Fluorescent microtubules break up under illumination

The dynamic behavior of microtubules (MTs) in living cells has recently been elucidated by the use of tubulin tagged with a fluorescent molecule and then injected into cultured cells. The dynamics of the labelled tubulin are then followed either by watching the incorporation of the fluorescence immediately after injection, or by monitoring fluorescence redistribution after photobleaching (FRAP). Until recently, the best fluorescent analogue of tubulin appeared to be dichlorotriazinyl-amino-fluorescein tubulin (DTAF-tubulin). We have now made fluorescein, rhodamine and X-rhodamine n-hydroxy-succinimidyl derivatives of tubulin. These analogues all have a higher fluorescence to protein (f-to-p) ratio and better in-vitro assembly characteristics than the DTAF-tubulin previously used. They have been injected into cultured PtKl cells, where they incorporate well into the microtubule cytoskeltons of the cells. However, while characterising the properties of these four analogues we discovered that they all suffer from a major problem: Microtubules formed from them, either in vitro or in vivo, are destroyed as the bound fluorophore becomes photobleached. We have characterised the photolability of the fluorescent microtubules both in vivo, using epifluorescence microscopy, and in vitro with DIC microscopy.