots correspond to single reporter mRNA molecules in the cytoplasm that are visible because their MS2 stem loops are bound to molecules of mKATE-MCP.

Imaging mRNA in dividing live yeast cellsEnlarge
Imaging translation in a live dividing yeast cell.

The cell is expressing a reporter mRNA with GCN4 repeats and a second mRNA encoding GFP linked to a Gcn4 antibody fragment (SunTag). Bright areas include sites of single polysomes where SunTags are bound to nascent Gcn4 peptides.

Imaging translation in a live dividing yeast cellEnlarge
Splitting mammalian cell cultures.

Biological safety cabinet used to develop and maintain mammalian cell lines for ribosome profiling and microscopy experiments.

Splitting mammalian cell culturesEnlarge
Imaging gels and western blots.

This apparatus is required for detecting reporter proteins and imaging gels for ribosome profiling.

Imaging gels and western blotsEnlarge
Running sucrose gradients.

Sucrose gradients are required for separating ribosomes from other heavy complexes in the cell for ribosome profiling experiments.

Running sucrose gradientsEnlarge
Keynote at the 2017 NIDDK Scientific Conference.

Talk given by Dr. Guydosh on major focus areas of the lab.

Keynote at the 2017 NIDDK Scientific ConferenceEnlarge
Injection of C. elegans Gonads

In this video, Amy Fabritius, a post-doctoral fellow in Dr. Andy Golden's lab, demonstrates microinjection of DNA into the gonad of C. elegans hermaphrodites. This is an essential skill one needs to master to edit the genome using the CRISPR/Cas9 tools.

mel-15 embryos lack paternal DNA

Wild type and mel-15(it7) hermaphrodites were shifted to 24°C as L4s. After ~24 hours, hermaphrodites were dissected and embryos imaged with a confocal microscope. Both maternal and paternal DNA are present in wild type embryos (paternal DNA, arrow). Embryos fertilized by mel-15(it7) males do not have paternal DNA, but other paternal products (i.e. centrosomes, arrowheads) are transferred to the embryo during fertilization. M=maternal DNA, P=paternal DNA, white circle demarks area of missing paternal DNA. Red: Histone::mCherry. Green: nuclear pore::GFP, tubulin::GFP. Scale bar= 30 μm.

images displaying mel-15 embryos which lack paternal DNAEnlarge
Spectra of poly-L-lysine.Circular dichroism, absorbance, and g-factor spectra of poly-L-lysine, in the random coil (H2O), alpha-helical (TFE), and beta-sheet (SDS) forms.Spectra of poly-L-lysine.Enlarge
Figure 3. Model of yeast prion amyloid structure

The parallel in-register architecture of yeast prion amyloids features the favorable interaction between aligned identical amino acid side-chains. This structure can explain how proteins can template their own conformation and how a protein can act as a gene.

Figure 3. Model of yeast prion amyloid structureEnlarge
Figure 2. Prion domains of yeast prion proteins

For each of the yeast and fungal prions, a restricted portion of the protein is necessary and sufficient for generation and propagation of the prion. These domains are generally rich in glutamine and asparagine and are the part of the protein that comprises the core of the amyloid in an in-register parallel structure.

Figure 2. Prion domains of yeast prion proteinsEnlarge
Figure 1. Three genetic criteria for a yeast prion

[URE3] is a non-Mendelian genetic element of S. cerevisiae that makes cells able to take up ureidosuccinate when ammonia is the nitrogen source. [PSI] is a non-Mendelian genetic element that increases the efficiency with which weak suppressor tRNAs allow read-through of translation termination codons. Three properties of these genetic elements indicated to us that they were prions of Ure2p and Sup35p, respectively: (1) reversible curability, (2) overproduction of the prion protein inducing prion formation de novo, and (3) similarity of prion phenotype and phenotype of mutation of the gene encoding the prion protein (with prion propagation depending on that gene). These three properties allowed us to first identify these yeast non-chromosomal genetic elements as prions.

Figure 1. Three genetic criteria for a yeast prionEnlarge
Ure2-GFP aggregates in a [URE3] prion cell.Ure2-GFP fluorescence in a strain containing [URE3] and in a strain lacking the prion ([ure-o])Ure2-GFP aggregates in a [URE3] prion cell.Enlarge