A Comparison of GFP‐Tagged Clathrin Light Chains with Fluorochromated Light Chains In Vivo and In Vitro |
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| Authors: | Anika Hoffmann Philip N. Dannhauser Stephanie Groos Lars Hinrichsen Ute Curth Ernst J. Ungewickell |
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| Affiliation: | 1. Department of Cell Biology, Center of Anatomy, Hannover Medical School, Carl‐Neuberg Str. 1, D‐30625 Hannover, Germany;2. Current address: Department of Pediatric Pulmology and Neonatology, Hannover Medical School, Carl‐Neuberg Str. 1, D‐30625 Hannover, Germany;3. These authors contributed equally to the work presented in this paper.;4. Current address: Lohmann Animal Health GmbH & Co. KG, Heinz‐Lohmann‐Str. 4, 27472 Cuxhaven, Germany;5. Department of Biophysical Chemistry, Center of Biochemistry, Hannover Medical School, Carl‐Neuberg Str. 1, D‐30625 Hannover, Germany |
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| Abstract: | Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)‐tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP‐tagged LC variants. Cy3‐labeled light chains (Cy3‐LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1–2 min the Cy3‐LC heavy chain complexes entered clathrin‐coated structures, whereas uncomplexed Cy3‐LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time–course of an endocytic cycle. To explore whether GFP‐tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild‐type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3‐ and Alexa 488‐labeled LC behaved similar to wild‐type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells. |
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| Keywords: | electron microscopy endocytosis FRAP live‐cell imaging microinjection |
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