School Papers

Introduction named proto-lysosomes, these tubular structures can contribute

Introduction

Autophagy occurs when a vesicle transporting of damaged organelles to
the lysosome for degradation. Autophagosome is a double layer membrane vesicle
which engulfs the damaged intracellular contents (organels). When autophagosome
fuses with lysosome they will form hybrid organelles named autolysosomes.  The digestion of the intracellular contents
occurs inside the autolysosome. The finally step of autophagy is autophagic
lysosome reformation (ALR). Increasing of autophagy process can be induced by
various stimuli such as starvation. Defects in autophagy have
been linked to cancer and various human diseases.

One of the key regulators of autophagy is the target
of rapamycin, TOR kinase, which is the major inhibitory signal that shuts off
autophagy in the presence of growth factors, cellular energy and nutrients.
(12)

 

 

Aim

Autophagy consists of several
sequential steps, sequestration, transport of cargo to lysosomes, degradation
of damage organelles, and utilization of degradation products and each step
have different function. The aim
of this study is to explain haw the autophagolysosome reformation occurs during
those several step.

 

Autophagic lysosome
reformation

During the process of autophagy, autophagosomes undergo a maturation
process consisting of multiple fusions with lysosomes. The terminal process of
autophagy named autophagic lysosome reformation (ALR) which
is essential for maintaining lysosome homeostasis during autophagy.  By using LAMP1-YFP as a lysosome marker and LC3-CFP
as an autophagosom marker after 4 h of starvations, non-lysosome was
represented while few large autolysosome could be observed. After 12 h of
starvation lysosome had normal size and number.

During ALR, tubules are formed from
autolysosomes, and small vesicles named proto-lysosomes, these tubular
structures can contribute autolysosomes to recycle of lysosome membrane
components. Eventually, proto-lysosomes can be functional lysosomes during
maturation stage.

 

What can triggers ALR?

Cell growth, nutrients, such as carbohydrates, amino
acids and ATP is regulated by mammalian target of rapamycin (mTOR). During
starvation, the target of mTOR, should be inhibited, thereby inducing
autophagy.  Recent studies have treated cell with rapamycin a specific inhibitor
of mTOR after long-lasting of starvation mTOR was
reactivated (13). During the starvation mTOR activity was blocked by rapamycin
which inhibited the mTOR reactivation. Knocking down mTOR increases autophagy
buy totally inhibits ALR leaving giant autolysosomes which is consistent with previously
study (13,11).  mTOR reactivation is dependent on lysosomal degradation
activity.

 

During
starvation lysosomal degradation activity is eliminated and mTOR reactivation
is reduced which leads to repeal ALR. However this mechanism leaves
autolysosome active under longer period in cell. However, the activities of mTOR and lysosomes are deeply interconnected and
mTOR reactivation is essential step for
ALR (14).

 

Spinster
is required for ALR and mTOR reactivation following starvation

Autolysosomes forms when autohagosome
fuses with lysosomes in which the highly acidic pH in the lysosome hydrolyses
cargo such as defective cellular organelles for recycling (16). The digested
products export by lysosome membrane protein (lysosomal efflux ) into the
cytosol or transported to the Golgi via endosomal trafficking for recycling
(15). Yeast Atg22 and Avt3, Avt4 vacuolar contribute for amino acid efflux
transporter which plays a role in autophagy.

Spin is a lysosomal
transmembrane protein and sugar transporter as a
regulator of ALR, where localized on late
endosomal/lysosomal membranes in mammalian cells. Previous studies have shown that
Spin is essential for ARL and mTOR reactivation during starvation. Drosophila
spin mutants entails to accumulation of enlarged
lysosomes and late endosomal inclutions. (17). Since, impaired
degradation capacity of autolysosomes occur when spin sugar transportation
activity is defected, resulting in defect of mTOR reactivation, an essential
signalling for ALR. This indicates that ALR is impaired in spin knockdown
cells.

The mechanism
of ALR after mTOR reactivation

Starvation-induced
autophagy, lysosomes are reformed from autolysosomal membranes through an
evolutionarily conserved cellular process ARL which initiate after mTOR reactivation. Previous studies have
predict that clathrin and
phosphatidylinositol-4,5-bisphosphate (4,5)P(2)) is essential for ALR regulation.  However, PI (4,5)P2 could be generated on
lysosomal membrane  in particular  lysosomal or autolysosomes by tubular structures
(13).  Autolysosome accumulation occurs
during starvation in clatrin knockdown cell which leads to impaired ALR. Adapter
protein 2 (AP2) function is to anchor clatrin to membranes during endocytosis
(25). Hence, PI (4,5)P2 can be membrane anchored with clatrin by  Adapter protein AP2.