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Negative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments.

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Date
2012-07
Authors
Milam, Sara L
Osawa, Masaki
Erickson, Harold P
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Abstract
FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by negative-stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for negative-stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments.
Type
Journal article
Subject
Escherichia coli
Mycobacterium tuberculosis
Lipid Bilayers
Bacterial Proteins
Cytoskeletal Proteins
Liposomes
Microscopy, Electron
Microscopy, Fluorescence
Fluorescence Recovery After Photobleaching
Negative Staining
Amino Acid Motifs
Molecular Conformation
Permalink
https://hdl.handle.net/10161/16457
Published Version (Please cite this version)
10.1016/j.bpj.2012.05.035
Publication Info
Milam, Sara L; Osawa, Masaki; & Erickson, Harold P (2012). Negative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments. Biophysical journal, 103(1). 10.1016/j.bpj.2012.05.035. Retrieved from https://hdl.handle.net/10161/16457.
This is constructed from limited available data and may be imprecise. To cite this article, please review & use the official citation provided by the journal.
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Scholars@Duke

Erickson

Harold Paul Erickson

James B. Duke Distinguished Professor Emeritus
Recent research has been on cytoskeleton (eukaryotes and bacteria); a skirmish to debunk the irisin story; a reinterpretation of proposed multivalent binders of the coronavirus spike protein. I have also published an ebook on "Principles of Protein-Protein Association" suitable for a course module or individual learning.

Masaki Osawa

Assistant Research Professor of Cell Biology
Alphabetical list of authors with Scholars@Duke profiles.
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