POSTERS - BLAST X - University of Utah

BLAST X Mon. Evening Session
IDENTIFYING NOVEL BACTERIAL CYTOSKELETAL ELEMENTS AND CYTOSKELETAL
INTERACTORS THROUGH HIGH-THROUGHPUT IMAGING
John Werner, Michael Ingerson-Mahar, Jonathan Guberman, and Zemer Gitai
Princeton University, Department of Molecular Biology, LTL-355 Washington Rd., Princeton, NJ
08540
Bacterial cytoskeletal proteins polymerize into filamentous structures that represent key
regulators of a wide array of cellular processes, including cell shape determination, cell division,
and cell polarity. While bacterial homologs of all three major eukaryotic cytoskeletal families
have already been described, the upstream regulators of bacterial cytoskeletal assembly and
downstream effectors of cytoskeletal function remain poorly understood. In addition, recent
studies have suggested that additional filament-forming proteins remain uncharacterized.
All known cytoskeletal elements in both bacteria and eukaryotes have distinct nonuniform
subcellular distributions. Focusing on the asymmetric bacterium, Caulobacter
crescentus, we thus employed a directed high-throughput imaging approach to identify both
novel bacterial cytoskeletal proteins and proteins that act upstream or downstream of the
previously-characterized cytoskeletons. We developed a pipeline of high-throughput methods
for generating fluorescent protein fusions, expressing them in Caulobacter, imaging their
subcellular distribution at high resolution, and quantitating the resulting imaging data. By using
this pipeline to analyze over 2,800 Caulobacter proteins as both N-and C-terminal mCherry
fusions, we identified a set of ~300 localized Caulobacter proteins. This set included the three
known Caulobacter cytoskeletons (the MreB actin homolog, FtsZ tubulin homolog, and
Crescentin intermediate-filament). We also identified a novel protein that localizes to a tight line
that hugs a short region of the inner curvature of Caulobacter cells. This protein may polymerize
on its own, as it is capable of forming linear structures when expressed in heterologous systems
such as E. coli or S. pombe. Preliminary studies suggests that this previously-uncharacterized
cytoskeletal element plays a role in cell shape determination and may exhibit crosstalk with
other cytoskeletal proteins.
To identify upstream regulators of cytoskeletal assembly, we modified our pipeline to
allow us to assay the effects of overexpressing genes of interest on the localization patterns of
MreB, FtsZ, and Crescentin. A pilot screen of ~200 conserved proteins with no known function
identified four proteins that perturbed FtsZ, one that perturbed MreB, one that perturbed both
MreB and FtsZ, and one that perturbed Crescentin. The functions and mechanisms of action of
these candidate cytoskeletal regulators are currently being examined. Finally, to identify
downstream cytoskeletal effectors, we imaged our library of localized Caulobacter proteins in
the presence of the MreB-delocalizing compound, A22. We found a large number of proteins
that are either delocalized or mislocalized by A22 and are currently determining the cellular
functions of these proteins as well as the nature of their direct or indirect associations with
MreB.
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