MCB 2050 -
Endomembrane
System with 100%
correct answers 2025
Endomembrane system - answer - dynamic, coordinated and
interconnected network of the cell's organelles and related structures
- large amounts of material are exchanged between each
organelle/structure via small, membrane-bound transport vesicles
- several distinct trafficking pathways exist and all rely on transport
vesicles
Vesicle coat proteins - answer select which donor membrane and soluble
cargo proteins enter/dont enter nascent transport vesicle and regulate
vesicle formation and budding
motor proteins - answer direct vesicle movement within cell by linking to
vesicle surface and cytoskeleton element
biosynthetic pathway - answer materials are transported from ER > Golgi
> endosomes > lysosomes/PM/ECS
constitutive secretion - answer - ER-derived materials are continually
transported from Golgi to PM and/or released via exocytosis outside of cell
- secretory transport vesicle membrane components are incorporated into
PM and vesicle lumenal cargo is released into ECS
regulated secretion - answer - occurs only in specialized cells
,- ER-derived materials from Golgi are stored in secretory granules
- in response to a cellular signal, secretory granules fuse with PM and
release (via exocytosis) lumenal cargo into extracellular space
endocytic pathway - answer - operate in opposite direction of secretory
pathway (materials into cell)
- materials from PM and/or ECS are incorporated into cell > endosomes
and lysosomes
- amount of secretion varies between different cell types
- pancreatic and intestine epithelial cells are highly polarized
- organelles organized in a distinct way
Autoradiography, Pulse-chase labeling experiments - answer -
demonstrated how proteins move through secretory pathway
- pancreatic tissue was incubated (pulse) with radioactive AA > nascent
proteins > washed and incubated (chase) > protein synthesis > tissue
killed and undergoes autoradiography
- brief chase = RER, intermediate chase = Golgi, long chase = secretory
granules
- results helped define secretory pathway and organization/coordination of
protein trafficking in endomembrane system
Live-cell imaging using autofluorescent proteins - answer - using
standard molecular biology techniques, gene encoding an autofluorescent
protein is linked to a gene of interest
- recombinant gene fusion is introduced into selected organism/tissue/cell
- intracellular localization and trafficking of the expressed fluorescent
fusion protein is visualized in the living specimen using fluorescence
microscopy
VSVG-GFP fluorescence microscopy labeling experiments - answer -
involves live cell imaging using autofluorescent microscopy
- mutation in VSVG is reversible that allows for turning on/off intracellular
transport
, - 40*C (restrictive temp) - nascent VSVG protein misfolded and remains in
ER = RER
- 32*C (permissive temp) - VSVG protein properly folds and transported
from ER = Golgi
- longer times = cell surface
- consistent with results rom pulse-chase labelling/autoradiography
studies
Subcellular fractionation using centrifugation - answer - techniques used
to separate and purify specific organelles on the basis of their varying
sizes and/or densities
- allows for study of organelles structure and functions
- isolation of organelles by centrifugation
- homogenate filtered and subjected to differential centrifugation
- microsomes are fragments of the ER network that fuse and reform into
small, spherical vesicles
- individual organelles in each pellet fraction can be further purified
Equilibrium density-gradient centrifugation - answer - separate intact
organelles/cellular component on the basis of density
- determine composition of the isolated organelles using proteo/lipidomic
and/or use in cell-free import and vesicle trafficking assays
Cell-free systems (biochem approach) - answer - characterization of the
activities of specific endomembrane protein components in vitro
- isolated proteins incubated with liposomes
- liposomes mixed with purified proteins
- allows for processes underlying protein/vesicle trafficking in
endomembrane system to be reconstituted in vitro
Mutant phenotype analysis (genetics approach) - answer - approaches to
identify genes/proteins and steps involved in protein/vesicle trafficking in
endomembrane system by screening for mutant phenotypes
Endomembrane
System with 100%
correct answers 2025
Endomembrane system - answer - dynamic, coordinated and
interconnected network of the cell's organelles and related structures
- large amounts of material are exchanged between each
organelle/structure via small, membrane-bound transport vesicles
- several distinct trafficking pathways exist and all rely on transport
vesicles
Vesicle coat proteins - answer select which donor membrane and soluble
cargo proteins enter/dont enter nascent transport vesicle and regulate
vesicle formation and budding
motor proteins - answer direct vesicle movement within cell by linking to
vesicle surface and cytoskeleton element
biosynthetic pathway - answer materials are transported from ER > Golgi
> endosomes > lysosomes/PM/ECS
constitutive secretion - answer - ER-derived materials are continually
transported from Golgi to PM and/or released via exocytosis outside of cell
- secretory transport vesicle membrane components are incorporated into
PM and vesicle lumenal cargo is released into ECS
regulated secretion - answer - occurs only in specialized cells
,- ER-derived materials from Golgi are stored in secretory granules
- in response to a cellular signal, secretory granules fuse with PM and
release (via exocytosis) lumenal cargo into extracellular space
endocytic pathway - answer - operate in opposite direction of secretory
pathway (materials into cell)
- materials from PM and/or ECS are incorporated into cell > endosomes
and lysosomes
- amount of secretion varies between different cell types
- pancreatic and intestine epithelial cells are highly polarized
- organelles organized in a distinct way
Autoradiography, Pulse-chase labeling experiments - answer -
demonstrated how proteins move through secretory pathway
- pancreatic tissue was incubated (pulse) with radioactive AA > nascent
proteins > washed and incubated (chase) > protein synthesis > tissue
killed and undergoes autoradiography
- brief chase = RER, intermediate chase = Golgi, long chase = secretory
granules
- results helped define secretory pathway and organization/coordination of
protein trafficking in endomembrane system
Live-cell imaging using autofluorescent proteins - answer - using
standard molecular biology techniques, gene encoding an autofluorescent
protein is linked to a gene of interest
- recombinant gene fusion is introduced into selected organism/tissue/cell
- intracellular localization and trafficking of the expressed fluorescent
fusion protein is visualized in the living specimen using fluorescence
microscopy
VSVG-GFP fluorescence microscopy labeling experiments - answer -
involves live cell imaging using autofluorescent microscopy
- mutation in VSVG is reversible that allows for turning on/off intracellular
transport
, - 40*C (restrictive temp) - nascent VSVG protein misfolded and remains in
ER = RER
- 32*C (permissive temp) - VSVG protein properly folds and transported
from ER = Golgi
- longer times = cell surface
- consistent with results rom pulse-chase labelling/autoradiography
studies
Subcellular fractionation using centrifugation - answer - techniques used
to separate and purify specific organelles on the basis of their varying
sizes and/or densities
- allows for study of organelles structure and functions
- isolation of organelles by centrifugation
- homogenate filtered and subjected to differential centrifugation
- microsomes are fragments of the ER network that fuse and reform into
small, spherical vesicles
- individual organelles in each pellet fraction can be further purified
Equilibrium density-gradient centrifugation - answer - separate intact
organelles/cellular component on the basis of density
- determine composition of the isolated organelles using proteo/lipidomic
and/or use in cell-free import and vesicle trafficking assays
Cell-free systems (biochem approach) - answer - characterization of the
activities of specific endomembrane protein components in vitro
- isolated proteins incubated with liposomes
- liposomes mixed with purified proteins
- allows for processes underlying protein/vesicle trafficking in
endomembrane system to be reconstituted in vitro
Mutant phenotype analysis (genetics approach) - answer - approaches to
identify genes/proteins and steps involved in protein/vesicle trafficking in
endomembrane system by screening for mutant phenotypes
