Continuity of, and Diffusion through, the Endoplasmic Reticulum
Diffusion through the Endoplasmic Reticulum
The endoplasmic reticulum (ER) is a meshwork of narrow membrane-bound tubes running through the cytoplasm of eukaryotic cells. It’s vitally important for the folding of secreted proteins and for calcium signaling (among other things).
Because it’s a narrow network involved in signaling, we wondered how quickly can proteins move through it. Does the meshwork geometry slow things down? And is the endoplasmic reticulum one single network, or are there separated islands?
Using FRAP to look at Diffusion
To figure this out, we expressed a form of GFP that was targeted to the ER lumen, then bleached a small part of the cell and looked how quickly unbleached GFP diffused back into the bleached spot—Fluorescence Recovery After Photobleaching (FRAP).
We found that the signal recovers rapidly, only about 6 times slower than it does in cytoplasm. We calculated that the geometric constraint accounts for a relatively small (~2-fold) slowing of the diffusion, and the rest (~3-fold) is due to the environment within the ER itself.
| Translational Diffusion | ||
| D(cm2/s) | D(water)/D | |
| Water | 8.7 × 10−7 | 1 |
| Cytoplasm | 2.5–3 × 10−7 | 2.9–3.5 |
| Mitochondria | 2–3 × 10−7 | 3–4 |
| ER | 0.5–1 × 10−7 | 9–18 |
Continuity of the ER
Because the diffusion is relatively rapid, we could use the photobleaching technique to tell whether the ER was continuous, by repeatedly bleaching one spot. The GFP from the rest of the ER would diffuse into that spot and be bleached—bleaching the whole ER if it were continuous, but only a small patch if it were made of many islands. We saw that the whole of the ER was bleached within 2 minutes, only the golgi remaining:
A single spot (at crosshairs) was repeatedly illuminated (200 ms illumination every 2 s) with an intense laser beam. Images were obtained after indicated number of illumination pulses. The arrow indicates an adjacent unbleached cell. Residual perinuclear, Golgi-like fluorescence is seen after 60 bleach pulses (2 minutes; right panel)