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In-vivo Centrifugation of Drosophila Embryos
Published on: June 23, 2010
Three-dimensional structural characterization of centrosomes from early Drosophila embryos
M Moritz1, M B Braunfeld, J C Fung
1Department of Biochemistry & Biophysics, University of California at San Francisco 94143-0448, USA.
The Journal of Cell Biology
|September 1, 1995
Summary
Researchers isolated functional centrosomes from fruit fly embryos to study microtubule (MT) nucleation. They found no large structures at MT minus ends, suggesting nucleation sites closely associate with MTs, and identified potential ring-like nucleation sites within the pericentriolar material (PCM).
Area of Science:
- Cell Biology
- Structural Biology
- Biophysics
Background:
- The mechanism and structure of microtubule (MT)-nucleating sites within the centrosome's pericentriolar material (PCM) remain poorly understood.
- Challenges include obtaining sufficient centrosome quantities and achieving high-resolution structural data using conventional electron microscopy (EM).
Purpose of the Study:
- To develop a protocol for isolating large quantities of functional centrosomes from early Drosophila embryos.
- To perform a three-dimensional structural characterization of these centrosomes using automated electron tomography.
Main Methods:
- Isolation of functional centrosomes from early Drosophila embryos.
- Automated electron tomography for 3D structural reconstruction.
- Analysis of centrosomes with and without regrown microtubules (MTs).
Main Results:
- High-resolution (6-8 nm) reconstructions revealed no large structures at MT minus ends, implying nucleation material closely conforms to the MT end.
- MT minus ends were distributed throughout the PCM, not solely near centrioles, and oriented in multiple directions.
- Ring-like structures, similar in diameter to MTs, were observed in the PCM of centrosomes lacking MTs, potentially representing nucleation sites.
Conclusions:
- The study provides a method for obtaining centrosomes for detailed structural analysis.
- Findings suggest that MT nucleation sites are adaptable and distributed within the PCM, with potential ring-like structures acting as nucleation centers.
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