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© Emeline Camand
Marquage par immunofluorescence d'astrocytes tumoraux ou astrocytomes (lignée cellulaire humaine U373), montrant en rouge, APC et en vert, la tubuline des microtubules. APC est un supresseur de tumeur qui est impliqué dans la polarisation des astrocytes normaux. La localisation d'APC est altérée dans des lignées de gliomes. Pour essayer de corriger, les dérèglements observés lors de la migration des cellules d'astrocytes tumuraux ou gliomes on cherche à connaitre les mécanismes moléculaires fondamentaux qui controlent la polarisation et la migration cellulaire.
Publication : Methods in cell biology

Studying kinesin motors by optical 3D-nanometry in gliding motility assays

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Methods in cell biology - 01 Jan 2010

Nitzsche B, Bormuth V, Bräuer C, Howard J, Ionov L, Kerssemakers J, Korten T, Leduc C, Ruhnow F, Diez S

Link to Pubmed [PMID] – 20466139

Methods Cell Biol. 2010;95:247-71

Recent developments in optical microscopy and nanometer tracking have facilitated our understanding of microtubules and their associated proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules, mainly using a geometry in which labeled motors move on surface-immobilized microtubules. Yet, we think that the historically older gliding geometry, in which motor proteins bound to a substrate surface drive the motion microtubules, offers some unique advantages. (1) Motility can be precisely followed by coupling multiple fluorophores and/or single bright labels to the surface of microtubules without disturbing the activity of the motor proteins. (2) The number of motor proteins involved in active transport can be determined by several strategies. (3) Multimotor studies can be performed over a wide range of motor densities. These advantages allow for studying cooperativity of processive as well as nonprocessive motors. Moreover, the gliding geometry has proven to be most promising for nanotechnological applications of motor proteins operating in synthetic environments. In this chapter we review recent methods related to gliding motility assays in conjunction with 3D-nanometry. In particular, we aim to provide practical advice on how to set up gliding assays, how to acquire high-precision data from microtubules and attached quantum dots, and how to analyze data by 3D-nanometer tracking.

http://www.ncbi.nlm.nih.gov/pubmed/20466139