A highly-sensitive high throughput assay for dynamin's basal GTPase activity

Aparna Mohanakrishnan, Triet Vincent M. Tran, Meera Kumar, Hong Chen, Bruce A. Posner, Sandra L. Schmid

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Clathrin-mediated endocytosis is the major pathway by which cells internalize materials from the external environment. Dynamin, a large multidomain GTPase, is a key regulator of clathrin-mediated endocytosis. It assembles at the necks of invaginated clathrin-coated pits and, through GTP hydrolysis, catalyzes scission and release of clathrin-coated vesicles from the plasma membrane. Several small molecule inhibitors of dynamin's GTPase activity, such as Dynasore and Dyngo-4a, are currently available, although their specificity has been brought into question. Previous screens for these inhibitors measured dynamin's stimulated GTPase activity due to lack of sufficient sensitivity, hence the mechanisms by which they inhibit dynamin are uncertain. We report a highly sensitive fluorescence-based assay capable of detecting dynamin's basal GTPase activity under conditions compatible with high throughput screening. Utilizing this optimized assay, we conducted a pilot screen of 8000 compounds and identified several "hits" that inhibit the basal GTPase activity of dynamin-1. Subsequent dose-response curves were used to validate the activity of these compounds. Interestingly, we found neither Dynasore nor Dyngo-4a inhibited dynamin's basal GTPase activity, although both inhibit assembly-stimulated GTPase activity. This assay provides the basis for a more extensive search for more potent and chemically desirable dynamin inhibitors.

Original languageEnglish (US)
Pages (from-to)e0185639
JournalPLoS One
Volume12
Issue number9
DOIs
StatePublished - 2017

Fingerprint

Dynamins
GTP Phosphohydrolases
guanosinetriphosphatase
Assays
Throughput
Clathrin
clathrin
assays
Endocytosis
endocytosis
Dynamin I
Clathrin-Coated Vesicles
coated vesicles
Cell membranes
Guanosine Triphosphate
dynamins
Hydrolysis
Screening
neck
Neck

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

A highly-sensitive high throughput assay for dynamin's basal GTPase activity. / Mohanakrishnan, Aparna; Tran, Triet Vincent M.; Kumar, Meera; Chen, Hong; Posner, Bruce A.; Schmid, Sandra L.

In: PLoS One, Vol. 12, No. 9, 2017, p. e0185639.

Research output: Contribution to journalArticle

Mohanakrishnan, Aparna ; Tran, Triet Vincent M. ; Kumar, Meera ; Chen, Hong ; Posner, Bruce A. ; Schmid, Sandra L. / A highly-sensitive high throughput assay for dynamin's basal GTPase activity. In: PLoS One. 2017 ; Vol. 12, No. 9. pp. e0185639.
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