Negative-stain electron microscopy of inside-out FtsZ rings reconstituted on artificial membrane tubules show ribbons of protofilaments.
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 articleSubject
Escherichia coliMycobacterium tuberculosis
Lipid Bilayers
Bacterial Proteins
Cytoskeletal Proteins
Liposomes
Microscopy, Electron
Microscopy, Fluorescence
Fluorescence Recovery After Photobleaching
Negative Staining
Amino Acid Motifs
Molecular Conformation
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https://hdl.handle.net/10161/16457Published Version (Please cite this version)
10.1016/j.bpj.2012.05.035Publication 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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Show full item recordScholars@Duke
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
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