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Microtubule organization and the effects of GFP-tubulin expression in dictyostelium discoideum

M Kimble1, C Kuzmiak, K N McGovern

  • 1Department of Biology, University of South Florida, Tampa 33620-5150, USA. mkimble@chuma.cas.usf.edu

Cell Motility and the Cytoskeleton
|September 26, 2000
PubMed

Insights

Green fluorescent protein alpha-tubulin labels microtubules in Dictyostelium amoebae, revealing motility and potential motor protein interactions. However, it can interfere with microtubule nucleation and increase light sensitivity.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biophysics

Background:

  • Microtubules are essential cytoskeletal components involved in cell motility and structure.
  • Fluorescent protein tagging is a common technique for visualizing cellular structures in living organisms.

Purpose of the Study:

  • To label microtubules in living Dictyostelium amoebae using a GFP-alpha-tubulin fusion protein.
  • To investigate the dynamics and interactions of labeled microtubules.
  • To assess potential interference of the fusion protein with microtubule organization and function.

Main Methods:

  • Expression of GFP-alpha-tubulin fusion protein in Dictyostelium amoebae.
  • Live-cell imaging to observe microtubule motility.
  • Electron microscopy (EM) for ultrastructural analysis of microtubule distribution.
  • Immunofluorescence (IF) microscopy.
  • Protein blot analysis.

Main Results:

  • GFP-alpha-tubulin successfully incorporated into microtubules, which exhibited high motility.
  • EM analysis revealed close associations between cytoplasmic microtubules, suggesting a mechanism for motility generation.
  • Protein blot analysis showed lower incorporation efficiency of GFP-alpha-tubulin compared to endogenous alpha-tubulin.
  • EM and IF suggested interference of GFP-alpha-tubulin with microtubule nucleation.
  • Cells expressing GFP-alpha-tubulin showed increased sensitivity to blue light.

Conclusions:

  • GFP-alpha-tubulin serves as a viable marker for microtubules in living Dictyostelium amoebae.
  • The observed motility is potentially linked to motor protein activity along adjacent microtubules.
  • Potential interference with microtubule nucleation and increased photosensitivity should be considered.
  • The use of GFP-alpha-tubulin as a marker is not recommended for studies focusing on microtubule assembly dynamics.

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