Biomedical Engineering Reference
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of autophagy in Purkinje cells in mice causes axonal swelling and
degeneration of the axon terminals (
6
). The neurons in patients
with neuronal ceroid lipofuscinosis (NCL) show accumulation
of mitochondrial ATPase subunit c in lysosomes, indicating
that mitochondria are degraded via the autophagy-lysosomal
system in neurons, and that impairment of mitochondrial
protein-degradation occurred in the patients. Mutation of cln3,
one of the NCL-related genes, or a lack of cathepsin D, one of
the major lysosomal proteases, leads to neuronal ceroid lipofus-
cinosis with autophagic vacuolization (
7, 8
).
During autophagy, unique double-membrane autophagosomes
are formed for sequestration of intracellular components (AP in Fig.
2
).
According to their formation, cytosolic LC3 (LC3-I) is conjugated to
phosphatidylethanolamine (PE) to localize the resultant LC3-II (a
LC3-PE conjugate) on the autophagosomes (
9
). The autophagosomes
then fuse with lysosomes to form autolysosomes (AL in Fig.
2
), whereby
sequestered intra-autophagosomal components are degraded by lyso-
somal hydrolases. Concomitantly, intra-autophagosomal LC3-II is
also degraded by lysosomal proteases (Fig.
1
). Therefore, LC3-II itself
could be used as a marker of autophagosome formation, and its deg-
radation as a monitoring indicator of the late period in autophagic flux
(
10
). Monitoring of LC3-I and LC3-II by Western blotting and immu-
nofluorescence analyses is essential to investigate the mechanism of
mammalian autophagy (Figs.
3
and
4
).
When mammalian cells and tissues are deficient in autophagy,
the levels of ubiquitin and p62 are increased (
11, 12
). In addition
to ubiquitin, p62/SQSTM1 is useful as a marker of autophagic
Fig. 2. Typical autophagosomes and autolysosomes on electron microscopy. Mouse embryonic
fibroblasts were incubated in HBSS medium for 2 h and fixed as described in the text.
Autophagosomes (AP), nascent AP, and autolysosomes (AL) can be detected as indicated.
Note that the structure indicated as “AP” could also be nascent AP because a connection
point between the segregated portion and surrounding cytosol may be present in the front
or rear of this section. In autolysosomes, the segregated organelles lose their distinct
shape and are usually replaced by electron-dense materials. Bar indicates 1
m
m. Note that
the diameter of the typical autophagosomes (AP) is 0.5 -1
m
m.
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