Abstract
This unit describes approaches and tools for studying the dynamics and organization of endoplasmic reticulum (ER) membranes and proteins in living cells using fluorescence microscopy. The ER plays a key role in secretory protein biogenesis, calcium regulation, and lipid synthesis. However, study of these processes has often been restricted to biochemical assays that average millions of lysed cells or imaging of static fixed cells. With new fluorescent protein (FP) reporter tools, sensitive commercial microscopes, and photobleaching techniques, investigators can interrogate the behaviors of ER proteins, membranes, and stress pathways in single live cells. Solutions are described for imaging challenges relevant to the ER, including the mobility of ER membranes, a range of ER structures, and the influence of post-translational modifications on FP reporters. Considerations for performing photobleaching assays for ER proteins are discussed. Finally, reporters and drugs for studying misfolded secretory protein stress and the unfolded protein response are described.
| Original language | English (US) |
|---|---|
| Article number | 21.7 |
| Journal | Current Protocols in Cell Biology |
| Issue number | SUPPL.60 |
| DOIs | |
| State | Published - 2013 |
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Keywords
- Confocal microscopy
- Diffusion
- FLIP
- FRAP
- Live cell imaging
- Membrane
- Microtubule
- Superfolder green fluorescent protein
- Tubule
ASJC Scopus subject areas
- Cell Biology
Cite this
Probing endoplasmic reticulum dynamics using fluorescence imaging and photobleaching techniques. / Costantini, Lindsey; Snapp, Erik.
In: Current Protocols in Cell Biology, No. SUPPL.60, 21.7, 2013.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Probing endoplasmic reticulum dynamics using fluorescence imaging and photobleaching techniques
AU - Costantini, Lindsey
AU - Snapp, Erik
PY - 2013
Y1 - 2013
N2 - This unit describes approaches and tools for studying the dynamics and organization of endoplasmic reticulum (ER) membranes and proteins in living cells using fluorescence microscopy. The ER plays a key role in secretory protein biogenesis, calcium regulation, and lipid synthesis. However, study of these processes has often been restricted to biochemical assays that average millions of lysed cells or imaging of static fixed cells. With new fluorescent protein (FP) reporter tools, sensitive commercial microscopes, and photobleaching techniques, investigators can interrogate the behaviors of ER proteins, membranes, and stress pathways in single live cells. Solutions are described for imaging challenges relevant to the ER, including the mobility of ER membranes, a range of ER structures, and the influence of post-translational modifications on FP reporters. Considerations for performing photobleaching assays for ER proteins are discussed. Finally, reporters and drugs for studying misfolded secretory protein stress and the unfolded protein response are described.
AB - This unit describes approaches and tools for studying the dynamics and organization of endoplasmic reticulum (ER) membranes and proteins in living cells using fluorescence microscopy. The ER plays a key role in secretory protein biogenesis, calcium regulation, and lipid synthesis. However, study of these processes has often been restricted to biochemical assays that average millions of lysed cells or imaging of static fixed cells. With new fluorescent protein (FP) reporter tools, sensitive commercial microscopes, and photobleaching techniques, investigators can interrogate the behaviors of ER proteins, membranes, and stress pathways in single live cells. Solutions are described for imaging challenges relevant to the ER, including the mobility of ER membranes, a range of ER structures, and the influence of post-translational modifications on FP reporters. Considerations for performing photobleaching assays for ER proteins are discussed. Finally, reporters and drugs for studying misfolded secretory protein stress and the unfolded protein response are described.
KW - Confocal microscopy
KW - Diffusion
KW - FLIP
KW - FRAP
KW - Live cell imaging
KW - Membrane
KW - Microtubule
KW - Superfolder green fluorescent protein
KW - Tubule
UR - http://www.scopus.com/inward/record.url?scp=84896320328&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84896320328&partnerID=8YFLogxK
U2 - 10.1002/0471143030.cb2107s60
DO - 10.1002/0471143030.cb2107s60
M3 - Article
JO - Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
JF - Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
SN - 1934-2500
IS - SUPPL.60
M1 - 21.7
ER -