Section 1: Membranes + compartments Flashcards
membranes define ___ from ___
self from non-self
membranes contain _____
life-containing reactions
eukaryotic cells are bigger than prokaryotic cells which indicates ____
more complexity regarding possible rxns
large eukaryotic cells requires ____ for efficiency
compartmentalization
endomembrane system separates the cell into different compartments, or organelles, such as _____ (4)
the nucleus, the endoplasmic reticulum (ER), the Golgi apparatus, and lysosomes
compartmentalization happens through ____
endomembranes
compartmentalization: endomembranes have 2 advantages
-separate different classes of reactions from each other
3 reasons of using yeast cells as model organism
-easier to study (less complex)
-easier to grow
-easier for creating knockouts (gene deletion)
4 functions of membranes
-semi-permeable barrier
-assembly-line for metabolic reactions &enzymes
-gives cells & organelles identity
-signaling between organelles & cells
semi-permeable barrier: gases + small uncharged molecules
yes, they can move through unobstructed
semi-permeable barrier: ions + large molecules
No, they can’t move through unobstructed
glycosylation + ATP production in mitochondria both employ a ____
assembly-line for metabolic reactions within membranes (ER & mitochondria)
ER + mitochondria interact through ___ to facilitate ____
-ER tubules wrapping around mitochondria
-mitochondria fission
biological membranes consist of a bilayer sheet that is ___thick
5nm (large variation though)
membrane lipids are _____(3)
-primarily phospholipids, sphingomyelin + cholesterol, small amount of glycolipids
phospholipids in membranes (4)
-phosphatidylcholine/PC
-phosphatidylethanolamine/PE
-phosphatidylinositol/PI
-phosphatidylserine/PS
phospholipids in membranes in order of abudance
PC > PE > PI > PS
headgroup of phospholipids determines ____
charge
shape of phosphatidylethanolamine/PE
-more conical, limits fluidity, and adds curvature to membranes
shape of phosphatidylcholine/PC
-cylindrical, less curvature
membrane composition affects ___ & _____
thickness & curvature
function of cholesterol
-makes lipid rafts that increases thickness, attracts proteins and stiffens membranes
Membrane lipids can be isolated using which technique?
Thin-layer chromatography (TLC)
Transverse membrane asymmetry (_____) depends on ____(2)
-movement across the two leaflets
-asymmetric lipid composition
-protein orientation during insertion
PS and PE are on the ____ leaflet via ___
-inner
-ATP-dependent flippase
Glycolipids are on the ____ leaflet
-outer
protein insertion into membranes _____
confines functions to either side
what happens to membrane asymmetry when cells die?
membrane asymmetry is lost
Lateral asymmetry is ____
movement witin one leaflet
Lateral asymmetry is set by ____
lipids rafts forming thicker areas in membrane, attracting proteins
Lipid rafts are rich in __ & ____
-cholesterol & sphingomyelin
which phospholipid is present around curves of membranes
PE
Organelles membrane identity determined by ___
phospholipid type
morphological methods in cell biology describes _______ (4) of cells
the shape, structure, form, and size
3 main types of morphological/microscopical techniques
-light microscopy
-fluorescence microscopy (similar to light)
-electron microscopy
The different types of morphological/microscopical techniques offer ____
different resolution
Highest resolution: electron or light microscopy
electron microscopy
5 methods for the characterization of intracellular compartments
- Histology
- Immunofluorescence microscopy
- Electron microscopy
- Live cell imaging by phase contrast
- Live cell imaging by fluorescence of GFP-tagged proteins
Histology gives insight into ______
tissue structure
Immunofluorescence microscopy allows us to detect the _____
location of a specific protein
Electron microscopy gives insight to _____
fine structure + localization of proteins
Live cell imaging by phase contrast or fluorescence of GFP-tagged proteins allows us to ____
monitor cells in real time
Histology general protocol
- Chemical fixation
- Washing samples with alcohols
- Cleaning with xylol/toluol to remove alcohols
- Cutting with microtome and mounting on coverslip
- Staining with hematoxylin & eosin
Hematoxylin stains _____ a ___ color
-the nucleus
-deep blue-purple
Eosin stains _____ a ____ color.
-the cytoplasm
-orange-pink-red
Cutting of samples for analysis performed by _____
-microtome
Immunofluorescence general protocol
- Chemical fixation
- Washing samples, block excess proteins, (optional: permeabilizing)
- Incubate with primary antibodies + washing
- Incubate with secondary, fluorescent antibodies + washing
- Mounting + imaging
In immunofluorescence, which antibodies are fluorescent?
secondary antibodies
Immuno-localization reveals both _____and _____of organelles
shape and protein content
With immuno-localization, controls are needed to ____
determine the proteins visualized
evolution of fluorescence microscopy
widefield -> confocal (sharper images) -> STED (see antibodies)
electron microscopy
- Chemical fixation
- Staining with uranyl acetate + dehydration
- Cutting in thin slices
- (Opt.) Incubate with primary + secondary, gold-labelled antibodies
- Mounting + imaging
electron microscopy: sizes of gold particles on antibodies indicates ___
different antigens (proteins, organelles markers)
3D analysis electron microscopy
tomography
Live cell imaging protocol
1.Transfect cDNA expressing GFP-fused protein of interest
2. Mount on microscope with growth medium
3. Imaging
Live cell imaging reveal both ___ and ___
shape and time-dependent movement
Live cell imaging: incident light ____
must be controlled
EM best for live or fixed cells?
fixed cells
Fluorescence microscopy best for live or fixed cells?
live (GFP) + fixed cells (Immunofluorescence)
Things that affect morphological methods
- Native structures can’t be affected by methods
- Specificity of primary antibodies
- GFP hybrids don’t affect protein + organelle function
4.Detergents that permeabilize membranes + make structure accessible to antibodies
homogenization involves ____
breaking cells in a mechanical way to produce homogenate
homogenization: ER
ER unlikely to be intact and becomes microsomes
Microsomes (def.)
ER fragments
Following homogenization, membranes can be separated based on _____
- size
- density
- protein composition
Differential centrifugation (def.)
-Start from tissue/cell homogenate
-Spin at different speeds for various times
-pellets at different speeds contain cell components of various sizes
Velocity centrifugation (also called rate-zonal sedimentation) (def.)
-cell homogenate on shallow sucrose gradient (prevents mixing)
-Centrifuge to separate organelles on the basis of their relative masses
-Collect fractions from bottom of tube
-Recover organelles of different sizes
Velocity centrifugation: slow-sedimenting component
smaller organelles (relatively higher up)
Velocity centrifugation: fast-sedimenting component
bigger organelles (relatively lower down)
Equilibrium density centrifugation
-cell homogenate on steep sucrose gradient
-Centrifuge to equilibrium
-Collect fractions from bottom of tube
-Recover organelles of different density
Velocity centrifugation vs. Equilibrium density centrifugation
masses vs. density
Equilibrium density centrifugation organization
low-density (top) –> high density (bottom)
Immuno-isolation of specific organelles
1.Mix cellular homogenate with antibody against specific organelle
2.Incubate immuno complex with beads coated with protein-A
3.Recover beads by low speed centrifugation
Membrane proteins are soluble only in ____ ( in _____) for analysis outside native membranes + structures
detergents (in excess)
Shape of detergents vs shape of phospholipids
conical vs cylindrical
critical micelle concentration (CMC) (def.)
the concentration of surfactants above which micelles form
Detergents disrupt the protein:lipid interactions that could be ____ so in the presence of excess lipids, sealed compartments can be reconstituted to yield ______
-critical for activity
-proteoliposomes
proteoliposomes (def.)
-systems that mimic lipid membranes (liposomes) with a protein
SDS Gel Electrophoresis
- Denature with SDS, heat & reducing agent (loses 3D structure and has - charge)
- Load on polyacrylamide gel
- Voltage to separate (-ve proteins -> + electrode, smaller moves faster)
- Visualize
Mercaptoethanol function
reduces disulfide bonds
Visualization with SDS Gel Electrophoresis
-Coomassie blue (CB) detects protein with dye
-Autoradiography (AR) detects radioactive proteins on film
Membrane fluidity determined by ____
organellar lipid composition, cytoskeleton, protein-protein interactions
Membrane fluidity is typically measured via ____
GFP-based protein indicators inserted into specific cellular membranes
Photoactivation (def.)
-Molecule modified with fluorescent tag
-blurry image indicates protein movement over time (Kinetics of signal spread yields rate of diffusion)
Fluorescence Recovery After Photobleaching (FRAP)
-bleach and recovery indicates rate of diffusion
