Cell Structure and function homework

Color is

Objects that are larger than the shortest wavelength of visible light

Nanometer nm is `

10^-19 m

Light microscopy can detect light

object that is less than 200nm but it will be blurry

Conventional light microscopy resolve

2 objects less than 200 nm apart

The greater the numerical aperture (n sin theta)

the better the resolution (resolve objects closer together)

Numerical aperture oil vs air

Oil is higher

Longer wavelength light from microscope light source

the worse the resolution (objects far apart to resolve)

Noise in a light image

random fluctuation in distribution of photos that pass through specimen

Not a good way to visualize living cell, but best for different feature distinguishing

Light microscopy of fixed and stained specimen

Dye hematoxylin has affinity for negatively charged macromolecules can reveal subcellular localization of

dna, rna, acidic proteins

In situ hybridization detects specific

RNA

In situ hybridization uses probes made out of

DNA

Fluorescence microscopy used to detect specific protein if

Protein fluorescent domain

antibody binds protein is attached to fluorophore

Fluorescent probes used in fluorescence microscopy

absorb light and then emit light at longer wavelength

Light absorbed by fluorophore, leading to later emission of light is

excitation light

Indirect immunocytochemistry, marker molecule leads to detection is attached to

secondary antibody that binds to primary antibody that binds a specific antigen

Confocal microscopy produced optical sections of specimen by

excluding out of focus light

Cause least amount of damage to specimen and allow deep imaging into sepectrum

infrared light in 2 photon applications

FRET absorbs what wavelength by one component and secon leads to emission of what wavelength

from shorter to longer

FRET signal

close together

Fluorescence recovery after photobleaching detects

movement of tagged proteins or tagged molecules into bleached area

Method detect only targets close to surface of cover slip

TIRF

Switching fluorophores off and on can lead to pinpoint localization

Better than conventional or confocal fluorescence microscopy

Wavelength electron in electron microscope with 100,000 volts is 0.002 nm. So visible light with wavelength 400 nm has

200,000 fold longer wavelength than the wavelength of electrons

Which is better electron or light microscope

Electron because resolves objects closer together

Practical resolution of electron microscope for biological objects are 1 nm because

problems with specimen preparation

contrast with specimen

damage to specimen from high velocity electrons

practical resolution for electron microscopy is

200 times better than light microscopy with 200 nm wavelength

Beam electrons travels through specimen in

TEM transmission electron microscopy

Immunogold microscopy would be appropriate for

electron microscopy (gold particles)

3D images of specimen by

multiple TEM images taken at different angles then combined by image processing (em topography)

Taking images of identical molecules then combining them to product averaged image is

single particle reconstruction

Phospholipid

Blue:polar end/ OH group/ WATER interacts with polar heads
Green: phosphate group/ WATER interacts with polar heads
Red: who knows
cis disulfite bonds: INCREASES FLUIDITY

How to know which is NOT phospholipid

The one without charge

Micelle

Least likely to move in lipid bilayer is

flipflop

Phospholipids synthesized on one leaflet of lipid bilayer, movement of one of these lipids to other leaflet

requires flippases or translocases (transport)

Lipid bilayer with greater distance/ longer membrane spanning proteins?

amphipathic lipid with SATURATED fatty acids. Saturated is straight, unsaturated is bend with cis bond

Greater fluidity in lipid bilayer

amphipathic lipid with UNSATURATED fatty acid tails (prevent tight packing)

Extracellular side has blue

Because GPI anchor is on top, extracellular on top

Protein that is integral membrane by means of lipid anchorage (no transmembrane) on cytosolic side

Number 5

Peripheral membrane in cytosol

Number 7

Protein anchored in membrane by a-helix interacts with only one membrane leaflet

Number 4

GPi anchored protein attached to

inside (lumen) of the ER on non-cytosolic side

anchors are hydrophobic so they stick to membrane bilayer

GTPases of Rab protein can anchor in lipid bilayer because binding to GTP causes exposure of

hydrophobic peptide of lipid anchor domain of protein

Most common membrane spanning structure is

alpha helix. First is one membrane spanning, second is 7 membrane s[anning

Which occurs first?

Insert hydrophobic helices
associate membrane spanning helices, displaces interactions

Insert hydrophobic helices

Amphipathic helices have hydrophobic side chains facing one side and semi-hydrophilic facing other.

The lipid portion is rich in hydrophobic side chains

Series of 10 amino acids to span membrane

beta barrel

Proteins glycosylated in lumen of ER, if in plasma membrane then sugar faces

extracellular space

Disulfide bonds least likely to occur on surfaces of proteins

exposed to reducing environment of cytosol

Strongest detergent (polar end interacting strongly with water)

SDS

Detergents do

concentration increases linearly but then no longer rises once concentrations hit critical micelle concentration

1Hydrophobic portions of detergents interact with/ 2water react with

1hydrophobic amino acid side chains or lipids/ 2polar or charged groups

SDS is strong detergent because

interferes with protein secondary and non-covalent tertiary structure, denature proteins

Experiment fused mouse cell with human cells show

proteins diffuse around leaflet of lipid bilayer

Access movement of membrane component into area of membrane bleaches with fluorescence of fluorescently tagged molecules is

FRAP= photobleaching

Which one restrict lateral diffusion of plasma membrane protein by cell to cell contact by cell surface proteins?

last one

Which prevents protein diffusion around lipid bilayer by self association of particular protein into large aggregates

First one

Which prevent lateral diffusion of transmembrane protein by anchorage to elements of cytokeleton

Third one

Membrane curvature caused by curved membrane surface

Third one

Microtubules have diameter 25 nm. Confocal microscopy, diameters appear to be 10x greater why?

best resolution with violet light is around 200 nm

If condenser lens move closer to specimen, what happens to theta and resolution

increases theta and improves resolution

Combined fluorescence image of all that

Sequential collection of signa using 3 different excitation/emission filter sets then combining data into one image?

Good pair for fluorescence resonance energy transfer to assess interactions between 2 proteins?

CFP/GFP

Phase contrast imaging

looking at living cells without stan or fluorophore

TEM image/ forms image from electrons that

PASS THROUGH SPECIMEN

electron microscopy approach to generating 3d reconstruction

many TEM images of a single specimen that tilted between images.

2 different proteins using immunogold electron microscopy

2 different sized gold particles are used

Plasma membrane has low cholesterol content, so this membrane

is NOT lipid draft

Amphipathic lipid monolayer of intracellular lipid droplets is

derived from the cytosolic leaflet of the ER membrane

Which anchored by single amphipathic helix?

protein 4

Glyco—-lol anchors insert into leaflet of membrane because

hydrophobic groups

Peripheral membrane protein covalently linked by disulfite bond to integral protein

in the extracellular space

Cytisolic, peripheral membrane protein?

protein 7

Which can be released into extracellular fluid by removing its anchor?

protein 6

NOT resolvable by conventional light microscopy?

ribosome

Detection of mRNA or base pairs by

in situ hybridization

Involve specimen staining before imagine?

bright field microscopy

Proteins in lipsosome

get their facing that way by random inforporation

Not required for Na=/k+ atphase moving Na+ out of liposome

extra liposomal na+

Ions

Extracellular: Na+, Ca2+

Cytosol: K+

pH higher in the

extracellular fluid in the cell

Cross a lipid bilayer

Easy: Non polar molecules like o2,co2

Hard: ions like H+,Na+,K+,

Limiting factor of simple diffusion of molecule across lipid bilayer is

ability to interact with hydrophobic interior of lipid bilayer

Hydrophobic group?

CH3

Carrier or transporter?

Left

Direction of net movement of solutes shown is down concentration gradient, which way solutes move?

either in or out of the cell, depend on concentration gradient

Kind if transporter/carrier- or channel mediated movement that goes in direction of concentration gradient from high to low

passive transport

Active transport channel is on thr

right

Simple diffusion

direct diffusion of molecule across lipid bilayer without involve membrane protein

Researcher does experiment involve rate of movement across lipid bilayer. Movement rate keeps going up as solute concentration increases and never saturated. This movement is

Simple diffusion or channel mediated transport

When protein couples ATP hydrolysis with pumping a solute across lipid bilayer to create chemical or electrochemical gradient for that solute, then solute back across lipid bilayer down gradient of another solute

secondary active transport

Common feature of

coupled to some potential energy source like atp

UNIPORTER SYMPORTER ANTIPORTER IN THAT ORDER

Sodium dependent glucose transporter is

symport

Na/K pump created gradient that drives glucose uptake by sodium dependent glucose is

secondary active transport

No need for active transport to create sodium gradient

small intestine after ingestion of foods or sodium beverages