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

Section 1: Membranes + compartments Flashcards

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
Q

PS and PE are on the ____ leaflet via ___

A

-inner
-ATP-dependent flippase

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26
Q

Glycolipids are on the ____ leaflet

A

-outer

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27
Q

protein insertion into membranes _____

A

confines functions to either side

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28
Q

what happens to membrane asymmetry when cells die?

A

membrane asymmetry is lost

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29
Q

Lateral asymmetry is ____

A

movement witin one leaflet

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30
Q

Lateral asymmetry is set by ____

A

lipids rafts forming thicker areas in membrane, attracting proteins

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31
Q

Lipid rafts are rich in __ & ____

A

-cholesterol & sphingomyelin

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32
Q

which phospholipid is present around curves of membranes

A

PE

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33
Q

Organelles membrane identity determined by ___

A

phospholipid type

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34
Q

morphological methods in cell biology describes _______ (4) of cells

A

the shape, structure, form, and size

35
Q

3 main types of morphological/microscopical techniques

A

-light microscopy
-fluorescence microscopy (similar to light)
-electron microscopy

36
Q

The different types of morphological/microscopical techniques offer ____

A

different resolution

37
Q

Highest resolution: electron or light microscopy

A

electron microscopy

38
Q

5 methods for the characterization of intracellular compartments

A
  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
Q

Histology gives insight into ______

A

tissue structure

40
Q

Immunofluorescence microscopy allows us to detect the _____

A

location of a specific protein

41
Q

Electron microscopy gives insight to _____

A

fine structure + localization of proteins

42
Q

Live cell imaging by phase contrast or fluorescence of GFP-tagged proteins allows us to ____

A

monitor cells in real time

43
Q

Histology general protocol

A
  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
Q

Hematoxylin stains _____ a ___ color

A

-the nucleus
-deep blue-purple

45
Q

Eosin stains _____ a ____ color.

A

-the cytoplasm
-orange-pink-red

46
Q

Cutting of samples for analysis performed by _____

A

-microtome

47
Q

Immunofluorescence general protocol

A
  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
Q

In immunofluorescence, which antibodies are fluorescent?

A

secondary antibodies

49
Q

Immuno-localization reveals both _____and _____of organelles

A

shape and protein content

50
Q

With immuno-localization, controls are needed to ____

A

determine the proteins visualized

51
Q

evolution of fluorescence microscopy

A

widefield -> confocal (sharper images) -> STED (see antibodies)

52
Q

electron microscopy

A
  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
Q

electron microscopy: sizes of gold particles on antibodies indicates ___

A

different antigens (proteins, organelles markers)

54
Q

3D analysis electron microscopy

A

tomography

55
Q

Live cell imaging protocol

A

1.Transfect cDNA expressing GFP-fused protein of interest
2. Mount on microscope with growth medium
3. Imaging

56
Q

Live cell imaging reveal both ___ and ___

A

shape and time-dependent movement

57
Q

Live cell imaging: incident light ____

A

must be controlled

58
Q

EM best for live or fixed cells?

A

fixed cells

59
Q

Fluorescence microscopy best for live or fixed cells?

A

live (GFP) + fixed cells (Immunofluorescence)

60
Q

Things that affect morphological methods

A
  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
Q

homogenization involves ____

A

breaking cells in a mechanical way to produce homogenate

62
Q

homogenization: ER

A

ER unlikely to be intact and becomes microsomes

63
Q

Microsomes (def.)

A

ER fragments

64
Q

Following homogenization, membranes can be separated based on _____

A
  • size
  • density
  • protein composition
65
Q

Differential centrifugation (def.)

A

-Start from tissue/cell homogenate
-Spin at different speeds for various times
-pellets at different speeds contain cell components of various sizes

66
Q

Velocity centrifugation (also called rate-zonal sedimentation) (def.)

A

-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
Q

Velocity centrifugation: slow-sedimenting component

A

smaller organelles (relatively higher up)

68
Q

Velocity centrifugation: fast-sedimenting component

A

bigger organelles (relatively lower down)

69
Q

Equilibrium density centrifugation

A

-cell homogenate on steep sucrose gradient
-Centrifuge to equilibrium
-Collect fractions from bottom of tube
-Recover organelles of different density

70
Q

Velocity centrifugation vs. Equilibrium density centrifugation

A

masses vs. density

71
Q

Equilibrium density centrifugation organization

A

low-density (top) –> high density (bottom)

72
Q

Immuno-isolation of specific organelles

A

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
Q

Membrane proteins are soluble only in ____ ( in _____) for analysis outside native membranes + structures

A

detergents (in excess)

74
Q

Shape of detergents vs shape of phospholipids

A

conical vs cylindrical

75
Q

critical micelle concentration (CMC) (def.)

A

the concentration of surfactants above which micelles form

76
Q

Detergents disrupt the protein:lipid interactions that could be ____ so in the presence of excess lipids, sealed compartments can be reconstituted to yield ______

A

-critical for activity
-proteoliposomes

77
Q

proteoliposomes (def.)

A

-systems that mimic lipid membranes (liposomes) with a protein

78
Q

SDS Gel Electrophoresis

A
  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
Q

Mercaptoethanol function

A

reduces disulfide bonds

80
Q

Visualization with SDS Gel Electrophoresis

A

-Coomassie blue (CB) detects protein with dye
-Autoradiography (AR) detects radioactive proteins on film

81
Q

Membrane fluidity determined by ____

A

organellar lipid composition, cytoskeleton, protein-protein interactions

82
Q

Membrane fluidity is typically measured via ____

A

GFP-based protein indicators inserted into specific cellular membranes

83
Q

Photoactivation (def.)

A

-Molecule modified with fluorescent tag
-blurry image indicates protein movement over time (Kinetics of signal spread yields rate of diffusion)

84
Q

Fluorescence Recovery After Photobleaching (FRAP)

A

-bleach and recovery indicates rate of diffusion

z:Cell 300 (17 decks)

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