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z:Cell 300 > Section 1: Membranes + compartments > Flashcards

Section 1: Membranes + compartments Flashcards

(84 cards)

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1
Q

membranes define ___ from ___

A

self from non-self

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2
Q

membranes contain _____

A

life-containing reactions

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3
Q

eukaryotic cells are bigger than prokaryotic cells which indicates ____

A

more complexity regarding possible rxns

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4
Q

large eukaryotic cells requires ____ for efficiency

A

compartmentalization

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5
Q

endomembrane system separates the cell into different compartments, or organelles, such as _____ (4)

A

the nucleus, the endoplasmic reticulum (ER), the Golgi apparatus, and lysosomes

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6
Q

compartmentalization happens through ____

A

endomembranes

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7
Q

compartmentalization: endomembranes have 2 advantages

A

-separate different classes of reactions from each other

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8
Q

3 reasons of using yeast cells as model organism

A

-easier to study (less complex)
-easier to grow
-easier for creating knockouts (gene deletion)

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9
Q

4 functions of membranes

A

-semi-permeable barrier
-assembly-line for metabolic reactions &enzymes
-gives cells & organelles identity
-signaling between organelles & cells

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10
Q

semi-permeable barrier: gases + small uncharged molecules

A

yes, they can move through unobstructed

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11
Q

semi-permeable barrier: ions + large molecules

A

No, they can’t move through unobstructed

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12
Q

glycosylation + ATP production in mitochondria both employ a ____

A

assembly-line for metabolic reactions within membranes (ER & mitochondria)

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13
Q

ER + mitochondria interact through ___ to facilitate ____

A

-ER tubules wrapping around mitochondria
-mitochondria fission

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14
Q

biological membranes consist of a bilayer sheet that is ___thick

A

5nm (large variation though)

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15
Q

membrane lipids are _____(3)

A

-primarily phospholipids, sphingomyelin + cholesterol, small amount of glycolipids

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16
Q

phospholipids in membranes (4)

A

-phosphatidylcholine/PC
-phosphatidylethanolamine/PE
-phosphatidylinositol/PI
-phosphatidylserine/PS

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17
Q

phospholipids in membranes in order of abudance

A

PC > PE > PI > PS

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18
Q

headgroup of phospholipids determines ____

A

charge

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19
Q

shape of phosphatidylethanolamine/PE

A

-more conical, limits fluidity, and adds curvature to membranes

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20
Q

shape of phosphatidylcholine/PC

A

-cylindrical, less curvature

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21
Q

membrane composition affects ___ & _____

A

thickness & curvature

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22
Q

function of cholesterol

A

-makes lipid rafts that increases thickness, attracts proteins and stiffens membranes

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23
Q

Membrane lipids can be isolated using which technique?

A

Thin-layer chromatography (TLC)

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24
Q

Transverse membrane asymmetry (_____) depends on ____(2)

A

-movement across the two leaflets
-asymmetric lipid composition
-protein orientation during insertion

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25
PS and PE are on the ____ leaflet via ___
-inner -ATP-dependent flippase
26
Glycolipids are on the ____ leaflet
-outer
27
protein insertion into membranes _____
confines functions to either side
28
what happens to membrane asymmetry when cells die?
membrane asymmetry is lost
29
Lateral asymmetry is ____
movement witin one leaflet
30
Lateral asymmetry is set by ____
lipids rafts forming thicker areas in membrane, attracting proteins
31
Lipid rafts are rich in __ & ____
-cholesterol & sphingomyelin
32
which phospholipid is present around curves of membranes
PE
33
Organelles membrane identity determined by ___
phospholipid type
34
morphological methods in cell biology describes _______ (4) of cells
the shape, structure, form, and size
35
3 main types of morphological/microscopical techniques
-light microscopy -fluorescence microscopy (similar to light) -electron microscopy
36
The different types of morphological/microscopical techniques offer ____
different resolution
37
Highest resolution: electron or light microscopy
electron microscopy
38
5 methods for the characterization of intracellular compartments
1. Histology 2. Immunofluorescence microscopy 3. Electron microscopy 4. Live cell imaging by phase contrast 5. Live cell imaging by fluorescence of GFP-tagged proteins
39
Histology gives insight into ______
tissue structure
40
Immunofluorescence microscopy allows us to detect the _____
location of a specific protein
41
Electron microscopy gives insight to _____
fine structure + localization of proteins
42
Live cell imaging by phase contrast or fluorescence of GFP-tagged proteins allows us to ____
monitor cells in real time
43
Histology general protocol
1. Chemical fixation 2. Washing samples with alcohols 3. Cleaning with xylol/toluol to remove alcohols 4. Cutting with microtome and mounting on coverslip 5. Staining with hematoxylin & eosin
44
Hematoxylin stains _____ a ___ color
-the nucleus -deep blue-purple
45
Eosin stains _____ a ____ color.
-the cytoplasm -orange-pink-red
46
Cutting of samples for analysis performed by _____
-microtome
47
Immunofluorescence general protocol
1. Chemical fixation 2. Washing samples, block excess proteins, (optional: permeabilizing) 3. Incubate with primary antibodies + washing 4. Incubate with secondary, fluorescent antibodies + washing 5. Mounting + imaging
48
In immunofluorescence, which antibodies are fluorescent?
secondary antibodies
49
Immuno-localization reveals both _____and _____of organelles
shape and protein content
50
With immuno-localization, controls are needed to ____
determine the proteins visualized
51
evolution of fluorescence microscopy
widefield -> confocal (sharper images) -> STED (see antibodies)
52
electron microscopy
1. Chemical fixation 2. Staining with uranyl acetate + dehydration 3. Cutting in thin slices 4. (Opt.) Incubate with primary + secondary, gold-labelled antibodies 5. Mounting + imaging
53
electron microscopy: sizes of gold particles on antibodies indicates ___
different antigens (proteins, organelles markers)
54
3D analysis electron microscopy
tomography
55
Live cell imaging protocol
1.Transfect cDNA expressing GFP-fused protein of interest 2. Mount on microscope with growth medium 3. Imaging
56
Live cell imaging reveal both ___ and ___
shape and time-dependent movement
57
Live cell imaging: incident light ____
must be controlled
58
EM best for live or fixed cells?
fixed cells
59
Fluorescence microscopy best for live or fixed cells?
live (GFP) + fixed cells (Immunofluorescence)
60
Things that affect morphological methods
1. Native structures can't be affected by methods 2. Specificity of primary antibodies 3. GFP hybrids don't affect protein + organelle function 4.Detergents that permeabilize membranes + make structure accessible to antibodies
61
homogenization involves ____
breaking cells in a mechanical way to produce homogenate
62
homogenization: ER
ER unlikely to be intact and becomes microsomes
63
Microsomes (def.)
ER fragments
64
Following homogenization, membranes can be separated based on _____
- size - density - protein composition
65
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
66
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
67
Velocity centrifugation: slow-sedimenting component
smaller organelles (relatively higher up)
68
Velocity centrifugation: fast-sedimenting component
bigger organelles (relatively lower down)
69
Equilibrium density centrifugation
-cell homogenate on steep sucrose gradient -Centrifuge to equilibrium -Collect fractions from bottom of tube -Recover organelles of different density
70
Velocity centrifugation vs. Equilibrium density centrifugation
masses vs. density
71
Equilibrium density centrifugation organization
low-density (top) --> high density (bottom)
72
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
73
Membrane proteins are soluble only in ____ ( in _____) for analysis outside native membranes + structures
detergents (in excess)
74
Shape of detergents vs shape of phospholipids
conical vs cylindrical
75
critical micelle concentration (CMC) (def.)
the concentration of surfactants above which micelles form
76
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
77
proteoliposomes (def.)
-systems that mimic lipid membranes (liposomes) with a protein
78
SDS Gel Electrophoresis
1. Denature with SDS, heat & reducing agent (loses 3D structure and has - charge) 2. Load on polyacrylamide gel 3. Voltage to separate (-ve proteins -> + electrode, smaller moves faster) 4. Visualize
79
Mercaptoethanol function
reduces disulfide bonds
80
Visualization with SDS Gel Electrophoresis
-Coomassie blue (CB) detects protein with dye -Autoradiography (AR) detects radioactive proteins on film
81
Membrane fluidity determined by ____
organellar lipid composition, cytoskeleton, protein-protein interactions
82
Membrane fluidity is typically measured via ____
GFP-based protein indicators inserted into specific cellular membranes
83
Photoactivation (def.)
-Molecule modified with fluorescent tag -blurry image indicates protein movement over time (Kinetics of signal spread yields rate of diffusion)
84
Fluorescence Recovery After Photobleaching (FRAP)
-bleach and recovery indicates rate of diffusion

z:Cell 300 (17 decks)

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