September 22, 2017

During cell division, a ring of proteins forms around the equator of the cell. Then, after the chromosomes have been distributed to each pole of the mitotic spindle, the ring constricts, separating the cell into two daughter cells. This contractile ring is a complex structure that uses the mechanical energy of myosin motors to constrict a plasma membrane-associated network of F-actin fibers. Numerous studies have delineated the steps of contractile ring formation, starting with membrane-bound precursor nodes that eventually coalesce into a contiguous ring prior to constriction. However, a detailed understanding of contractile ring architecture is lacking, thereby preventing a complete understanding of its composition, structure and function. To fill this knowledge gap, Basic Sciences investigator Kathleen Gould and her laboratory utilized resources provided by Vanderbilt’s new Nikon Center of Excellence to obtain high resolution structural data on contractile ring structure in dividing Schizosaccharomyces pombe. The investigators individually expressed in yeast cells a total of 29 known contractile ring proteins linked at either their N- or C-terminus to a photoactivatable probe. The cells also expressed a distinct membrane-associated photoactivatable probe. This enabled the researchers to use fluorescence photoactivation localization microscopy (fPALM), a super-resolution microscopy technique to localize each protein in terms of its distance from the membrane. The results demonstrated three structurally distinct regions at 0-80 nm, 80-160 nm, and 160-400 nm from the membrane’s cytosolic surface. In the layer closest to the membrane were membrane-associated scaffold proteins and the tail of myosin II. The intermediate layer contained a complex network of accessory proteins and signaling molecules. The layer most distant from the membrane comprised predominantly F-actin along with the motor heads of myosin and an F-actin cross-linker. The researchers also found that some proteins, particularly those close to the membrane, formed clusters of various sizes, whereas others were more uniformly distributed, especially in the most distant layer. These findings provide a critical foundation for the better understanding of contractile ring assembly and function. The work is published in the journal eLife [N. A. McDonald, et al., (2017) eLife., 6, e28865].