Publications about Nuclear Organization and DNA Repair
1. Muzaffar, A., Daze, K.D., Strongin, D.E., Rothbart, S.B., Hector Rincon-Arano, H., Allen H.F., Li, J, Brian D. Strahl, B.D., Hof, F. and Kutateladze, T.G. (2015), In review
2. Strongin, D.E., Groudine, M., Ritland Politz, J.C. (2014) Nucleolar tethering mediates pairing between the IgH and Myc loci. . Nucleus. 5(5):38-45. PubMed Open Access
Genes (i.e. the sequences of DNA on chromosomes that encode proteins) adopt statistically preferred positions within the nucleus. For example, the IgH and Myc genes which we studied here are on average located closer together in B-cells (a type of immune cell) than would be predicted if they were positioned randomly. These genes are noteworthy because they a frequently translocation partners. Translocations are events where DNA breaks are incorrectly repaired and two chromosomes improperly fuse together. In the case of IgH and Myc that translocation causes specific forms of cancer. We showed that the reason IgH and Myc are positioned close together is they are both tethered to an organelle (a structure within a cell that performs a specific function) called the nucleolus.
Publications about DsRed
3. *Strack, R.L., *Strongin, D.E., Mets, L., Glick, B.S., and Keenan, R.J. (2010) Chromophore Formation in DsRed Occurs by a Branched Pathway. J. Am. Chem. Soc. 132:8496-505. PubMed
In this paper we proposed a chemical pathway describing the way a red fluorescent protein called DsRed becomes red. There is always a little green mixed in with the red and previous proposals suggested a linear pathway where the green was just a point along the way to red where a few molecules got stuck. We proposed that the pathway was actually branched and at the branch point there is what is known as kinetic competition, that is whichever reaction happens first wins. We altered the chemical environment to favor one reaction or the other at the branch point and validated our new branched pathway model.
4. *Strack, R.L., *Strongin, D.E., Bhattacharyya, D., Tao, W, Berman, A, Broxmeyer, H.E., Keenan, R.J. and Glick, B.S. (2008) A non-cytotoxic DsRed variant for whole-cell labeling. Nat. Methods. 5:955-957. PubMed
In this paper we engineered new variants of a red fluorescent protein called DsRed. Fluorescent proteins are used to label cells or tag proteins. We want them to be as inert as possible because they are there as markers and not to influence the cells they put into. However, a lot of different fluorescent proteins show toxicity towards cells. We used random mutagenesis followed by screening as well as making rational site directed mutations to engineer new variants of DsRed that were no longer toxic to cells. We validated our improvements using a variety of analyses in bacteria, yeast and mammalian cells.
5. Strongin, D.E., Bevis, B., Khuong, N., Downing, M.E., Strack, R.L., Sundaram, K., Glick, B.S., and Keenan, R.J. (2007) Structural rearrangements near the chromophore influence the maturation speed and brightness of DsRed variants. Protein Eng. Des. Sel. 20:525-534. PubMed
The red fluorescent protein called DsRed is naturally a tetramer, meaning four copies of the protein always bind together. One of the ways we use fluorescent proteins is to genetically attach a copy as a fusion tag to other proteins which we are interested in studying. We want this tag to be inert, however if it is a tetramer the fluorescent protein tag will drive the proteins it is fused to into tetramers as well, potentially disrupting their function. I used a combination of random mutagenesis and rational site directed mutations to engineer a monomeric (i.e. single copy, no longer a tetramer) version of DsRed. The final DsRed monomer has 45 mutations compared to the original protein. Besides now being monomeric, DsRed-monomer has a few other slight differences in its physical properties, e.g it isn’t as bright as the tetramer. One way to understand a given property is to compare two variants at the molecular level. We solved x-ray crystal structures of DsRed-monomer and another DsRed variant and used the crystal structures (which show the position of individual atoms) to surmise the molecular basis for those differences in physical properties, e.g. brightness.
Publications about the Endoplasmic Reticulum
6. Losev, E., Reinke, C.A., Jellen, J., Strongin, D.E., Bevis, B.J., and Glick, B.S. (2006) Golgi maturation visualized in living yeast. Nature. 441:1002-1006. PubMed
A longstanding debate in cell biology was how proteins that are destined to be secreted from the cell move through the Golgi apparatus. In most eukaryotic organisms the Golgi is a stack of flattened cisternae, basically like a stack of pancakes. The secretory cargo acquires sequential sugar modifications as it moves through the Golgi because the enzymes that make those modifications are in discrete parts of the stack, e.g. there are early enzymes, middle enzymes and late enzymes. It was not clear if the cargo moved from cisterna to cisterna, like a package moving from shipping center to shipping center before being delivered to you or if the cisternae themselves moved up through the stack and the enzymes moved backwards like walking the wrong way on an escalator to stay in place. The technical problem is that the cisternae in a stack are too close together to tell them apart in a light microscope. Interestingly in bakers yeast, which is a common model organism, the cisternae are spread out. Eugene Losev labeled one enzyme green and another red. If the shipping center model was correct we would see cisternae stay the same color over time because the enzymes stay in place while the cargo moves. If the escalator model, called cisternal maturation, was correct we would see individual cisternae turn from green to red, which is what we saw confirming the cisternal maturation model. My direct contribution to this paper was engineering the red fluorescent protein that enabled us to do the experiment.
7. Connerly P.L., Esaki M., Montegna E.A., Strongin D.E., Levi S., Soderholm J., Glick B.S. (2005) Sec16 is a determinant of transitional ER organization. Curr Biol. 15:1439-47. PubMed
My thesis lab was particularly interested in how a subdomain of the endoplasmic reticulum(ER), namely transitional ER (tER) sites (also called ER exit sites) are formed. Pam Connerly performed a genetic screen in the budding yeast called Pichia pastoris, to look for mutants that had disrupted tER sites. The mutation was in a gene called Sec16. Pam went on to validate Sec16 as a mediator of tER site organization using a variety of approaches. My contribution to this paper was quantitative imaging to determine the stoichiometry (that is the ratio of the number of molecules) of Sec16 to other tER site proteins.
8. Soderholm, J., Bhattacharyya, D., Strongin, D., Markovitz, V., Connerly, P.L., Reinke, C.A., and Glick, B.S. (2004) The transitional ER localization mechanism of Pichia pastoris Sec12. Developmental Cell. 6:649-59. PubMed
The protein Sec12 is an important part of the machinary that gives tER sites (see summary of Connerly et al, above) their function. However, it was not clear how Sec12 got into tER sites and stayed there. Jon Soderholm showed that dimerization (two copies of the protein binding to each other) of Sec12 is necessary and sufficient to localize it to tER sites. My contribution to this paper was quantitative imaging using a technique called fluorescence recovery after photobleaching (FRAP) to show that Sec12 dynamically associated with tER sites, meaning it was concentrated at those sites but at the molecular level is was hanging out there a while and then moving to other tER sites and back and forth. Think of people looking at paintings in a museum. A snap shot at any given point in time will show concentrations of people in front of each painting because they stop to look for a while but over time individuals move around among the paintings.
Publications about tissue engineering
9. Zaleske D., Peretti G., Allemann F., Strongin D., MacLean R., Yates K.E., Glowacki J. (2003) Engineering a joint: a chimeric construct with bovine chondrocytes in a devitalized chick knee. Tissue Engineering 9:949-956. PubMed
10. Glowacki, J., Yates, K.E., Warden, S., Allemann, F., Peretti, G., Strongin, D., MacLean, R. and Zaleske, D. (2002) Engineering a Biological Joint. Annals of the New York Academy of the Sciences 961:123-125. PubMed
I contributed to these two papers when I was a lab tech between college and grad school. The lab was interested in creating artificial joints by creating an inert 3D scaffold to seed with the cells that make cartilage, called chondrocytes. We used the knees from embryonic chickens which were stripped of chicken cells and seeded with cow chondrocytes in a 3D mesh. I worked out the micro-surgical techniques and some culture conditions, but mostly contributed the histology, i.e. I preserved the samples, cut them into really thin slices that we could mount on microscope slides and then stained the slides to look at how the artificial joint was developing.