Endocytosis and Intracellular vesicle transfer Flashcards
Describe cytosolic vesicles
Cytosolic vesicles are
single membrane bound
vesicles
Trafficking of these
vesicles involves
processes:
- Budding
- Scission
- Fusion
- Linked to the ER and
- Golgi membrane
system
- Lysosomes
- Peroxisomes
Describe absorption mechanisms
Living cells and tissues show a great capacity for absorbing a variety of substances from the surrounding
medium.
Large molecules, small molecules, electrolytes and nonelectrolytes can all be absorbed. Sometimes
substances are absorbed by diffusion or by active transport
What are the three mechanisms which allows the cell to take up substances
Phagocytosis, pinocytosis, receptor-mediated endocytosis
Describe phagocytosis
- “cell eating”
- occurs only in specialized cells
macrophages, dendritic cells and
neutrophils - capture and destroy pathogens
and particulate antigens - essential component of the innate
immune response - Receptors are involved – but not
specific - Fc- and complement-receptor
mediated phagocytosis, named for
binding specificity for antibody tail
region called Fc (Fragment,
crystallizable) - clearance of apoptotic bodies
- some bacteria “hijack” this process
in non-phagocytic cells to enter
and infect them
The process by which cells engulf solid matter
Describe pinocytosis
- “cell drinking”
- Unspecific, continuous
- All cells, extracellular fluid including
molecules - micropinocytosis within vesicles
(0.1-0.4 µm diameter) - macropinocytosis within vacuoles
(0.5-5.0 µm) named
macropinosomes
The process by which cells take up fluids and solute
Receptor mediated endocytosis
- General term for all mechanisms of
absorption extracellular fluid and
substances - Generates small vesicles, 40-120nm
- substances bind to receptor sites
- vesicle called endosome
- can be utilized by viruses to enter
cells - Endocytosis can be
- Clathrin-dependent or
- Clathrin-independent
process by which cells absorb metabolites, hormones, proteins – and in some cases
viruses – by the inward budding of the plasma membrane (invagination).
Describe clathrin-mediated endocytosis
Initiation: Clathrin coat (red) module recruited to
the bud deforms the membrane at the inner leaflet.
Assembly: The resulting curvature rapidly recruits
dynamin to the neck first. Dynamin (green) further
deforms membrane.
Maturation 1: Actin (blue) begins to polymerise and
dynamin further accumulates at the neck, resulting
in an even higher membrane curvature that recruits
phosphatase to the endocytic site.
Maturation 2: Dynamic protection of the neck.
Phosphatidylinositol-4,5-bisphosphate (PIP2,
orange) creates a PIP2 phase segregation. The
resulting interfacial force squeezes the neck,
creating even higher membrane curvature that
induces more PIP2 hydrolysis.
Scission: Rapid vesicle scission and disassembly of
endocytic apparatus.
Adaptor proteins Ap1-4
- Ap’s are tetrameric adaptor
complexes - Ap2 is required for CME
- The A2 subunits µ2 and σ2 bind to
the activated receptor via the YXXφ
domain - The Ap2 subunits α and β2 binds to
Clathrin
Descirbe Phosphoinositides
Mark endocytic vesicles and membrane domains
- Phosphoinositides (PIPs) mark
organelles and are linked to
vesicular transport events. - PIPs can be phosphorylated at the
position 3,4,5. - PIs recruit specific proteins to
regions of membranes - PI(5)-kinase converts PI(4)P to
PI(4,5)P2. - PIP2 recruits the adaptor protein
AP2 for Clathrin mediated
endocytosis - PI(5)-phosphatase hydrolyses
PI(4,5)P2 -> weakens the binding of
AP2 -> uncoating of the Clathrin
coat.
Describe the clathrin coat
- Clathrin is a triskelion-shaped
scaffold protein composed of three
heavy and three light chains - Outer Diameter: 70–80nm, contain
about 35–40 triskelion - Inner diameter 40nm
- Initiation of the Clathrin complex
formation requires the accumulation
of PIP2 and adaptor proteins. - Adaptor proteins link the activated
receptors to Clathrin. - Clathrin polymerize and act as a
reinforced mould in which the
membrane vesicle may form
without direct association with the
membrane.
Clathrin is a protein that plays a major role in the formation of coated vesicles. It
forms a cage around a vesicle
Describe clathrin cage disassembly
- The vesicles are then transported
and sorted, based on receptor type
or membrane composition, to the
various destinations. - Dissociation of the coat occurs
rapidly following scission of the
vesicle from the membrane. - Auxilin binds to the triskelion.
- 3 Hsc70:ATPs are hydrolysed to 3
Hsc70:ADPs - Hsc70:ADP removes the Clathrin
triskelion from the cage structure - Clathrin-coat has to be removed to
allow fusion with other cell
organelles (e.g. early endosomes)
What other coating mechanisms are there?
- Common principle for coating:
- Coat formation, membrane budding,
uncoating. - For internal transport COPI (TGN), COPII
(ER), retromer (coats, etc. - Example: Caveolin-mediated endocytosis
- involves the formation of 50-100nm
plasma invaginations - Membrane deformation is driven by
Caveolin and associated Cavin proteins - It requires GTPase dynamin function for
vesicle scission - Caveolin: three homologues : Cav1-3
- Each caveolae contains ca. 150 Cav
molecules - Caveolin are well described for virus
internalization
Describe the early endosome
- After endocytosis, vesicles fuse with
the early endosome.
The early endosome collects cargo and
distributes it to recycling endosome or
towards the late endosome for
degradation
pH of early endosomes 6.0
The RabGTPases can be used to
identify individual endosomes.
Rab5 marks early endosomes.
Rab4/11 mark recycling endosomes
Rab7 marks late endosomes
Describe the Rab proteins
The Rab proteins are monomeric Gproteins
70 different Rab proteins
Contain a GTPase fold
Rabs are anchored to the membrane
by a lipid group
Rab switch between the inactive GDP
(guanosine diphosphate) and an active
form bound to GTP (guanosine
triphosphate)
The conversion is regulated by
GTP exchange factors (GEF)
-> Rab activation
GTPase activating proteins (GAP)
-> Rab inactivation
Describe the maturation of endosomes
The Rab proteins occupy distinct
membrane domains or “Rabdomains” on early endosomes.
Endosomes are organized in a
modular system, each module or
Rab-domain fulfilling a set of
functions.
Rab GTPases allow the sequential
‘transport’ of cargo between
adjacent Rab-domains. Rab5 early
endosome to the Rab4 recycling
endosome, or to Rab7 late
endosome.
Transport or maturation?
Describe the recycling endosome
The recycling endosome (RE) displays a
heterogeneous tubular-vesicular
morphology
The most prominent RE markers to date
are the small GTPase Rab4 and Rab11.
Rab4 positive vesicles allow fast recycling
of cargo to plasma membrane.
Rab11 allows slower recycling.
Rab11 RE can be seen as “storage
endosomes” for receptors
The RE is a dynamic tubular endosomal compartment, which transports and stores e.g.
membrane receptors by receiving and releasing vesicles
Describe late endosomes / multivesicular bodies
Multivesicular bodies (MVB) or late
endosomes contain membrane-bound
intraluminal vesicles.
Vesicles form by budding into the lumen
(->ESCRT complex)
MVB pH 5.0-6.0
MVBs can fuse with lysosomes for
degradation of content
MVBs can fuse with the plasma membrane
to release vesicles via exocytosis (->
intercellular transport)
Describe lysosomes
Function : “lys” stands for cut or digest
Membrane bound organelle
Contain hydrolytic enzymes
pH 4.0-5.0 – optimal for acid hydrolases
Digest proteins from the cell, e.g.
organelles which are not functioning
anymore get digested
E.g. macrophages engulf bacteria and
break them down in lysosomes
Lysosomes break down hormones,
signalling proteins, etc. in their building
blocks (amino acids).
These will be released in cytoplasm and
they will be re-used.
LDL as an example for RME
Low-density lipoproteins (LDLs) are taken up
by the cell so that cholesterol can be used in
construction of membranes, etc.
LDL binds to LDL receptors.
Receptor and ligand are internalised into early
endosomes by RME.
LDL is transported to early endosomes
From the early endosome the LDL receptors
are transported to the recycling endosome
(Rab11) or transported to the plasma
membrane directly via Rab4-positive vesicles.
LDLs are routed to the late endosome and
lysosome for “digestion”. Cholesterol is
released in the cytosol and used to build new
membranes e.g. in the TGN.
