Remodeling the membrane during cellularization in Drosophila

Research Areas:

Cell biology, Cellularization, Drosophila

Imaging Needs:

High sensitivity

Imaging System (immunofluorescence):
  • Nikon Eclipse TE2000-E inverted microscope
  • Plan Apochromat 60× 1.5/numerical aperture (NA) 1.0 oil immersion lens
  • PerkinElmer spinning disc confocal
  • Hamamatsu ImagEM EM-CCD camera
  • Universal Imaging MetaMorph software
Imaging System (DIC time-lapse microscopy):
  • Nikon E-800 microscope
  • Harvard Apparatus temperature-controlled stage
  • Plan Apochromat 40× 1.5/NA 1.0 oil immersion lens
  • Hamamatsu ORCA-ER CCD camera
  • MetaMorph 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 is cell cleavage organized in developing embryos?

In early development, Drosophila embryos produce thousands of epithelial cells. During division, cells form a deep furrow in the plasma membrane, enclosing each nucleus and eventually cleaving the cell. Endocytic microtubules form at the tip of this cleavage furrow, aiding membrane remodeling, then disappearing as the furrow narrows and mitosis completes. Little is known about these tubules and their role in cellularization outside of an association with a protein implicated in membrane reogranization.

THE BARRIERS

The tubules of Drosophila cleavage furrows are marked by amphiphysin, a protein involved in membrane bending and curvature during endo- or exocytosis. Its structure includes an N-terminal lipid interaction and membrane bending BAR domain. Found on the cytoplasmic side of synaptic vesicles, amphiphysin is thought to recruit the GTPase dynamin to sites of endocytosis in eukaryotic cells. Dynamin, in turn, is known to perform scission of new vesicles from membranes.

In cellularization, a lack of dynamin results in elongated cleavage furrow tubules. It’s not clear what drives this tubule growth, but proteins such as amphiphysin and septins, which can tubulate liposomes in vitro, are likely candidates. Further answers require a close look at Drosophila cleavage furrow tubules and associated proteins, but a shortage of markers for the tubules has made such studies difficult.

THE SOLUTION

The BAR domain of amphiphysin is required for cleavage furrow tip–tubule formation during cellularization in Drosophila embryos
Jing Su, Brenda Chowa, Gabrielle L. Boulianne, and Andrew Wilde
Mol Biol Cell. 2013 May 24(9):1444–53. PMCID: PMC3639055.

Su, et al,1 sought new tubule markers in Drosophila lines expressing green fluorescent protein (GFP) fusion proteins, immunostained to locate cleavage furrows. Immunofluorescence using a Hamamatsu ImagEM EM-CCD camera revealed that all septin-GFP fusion proteins tested localized to cleavage furrow tubules, identifying septins as a new tubule marker.

Interestingly, the authors found three different populations of tubules, each marked by characteristic combinations of amphiphysin and septins. Specific populations required dynamin for tubule formation. All tubule formation required amphiphysin’s BAR domain specifically.

Amphiphysin and dynamin showed opposing effects in cleavage furrow formation. Without amphiphysin, formation accelerated. In contrast, dynamin disruption resulted in longer tubules and slower furrow development. For a closer look, the research team used differential interference contrast (DIC) microscopy at 20°C, taking time-lapse images every 10 seconds using a Hamamatsu ORCA-ER camera. They found three distinct phases of furrow progress, each differently affected by amphiphysin or dynamin disruption. The authors propose a model for amphiphysin and dynamin actions in membrane remodeling during cellularization.

THE POSSIBILITIES

Su, et al.1 were able to co-localize key proteins and watch their activities during cellularization, using Hamamatsu’s ImagEM EM-CCD and ORCA-ER cameras. 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. Su, et al. The BAR domain of amphiphysin is required for cleavage furrow tip–tubule formation during cellularization in Drosophila embryos. Mol Biol Cell. 2013 May 24(9):1444–53. PMCID: PMC3639055.
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