Vimentin Mediates Uptake of C3 Exoenzyme

Astrid Rohrbeck,Ingo Just,Markus Höltje,Gudrun Ahnert-Hilger,Sandra Hagemann,Anke Schröder,Andreas Pich,Daniel Gillet

doi:10.1371/journal.pone.0101071

Astrid Rohrbeck, Ingo Just + Show 6 more

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https://doi.org/10.1371/journal.pone.0101071

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Abstract

Clostridium botulinum C3 exoenzyme (C3) selectively inactivates RhoA/B/C GTPases by ADP-ribosylation. Based on this substrate specificity C3 is a well-established tool in cell biology. C3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. Based on different approaches vimentin was identified as membranous C3-interaction partner by mass spectrometry. Vimentin in fact was partly localized at the outer surface of hippocampal HT22 cells and J744A.1 macrophages. Domain analysis identified the rod domain as binding partner of C3. Vimentin was also involved in uptake of C3 as shown by knock down of vimentin in HT22 and J774A.1 cells. The involvement of vimentin in uptake of C3 was further supported by the findings that the vimentin disruptor acrylamide blocked uptake of C3. Vimentin is not only a major organizing element of the intermediate filament network but is also involved in both binding and uptake of C3 exoenzyme.

Highlights

Exoenzyme C3 from Clostridium botulinum is the prototype of Rho-ADP-ribosylating transferases produced by different bacterial strains such as Clostridia, Bacilli and Staphylococci [1,2,3]
C3 binds to proteinaceous structures at intact HT22 cells To check whether C3 binding to intact HT22 cells was dependent on proteinaceous cell surface structures or not, cells were treated with pronase prior to binding to C3
Binding of C3 to HT22 cells was detected by Western blot using C3-specific antibody (Fig. 1A) and flow cytometry by fluorescently labeled C3 (Fig. 1B)

Summary

Introduction

Exoenzyme C3 from Clostridium botulinum is the prototype of Rho-ADP-ribosylating transferases produced by different bacterial strains such as Clostridia, Bacilli and Staphylococci [1,2,3]. The transferred ADP-ribose moiety at acceptor amino acid Asn-41 renders Rho functionally inactive and halts Rho-dependent downstream signaling [4,5]. ADP-ribosylation is highly specific as preferentially RhoA, B and C from the Ras superfamily of small GTPases are modified. This specificity is the basis for its use as cell biological tool to inactivate RhoA/B/C function in intact cells, still in the time of knock down techniques [8,9]. In addition to its enzymatical activity, C3 exhibits an enzyme-independent biological activity to stimulate axonal and dendritic growth as well as branching in primary hippocampal neurons [10]

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