Research News

Lights… Camera… Action… and Cut! A new system to visualize and analyze membrane fission

The plasma membrane encloses and defines a cell, the basic unit that living organisms are made of. To facilitate cellular processes like transport of metabolites or signal transmission, the plasma membrane needs to pinch off parts of itself into the cell (endocytosis). This process of membrane fission is highly regulated so that the cut allows nothing more than the intended contents into the cell. Dynamin and a few other proteins with similar characteristics represent a group of specialized protein machines that cut membranes by using energy from nucleotide hydrolysis.

A new study from Dr. Thomas Pucadyil’s group at IISER Pune, published this week in Nature Cell Biology, describes a novel model membrane assay system with which to observe the dynamin-catalyzed membrane fission reaction using a standard wide field fluorescence microscopic approach.

The membrane system the team has devised comprises of an array of narrow ~40 nm wide supported membrane tubes, dubbed SMrT templates, laid out on a non-reactive glass surface. This system allowed them to visualize dynamin-catalyzed membrane fission reaction as a single membrane tube getting cut at multiple independent sites.

SMrT

Montage of a single membrane tube of ~40 nm in diameter seen under the fluorescence microscope undergoing multiple cuts in response to dynamin and GTP addition (Image Credit: Thomas Pucadyil)

The advantage of this assay system amenable to fluorescence microscopy is that a single experiment allows the analysis of scores of independent membrane fission events along with potential information on membrane intermediates generated during the membrane fission reaction. Using this assay system, the Pucadyil lab has proposed a mechanism by which the dynamin scaffold assembly only imposes a moderate degree of curvature stress on the underlying membrane tube. Their analysis suggests GTP hydrolysis to be necessary to further constrict the membrane tube down to dimensions that cause the generation of tube intermediates, which resolve spontaneously into cuts on the tube.

Dynamin action

Analysis of tube fluorescence changes during the fission reaction reveals the dimensions of the membrane intermediates generated during the fission reaction and thereby reveals the mechanism of dynamin-catalyzed membrane fission (Image Credit: Thomas Pucadyil)

The paper titled “A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function” and authored by Srishti Dar, Sukrut Kamerkar and Thomas Pucadyil has appeared as an advance online publication of Nature Cell Biology.

This work received funding from the Wellcome Trust-DBT India Alliance and CSIR India.

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