Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Biological and Environmental Sciences

Specialization

Cell and Molecular Biology (CMB)

Department

Cell & Molecular Biology

First Advisor

Jodi L. Camberg

Abstract

It is an immense undertaking for any organism, including a bacterium, to self-replicate. Accurate and rapid division is essential for bacteria to proliferate and produce progeny, especially in environments where there is competition for resources. The nucleoid, the region of the cell that contains the DNA, harbors the genetic information required to achieve robust division, but it is the assembly of the cell division machinery, collectively referred to as the divisome, that produces and coordinates the mechanical force necessary to divide a cell. The hallmark of prokaryotic cell division is the formation of a protein super-structure, called the Z-ring, which spans the circumference of the cell division site on the interior of the cytoplasmic membrane. A nascent Z-ring, or proto-ring, is minimally comprised of polymerized FtsZ tethered to the cell membrane by FtsA, which is anchored directly to the membrane. The proto-ring acts as a scaffold for a sequential pathway of at least 10 essential cell division proteins and more than 15 other accessory proteins, which collectively regulate the spatiotemporal assembly of the mature Z-ring. Once fully assembled, the Z-ring and accessory proteins comprise the divisome that coordinates constriction of the mother cell into two identical daughter cells.

In Manuscript I, I review how proteins physically perturb lipid membranes to achieve a morphological cellular event, with emphasis on how prokaryotes assemble and utilize the Z-ring during division. I discuss current models for how division occurs and describe how dynamic protein conformational changes induce various cellular architectures.

In Manuscript II we discovered a new protein polymer comprised of the prokaryotic cell division proteins MinC and MinD, which copolymerize in the presence of ATP and rapidly depolymerizes in the presence of MinE. We report mechanistic insight into regulation of the protein assembly and propose a model of function for the polymer.

In Manuscript III we report the first natively purified and enzymatically active preparation of FtsA. We show that FtsA intercalates its membrane targeting sequence (MTS) into the lipid bilayer and uses ATP to directly remodel membrane architecture. These observations led us to propose a new model of the cell division pathway whereby FtsA directly exerts constrictive force at the division septum. Using our understanding from the work in Manuscript III we demonstrated in Manuscript IV that FtsA binds to phospholipids in an ATP-dependent manner through a mechanism that is distinct from the MTS. Additionally, we show that FtsA forms polymers in the presence of ATP and that polymerization is concentration dependent. This manuscript advances our model of FtsA-mediated lipid reorganization through mechanistic insight of how FtsA associates with the membrane and forms ATP-dependent polymers.

In conclusion, we report novel protein assemblies of MinC with MinD and, separately, assemblies of FtsA and we propose a model for how each polymer may affect cell division. Altogether, this work elucidates the role of protein polymers during prokaryotic cell division, and more generally, broadens our perspective of how protein polymers affect diverse cellular activities.

Available for download on Sunday, October 11, 2020

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