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Tau-based fluorescent protein fusions to visualize microtubules
Paul Mooney1,2,3, Taylor Sulerud1,2,3, James F Pelletier3,4
1Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, 82071, USA.
Abstract:
The ability to visualize cytoskeletal proteins and their dynamics in living cells has been critically important in advancing our understanding of numerous cellular processes, including actin- and microtubule (MT)-dependent phenomena such as cell motility, cell division, and mitosis. Here, we describe a novel set of fluorescent protein (FP) fusions designed specifically to visualize MTs in living systems using fluorescence microscopy. Each fusion contains a FP module linked in frame to a modified phospho-deficient version of the MT-binding domain of Tau (mTMBD). We found that expressed and purified constructs containing a single mTMBD decorated Xenopus egg extract spindles more homogenously than similar constructs containing the MT-binding domain of Ensconsin, suggesting that the binding affinity of mTMBD is minimally affected by localized signaling gradients generated during mitosis. Furthermore, MT dynamics were not grossly perturbed by the presence of Tau-based FP fusions. Interestingly, the addition of a second mTMBD to the opposite terminus of our construct caused dramatic changes to the spatial localization of probes within spindles. These results support the use of Tau-based FP fusions as minimally perturbing tools to accurately visualize MTs in living systems.
Insights
Researchers developed novel fluorescent protein fusions to visualize microtubule (MT) dynamics in living cells. These Tau-based probes offer a minimally perturbing method for studying MTs, crucial for cell division and motility.
Area of Science:
- Cell Biology
- Molecular Imaging
- Biophysics
Background:
- Visualizing cytoskeletal proteins and their dynamics in living cells is crucial for understanding cellular processes.
- Microtubules (MTs) play vital roles in cell motility, division, and mitosis.
Purpose of the Study:
- To develop and characterize novel fluorescent protein (FP) fusions for visualizing MTs in living systems.
- To assess the suitability of these probes for studying MT dynamics with minimal perturbation.
Main Methods:
- Construction of FP fusions with a modified, phospho-deficient MT-binding domain of Tau (mTMBD).
- Expression and purification of FP-mTMBD constructs.
- Visualization of MTs in Xenopus egg extract spindles using fluorescence microscopy.
- Comparison with Ensconsin-based probes and assessment of MT dynamics.
Main Results:
- Single mTMBD fusions decorated Xenopus spindles more homogeneously than Ensconsin-based constructs.
- Tau-based FP fusions minimally perturbed MT dynamics.
- Dual mTMBD constructs significantly altered probe localization within spindles.
Conclusions:
- Tau-based FP fusions are effective and minimally perturbing tools for visualizing MTs in living systems.
- mTMBD binding affinity is robust to mitotic signaling gradients.
- Probe design, including the number of mTMBD domains, influences spatial localization.
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