Tuesday, February 3, 2009
- We continue with vesicle transport, we’re moving away from the Golgi, the
trans Golgi network, that is basically the major branching point in vesicle
transport and how do vesicles now make it to the outside or the plasma
membrane and release their contents into the outside of the cell (extracellular
- Just to recap, this slide is what we’ve been looking at in terms of vesicle
transport. We looked at vesicle transport last lecture from the trans Golgi
network to the lysosome and how there’s a signal that is added on in the cis
Golgi onto proteins that are to be directed to the lysosome. These are for
example, lysosomal hydrolases that need to end up in the lysosome and
provide the functions of the lysosome in terms of degrading macromolecules.
- Just to recap, the trans Golgi network is a major branching point for proteins
that are to be sorted to different compartments in vesicles. Proteins that move
through the Golgi will end up in the trans Golgi network and the vesicles that
bud off the trans Golgi network can have different destinations.
- We looked at the lysosome, going through either the late endosome or early
endosome, these will eventually mature or become the lysosome.
- But vesicles can also bud off the TGN and make it to the plasma membrane,
this is called exocytosis. There are two major forms of exocytosis that can
occur. One is regulated and one is constitutive and basically what that means
is that when the vesicle buds off, it either goes to the plasma membrane and
fuses automatically, and that is the constitutive version or the vesicle can sit
around in the cytoplasm and wait for a signal in order to fuse to the plasma
membrane. That is called the regulated secretory pathway.
- We’ll look at those two examples so basically, proteins that are going
through the secretion pathway enter the ER, go through the Golgi, there are
two different ways they can go through the Golgi that we looked at and then
they’re sorted out at the major branching point which is the trans Golgi
network to either the lysosome via the early endosomes or to the plasma
membrane in a process called exocytosis.
- We’ll now look at a small animation that goes through the process till where
Animation - The signal sequence of a membrane protein directs the ribosome to the
rough ER. The growing polypeptide chain enters the lumen of the ER through
a protein import channel in the membrane. There the signal sequence is
cleaved, sugars are added and the protein folds. Some enzymes or structural
proteins remain in the ER. Most proteins that are soluble in the ER are
transported in vesicles to the cis Golgi.
- In the cisternal maturation model, the cis Golgi matures to become the
medial Golgi, a new cis Golgi is formed by fusion of the ER vesicles. The
maturation process includes retrograde vesicular transport of resident Golgi
- Proteins destined for secretion after modification in the Golgi are
transported from the trans Golgi to the plasma membrane. When the
membranes fuse, the proteins are released into the extracellular space.
- Soluble proteins on the outside of a cell can reenter by endocytosis and are
sorted to lysosomes for degradation. Other proteins, including lysosomal
enzymes sort directly to lysosomes from the trans Golgi. - Looking at exocytosis, the fusion of vesicles to the plasma membrane, the
uptake or formation of vesicles at the plasma membrane & uptaking
extracellular components and maybe we’ll get into cell communication if we
Secreted soluble proteins
- The first process we’ll look at is exocytosis and basically this is the
transport from the trans Golgi network to the plasma membrane. You can see
it there and there are two major versions of this: one is again the constitutive
or the regulated.
- Basically exocytosis is the fusion of transport vesicles with the PM.
Obviously these vesicles carry something with them & these transport
vesicles are carrying with them lipids. Recall that lipids are formed or
synthesized in the ER so they need to be transported to the PM if the plasma
membrane is to acquire any of these newly synthesized lipids. These vesicles
will contain lipids that will fuse and become part of the plasma membrane.
- They also contain transmembrane proteins that we saw in the animation, the
squiggly lines going through the plasma membranes and those will, once
they’re in the vesicle, once the vesicle fuses with the plasma membrane, it
will become plasma membrane associated proteins.
- They also include secreted soluble proteins. The vesicles will contain these
secreted soluble proteins and once they fuse with the PM, they’ll release
these proteins into the extracellular space. Unlike the membrane proteins,
these will not remain associated with the membranes and then they’re free to
move around outside of the cell.
- Now he mentioned that there are 2 different types of exocytosis. One of
them is called the constitutive secretory pathway & the other is called the
regulated secretory pathway. Basically the major difference b/w the two, is
that the vesicles will bud off the TGN, then you’ll have a vesicle that contains
different proteins either soluble or membrane proteins, as well as lipids
synthesized in the ER. This vesicle will right away fuse with the PM in an
unregulated fashion, that is why it is called constitutive, b/c this vesicle won’t
be regulated in terms of time when it fuses with the PM so it will form and
fuse with the PM.
- In contrast the regulated secretory pathway is very similar where you have
again a vesicle will bud off the trans Golgi network, shown in the slide and
this time the secretory vesicle with its contents, usually these are soluble
proteins, that will be released outside of the cell. An example of this we’ll
cover is insulin so insulin goes into this regulated secretory pathway and then
these vesicles await a signal in order to fuse with the PM. This signal might
be a hormone or a neurotransmitter that is recognized at the cell surface and
induces an intracellular signaling pathway which might include an increase in
cytosolic calcium for example. This intracellular signaling pathway regulates
the fusion of this vesicle to the PM so the vesicle will form and await a signal
before fusing with the PM. That is the regulated secretory pathway. - The constitutive secretory pathway is basically the default pathway, proteins
that don’t have a sorting signal will go through this pathway so if you’ll
recall, proteins that are destined to remain in the ER have a specific AA
sequence called the KDEL sequence or the AAs K D E and L. If you were to
remove that KDEL sequence on an ER localized protein, so now you take
that same protein, you mutate that KDEL sequence so it no longer has it, that
protein without a KDEL sequence, assuming it doesn’t have any other sorting
signals, would end up in the constitutive secretory pathway and go outside
the cell. It is the KDEL sequence that will retain it in the ER but if it doesn’t
have that sequence, it will go through the Golgi into a vesicle and out the cell,
it is the default pathway for proteins that enter the ER and don’t have any
other sorting sequence.
- An example is antibodies by activated B lymphocytes. The antibodies will
be produced and released automatically by the B lymphocytes – they aren’t
retained or wait for any signal.
Vesicles fuse with plasma membrane
Found as aggregates in TGN (trans-Golgi network)
- The constitutive secretory pathway is the default pathway for proteins
without a sorting signal. The regulated secretory pathway on the other hand is
basically the vesicles coming out of this pathway are stored near the PM so
again they’ll bud off the trans Golgi network and they will accumulate next to
the PM and then they wait for a signal such as an increase in intracellular
calcium. Once this signal is perceived in the cell, the vesicle will fuse with
the PM and then release their contents into extracellular space.
- How are these proteins sorted to these types of vesicles? The exact signal
that leads to proteins ending up in the regulated secretory pathway is not very
well characterized in terms of what is the specific sequence that would lead
into this pathway, however it has been observed that the proteins that do end
up in this pathway are found as aggregates in the trans Golgi network.
They’re usually found in large aggregates in the trans Golgi network & these
aggregates just keep getting larger and larger as the secretory vesicles form.
Vesicles that are budding off the trans Golgi network will actually fuse
together. As the trans Golgi vesicles are budding off, you can see the proteins
are already aggregating in the trans Golgi network, they will bud off and form
an immature secretory vesicle. What happens here is basically vesicles are
continuously adding on components into this immature secretory vesicle and
at the same time, clathrin coated vesicles are removing membranes and
recycling back to the trans Golgi network.
- What you’re getting is an increased concentration of proteins into the
secretory vesicle that will form very large aggregates and eventually result in
this mature secretory vesicle that is usually packed with a specific type of
protein such as insulin.
- One of the major cues that leads a protein into the regulated secretory
pathway is usually they start to aggregate at the trans Golgi network.
- This is the example of insulin. You can see here, here is a regulated
secretory vesicle and it is a large crystalline structure of insulin. There are
lots of insulin molecules aggregating together forming this fuzzy ball we can
see under the electron microscope. There is a lot of aggregation of proteins
that occur there.
- You’ll recall that insulin is secreted from pancreatic beta cells. These are
cells of the pancreas & remember the GLUT2 that results when you increase
the blood glucose levels, there is substantial increase in the rate of glucose
transport into these pancreatic beta cells & that increase is perceived & will
lead to release of insulin by the regulated secretory pathway. - The elevation in blood glucose is perceived by pancreatic beta cells and this
increase in blood glucose is actually translated into an increase in intracellular
calcium. Increase in blood glucose = increase in intracellular calcium
concentrations, this is the signal perceived to induce these vesicles to fuse
with the plasma membrane and release insulin into the bloodstream.
- Then you’ll recall that in fat & muscle cells, there is actually another
glucose transporter, the glucose transporter 4 that when these cells perceive
insulin, there is also regulated secretion of this glucose transporter 4 to the
PM & then fat & muscle cells will increase their uptake of glucose.
- This is an example of the regulated secretory pathway.
- That is the 2 examples of exocytosis. Now onto the reverse process:
endocytosis. Endocytosis is the transport into the cell from the PM.
- Here is the PM and vesicles will bud off the PM and in these vesicles will
be content from the extracellular space, the process involved in taking up
macromolecules from the outside. One of these in example we’ll go into is
cholesterol uptake & how cholesterol is taken up into the cell by endocytosis.
- Another junction of endocytosis is to down regulate signaling pathways. A
way that this is done is that there are receptors at the PM that will often
respond to extracellular signals. These may be hormones for example. Once
the signal is perceived, that receptor will signal the signaling pathway that is
downstream of it & then in order to down regulate that pathway or turn it off,
endocytosis can uptake receptors from the PM & bring them to the lysosome
for degradation. There is degradation of the receptor that is recognizing a
hormone and that can lead to down regulation of the signaling pathway.
- Another example of this that we’ll also go into for the cholesterol receptor is
the receptor can actually be uptaken by endocytosis and then recycled back to
the plasma membrane and not diverted to the lysosome for degradation. The
receptor can have 2 fates, either it can go to the lysosome, be degraded or by
recycled back to the PM and continue on with its signaling.
- Of course when you’re uptaking lipids for forming vesicles from the PM
you’re losing lots of lipids because vesicles are forming, taking with them a
little chunk of PM as they bud off and go to their intracellular compartments.
If this continued indefinitely without replenishing the lipids then the PM
would just get smaller and smaller until the cell disappeared. The lipids in the
plasma membrane are replenished by exocytosis. You have endocytosis
where the membrane is being taken up, the membrane is shrinking but
exocytosis is also replenishing these lipids in the plasma membrane.
- If you’ll notice in the slide that as things are coming in from the PM, they
first go into the early endosome, and like the trans Golgi network can be
thought of as a branching point for the secretion pathway, the early endosome
can be thought of as a branching point for endocytosis b/c once things get
into the early endosome, they have a number of different fates they can go
through. One is to go to the lysosome for degradation, we’re going to see how
receptors can do that. Another fate is to be recycled back to the PM so once a
protein ends up in the early endosome, it can actually be recycled back to the
plasma membrane. There are even some proteins that go directly from the
early endosome back to the trans Golgi. You can see that the early endosome
is a major branching point in endocytosis like the trans Golgi network is a
major branching point for the secretion pathway.
- VIDEO: So that is an example of phagocytosis by a neutrophil so you can
see that it is ingesting large particles that may give you gas like a bacteria.
Then the other example is pinocytosis & this is the example we’ll see in more
detail. We won’t go into the details of phagocytosis, we will look more at
pinocytosis and see what type of uptake is taken up through pinocytosis and basically in contrast to phagocytosis, pinocytosis is the uptake or the
formation of small vesicles that ingest fluids and solutes. One of the major
differences between the two is that phagocytosis is large vesicles or large
particles that are being uptaken & pinocytosis are smaller vesicles ingesting
smaller contents so basically proteins, or even solutes that are in the
extracellular space. Both are different types of endocytosis though.
Large vesicles that ingest large particles, microorganisms, dead cells
Small vesicles that ingest fluids and solutes
- So the 2 major types of endocytosis. The 1 one is phagocytosis & basically
phagocytosis is the ingestion of large or the formation of large vesicles that
ingest large particles & these large particles can actually be bacteria or even
dead cells. These are huge particles ingested by macrophages for example.
- In this case we will look at a type of white blood cell called a neutrophil
which accumulate at sites of infection or inflammation and can ingest bacteria
at these sites. This is an interesting movie to give us an idea of what
phagocytosis looks like. Don’t forget that there are vesicles forming to uptake
an entire bacterium into this neutrophil cell.
- VIDEO: It shows the neutrophil chasing a bacterium around. Neutrophils
are white blood cell that hunt and kill bacteria. In this spread, a neutrophil is
seen in the midst of red blood cells. Staphylococcus bacteria have been
added, this bacteria release a kemoattractant that is sensed by the neutrophil.
The neutrophil becomes polarized and starts chasing the bacteria. The
bacteria bounced around by thermal energy move in a random path seeming
to avoid their predator. Eventually, the neutrophil catches up with the bacteria
and engulfs it by phagocytosis.
- The two examples are clathrin coated vesicles forming at the PM in what
are called clathrin coated pits. The plasma membrane if you look at one, have
certain areas that you can see pits forming and these pits are just basically
indentations in the plasma membrane and at the bottom of these indentations,
you can see molecules of clathrin forming and these clathrin coated pits will
eventually form clathrin coated vesicles. These are sites of pi