MB207 Molecular Cell Biology

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MB207 Molecular Cell Biology

• Intracellular Vesicular Traffic

• Outline
• Principles of vesicular traffic
• Transport from ER through Golgi Apparatus
• Transport from trans Golgi network to lysosomes
• Transport into cell from plasma membrane
  endocytosis
• Transport from trans Golgi network to the cell
  exterior exocytosis

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Endocytosis and exocytosis
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Vesicular Transport

• Process in which membrane-enclosed transport
  vesicles transport proteins from one
  membrane-enclosed compartment to another.
• Shape spherical, larger irregular-shaped
  vesicles.
• Proteins do not move across the lipid bilayer of
  any membranes. But only move between
  topologically equivalent compartments (eg. Lumen
  of ER to lumen of Golgi to exterior of the cell).

Principles of vesicular transport

1. A protein-coated membrane-enclosed transport
   vesicle buds off from the membrane of donor
   compartment carrying a variety of specifically
   selected cargo molecules.
2. Transport vesicle binds to the target compartment
   and fuse with the membrane of the target
   compartment.
3. Cargo molecules transfer into lumen of the target
   compartment and inserting the vesicular membrane
   components into the target compartment membrane.

• Overview of vesicle transport
• Budding
• Uncoating
• Transport
• Docking
• Fusion

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Question 1 How do transport vesicles form?
(Budding) Question 2 What deforms
the membrane to cause a vesicle form?
(A planar phospholipid lipid bilayer wants
to remain flat. But, the small transport vesicles
that are seen in cells are small and highly
curved. It is this protein coat that causes the
membrane to deform and form a transport vesicle.)
Coat proteins assemble on the membrane surface
and curve the membrane into a vesicle
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- new synthesized ER molecules are sorted and delivered to either other organelles or the cells plasma membrane - molecules from exterior of the cell are taken up into the cell and trafficked to an appropriate intracellular compartment

• exchange of membrane material and vesicular
  lumenal contents, each organelle maintains its
  own highly specialized characteristics.

1. Helps select the cargo by concentrating specific membrane proteins into specialized membrane patches that give rise to the vesicle membrane. 2. Assembly of the coat proteins into curved basket-like lattices deforms the membrane in a manner that helps form vesicles of uniform size
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Three types of protein-coated vesicles
(A) Clathrin-coated vesicles (B) COPI-coated vesicles (C) COPII-coated vesicles
-mediate transport from 1. The plasma membrane to early endosomes 2. Immature secretory vesicles to golgi apparatus 3. Golgi apparatus to late endosomes -mediate transport from 1. Early Golgi back to ER 2. Golgi apparatus to plasma membrane 3. Between various Golgi cisternae -mediate transport from 1. ER to the Golgi apparatus
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• 3 main types of coated vesicles (each type use
  for different transport steps in the cell)
• COP II-coated (Sec complex)
• Move material from ER to the cis-Golgi complex (5
  protein complex, including SARI GTPase)
• COP I-coated (7 different COP subunits)
• Retrograde movement from Golgi cisternae to ER (8
  protein complex, including ARF GTPase)
• Clathrin-coated (Clathrin adaptin)
• Move from the TGN (trans Golgi network) to
  endosome
• Move from plasma membrane (endocytosis) to
  endosome
• Golgi to lysosome

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• Clathrin-coated vesicles
• Major proteins in clathrin-coated vesicle are
  clathrin and adaptin
• Each clathrin subunit composes of heavy and light
  chain
• Three clathrin subunits form a three-legged
  structure called triskelion.
• Adaptin bind to both clathrin and transmembrane
  cargo receptor, which in turn bind to the target
  cargo
• As coated bud grows, a GTP-binding protein,
  dynamin, assemble around the neck of each bud.
  Hydrolysis of GTP will regulate the speed of the
  pinching-off process for membrane fusion.
• Shortly after vesicle formed, energy required to
  remove clathrin coat

Clathrin triskelion ( 3 legged structure)
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The assembly and disassembly of a clathrin coat

• Curvature was introduced into the membrane which
  leads to the formation of uniformly sized coated
  buds.
• The adaptins bind both clathrin trikelions and
  membrane-bound cargo receptors, thereby mediating
  the selective recruitment of both membrane and
  cargo molecules into the vesicle.
• The pinching-off of the bud to form a vesicle
  involves membrane fusion, facilitated by the
  GTP-binding protein dynamin which assembles
  around the neck of the bud.
• The coat of clathrin-coated vesicles is rapidly
  removed shortly after the vesicle forms.

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Small GTPases regulate budding

• 2 conformational states
• GDP (off)
• GTP (on)

• SarI proteins is a coat recruitment GTPase.
• Inactive, soluble SarI-GDP binds to a GEF
  (called SecI2) in the ER membrane, causing SarI
  to release its GDP and bind to GTP.
• A GTP-triggered conformational change in SarI
  exposes its fatty acid chain, which inserts into
  the ER membrane.
• Membrane-bound, active SarI-GTP recruits COPII
  subunits to the membrane. This causes the
  membrane to form a bud which includes selected
  membrane proteins.
• A subsequent membrane-fusion event pinches off
  and releases the coated vesicle.

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Transport vesicle docking

• Specificity in targeting ensures that membrane
  traffic proceeds in an orderly way.
• Therefore, transport vesicles must be highly
  selective in recognizing the correct target
  membrane with which to fuse.
• ? transport vesicles display surface markers that
  identify them according to their origin and type
  of cargo while target membrane display
  complementary receptors that recognize the
  appropriate markers.
• The recognition step is thought to be controlled
  mainly by two classes of proteins
• i) SNAREs (Soluble NSF Attachment Protein
  Receptor)
• - central role both in providing specificity
  and in catalyzing the fusion of
• vesicles with the target membrane.
• ii) Rabs (GTPase)
• - work together with other proteins to
  regulate the initial docking and
• tethering of the vesicle to the target
  membrane.

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SNAREs

• compartment identifiers - required for vesicle
  docking and fusion
• transmembrane protein that exist as
  complementary sets to ensure correct vesicle
  targeting
• i) vesicle membrane SNAREs, v-SNAREs
• ii) target membrane SNAREs, t-SNAREs

• Complementary sets of v-SNAREs and t-SNAREs
  contribute to the selectivity of
  transport-vesicle docking and fusion
• The v-SNAREs are packaged together with the coat
  proteins during the budding of transport vesicles
  from the donor membrane and bind to complementary
  t-SNAREs in the target membrane.
• After fusion, the v- and t-SNAREs remain
  associated in a tight complex.
• The complexes have to be dissociated before the
  t-SNAREs can accept a new vesicle or the v-SNAREs
  can be recycled to the donor compartment for
  participation in a new round of vesicular
  transport.
• Different v-SNAREs can be packaged with
  different cargo molecules when leaving the donor
  compartment.

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Dissociation of SNARE pairs

• Complexes have to be disassembled before the
  SNAREs can mediate new rounds of transport.
• NSF protein cycles between membranes and the
  cytosol and catalyzes the disassembly process.
  NSF (an ATPase) uses the energy of ATP hydrolysis
  to solubilize and facilitates refolding of
  denatured proteins.
• After the v-SNAREs have mediated the fusion of a
  vesicle on a target membrane, the NSF binds to
  the SNARE complex via adaptor proteins and
  hydrolyzes ATP to interfere the SNAREs apart.

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Coiled-coil SNARE complexes

• This complex are formed hold the vesicle close to
  the target membrane. Numerous noncovalent
  interactions between SNARE protein stabilize the
  coiled-coil structure.
• The SNAREs responsible for docking synaptic
  vesicles at the plasma membrane of nerve terminal
  consists of three proteins.
• The v-synaptobrevin and t-SNARE syntaxin are
  both transmembrane proteins and each contributes
  one a-helix to the complex. The t-SNARE Snap25 is
  a peripheral membrane protein that contributes
  two a-helices to the four-helix bundle.
• Trans-SNARE complexes always consists of four
  tightly intertwined a-helices, three contributed
  by t-SNARE and one by a v-SNARE.

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Rab protein Docking of transport vesicle

• Rab proteins are monomeric GTPases.
• Rab proteins facilitate and regulate the rate of
  vesicle docking and the matching of v-SNAREs and
  t-SNAREs.
• Rab proteins cycle between a membrane and the
  cytosol. In their GDP-bound state, they are
  inactive. They are active in the cytosol and in
  their GTP-bound state .
• Rab protein hops onto the vesicle during
  budding.
• SNARE proteins are tightly capped or turned
  off by association with other proteins.
• Rab protein are specifically distributed through
  the secretory pathway.

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Subcellular locations of some Rab proteins
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Fusion of membranes mediated by SNAREs
Tight SNARE pairing forces lipid bilayers into
close apposition so that H2O are expelled from
the interface.
Lipid of 2 interaction leaflets of the barriers
then flow between the membrane to form a
connecting stalk.
Lipid of the other 2 leaflets then contact each
other forming a new bilayer, which widens the
fusion zone (hemifusion).
Split of the new bilayer completes the fusion
reaction.
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Entry of enveloped viruses into cells HIV
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Transport from the ER through the Golgi apparatus

• Transport process from ER to Golgi apparatus and
  from Golgi apparatus to the cell surface and
  elsewhere.
• ? the proteins pass through a series of
  compartments where they are successively
  modified.
• Transport vesicles select cargo molecules and
  move them to the next compartment in the pathway,
  while others retrieve escaped proteins and return
  them to the previous compartment where they
  normally function.
• Golgi apparatus ? major site of carbohydrate
  synthesis as well as
• sorting and
  dispatching station for the products
• of ER.
• Most proteins and lipids (acquired their
  appropriate oligosaccharides in the Golgi
  apparatus) are recognized in other ways for
  targeting into the transport vesicles going to
  other destinations.

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The recruitment of cargo molecules into ER
transport vesicles

• By binding to the COPII coat, membrane and cargo
  proteins become concentrated in the transport
  vesicles as they leave the ER.
• Membrane proteins are packaged into budding
  transport vesicles through the interactions of
  exit signals on their cytosolic tails with the
  COPII coat.
• Some of the membrane proteins trapped by the
  coat in turn function as cargo receptors, binding
  soluble proteins in the lumen and helping to
  package them into vesicles.
• Unfolded or imcompletely assembled proteins are
  bound to chaperones and are thereby retained in
  the ER compartment.

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Protein leave ER in COPIl -coated transport
vesicles

• Vesicular tubular clusters are the structures
  formed when ER-derived vesicles fuse with one
  another. The clusters are relatively short-lived
  because they quickly move along microtubules to
  the Golgi apparatus.
• COPII-coated vesicles leave the ER, uncoat and
  begin to fuse with one-another to form
  vesicular-tubular clusters.
• These clusters associate with motor proteins
  that drag them along microtubules in an
  ATP-dependent process.
• Meanwhile, retrograde transport removes certain
  components, purifying and concentrating the
  secretory cargo further.

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Retrieval of ER resident proteins is
receptor-mediated

• Escaped ER resident proteins are retrieved from
  the Golgi by KDEL receptors that recognize
  specific retrieval signals in ER proteins.
• The KDEL receptor present in the vesicular
  tubular clusters and the Golgi apparatus,
  captures the soluble ER resident proteins and
  carries them in COPI-coated transport vesicles
  back to the ER,
• Upon binding its ligands in this low pH
  environment, the KDEL receptor may change
  conformation, so as to facilitate its recruitment
  into budding COPI-coated vesicles.

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A model for the retrieval of ER resident proteins

• The retrieval of ER proteins begins in vesicular
  tubular cluster and continues from all
  environment of the ER, the ER resident proteins
  dissociate from the KDEL receptor, which is
  returned to the Golgi apparatus for reuse.
• KDEL-receptors bind to KDEL-bearing proteins in
  the low pH environment of the Golgi and release
  that Cargo in the neutral pH of the ER.
• pH probably alters KDEL receptor conformation -
  regulating cargo binding and inclusion in COPI
  vesicles.

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Golgi apparatus

• The Golgi complex is typically disc-shaped with a
  stack of 4 - 6 cisternae.
• Individual stacks may be interconnected in a
  large complex.
• Golgi complex processes protein (glycosylation
  sulfation) and synthesizes some polysaccharides.
• Functionally distinct compartments, arranged from
  cis face (closest to ER) to trans (exit) face
• Cis-Golgi network sorts new proteins, separating
  those for return to the ER from those to pass to
  the next Golgi station
• Trans-Golgi network sorts proteins into vesicles
  bound for specific destinations i.e. lysosomes,
  secretory vesicles or the cell surface.

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Functional compartmentalization of the Golgi
apparatus

• Post-translational modifications in the Golgi
• Specific modifications occur in specific
  subcompartments, because modifying enzyme
  localization is tightly controlled.
• The state of protein modification can identify
  how far a protein has proceeded in transport.

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• Two models for movement

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Two Models For Cis to Trans-Golgi Progression
Traditional Model - Golgi is a static organelle.
Secretory proteins move forward in small
vesicles. Golgi resident proteins stay where
they are.
Radical Model - Golgi is a dynamic structure.
It only exists as a steady-state representation
of transport intermediates. Secreted molecules
move ahead with a cisterna. Golgi resident
proteins move backward to stay in the same
relative position.
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Functions of Golgi apparatus

• Sorting and processing of proteins and lipids
  coming from ER
• Example mannose 6-phosphate is added to proteins
  that are destined for lysosomes
• 2. Oligosaccharide synthesis
• Many protein from ER are glycosylated during
  their transit through the Golgi
• Pectin and hemicellulose for the cell wall of
  plants proteoglycans for the extracellular
  matrix in animal.

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Transport from trans Golgi network to lysosomes

• Lysosomes are organelles filled with hydrolytic
  enzymes that are used for controlled
  intracellular digestion of macromolecules
• Turnover of cells macromolecules
• Extracellular macromolecules from other cells
• Bacteria (macrophages)
• The interior of a lysosome is acidic (pH 4.6)
• Unique enzymes of lysosomes are adapted to low pH
• Advantages
• Digestive reactions more rapid at low pH
• Leaked lysosomal enzymes do not destroy the cell
  (pH 7 to 7.3)
• Low pH is maintained by a ATPase
• pH decreases from ER to Golgi to endosome

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Three pathways to degradation in lysosomes

• Lysosome can be small (25 to 50 nm) or large
  (over 1 µm).
• Multiple paths to deliver material to lysosomes.
• Each pathway leads to intracellular digestion of
  materials derived from different source
• i) Through Late endosome - Developed from
  endosomes, an endocytotic vesicle derived from
  the plasma membrane.
• ii) Through Phagosome - A vacuole formed around a
  particle absorbed by phagocytosis by the fusion
  of the cell membrane around the particle.
• iii) Through Autophagosome - A membrane-bound
  body occurring inside a cell and containing
  decomposing cell organelles.
• Materials delivered to lysosome may be degraded
  in the lysosome or expelled (exocytosis)

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Transport of newly sythesized lysosomal
hydrolases to lysosomes

• Lysosomal proteins are synthesized in the RER and
  transported to the Golgi complex, just like
  secreted proteins. However, enzymes in the Golgi
  recognize and tag lysosome-bound proteins by
  phosphorylating the mannose residues.
• The mannose-6-phosphate groups are recognized by
  the mannose-6-phosphate receptors (MPR) at the
  trans Golgi network and hence the recognition
  signals for packaging.
• At late endosome / lysosome, the protein will
  dissociate from the receptors and the receptors
  can then be recycled.

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Endocytosis Transport into the cell from the
plasma membrane

• Internalization of external material including
  proteins located on the plasma membrane
• Purpose Cellular uptake of macromolecules
  usually en route to the lysosome. This could
  include the ingestion of metabolites, lipids like
  cholesterol through LDL and the LDL receptor or
  iron via the transferring receptor. Other reasons
  for endocytosis include the termination of cell
  surface events (i.e. signaling).

• Types of endocytosis
• Phagocytosis or cellular eating Ingestion of
  large particles such as microorganisms or dead
  cells (usually gt250 nm in diameter) by phagocytes
  such as macrophages neutrophils.
• Pinocytosis or cellular drinking Fluid-phase
  endocytosis primary for the uptake of fluids and
  solutes (100 nm in diameter).

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• Mechanisms of Endocytosis
• Similar to vesicle budding from the ER.
• Coat protein clathrin is primarily responsible
  for the majority of vesicular traffic between the
  trans-golgi network and plasma membrane in both
  directions.
• Another type of vesicles called caveolae can also
  be used for pinocytosis. Caveolae collect cargo
  by the lipid composition of the caveolar membrane
  rather than the protein coat.

Formation of clathrin coated vesicles from the
plasma membrane
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Endocytotic pathway from the plasma membrane to
lysosomes

• Maturation from early to late endosomes occurs
  through the formation of multivesicular bodies,
  which contain large amounts of invaginated
  membrane and internal vesicles.
• These bodies move inward along microtubules, and
  recycling of components to the plasma membrane
  continues as the bodies move.
• The multivesicular bodies gradually turn into
  late endosomes, either by fusing with each other
  or by fusing with pre-existing late endosomes.
• The late endosomes no longer send vesicles to the
  plasma membrane but communicate with the trans
  Golgi network via transport vesicles, which
  deliver the proteins that will convert the late
  endosome into a lysosome.

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Receptor-mediated endocytosis fate of the
receptors
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An example of receptor-mediated endocytosis
cholesterol uptake

• Low density lipoproteins (LDL) is a storage form
  of cholesterol and the primary vehicle for
  cholesterol transport in the blood.
• Cells need cholesterol for its function and
  endocytose these LDL particles to bring them into
  the cells
• Cell surface LDL receptor binds to LDL.
• The cytosolic portion of the LDL receptor
  associates with a specific set of clathrin
  adapters and nucleates the binding of a few
  clathrin triskelions to form a coated pit.
• As more receptors enter the coated pit, more
  adaptors and clathrin is recruited to form a
  coated vesicle.
• The LDL-containing coated vesicles uncoat and
  fuse with the early endosome.
• The receptor disengages the LDL particle due the
  pH change and is recycled to the plasma membrane.
  
• The LDL particles are transported to the
  lysosomes where it is degraded and utilized.

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Normal and mutant LDL receptors

• LDL receptor proteins binding to a coated pit in
  the plasma membrane of a normal cell.

• A mutant cell in which the LDL receptor proteins
  are abnormal and lack the site in the cytoplasmic
  domain that enables them to bind to adaptins in
  the clathrin-coated pits.
• Such cells bind LDL but cannot ingest it.
  Increased risk of a heart attack caused by
  atherosclerosis.

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Transport from the trans Golgi netowrk to the
cell exterior Exocytosis

• 2 forms of secretory pathways for proteins,
  lipids, or modified polysaccharides
• Constitutive secretion
• This is a continuous pathway for the synthesis
  and export of proteins, such as those of the ECM
  (extracellular matrix) or plasma membranes.
  Operates in all cells.
• Regulated secretion
• In specialized secretory cells. Vesicles
  (secretory granules) are prepared and stored for
  export in response to a signal. e.g. release of
  hormones, histamine by Mast cells, digestive
  enzymes neuro-transmitters at the synapses.

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Constitutive Secretory pathway Regulated Secretory pathway
Nonselective, default pathway- signal is not required Contents are released immediately upon contact with the plasma membrane. Secretory vesicles, released on demand (eg. Hormones, neurotransmitters, digestive enzymes).

• Properties of regulated exocytosis
• Proteins are typically very dense (concentrated).
• Proteins are selected by signal patches.
• Pre-pro-proteins and polyproteins (delayed
  activation) eg. Preproinsulin activated in
  secretory granules, trysinogen activated after
  secretion.
• SNARE catalyzed fusion with plasma membrane is
  inhibited until appropriate signal is received
  (eg. Hormone, electrical excitation).

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Formation of secretory vesicles

• Secretory proteins become aggregated and highly
  concentrated in secretory vesicles by two
  mechanisms
• They aggregate in the ionic environment of the
  trans Golgi network (often the aggregates become
  more condensed as secretory vesicles mature and
  their lumen becomes more acidic.
• Excess membrane and lumenal content present in
  immature secretory vesicles are retrieved in
  clathrin-coated vesicles as the secretory
  vesicles mature.

• Secretory vesicles bud from the TGN (with
  clathrin coats), uncoat, move near the plasma
  membrane, tether, dock, and fuse using PM
  specific proteins.

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Polarized cells direct proteins from the trans
Golgi network to the appropriate domain of the
plasma membrane

• Polarized cells cell with two compositionally
  distinct and different plasma membranes, the
  apical and basolateral plasma membrane.
• The two different membrane domains are separated
  by a molecular fence (tight junctions) which
  prevents proteins and lipids from diffusing
  between the two domains, so that the compositions
  of the two domains are different.

• The plasma membrane of the nerve cell body and
  dendrites resembles the basolateral plasma
  membrane domain of a polarized epithelial cell,
  whereas the plasma membrane of the axon and its
  nerve terminals resembles the apical domain of an
  epithelial cell.
• The different membrane domains of both
  epithelial cell and the nerve cell are separated
  by a molecular fence, consisting of a meshwork of
  membrane proteins tightly associated with the
  underlying actin cytoskeleton (tight
  junction/axonal hillock).

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The formation of synaptic vesicles