Midterm 1 Flashcards
Dorsal root ganglion (DRG)
-contains sensory neuronal cell bodies
-one major incoming projection from periphery and one major outgoing projection enters CNS
Experimental Strategies
- Whole animals
- Cultured tissues from animals (explants)
- Cultured cells from animals
- primary cells: dissect out tissue from animals
- transformed cells: comes from tumors, cells are always dividing, immortal
Polyclonal antibodies
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
Monoclonal antibodies
B cells + transformed cells
-cells are immortal
-from same B cell, specific to one epitope only
-more time intensive
Immune fluorescence microscopy
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
How do you get antibodies to localize to an intracellular protein?
-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
How do you visualize a protein of interest in living cells?
-live cell imaging via GFP-fusion proteins
-viewed using fluorescence microscopy
-plasmid that encodes fused GFP+protein of interest into animal/cells of interest
Transgenic animals
Adding a gene (GOF)
-over expression of gene
Transfection
-adding a gene (GOF)
-plasmid (w/ cDNA of protein)+transfection reagent is added to media
-the DNA (gene of interest enters cells)
Delete a protein of interest
Knockouts (LOF) or CRISPR/Cas9
Dominant negatives (LOF)
-deletion of a protein
-positing of a multimeric complex
-mutant protein poisons normal protein complex, making it inactive
Function blocking antibody
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
SDS-PAGE
Linearize protein and assign negative charge, then separate by molecular size
Western blot/immunoblotting
-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
Hi
-using antibodies to identify specific proteins on gels
-coomassie blue: visualizes all proteins
Asymmetric division
-sister cells born different
-fertilized egg cytoplasm isn’t homogenous
-daughter cells have 2 different sets of genes
Symmetric division
-sister cells become different as result of influences acting on them after birth
-different environments/signals tell cells to change
Early frog development: up to blastula stage
-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
What are the key mechanisms to drive gastrulation?
- Changes in cell shape
- Cells rearrange their positions with respect to one another
- Filopodial-ECM interactions
Gastrulation (blastula to gastrula)
- Blastopore: epithelial cells at bottom invaginate, making a flat dome
-actin fibers begin to constrict, resulting invagination
-cytochalasin disrupts this - Cells extend filopodia to grab ECM, pulling the cell upwards/inwards
-strong lateral attachment btwn cells, receptors to ID the filopodia - Surface cells migrate towards blastopore, then inside embryo (tight lateral associations)
- End of gut tube contacts epithelial cells, making future mouth (other end is future anus)
- Cells on surface=ectoderm-> will either become epidermis or nervous system
Gastrula to Neurula State
-cells involving the IMZ migrate underneath neurogenic region (gastrulation)
-ectoderm: epidermal or neuronal fate
Colcemid
Blocks microtubule elongation in neural plate cells
Cytochalasin
Stops actin filaments from contracting
Cell determination
A cell is committed to a particular eventual fate, even though it might not exhibit any differentiated properties
Cell differentiation
Overt acquisition/ expression of differentiated properties
-ex: neurotransmitters by a neuron
Determination experiment
Not determined: donor cells conforms to host cells
Determined: donor cells remains the same, even though surrounding host cells are different
Amyloid precursor protein (APP)
-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
Semaphorin 3A
-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
Growth cone
-filopodia extend out ~50-100 um in length
-lamellipodial veils btwn filipodia
-both extend and retract-very dynamic
Different speeds and shapes of growth cones
-changes as it advances through different environments
-at target: sends out more filipodia once in target region
What are Ti axons?
-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
How does navigation work?
-single filopodia can direct growth cones
-filipodia sense environmental cue
-growth cones moves closer to environmental cue
Navigation by differential preferences of growth cones for INSOLUBLE ROADWAYS
-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”
Can growth cones prefer one insoluble substrate over another insoluble substrate?
Yes, grew towards polyornithine
-grew away from palladium
Insoluble “molecular roadways”
- Extracellular matrix: roadway produced by cells at an earlier time and then deposited extracellularly
- CAMs or Cadherins: roadway is cell along desired route expressing a corresponding CAM or cadherin
Integrin receptors
“Integrate” the ECM with the cytoskeleton
Guidance by gradients of diffusable molecules
-chemo-attractants and chemo-repellants
Soluble factors for chemotaxis
- growth cones use for chemoattractants and chemorepellants
- growth cones follows origin of attractant (NGF) and grows towards it
