Biomedical Engineering Reference
In-Depth Information
3. Biotinylation of cell-surface proteins varies by cell type and
protein. However, cell-surface biotinylation of the same plasma
membrane protein in the same cells is a useful technique for
comparing treatments that affect cellular traffi cking, provided
that identical conditions are used to treat the samples.
4. In the endocytic and recycling assays, maintaining cells at
0-4 °C during biotin labeling, antibody incubations, and
washing steps is important for minimizing both the endocyto-
sis and exocytosis of cell-surface molecules. It is also essential
to keep the cells viable and intact during the different incuba-
tion and washing steps. Addition of 0.1 % BSA or 5-10 % FBS
to the washing buffer may increase cell viability and recovery.
5. The protocols described here can be combined with plasmids
or small molecules available to interfere with intracellular traf-
fi cking. This helps to decipher the route of a particular cell-
surface protein follows. Some common drugs and plasmids to
inhibit membrane traffi cking are described below.
(a) ER-to-Golgi transport: brefeldin A (BFA) and overexpres-
sion of Sar1-H79G, Sar1-T39N, Arf1-Q71L, Rab1-
N124I, and Syntaxin5 [
14
-
16
]. BFA blocks nucleotide
exchange on Arf1 by stabilizing a transient complex
between Arf1-GDP and Sec7 domain-containing GEFs.
Treating cells with 5
g/mL BFA causes the Golgi to dis-
assemble and redistribute into the ER.
(b) Golgi to plasma membrane transport inhibitor: sulfon-
amides [
17
].
(c) Microtubule disruptor: nocodazole (5
ʼ
ʼ
g/mL) and colchi-
(d) Actin depolymerizing drugs: cytochalasin B (1-30
cine (1
ʼ
M) [
18
].
ʼ
M)
g/mL) [
18
].
(e) Membrane fusion inhibitor: N-ethylmaleimide (NEM,
0.5 mM). NEM inhibits membrane fusion by disrupting
the function of the soluble NEM-sensitive factor attach-
ment protein receptor (SNARE) [
12
].
(f) ATP depletion: Intracellular traffi cking is sensitive to ATP
depletion. ATP depletion can be achieved by incubating
cells in glucose-free media containing the metabolic inhib-
itors cyanide (5 mM) and 2-deoxy-
D
-glucose (5 mM)
[
19
].
(g) Protein synthesis inhibitor: cycloheximide (10 mg/mL)
and puromycin (0.2-1 mM).
(h) ER-associated degradation (ERAD) inhibitor: eeyarestatin
1 (10
and latrunculin A (0.2-10
ʼ
M) [
20
]. Eeyarestatin 1 specifi cally inhibits Sec61-
mediated protein translocation at the ER.
ʼ
