In the last year, we have continued our studies that investigate how cells divide and differentiate in an effort to understand how these processes may fail during diseases like cancer. Specifically, this report will outline progress that we have made in the past year that extend our studies on how proteins localize and assemble during growth and development of the model organism Bacillus subtilis. First, we have investigated the irreversible assembly of a novel cytoskeletal protein called SpoIVA that, unlike proteins which form dynamic structures, uses ATP hydrolysis to drive its assembly (not disassembly). Second, we are extending our analysis of a pathway that we discovered in B. subtilis that mediates the orchestrated morphogenesis of two large structures during spore formation. Third, we have discovered that a well characterized cell division protein in B. subtilis has a previously unappreciated role at the onset of spore formation that dictates the decision of the cell to enter this differentiation pathway. Mature spores of B. subtilis are encased in a proteinaceous shell called the coat that is composed of about seventy different proteins. These proteins assemble atop a platform created by a single structural protein called SpoIVA. Our previous studies had shown that SpoIVA is an ATPase, which we thought was an unusual activity for a static morphogenetic protein, and proposed that ATP hydrolysis may drive the assembly into a stable structure surrounding spores. To begin to rigorously test this hypothesis, we initiated a multidisciplinary collaboration with the bioinformatics group of L. Aravind in NCBI, who were able to construct a putative topology diagram of the protein and predict key residues that were specifically required for ATP hydrolysis, and not ATP binding. We then established two in vitro assays, including a biophysical assay that was done in collaboration with a postdoctoral fellow in the group of James Sellers in NHLBI, to measure the assembly of wild type SpoIVA and several variants that were deficient in ATP hydrolysis. Our results indicated that SpoIVA assembles into a static structure that does not readily disassemble;that this assembly requires ATP hydrolysis, not simply ATP binding;and that ATP hydrolysis results in a structural change in SpoIVA that drives polymerization into a largely nucleotide-free polymer. Moreover, bioinformatic analysis of SpoIVA revealed that the protein is a newly identified member of the TRAFAC class of GTPases (which includes signaling proteins like Ras, membrane remodeling proteins like dynamin, and motor proteins like myosin and kinesin) and that SpoIVA likely evolved as a result of an ancient gene duplication event of a highly conserved GTP-binding translation factor, followed by rapid divergent evolution. This divergence included the loss of GTP binding (and subsequent acquiring of ATP-binding specificity) and the addition of both a C-terminal polymerization domain and a membrane anchor domain. We proposed that nucleotide hydrolysis-dependent polymerization of static polymers may be a conserved mechanism by which durable biological structures may be built. The results of this work were published in January, 2013 in The Proceedings of the National Academy of Sciences. SpoIVA is anchored onto the surface of the developing spore by a small amphipathic helical protein called SpoVM. Previously, we reported that the proper subcellular localization of this small protein is dictated by a geometric cue: in this case, the convex membrane curvature present only on the surface of the developing spore. Spores are encased in a second shell, made of peptidoglycan, which lies beneath the coat and is called the cortex. For over four decades, it has been known that cortex assembly only initiates after proper initiation of coat assembly, but the mechanisms that mediate this orchestrated assembly were not known. Our lab recently discovered a previously unannotated gene which we named cmpA, that we proposed participates in a checkpoint that represses cortex assembly until coat assembly properly initiates. In the past year, our efforts have been directed at identifying the downstream target of CmpA inhibition, in an effort to understanding how CmpA mediates the morphogenesis of both structures. Additionally, we envision that identification of the CmpA target may reveal a novel antimicrobial target along with a demonstrated mechanism by which to inhibit it. Genetic approaches that we have employed thus far have revealed two other genes which participate in this pathway. During cell division in B. subtilis, a group of proteins that comprise the Min system assembles at mid-cell in order to ensure that only a single septum forms. Recently, we proposed that the cell division protein DivIVA, which recruits the Min system, preferentially localized to regions displaying high concave membrane curvature. Consistent with this notion, we found that DivIVA-GFP using deconvolution microscopy and discovered that DivIVA localized to mid-cell only after the onset of membrane invagination and that DivIVA did not localize to division sites where the division machinery had simply assembled but was not functional. In the past year, we have extended our analysis to study the effect of DivIVA localization at an asymmetrically-positioned division septum that is elaborated at the onset of spore formation. We have discovered that depletion of DivIVA at the onset of sporulation results in a block in polar septum formation. Concomitantly, this also results in the uncompartmentalized activation of a normally compartment-specific transcription factor that is absolutely required for progression through the sporulation program. We propose that DivIVA has two additional, previously unappreciated, roles at the onset of sporulation. First, it is required for the redeployment of the cell division machinery to polar sites. Second, it is required for anchoring a phospatase at the polar septum so that it does not constrict with the cell division machinery. We are currently employing super resolution microscopy techniques (in collaboration with the group of Hari Shroff in NIBIB) to examine the localization of DivIVA and the phosphatase that it anchors at the polar septum in order to resolve which side of the septum they reside. A manuscript describing these results is currently in preparation for submission.

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Wu, I-Lin; Narayan, Kedar; Castaing, Jean-Philippe et al. (2015) A versatile nano display platform from bacterial spore coat proteins. Nat Commun 6:6777
Fu, Riqiang; Gill Jr, Richard L; Kim, Edward Y et al. (2015) Spherical nanoparticle supported lipid bilayers for the structural study of membrane geometry-sensitive molecules. J Am Chem Soc 137:14031-4
Tan, Irene S; Weiss, Cordelia A; Popham, David L et al. (2015) A Quality-Control Mechanism Removes Unfit Cells from a Population of Sporulating Bacteria. Dev Cell 34:682-93
Gill Jr, Richard L; Castaing, Jean-Philippe; Hsin, Jen et al. (2015) Structural basis for the geometry-driven localization of a small protein. Proc Natl Acad Sci U S A 112:E1908-15
Eswaramoorthy, Prahathees; Winter, Peter W; Wawrzusin, Peter et al. (2014) Asymmetric division and differential gene expression during a bacterial developmental program requires DivIVA. PLoS Genet 10:e1004526
Ramamurthi, Kumaran S; Storz, Gisela (2014) The small protein floodgates are opening; now the functional analysis begins. BMC Biol 12:96
Tan, Irene S; Ramamurthi, Kumaran S (2014) Spore formation in Bacillus subtilis. Environ Microbiol Rep 6:212-25
Renner, Lars D; Eswaramoorthy, Prahathees; Ramamurthi, Kumaran S et al. (2013) Studying biomolecule localization by engineering bacterial cell wall curvature. PLoS One 8:e84143
Castaing, Jean-Philippe; Nagy, Attila; Anantharaman, Vivek et al. (2013) ATP hydrolysis by a domain related to translation factor GTPases drives polymerization of a static bacterial morphogenetic protein. Proc Natl Acad Sci U S A 110:E151-60
Tan, Irene S; Ramamurthi, Kumaran S (2013) Membrane remodeling: FisB will do in a pinch. Curr Biol 23:R251-3

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