Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they provide structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. Here, using in vitro reconstitution assays, we show that GDP-tubulin, usually considered inactive, can itself assemble into microtubules, preferentially at the minus end, and promote persistent growth. GDP-tubulin-assembled microtubules are highly stable, displaying no detectable spontaneous shrinkage. Strikingly, islands of GDP-tubulin within dynamic microtubules stop shrinkage events and promote rescues. Microtubules thus possess an intrinsic capacity for stability, independent of accessory proteins. This finding provides novel mechanisms to explain microtubule dynamics.
Guanosine Diphosphate , Microtubules , Tubulin , Microtubules/metabolism , Tubulin/metabolism , Tubulin/genetics , Guanosine Diphosphate/metabolism , Animals , Guanosine Triphosphate/metabolism , Humans
Microtubule architecture depends on a complex network of microtubule-associated proteins (MAPs) that act in concert to modulate microtubule assembly/disassembly and spatial arrangement. In vitro reconstitution of cytoskeleton dynamics coupled to single-molecule fluorescence assays has opened new perspectives to quantify the interaction of MAPs with microtubules. Here, we present a Total Internal Reflection Fluorescence (TIRF) microscopy-based assay enabling the characterization of Tau interaction with dynamic microtubules at the single-molecule level. We describe protein sample preparation in flow cells, single-molecule acquisitions by TIRF microscopy, and quantitative analysis of Tau oligomerization states and dwell time on microtubules.
Microscopy, Fluorescence , Microtubules/metabolism , Molecular Imaging , Single Molecule Imaging , tau Proteins/metabolism , Glass/analysis , Glass/chemistry , Lab-On-A-Chip Devices , Microscopy, Fluorescence/methods , Microtubules/chemistry , Molecular Imaging/methods , Photobleaching , Protein Binding , Single Molecule Imaging/methods , tau Proteins/chemistry
We report an original MS-based hyphenated method for the elucidation of the epimerization in GAG fragments. It consists of measuring simultaneously the MS/MS spectrum and the gas phase IR spectrum to gain direct structural information. This is possible using a customized MS instrument, modified to allow injection of a tunable IR laser inside of the instrument for in situ spectroscopy of trapped ions. The proof of principle of this approach is performed in the case of a hyaluronic acid tetrasaccharide standard. In addition, we provide the reference IR fingerprint of glucuronic and Iduronic monosaccharide standards. Remarkably, we show that the gas phase IR fingerprint of reference hexuronic acid monosaccharides proves to be transposable to oligosaccharides. Therefore, the method presented here is predictive and allows structural elucidation of unknown GAG fragments, even in the absence of referenced standards.
Hyaluronic Acid/isolation & purification , Iduronic Acid/isolation & purification , Spectrophotometry, Infrared/methods , Tandem Mass Spectrometry/methods , Hyaluronic Acid/chemistry , Iduronic Acid/chemistry , Monosaccharides/chemistry , Oligosaccharides/chemistry , Solutions , Spectrophotometry, Infrared/instrumentation , Spectrophotometry, Infrared/standards , Tandem Mass Spectrometry/standards
