Midterm 1

Question 1

Dorsal root ganglion (DRG)

Answer 1

-contains sensory neuronal cell bodies
-one major incoming projection from periphery and one major outgoing projection enters CNS

Question 2

Experimental Strategies

Answer 2

1. Whole animals
2. Cultured tissues from animals (explants)
3. Cultured cells from animals
   - primary cells: dissect out tissue from animals
   - transformed cells: comes from tumors, cells are always dividing, immortal

Question 3

Polyclonal antibodies

Answer 3

Antibodies produced by multiple clones of B cells and bind to variety of epitopes
-from different B cells, different epitopes on the same protein
-less time intensive

Question 4

Monoclonal antibodies

Answer 4

B cells + transformed cells

-cells are immortal
-from same B cell, specific to one epitope only
-more time intensive

Question 5

Immune fluorescence microscopy

Answer 5

Direct: antibody that is directly labeled with fluorescence binds to protein of interest
Indirect: primary antibody recognizes target, secondary antibody tarted constant region of primary Ab

Question 6

How do you get antibodies to localize to an intracellular protein?

Answer 6

-antibodies can’t normally pass through plasma membrane
-treat cells with non-ionic detergent to solubize protein
-treat with a fixative to lock proteins in place
-proteins are now dead
-antibodies can now localize to the intracellular proteins

Question 7

How do you visualize a protein of interest in living cells?

Answer 7

-live cell imaging via GFP-fusion proteins
-viewed using fluorescence microscopy
-plasmid that encodes fused GFP+protein of interest into animal/cells of interest

Question 8

Transgenic animals

Answer 8

Adding a gene (GOF)
-over expression of gene

Question 9

Transfection

Answer 9

-adding a gene (GOF)
-plasmid (w/ cDNA of protein)+transfection reagent is added to media
-the DNA (gene of interest enters cells)

Question 10

Delete a protein of interest

Answer 10

Knockouts (LOF) or CRISPR/Cas9

Question 11

Dominant negatives (LOF)

Answer 11

-deletion of a protein
-positing of a multimeric complex
-mutant protein poisons normal protein complex, making it inactive

Question 12

Function blocking antibody

Answer 12

Antibody that binds to and directly interferes with the function of its target
-difficult to find an exact function blocking antibody that works for a specific protein of interest

Question 13

SDS-PAGE

Answer 13

Linearize protein and assign negative charge, then separate by molecular size

Question 14

Western blot/immunoblotting

Answer 14

-run SDS-PAGE then blot onto membrane and probe with antibodies that are specific to a protein
-used to identify specific proteins on gels
-coomassie blue: visualizes all proteins

Question 15

Hi

Answer 15

-using antibodies to identify specific proteins on gels
-coomassie blue: visualizes all proteins

Question 16

Asymmetric division

Answer 16

-sister cells born different
-fertilized egg cytoplasm isn’t homogenous
-daughter cells have 2 different sets of genes

Question 17

Symmetric division

Answer 17

-sister cells become different as result of influences acting on them after birth
-different environments/signals tell cells to change

Question 18

Early frog development: up to blastula stage

Answer 18

-fertilized egg isn’t homogenous/ is asymmetrical
-1st and 2nd cleavage furrow (N to S direction)
-3rd (W to E direction)
-blastocoel: fluid filled cavity
-epithelial layer;tight lateral associations; CAMS

Question 19

What are the key mechanisms to drive gastrulation?

Answer 19

1. Changes in cell shape
2. Cells rearrange their positions with respect to one another
3. Filopodial-ECM interactions

Question 20

Gastrulation (blastula to gastrula)

Answer 20

1. Blastopore: epithelial cells at bottom invaginate, making a flat dome
   -actin fibers begin to constrict, resulting invagination
   -cytochalasin disrupts this
2. Cells extend filopodia to grab ECM, pulling the cell upwards/inwards
   -strong lateral attachment btwn cells, receptors to ID the filopodia
3. Surface cells migrate towards blastopore, then inside embryo (tight lateral associations)
4. End of gut tube contacts epithelial cells, making future mouth (other end is future anus)
5. Cells on surface=ectoderm-> will either become epidermis or nervous system

Question 21

Gastrula to Neurula State

Answer 21

-cells involving the IMZ migrate underneath neurogenic region (gastrulation)
-ectoderm: epidermal or neuronal fate

Question 22

Colcemid

Answer 22

Blocks microtubule elongation in neural plate cells

Question 23

Cytochalasin

Answer 23

Stops actin filaments from contracting

Question 24

Cell determination

Answer 24

A cell is committed to a particular eventual fate, even though it might not exhibit any differentiated properties

Question 25

Cell differentiation

Answer 25

Overt acquisition/ expression of differentiated properties
-ex: neurotransmitters by a neuron

Question 26

Determination experiment

Answer 26

Not determined: donor cells conforms to host cells
Determined: donor cells remains the same, even though surrounding host cells are different

Question 27

Amyloid precursor protein (APP)

Answer 27

-transmembrane protein
-RNAi knockdown leads to aberrant migration of newly born neurons from the VZ and aberrant layer formation
-if excess APP is added, migration proceeds too far and goes past end of radial glia

Question 28

Semaphorin 3A

Answer 28

-not a CAM, is a diffusable protein
-forms a gradient, high levels at the exterior of the neural tube to lower levels at VZ
-chemoattractant for migrating neurons

Question 29

Growth cone

Answer 29

-filopodia extend out ~50-100 um in length
-lamellipodial veils btwn filipodia
-both extend and retract-very dynamic

Question 30

Different speeds and shapes of growth cones

Answer 30

-changes as it advances through different environments
-at target: sends out more filipodia once in target region

Question 31

What are Ti axons?

Answer 31

-use local cues to navigate
-closely spaced landmark cells that have a strong affinity to growth cone
-sequential guide post cells are spaced close enough to one another for the filopodia to find them

Question 32

How does navigation work?

Answer 32

-single filopodia can direct growth cones
-filipodia sense environmental cue
-growth cones moves closer to environmental cue

Question 33

Navigation by differential preferences of growth cones for INSOLUBLE ROADWAYS

Answer 33

-growth cones encounter different insoluble molecular micro environments (ex: CAMS)
-growth cones are attracted/blind to some and are repelled by others
-attractants are like “molecular roadways”
-repellants are “guardrails”

Question 34

Can growth cones prefer one insoluble substrate over another insoluble substrate?

Answer 34

Yes, grew towards polyornithine
-grew away from palladium

Question 35

Insoluble “molecular roadways”

Answer 35

1. Extracellular matrix: roadway produced by cells at an earlier time and then deposited extracellularly
2. CAMs or Cadherins: roadway is cell along desired route expressing a corresponding CAM or cadherin

Question 36

Integrin receptors

Answer 36

“Integrate” the ECM with the cytoskeleton

Question 37

Guidance by gradients of diffusable molecules

Answer 37

-chemo-attractants and chemo-repellants

Question 38

Soluble factors for chemotaxis

Answer 38

• growth cones use for chemoattractants and chemorepellants
• growth cones follows origin of attractant (NGF) and grows towards it