We are located in Castetter Hall, Room 207!

We are looking for motivated graduate students interested in biochemistry, cell biology, or Drosophila genetics. Check out our research description to learn more about what we do around here!

 

Contact information:

Christopher A. Johnston

206-B Castetter Hall

University of New Mexico

Albuquerque, NM, 87131

Office: 505-277-2914

email: johnstca@unm.edu
 



Research in the Johnston Lab

Our research focuses on mechanisms of mitotic spindle orientation. During development, animals must generate an impressive degree of cellular diversity. Moreover, the organsim must properly arrange cells in 3-dimensions such that proper tissue architecture is established and maintained. Both of these fundamental developmental processes are regulated by oriented cell divisions, which are achieved by precise positioning of the mitotic spindle during mitosis. Our lab uses a multidisciplinary approach spanning biochemistry, structural biology, cell biology, and Drosophila genetics to investigate the molecular pathways that regulate spindle orientation. Specifically, we are interested in several over-arching questions:

1. What are the signaling pathways that link cell polarity to spindle positioning during asymmetric cell division?

2. Which spindle orientation components are conserved among various cell types, and which pathways are unique? How might diverse spindle orientation pathways be utilized within a cell type-specific context?

3. How are spindle orientation complexes regulated at the biochemical level? What are the structural determinants for the formation of these complexes?

4. What is the evolutionary basis for spindle orientation regulators, especially as it pertains to multicellularity in animals?

5. What is the role for spindle orientation in development and disease?

 

Recent Publications:

1. Johnston CA, Doe CQ, and Prehoda KE. Structure of an enzyme-derived phosphoprotein recognition domain. (2012) Plos One. 7(4).

2. Wee B, Johnston CA, Prehoda KE, and Doe CQ. Canoe binds RanGTP to promote Pins(TPR)/Mud-mediated spindle orientation. (2011) J. Cell Biol. 195(3):369-376.

3. Johnston CA, Whitney DS, Volkman BF, Doe CQ, and Prehoda KE. Conversion of the enzyme guanylate kinase into a mitotic-spindle orienting protein by a single mutation that inhibits GMP-induced closing. (2011) PNAS. 108(44):E973-978.

4. Segalen M, Johnston CA, Martin CA, Dumortier JG, Prehoda KE, David NB, Doe CQ, and Bellaiche Y. The Fz-Dsh planar cell polarity pathway induces oriented cell division via Mud/NuMA in Drosophila and zebrafish. (2010) Dev. Cell. 19(5):740-752.

5. Johnston CA, Hirono K, Prehoda KE, and Doe CQ. Identification of an Aurora-A/PinsLINKER/Dlg spindle orientation pathway using induced cell polarity in Drosophila S2 cells. (2009) Cell. 138(6):1150-1163.

 
 

 

The ability of cells to regulate the orientation of division during mitosis is a fascinating biological phenomenon. The choice between expanding the population of a given cell type and generating diversity between siblings can be decided by the orientation of the miotic spindle. This is especially important to multipotent progenitor stem cells, which must maintain a viable pool of stem cells (i.e. self-renewal), while also generating diversity of cell types during development (i.e. differentiation). Improper balance between self-renewal and differentiation can lead to several pathologies, including developmental defects as well as cancer. We are interested in understanding this biological process from a basic, fundamental framework. To that end, we aim to investigate spindle orientation pathways using a combinatorial approach of methodologies spanning in vitro and in vivo contexts. Below are representative results from our most used techniques; together they demonstrate how this combined approach can tell a complete story of newly identified spindle orientation pathways.

Anyone interested in participating in our research program is encouraged to contact us (see left column for information)!

Figure 1. Spindle orientation in Drosophila S2 cells. Although genetic approaches in model organisms (e.g. Drosophila) have been instrumental in defining a 'parts list' of spindle orientation components, they have several inherent drawbacks. Most importantly, genetic alterations in the animal often have deleterious effects on other components in a given pathway, making it difficult to define the sufficiency of a given gene and to ascribe a molecular mechanism of its action. To overcome this, we have developed a novel 'induced polarity' assay in S2 cells. We use the cell-adhesion protein, Echinoid (Ed), to induce polarization of components we are interested in studying (shown in GREEN). We then examine how that component regulates the position of the mitotic spindle (shown in RED). Mutational analysis along with RNAi-mediated loss of function studies can be rapidly performed in S2 cells. In this image, a spindle orientation protein called, Pins, is attached to Ed (Ed:Pins, GREEN), and is capable of orienting the spindle during cell division.

Figure 2. Spindle orientation in Drosophila neural stem cells (neuroblasts). Studies in S2 cells can rapidly identify new regulators of spindle orientation; however, as S2 cells are immortalized cultured cells far removed from a whole organism, we use standard genetic approaches in Drosophila to complement S2 cell studies in order to firmly place newly-identified pathways in a bona fide in vivo context. In this image, a Drosophila neuroblast from the larval central brain is shown with apically polarized Pins (RED) orienting the mitotic spindle (GREEN). Note the similarities between this in vivo system and our S2 cell culture system in Figure 1.

Figure 3. Biochemical analysis of protein-protein interactions. The experimental approaches described in Figures 1 and 2 can often define the linear order of components acting in a given pathway (called epistasis). However, to definitively understand how each component fits within complicated signal transduction pathways, we perform protein-protein interaction experiments in vitro. We use purified components isolated from a variety of sources-- mostly from expression in E. coli. These studies can define the nature of each physical interaction in a pathway and define existence of mutualism or antagonism between binding partners, which might act in more sophisticated feedback loops within the pathway. In this image, the ability of Pins to directly bind a downstream component, Dlg, is shown. Interestingly, this interaction depends on a phosphorylation of Pins by the Aurora-A kinase. Notably, each of these 3 components (Pins, Dlg, and Aurora-A) are necessary for spindle orientation in S2 cells and neuroblasts, and this study provided the molecular mechanism for how they act together at the biochemical level.

Figure 4. Structural analysis of spindle orientation complexes. The biochemical analysis in Figure 3 revealed a new level of regulation in Pins-mediated spindle orientation. We therefore sought to understand how Pins phosphorylation regulated binding to Dlg at the structural level. Using highly pure and concentrated preparations of the Pins/Dlg complex, we obtained protein crystals (not shown) that were subjected to a snychrotron energy source. The x-ray structure was then determined to 1.6 angstroms. The Dlg molecule is shown with its 3 subdomains color coded (BLUE: 'lid domain'; GREEN: 'core domain'; RED: 'GMP-binding domain'). A fragment of Pins is shown in CYAN. The structure defines the atomic details of the Pins/Dlg interaction. Of particular interest, we found that the Pins phosphorylated amino acid binds to the 'GMP-binding domain' of Dlg. Interestingly, this same region of a related protein, guanylate kinase (Guk), is used to bind the nucleotide GMP. Whereas all known life forms contain the Guk enzyme, only multicellular animals appear to contain Dlg. Thus, this study provided the basis for an evolutionary analysis of Dlg function (described below).

Figure 5. Evolutionary analysis of spindle orientation regulators. As noted in Figure 4 above, the GK domain (BLUE line) of Dlg is found exclusively in animals. In contrast, the GK enzyme (GREEN line) is found in all living organisms. The GK domain is thought to have evolved following a whole genome duplication event. Although the GK enzyme and domain share relatively high homology in both primary sequence and 3-dimensional structure, they exhibit drastically different biological functions. The enzyme catalyzes the conversion of GMP to GDP during purine metabolism; the domain serves as a protein-protein interaction domain (e.g. with Pins). Using several of the techniques described in the above figures, we recently identified a remarkably short sequence path that converts the enzyme to a functional 'domain'. The basis for this mutation-induced change in function relied on altering the conformational flexibility of the protein during ligand binding.