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TIP activity of Alexa 488–tagged EB1 protein (a plus end tracking protein). This movie shows pseudocolored overlaid images of rhodamine-labeled microtubules (red) and Alexa 488–tagged EB1 protein (green). EB1–Alexa 488 specifically decorates growing microtubule plus-ends and is lost during microtubule shortening phases. (Scale bar, 2 micron.)
Microtubule dynamics were visualized at 22 °C using a TIRF microscope ﬁtted on an inverted microscope (Nikon TE-2000U). TIRF excitation was achieved using a 488-nm optically pumped semiconductor laser (Sapphire, Coherent) to visualize Alexa 488 and a 532-nm diode-pumped solid-state laser (CrystaLaser) to visualize rhodamine. The laser light intensities used for imaging were kept low, so that the photobleaching rates were slow compared with the dwell times. Images were captured with a backilluminated electron-multiplying CCD camera (Cascade-512B, Photometrics) using time-lapse capture at 2-sec intervals for 2-color microscopy. Video corresponds to Movie S1 from Proc Natl Acad Sci U S A. 2009 Jan 13;106(2):492-7. Epub 2009 Jan 6.
Jacob E. Lazaru
Yale E. Goldman
Erika L. F. Holzbaur
Proc Natl Acad Sci U S A. 2009 Jan 13;106(2):492-7. Epub 2009 Jan 6.
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in vitro reconstitution
- Relation To Intact Cell