Getting a clear message: Duo regulates signal protein levels in cilia

Research Areas:

Cilia, Protein traffic, Signaling proteins, Ciliary membrane

Imaging Needs:

Live-cell microscopy

Imaging System (live-cell fluorescence microscopy):
  • Zeiss Axiovert 200M microscope
  • PlanNeofluar 100×/1.3 numerical aperture
  • PlanApoChromat 63×/1.40 NA objective lens
  • Hamamatsu ORCA-ER CCD camera
  • PerkinElmer OpenLab and ImageJ software
Imaging System (Rilpl2 LAP-tube imaging):
  • Olympus epifluorescence inverted microscope
  • Olympus 100×/1.35 NA or 60×1.40 NA objective lens
  • Photometrics CH350 camera
Imaging System (spinning confocal microscopy):
  • PerkinElmer spinning disk confocal microscope (UltraVIEW VoX with a Zeiss AxioObserver)
  • 63×/1.40 NA objective lens
  • Hamamatsu ORCA-R2 CCD camera
  • Volocity software
Seeing the brain in action

New imaging techniques are promising to reveal more about neural connectivities in the brain than ever before. See how Misha Ahrens, Philipp Keller and colleagues use lightsheet microscopy to visualize intact, living zebrafish brain in real time. Read now.

THE QUESTION

How do cells control movement of signaling proteins into and out of key organelles?

Most mammalian cell types include one or more cilia – most often a single, primary cilium, an important sensory and signaling organ in the cell. Malfunctions of cilia or their signaling proteins have been implicated in neuropathies, retinopathy, obesity, and kidney disease.

THE BARRIERS

Cilium function depends on the influence of signaling proteins in the ciliary membrane and interior. However, it is not clear how localization of signaling proteins to the cilia is regulated.

Two proteins, related to a known regulator of membrane traffic called Rab-interacting lysosomal protein (Rilp), present promising candidates. Rilp-like protein 2 (Rilpl2) is upregulated during differentiation of ciliated cells, and has known effects on the dendritic spines of neurons. Both Rilpl2 and a related protein Rilpl1 associate with Rab effectors, a clue that they might be involved in regulating protein movement across membranes. Could Rilpl1 and Rilpl2 have a role in cilia?

THE SOLUTION

The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content
Johanna R. Schaub and Tim Stearns
Mol Biol Cell. 2013 Feb. 24(4):453–464. PMCID: PMC3571868.

Through live-cell fluorescence imaging with the help of a Hamamatsu ORCA-ER camera, Schaub and Stearns1 showed that Rilpl1 and Rilpl2 localize to the primary cilium and centrosome in cultured tracheal cells. Moreover, Rilpl1 concentrates at the mother centriole.

For a closer look at tubule structures formed dynamically by Rilpl2, the researchers relied on spinning-disk confocal microscopy with a Hamamatsu ORCA-R2 camera. The Rilpl2 tubules resemble those formed by a known player in ciliary membrane traffic. Also, cells depleted of Rilpl1 and Rilpl2 accumulated signaling proteins inside their cilia, and failed to grow together into coordinated tissues, confirming a role for Rilpl1 and Rilpl2 in regulating signal protein levels in the cilium.

THE POSSIBILITIES

Schaub and Stearns counted on the Hamamatsu ORCA-ER and ORCA-R1 cameras to pinpoint to locations of key proteins in and near a cell’s primary cilium. Even newer lightsheet microscopy technology, supported by Hamamatsu’s ORCA-Flash4.0 camera and pioneered by Misha Ahrens, Philipp Keller, and colleagues, is helping locate functional indicators at the neuronal level in the whole brain—read Seeing the Living Brain.

References

  1. Schaub and Stearns. The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content. Mol Biol Cell. 2013 Feb. 24(4): 453–464. PMCID: PMC3571868.
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