Remodeling the cytoskeleton: Single-molecule imaging tracks binding of microtubule severing enzymes

Research Areas:

Cell biology, Microtubules, Severing enzymes, TIRF microscopy, Single-molecule imaging

Imaging Needs:

Single-molecule resolution

Imaging System (activity assay):
  • Hamamatsu ImagEM C9100 EM-CCD camera
  • Olympus CellR, NIH ImageJ, and Wavemetrics IGOR Pro software
  • Spectrophotometric measure of ATPase activity at 340 nm
Imaging System (TIRF microscopy):
  • Olympus IX71 microscope
  • 100×, NA= 1.45 objective lens
  • Hamamatsu ImagEM C9100 EM-CCD camera
  • Home-built solid-state laser excitation system; microtubules imaged using 535-nm
  • 200 ms exposure time with 0.229, 1 and 10 s frame intervals
  • 100 ms exposure time for dwell time measures, no frame delay
  • Olympus CellR, NIH ImageJ, and Wavemetrics IGOR Pro software
Imaging cellular events in real time

Find out how Fang Huang, Jeorg Bewersdorf and colleagues use the sCMOS technology in the ORCA-Flash4.0 camera to achieve video-rate imaging at nanometer scales. Read now.

THE QUESTION

How do enzymes bind and cleave microtubules during cytoskeletal remodeling?

A key component of the cytoskeleton, microtubules show tremendous plasticity through polymerization or depolymerization at the ends, or severing within the length of a tubule. Three ATPase enzyme families are known to catalyze microtubule severing: katanin, fidgetin and spastin. Though involved in differing cellular processes, the three share similarities in their catalytic regions that suggest common mechanisms of action.

THE BARRIERS

Severing requires physical association of the severing enzyme with the microtubule. However, technical challenges, such as potential alteration of molecules studied in vitro and fixation artifacts in electron microscopy, have made it difficult to characterize the severing mechanism.

THE SOLUTION

Spastin’s Microtubule-Binding Properties and Comparison to Katanin
Thomas Eckert, Doan Tuong-Van Le, Susanne Link, Lena Friedmann, and Günther Woehlke
PLoS One. 2012; 7(12): e50161. PMCID: PMC3521757.

Eckert, et al,1 characterized the microtubule binding properties of wild type and mutant spastin and katanin through a combination of biochemical assays, electron microscopy and fluorescence microscopy at the single-molecule level. Using total internal reflection fluorescence (TIRF) microscopy with a Hamamatsu ImagEM C9100 EM-CCD camera, the researchers were able to image individual GFP-labeled enzyme molecules and RFP-labeled microtubules, at frame rates sufficient to analyze enzyme landing rates, dwell times, and dissociation rates.

The analysis indicated an ionic bond between enzyme and microtubule, associated with a non-catalytic domain of the enzyme. Binding affinity was reduced with ADP compared to ATP availability, and was associated with formation of enzyme oligomers. The authors suggest that enzyme dimerization is crucial to microtubule severing.

THE POSSIBILITIES

Eckert, et al., were able to image the binding of severing enzymes with microtubules at the single-molecule level using TIRF microscopy and the high sensitivity and precise localization of Hamamatsu’s ImagEM C9100 EM-CCD camera. Imagine what’s possible with the even faster capabilities and wider fields-of-view of Hamamatsu’s ORCA-Flash4.0 technology. Learn more, read Exciting Advances Push the Limits of Visualization.

References

  1. Eckert, et al. Spastin’s Microtubule-Binding Properties and Comparison to Katanin. PLoS One. 2012; 7(12): e50161. PMCID: PMC3521757.
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