Lecture 13: Polarized cells Flashcards
epithelial tissue (def.)
One of the major tissues types in human body—covers all surfaces…inside and out as well as forming glands (mammary) and organs (liver, lung)
epithelial tissue functions
Protection
* Absorption,
* Secretion
* Sensory reception
apical surface of epithelial tissue faces ___
outside world
basolateral surface of epithelial tissue faces ___
ajacent cells and underlying CT and blood vessels
loss of cell polarity in epithelial tissue lead to ___
metastatic phenotype!
Carcinoma (def.)
type of cancer that develops from epithelial cells
adenocarcinoma (def.)
cancer that forms in the glandular tissue
cancer doesn’t form in ___
cells that don’t divide (ex. neuron)
apical and basolateral membrane of epithelial tissue are _____ because ____
compositionally distinct because they are separated by tight junctions (creating membrane polarity)
tight junctions do what?
restricts movement of macromolecules between cells
allow for directional movement of nutrients
Establishment and maintenance of cell polarity in epithelial tissue involves ____
sorting at the TGN to apical/basolateral side
studying epithelial cells requires ____ to identify Apical and Basolateral sorting signals
compartmentalized culture system
compartmentalized culture system (descri.)
has monolayer of MDCK cells (highly polarized)
apical medium and basal medium
sits porous filter
MDCK cells stands for ____
Madin-Darby Canine Kidney epithelial cells
limitations of MDCK cells
less dog reagents available
GH is a ___
non glycosylated secretory protein (usually)
adding glycosylation sites to GH does what?
increase secretion to apical side
blocking glycosylation of GH12 (glycos.) with tunicamycin results in what?
non-polarized secretion
apical sorting signals
-N or O-linked carbohydrate in ectodomain (membrane protein)
-transmembrane GPI domain (membrane protein)
ectodomain (def.)
part of membrane protein that faces extracellular space
mechanism of apical sorting
partitioning in glycosphingolipid-cholesterol (lipid rafts) rafts in TGN with TGN-localized lectin as receptors
Apical sorting is a ____ process in TGN which suggest that it uses ___
-saturable
-receptors to recruit cargo
Lectins are defined as ____
proteins that bind to carbohydrates
Lectin in TGN bind to ____ and partition into ____ for apical sorting
oligosaccharides
lipid rafts
other pathway for apical sorting
biosynthetic (direct) route- directly from protein production -NSF-independent , SNARES, tethering factors
PM is rich in ___ (from TGN)
lipid rafts
RNAi knockdown of Galectin-3 causes ____
apically targeted membrane proteins to be found on basolateral side, suggesting that it is an apical sorting receptor
Cation dependent M6P receptor (goes to
_______)
basolateral surface/endosomes
lysing cells with gentle non-ionic detergent retains ____
antibody-protein interactions
antibody-protein can be recovered with ___ + detected (radioactive proteins) with ____
protein A sepharose
SDS page + autoradiography
Basolateral sorting like apical soring is ____
saturable (uses receptors)
Basolateral sorting signals are found in ____
cytoplasmic regions of membrane proteins (need to be exposed to cytoplasm)
basolateral sorting motifs are ___ which are similar to ___
tyrosine and di-leucine-based motifs
endocytic/lysosomal targeting motifs
other route for basolateral sorting
biosynthetic (direct) route- directly from protein production -NSF-independent , SNARES, tethering factors
AP-1 adaptor protein + basolateral sorting
contain µ1B rather than µ1A which is part of “regular” AP1 complex
sorted to basolateral side
argeting to apical and basolateral domains requires ____Rabs, SNARES and tethering factors
different
sorting signal hierarchy
Basolateral signals dominant»>Apical sorting occurs in the absence of basolateral sorting signals
Caveolae (general def.)
invaginations of apical plasma membrane (distinct morphology)
Caveolae abundant on ____ and ___ cells
-endothelium and smooth muscle
Caveolae absent in ___
lymphocytes, RBCs
Caveolae coating description
striated
Caveolae are a type of ____ containing the protein ____
lipid raft
caveolin
lipid rafts are present at ___ and ___
PM and TGN
lipid raft properties
-rich in cholesterol and sphingolipids
-rich in GPI anchored proteins
-thicker regions
not all ___ are caveolae but all caveolae are _____
lipid rafts
_____ can determine lipid raft association
Membrane anchoring domains
influenza virus and caveolae
HA and NA proteins of flu virus concentrate in lipid rafts to bud off, important for virus entry too
Caveolin is a ____ that localizes at ___
- 21 kD phosphoprotein
caveolae
forms of caveolin
Cav-1,2 - form heterodimers
Cav-3 (present only in muscle cells)
expression of which caveolin promotes caveolae formation
Cav-1
Cav-3
disruption of cholesterol in membranes by this drug prevent caveolae formation?
filipin (flattened structures)
without ____ in membrane, caveolae can’t form
cholesterol
how caveolin forms caveolae
-caveolin forms hair pin in membrane (N/C termin. in cytoplasm)
-binds cholesterol & sphingolipids
-oligomerizes with 14-16 subunits
-palmitoylated near C-termin.
caveolae can form ____ structures
interconnected
knout out caveolin-1 gene in mice results
-no caveolae in vascular endothelial cells
-leaner, resistant to high fat
-more fatty acids in blood serum (enlarged heart)
-insulin resistance
-hypersensitivity to cancer
insulin resistance and caveolin-1
mutation in caveolin-binding domain of insulin receptor
caveolin stabilizes insulin receptor
caveolin and cancer
-caveolin-1 might be tumour suppressor
-dominant-negative mutation in gene in some breast cancer
are caveolae dynamic + involved in endocytosis?
yes
caveolae and endocytosis
-GTP hydrolysis required
-colocalize with intersectin (scaffolding) and dynamin (fission) at their necks
where does intersection and dynamin localize in caveolae
-necks of caveolae
Labelled _____can be used to follow caveolae-dependent transport
CTxB (Cholera toxin b-chain subunit )
_____ is required for budding of caveolae from PM
dynamin (interconnected structure still forms without dynamin)
