The C Terminus of FtsZ Regulates FtsZ Assembly Dynamics and Is Required for Bacillus Subtilis Cell Division

Bacterial cell division is initiated by the assembly of the tubulin homolog FtsZ into a ring: Z ring) at the nascent division site. Once formed, the Z ring serves as a scaffold for recruitment of the division machinery and helps provide some of the constrictive force for cytokinesis. In vitro FtsZ undergoes GTP-dependent assembly where individual subunits form single-stranded protofilaments and laterally-associated filament bundles. How the filamentous FtsZ structures observed in vitro translate into the behavior of the Z ring in vivo remains a fundamental question. In this dissertation I establish important roles for the previously uncharacterized FtsZ C-terminal domains during both FtsZ assembly and Z ring formation. My work provides significant insight into how FtsZ behavior at the protein level impacts its cellular function. Structurally, the FtsZ monomer is divided into 5 domains: an unstructured N-terminal peptide, a highly conserved N-terminal globular core, an unstructured C-terminal linker: CTL), a conserved set of ~11 residues referred to here as the C-terminal constant region: CTC), and a small, highly variable group of residues at the extreme C-terminus of FtsZ termed the C-terminal variable region: CTV). For simplicity, the N-terminal peptide and core are treated here as a single unit. The core shows a high degree of sequence conservation amongst bacterial species and contains residues required for GTP binding and hydrolysis as well as forming the contacts necessary to make filaments. The entire FtsZ C terminus consists of the CTL, CTC, and CTV. The CTL displays very little conservation between species both in primary sequence and length, is irresolvable by X-ray crystallography, and is presumed to be intrinsically disordered. The CTC and CTV are implicated in interactions between FtsZ and modulatory proteins. To reflect this function the combined domains have been termed the grappling hook peptide: GHP). Prior to this work, the roles that the C-terminal domains had in FtsZ assembly were unknown. In this dissertation, I demonstrate that these domains do have distinct functions. First I show the CTV is important for regulating lateral interactions between FtsZ protofilaments. B. subtilis FtsZ readily forms bundled structures in vitro. In contrast, I show E. coli FtsZ typically assembles into single-stranded protofilaments. Through deletion analysis and domain swapping, I determine these phenotypes to derive from differences in the CTVs of each species. I also establish that electrostatic interactions are a driving force behind FtsZ bundling. Alterations to the CTV sequence also greatly affect cell division in B. subtilis cells, suggesting filament bundling is important for a stable Z ring in vivo. Finally, I demonstrate the FtsZ CTL is essential for FtsZ protofilament assembly and cell division. I determine that a functional CTL must behave as an intrinsically disordered peptide with little primary sequence requirement but must be between 25 and 100 residues in length. These findings lead to a model for FtsZ in which the CTL behaves as a flexible tether anchoring FtsZ filaments to the membrane through interactions between the GHP and FtsZ modulatory proteins like FtsA. The linker can undertake different conformations and allow FtsZ filaments bundle through positioning the CTV near adjacent filaments and to respond to the curvature of the membrane, having implications for how the constrictive force for cytokinesis is generated

Mathematical and Statistical Techniques for Systems Medicine: The Wnt Signaling Pathway as a Case Study

The last decade has seen an explosion in models that describe phenomena in systems medicine. Such models are especially useful for studying signaling pathways, such as the Wnt pathway. In this chapter we use the Wnt pathway to showcase current mathematical and statistical techniques that enable modelers to gain insight into (models of) gene regulation, and generate testable predictions. We introduce a range of modeling frameworks, but focus on ordinary differential equation (ODE) models since they remain the most widely used approach in systems biology and medicine and continue to offer great potential. We present methods for the analysis of a single model, comprising applications of standard dynamical systems approaches such as nondimensionalization, steady state, asymptotic and sensitivity analysis, and more recent statistical and algebraic approaches to compare models with data. We present parameter estimation and model comparison techniques, focusing on Bayesian analysis and coplanarity via algebraic geometry. Our intention is that this (non exhaustive) review may serve as a useful starting point for the analysis of models in systems medicine.Comment: Submitted to 'Systems Medicine' as a book chapte

High levels of the adhesion molecule CD44 on leukemic cells generate acute myeloid leukemia relapse after withdrawal of the initial transforming event

Multiple genetic hits are detected in patients with acute myeloid leukemia (AML). To investigate this further, we developed a tetracycline-inducible mouse model of AML, in which the initial transforming event, overexpression of HOXA10, can be eliminated. Continuous overexpression of HOXA10 is required to generate AML in primary recipient mice, but is not essential for maintenance of the leukemia. Transplantation of AML to secondary recipients showed that in established leukemias, ∼80% of the leukemia-initiating cells (LICs) in bone marrow stopped proliferating upon withdrawal of HOXA10 overexpression. However, the population of LICs in primary recipients is heterogeneous, as ∼20% of the LICs induce leukemia in secondary recipients despite elimination of HOXA10-induced overexpression. Intrinsic genetic activation of several proto-oncogenes was observed in leukemic cells resistant to inactivation of the initial transformation event. Interestingly, high levels of the adhesion molecule CD44 on leukemic cells are essential to generate leukemia after removal of the primary event. This suggests that extrinsic niche-dependent factors are also involved in the host-dependent outgrowth of leukemias after withdrawal of HOXA10 overexpression event that initiates the leukemia

Notch Lineages and Activity in Intestinal Stem Cells Determined by a New Set of Knock-In Mice

The conserved role of Notch signaling in controlling intestinal cell fate specification and homeostasis has been extensively studied. Nevertheless, the precise identity of the cells in which Notch signaling is active and the role of different Notch receptor paralogues in the intestine remain ambiguous, due to the lack of reliable tools to investigate Notch expression and function in vivo. We generated a new series of transgenic mice that allowed us, by lineage analysis, to formally prove that Notch1 and Notch2 are specifically expressed in crypt stem cells. In addition, a novel Notch reporter mouse, Hes1-EmGFPSAT, demonstrated exclusive Notch activity in crypt stem cells and absorptive progenitors. This roster of knock-in and reporter mice represents a valuable resource to functionally explore the Notch pathway in vivo in virtually all tissues

Proteolysis-Dependent Remodeling of the Tubulin Homolog FtsZ at the Division Septum in \u3ci\u3eEscherichia coli\u3c/i\u3e

During bacterial cell division a dynamic protein structure called the Z-ring assembles at the septum. The major protein in the Z-ring in Escherichia coli is FtsZ, a tubulin homolog that polymerizes with GTP. FtsZ is degraded by the two-component ATP-dependent protease ClpXP. Two regions of FtsZ, located outside of the polymerization domain in the unstructured linker and at the C-terminus, are important for specific recognition and degradation by ClpXP. We engineered a synthetic substrate containing green fluorescent protein (Gfp) fused to an extended FtsZ C-terminal tail (residues 317–383), including the unstructured linker and the C-terminal conserved region, but not the polymerization domain, and showed that it is sufficient to target a non-native substrate for degradation in vitro. To determine if FtsZ degradation regulates Z-ring assembly during division, we expressed a full length Gfp-FtsZ fusion protein in wild type and clp deficient strains and monitored fluorescent Z-rings. In cells deleted for clpX or clpP, or cells expressing protease-defective mutant protein ClpP(S97A), Z-rings appear normal; however, after photobleaching a region of the Z-ring, fluorescence recovers ~70% more slowly in cells without functional ClpXP than in wild type cells. Gfp-FtsZ(R379E), which is defective for degradation by ClpXP, also assembles into Z-rings that recover fluorescence ~2-fold more slowly than Z-rings containing Gfp-FtsZ. In vitro, ClpXP cooperatively degrades and disassembles FtsZ polymers. These results demonstrate that ClpXP is a regulator of Z-ring dynamics and that the regulation is proteolysis-dependent. Our results further show that FtsZ-interacting proteins in E. coli fine-tune Z-ring dynamics

A user's guide to the Encyclopedia of DNA elements (ENCODE)

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome