Endocytosis I Flashcards
1
Q
Describe endocytosis - gen
A
Outside In
Late endosome, lysosome, early endoscope, recycling endosome
Clathrin dependent, clathrin independent, phagocytosis, caveolae, etc
= many ways to get into cell
2
Q
What does most endocytosis lead to
A
Early endosomes
3
Q
Name major endocytic compartments in order
A
Early endosome
Recycling endosomes
Late endosomes - multivesicualr bodies
Lysosomes
4
Q
Name all endocytic vesicles
A
Clathrin
Non clathrin
Phagosomes
Caveolae mediated endocytosis
5
Q
What does Clathrin fuse with
A
Clathrin vesicles fuse with early endosomes
6
Q
Describe non clathrin vesicles
A
Many kinds
Mostly deliver cargo to same early endosomes
7
Q
Describe clathrin triskelion
A
3 heavy chains
3 light chains
Polymerizes with itself - makes large vesicles bc membrane does not want to curve- ends up as big vesicle = 70-300nm
8
Q
Describe what clathrin forms
A
Lattice coat, similar to outer coat cop2 - sec13/31
Does not interact with cargo
Induce curvature to membrane for pit formation
9
Q
Describe legs of clathrin
A
Legs of clathrin intertwined with neighbours
10
Q
Describe clathrin mediated endocytosis
A
Clathrin = outer coat, doesn’t bind cargo
11
Q
Describe clathrin mediated endocytosis = what does clathrin bind
A
Adaptors - mostly ap2 on cell surface (equiv like inner layer cop2 coat) = which can recruit cargo receptors
12
Q
Describe clathrin mediated endocytosis = describe dynamin
A
Performs vesicle fission
After pit formed = polymerizes and squeezes and pinches off vesicle - once uncoated = becomes transport vesicle that can fuse with target membrane
*diff adaptors = depends on membranes/function of vesicle
13
Q
Describe clathrin mediated endocytosis = what can receptors do
A
Can concentrate cargo
Concentrate in clathrin pit and binds to proteins (lipoprotein) and pinches off
Polymerizes and brings lipoproteins to cytoplasm so can make yolk - in chick oocytes
14
Q
Name the 4 stages of vesicle formation in clathrin mediated endocytosis
A
Initiation
Propagation
Budding
Uncoating
15
Q
vesicle formation in clathrin mediated endocytosis = STEP 1
A
Initiation = formation of a clathrin coated pit, clathrin coat assembly, coat receptor recruitment
16
Q
vesicle formation in clathrin mediated endocytosis = STEP 2
A
Propagation = invagination of clathrin coated pit
17
Q
vesicle formation in clathrin mediated endocytosis = STEP 3
A
Budding = continual invaginatiin of clathrin coated pit scission of its neck
18
Q
vesicle formation in clathrin mediated endocytosis = STEP 4
A
Uncoatings = disassembly of clathrin coat
19
Q
Gga stands for
A
Golgi localizing
Gamma adaption ear domain homology
Arf binding proteins
20
Q
Describe ap complexes and gga
A
Interact with n terminal region of clathrin heavy chain
21
Q
At cell surface what is important
A
Ap2
(No arf)
22
Q
Describe arh
A
Monomeric arh (autosomal recessive hypercholesterolemia) for ldl receptor internalization
Binds to n terminal region of clathrin heavy chain
23
Q
Describe ap2 complex
A
Function at pm with clathrin
24
Q
Where do dynamins assemble
A
Assemble to form helix at stalk of budding vesicle
Polymerizes in rings
25
What happens when gtp hydrolysis dynamin
Causes conformational change = constricts neck = pinches off vesicle
26
Describe dynamin and synaptic vesicle recycling - exp set up
Fruit fly domain = shibire = has temp sensitive allele
At restrictive temp - 30c = flies paralyzed
At permissive temp - 20c = paralysis reversed, flies move
27
Describe dynamin and synaptic vesicle recycling - what is happening/why
At neuromuscular junction = synaptic vesicles of neurotransmitters are not being recycled = bc requires clathrin mediated endocytosis = clathrin vesicles stuck at 30c
28
Describe order of major endocytic components
Early endosome
Recycling endosome
Late endosome/mvb
Lysosome
29
Early endosome - gen
Cargo sorting station = deliver cargo to early endosome
Slightly acidic
Tubular and vacuolar domains
30
Early endosome - receptors/domains
Have receptors with cargos - sometimes receptors sorted to tubular domain and goes to cells reface
Globular domain remains and accumulates cargos and will be delivered further into endocytic pathway
31
Early endosome - what is it
First major compartment in endocytic pathway - aka sorting endosome
32
Early endosome - ph
Slightly acidic = ph 6-6.5
33
Early endosome - functions
Sorting function =
Some ligands detach from receptors bc low ph —> ligands remain in lumen of endosome and will reach Lyosome later
Receptors and much of the internalized membrane= sorted into tubular extensions of early endosome that detract and become recycling endosomes
34
Early endosome - membrane contains
Small gtpase rab 5
Tether protein eea1
Recycling and non recycling receptors
35
Early endosome - lumen contains
Contains cargo for lysosomal degradation
36
recycling endosome - gen
More than one pathway, recycling rib ranch
Fast vs slow recycling
Less acidic than early endosome
Back to cell surface
37
recycling endosome - from what
Tubaulr extensions of early endosomes
38
recycling endosome - function
Return membrane and most receptors to cell surface
Some cells = do not immediate return to cell surface but form a tubular recycling compartment - sometimes concentrated near Centrosome
39
recycling endosome - contains
Small gtpase rab11
Recycling receptors
40
late endosome / multivesicular bodies - gen
Degradative branch - start to have degradative enzymes
More acidic
Inward budding by escort-0 -I -ii - iii complexes = intraluminal vesicles
41
late endosome / multivesicular bodies - what is it
Membrane proteins targeted for degradation are budded into internal vesicles (do not know how)
42
late endosome / multivesicular bodies - what does compartment have
Some Degradative enzymes
43
late endosome / multivesicular bodies - ph
6.0-5.0
Also cargoes destined for degradation - like ldl
DoES not have recycling receptors
44
late endosome / multivesicular bodies - what can some proteins do
Some proteins can cycle between late endometrial and Golgi
Ex = m6pr and sortilin
45
late endosome / multivesicular bodies - has what proteins
Small gtpase rab7 in membrane and proteins recruited by rab7 = hops tethering complex, dyneins and kinesins
46
Lysosome - what is it
Terminal component for degradation- many things delivered to them
Heterologous population of electron dense organelles = reflecting various stages of cargo breakdown = high protein concentration
47
Lysosome - ph
Acidic = 4.5-5 = for degradative enzyme function = many diff enzymes
48
Lysosome - what happens to digested components
Digested components shuttled out by many specialized channels and transporters =transport broken Down material
49
Describe enzymes in lysosomes
Full of many = cathepsin d, acid phosphatase, saposin and others
Work only at acidic ph
If lysosome breaks = enzymes harmless in cytoplasm
Enzymes are capable of destroying lysosomal membrane tho
50
Describe immunogold lysosome
Large gold - protease
Small gold = labelling albumin
Not much cathepsin in late endosome
51
What does lysosomal membrane contain
Highly glycosulated proteins called lamps and limps = provide protection against lysosomal enzymes
52
Describe relationship between lysosomes and late endosomes
Exact reltionship between late endosomes and lysosomes unclear
Fuse tho
53
What is present on lysosomes
Small gtpase rab7
54
Where is vacuolar atpase found
Found in lysosomes and all endosome types
Also found in Golgi apparatus and Vtcs (not there is a diff ph in each of these compartments)
(Pumps h+ across membrane)
55
vacuolar atpase = where is it not found
Not found in mito = have diff mechanism for maintaining ph gradients
56
Name 3 methods to disrupt h+ gradient
Weak bases
Ionophores
Inhibitors of vtpase function
= will affect lysosomes, Golgi, endosomes
57
Describe weak bases - methods to disrupt h+ gradient
Ammonium chloride = grabs h+ and crosses membrane with it
Chloroquine = sops up protein so accumulates in compartment (malaria - chloroquine treatment, affects synaptic vesicle recycling but need very high amounts to deacidify enzymes - which could kill person)
58
DESCRIbe ionophores - methods to disrupt h+ gradient
Carrier (monensin) or channel forming = lets out h+ from membrane - collapses ph gradient = affects mitochondria too
59
Describe inhibitors of vtpase function - methods to disrupt h+ gradient
Bafilomycin = inhibit pump
60
Describe non clathrin endocytosis
About half of total endocytosis in many cell types
Several mechanisms
61
Name types of non clathrin endocytosis
Macropinocytosis -most cells, phagocytosis - special cells - macrophages - requires actin, not dynamin
Geec pathway = tubular endocytic vesicles, lipid rafts, gpi anchored proteins, folate receptor, does not use dynamin
Caveolin mediated endocytosis = requires dynamin = another coat
62
Describe freeze etch technique
Scrape with metal - visualized by ‘em, looking under membrane of cell = actin fibres, vesicles - rope liek structures = caveoli
63
Describe lipid rafts
Lipid micro domains on cell surface and in trans Golgi network
Enriched in sphingolipids and cholesterol
64
Where are lipid rafts assembled
At golgi
65
What are functions of lipid rafts
Compartmentalize proteins
Accommodate special Tm proteins, gpi-aps (glycosylphosphatidylinositol anchored proteins), signalling molecules
66
Where are lipid rafts smaller
Smaller on cell surface - 15nm
Than if in trans Golgi
67
Describe properties of lipid rafts
More rigid and thick
Less fluid
Proteins can be segregated into domains depending on length tm domain of integral membrane protein
68
Describe exp -adding cholesterol - lipid rafts
Phosphotidylcholine: sphinogomyelin (1:1) = long hdyrophobic tail
Phosphotidylcholine: sphinogomyelin: cholesterol (1:1:1) = if added cholesterol = forms, domains that segregate = phase separation when add cholesterol
69
Describe relationship between lipid rafts and methods of non clathrin endocytosis
Caveolae = tend to segregate raft domains under certain circumstances
Also geec = specializes in internalizing raft domains
70
Describe caveolae
Small flask shaped pits in pm - 70nm diameter
Coated with caveolin = unusual for coat protein bc it is an integral membrane protein
71
caveolae- enriched in
Cholesterol rich membrane concentrated in caveolae
Some signalling molecules can concentrate here = acts as platforms for signalling, favours signalling
72
Describe endocytsoed caveolae
Can fuse to form neutral ph compartments called caveosomes = distinct from endosomes describe prevsiously
Not common
73
What is one role of caveolae
Transcytosis of albumin and other proteins across endothelia = process is distinct from normal transcytosis describe
Is very rapid
Caveolae appear to pinch off from side facing capillary and cross cell and fuse with pm facing tissue = endocytosing at one end and exocytosing at other
74
What does caveolae Form
Makes hairpin - halfway through lipid bilayer and cannot uncoat = integral membrane protein
75
Where do caveolae Remain
In many cell types =appear to remain at cell surface
76
What is purpose of non endocytsoed caveolae
Caveolae may function as signalling platforms = as many signalling molecules on both inner and outer leaflets of pm prefer lipid raft liek membranes
So = serves as way to increase effective local concentration of signalling meolcules = increase efficiency of signalling
77
Describe albumin transport - caveolae ko mice
Mice ok but have leaky capillaries
Albumin not crossing capillary —> tissue so mice make capillaries leaky to compensate
78
Describe transcytosis of albumin
Albumin binds gp60 at apical endothelia —> activates transcytosis
Dynamin dependent
Finds target membrane
79
What does caveolin bind
Cholesterol
80
Describe caveolins
Integral membrane proteins that deform membrane to form flask shaped pits = caveolae = little caves
81
caveolae - found where
In most cells
82
What preferentially associate with caveolae
Lipid rafts
Caveolin 1 binds to cholesterol
83
Describe caveolin 1
Major component of caveolae
Other proteins for shape and function = Cavins, pacsin 2
84
Describe caveosome
Internalized compartment called caveosome = neutral ph, caveolin 1 positive
85
Describe caveolae as signalling platforms - gen
Not entirely essential bc recall = ko survives
Can make signalling more efficient if concentrated together
86
What types of structures are caveolae
Caveolae are normally static structures. They can sit on the surface for a long time without endocytosis.
87
What do some signalling molecules bind to (for caveolae)
Some signaling molecules bind to a conserved caveolin-scaffolding domain (CSD) on caveolin-1
88
Describe macropinocytosis - where happen
Every cells
89
Describe macropinocytosis - What is it
Nonspecific internalization of fluid, particles and membrane
90
Describe macropinocytosis - Depends on
Actin dependent and involves projections from pm = lamellipodia, circular ruffles or blebs
91
Describe macropinocytosis - Macropinosome
>500 nm diameter = very large endocytic vesicle = huge
92
Describe macropinocytosis - Final destination for cargo
Cargo eventually delivered to lysosomes
93
What does geec do
Swallows lipid rafts
94
Where are gpi anchored proteins found
Found mostly on apical surface polarized cells
Mainly resides on lipid rafts
In er
95
Describe what gpi anchored proteins Is
Attached to lipid anchor - goes halfway through membrane, w chain of sugars/some phosphate groups
Protein attachment to a sugar chain on gp1 anchor occurs at Er
Gpi more stable in lipid raft
96
gpi anchored proteins = function
Gpi direct anchored proteins to target membranes
Ex = folate receptor = binds folate at cell surface, gpi anchored
97
What is clic-geec - explain
Gpi anchored early endosome compartment
Tubular based, non coated, clathrin and dynamin independent
Polarized endocytsos at leading edge of migrating cells
98
What is clic-geec - stands for
Clic = uncoated ci tubulovesicualr carriers
Geec = gpi anchored protein enriched early endosomal compartment
99
What is clic-geec - explain Informally
Long tubule vesicle carriers - detaches
Enriched in cholesterol, spingolipids, gpi anchored proteins
100
What is clic-geec - explain Describe whole process
Internalize fluid, membrane and lipid raft associated gpi anchored proteins
Internalized tubulovesicular carreirs = clics = fuse together to form large vesicle = geec
After several minutes = fuse with normal early endosomes
101
Geec acidic?
More acidic than early endosomes
102
What is ap
Adaptor proteins
Often sorting receptors
Adaptor binds proteins with Tm domain
103
on golgi what is important
Other aps, gga, arf1 important
