Dynamin2 helps organize actin networks in cell migration

Research Areas:

Cell biology, Endoplasmic reticulum, Lipids, Fluorescence microscopy

Imaging Needs:

Live-cell video imaging

Imaging System (live-cell video):
  • Cells expressing GFP-myosin light chain 2, mCherry (mCh)-α-actinin, and/or fluorescent protein-tagged rat dynamin2aa
  • Nakagawa spinning disc and inverted microscope (Zeiss)
  • ×63, 1.4 n.a. objective lens
  • Laser excitation: 488 nm for GFP; 587 nm for mCherry
  • Dual 5126512 ImagEM X2 EM-CCD cameras or an ORCA-R2 camera (Hamamatsu)
  • Metamorph control software
  • Definite Focus controller (Carl Zeiss)
Imaging System (TIRF):
  • X71 inverted microscope (Olypmus)
  • ×60, 1.45 n.a. oil objective lens
  • Argon laser
  • ORCA-Flash4.0 CMOS camera (Hamamatsu)
  • Metamorph control software
Imaging System (immunofluorescence):
  • LSM510 confocal microscope (Zeiss)
  • ×63, 1.4 n.a. objective lens
  • ImageJ and Adobe Photoshop software
Imaging the living brain

What if the same quantitative in vitro cell assay could also be used in vivo? Takakura, et al., detect cellular events at both levels using firefly luciferase bioluminescence and the Hamamatsu ImagEM-1K EM-CCD camera. Read now.

THE QUESTION

How do cells migrate?

A network of interdependent actin filament structures shift and remodel to actuate movement in cell migration. Polymerization of an actin filament meshwork extends lamellipodia forward at the cell’s leading edge. Between the lamellipodium and cell nucleus—within the area called the lamella—actin filament bundles associated with myosin II form and contract, drawing the rest of the cell along. Actin filaments attach to focal adhesions, anchoring the cell to the extracellular matrix, providing traction.

The molecular mechanisms coordinating the dynamic assembly and disassembly of these actin structures during migration are not fully understood.

THE BARRIERS

The large GTPase dynamin2 has been shown to regulate actin, as well as microtubule components of the cytoskeleton, though its role and mechanisms of action are not clear. Dynamin2 associates directly with actin networks, but also with known actin regulatory proteins. Dynamin2 depletion or mutation alters actin and actomyosin cytoskeletal function. Many studies of dynamin–actin dynamics have observed fixed cells, limiting the view into this protein’s dynamic actions.

THE SOLUTION

Dynamin2 Organizes Lamellipodial Actin Networks to Orchestrate Lamellar Actomyosin
Menon M, Askinazi OL, Schafer DA
PLoS One. 2014 Apr 7; 9(4):e94330.PMCID: PMC3978067.

Menon, et al.1 examined dynamin2’s actions on actin networks in living U2-OS osteosarcoma cells, through a combination of live-cell time-lapse imaging, total internal reflection fluorescence (TIRF) microscopy, and immunofluorescence microscopy.

The research team captured time-lapse video of lamellar actomyosin dynamics in living U2-OS cells expressing GFP-myosin light chain and mCherry-α-actinin, markers for the relevant myosin and actin structures, and transiently expressing green fluorescent protein tagged dynamin2. The scientists captured movies of lamellar actomysin dynamics in the presence or absence of dynamin2. The video, taken at 10 seconds per frame over 10 minutes using dual Hamamatsu ImagEM X2 EM-CCD cameras, showed that dynamin2 depletion disrupted the normal formation of transverse actomyosin arcs in the lamella. Further video images, taken at 5 seconds per frame over 10 minutes using a Hamamatsu ORCA-R2 camera, showed dynamin2 strongly localized to advancing lamellipodia and associated with actin networks there.

To more closely monitor dynamin2’s actions in lammelipodia, the team collected images at even higher spatial and temporal resolution. Using live-cell TIRF images collected at 2 seconds per frame over 5–10 minutes using a Hamamatsu ORCA-Flash 4.0 camera, the team found actin enriched at the distal end of the lamellipod, flowing rearward toward the lamella. Again, dynamin2 depletion altered this normal pattern. The team further observed that dynamin2 loss changes the distribution of actin binding proteins in lamellipod. Through mutation experiments, they confirmed that the effects of dynamin on actin dynamics happen independently of its known role in endocytosis.

The authors conclude that dynamin2 regulates lamellipodial actin network formation and retrograde flow to the lamella. They propose that dynamin2 organizes actin filaments within lamellipodia, which in turn regulates assembly and flow of actiomysin structures in the lamella. That regulation aids coupling of actin at focal adhesions, helping to coordinate overall actin cytoskeleton dynamics in cell migration.

THE POSSIBILITIES

Menon, et al. characterized dynamin2 regulation of actin and actomyosin dynamics in living cells with the help of Hamamatsu’s ImagEM X2 EM-CCD, ORCA-R2, and ORCA-Flash 4.0 cameras. To correlate cellular observations with events deep inside grown, living organisms, Takakaura, et al. relied on Hamamatsu’s ImagEM camera, using luminescence in place of fluorescence. Learn more in Illuminating Activity—in vitro and in vivo.

References

  1. Menon, et al. Dynamin2 Organizes Lamellipodial Actin Networks to Orchestrate Lamellar Actomyosin. PLoS One. 2014 Apr 7; 9(4):e94330.PMCID: PMC3978067.
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