MCDB 153 Midterm 2 Experiments

What happened to a section of a growth cone with GFP-labeled tubulin that was "bleached out" with a laser beam over time? What did these results imply?

The bleached area did not move. This result implies that any new tubulin subunits were added at the end of the axon.

What happened to a section of a growth cone with GFP-labeled actin that was "bleached out" with a laser beam over time? What did these results imply?

The bleached area that was originally at the tip of the filopodia became incorporated into the axon cortex. This result implies that filamentous actin is assembled in the filopodia of the growth cone and some is left behind in the axon cortex.

What happened to a section of a growth cone with GFP-labeled membrane that was "bleached out" with a laser beam over time? What did these results imply?

The distances between different marked spots grew further apart over time. This result implies that new membrane gets added all along the stock.

How does the addition of Tau antisense to a growing neuroblast affect neurite outgrowth?

Addition of Tau antisense prevents any of the filopodia projected from becoming dominant and becoming an axon. They all remain dendrites.

When neurons in culture were exposed to NGF and vinblastine/colchicine (to disrupt MTs), what happened to the neurites?

The entire neurite collapsed. This suggested that microtubules were necessary to maintain neurite structure.

When neurons in culture were exposed to NGF and cytochalasin (to disrupt MFs), what happened to the neurites?

The entire neurite collapsed. This suggested that microfilaments were necessary to maintain neurite structure.

When neurons in culture were exposed to NGF and cytochalasin (to disrupt MFs) while in a polylysine dish, what happened to the neurites?

The growth cone of the neurite collapsed, but the neurite remained intact and grew slowly. This suggested that microfilaments were essential for growth cone structure and were likely involved in outgrowth but not solely responsible for outgrowth; something else likely contributed as well.

What was the question being asked in the limb bud removal experiment?

Is there a relationship between targets of innervation and the pool of innervating neurons?

In the limb bud removal experiment, how did they include a control?

They only removed the limb bud from one side of the chick embryo, so the other side was the control.

What was the result of the limb bud removal experiment?

There were many fewer neurons in the region of the spinal cord that would ordinarily innervate the "missing" limb.

What were some observations in Hamburger's supernumerary limb bud experiment?

The experimental (supernumerary) limb bud sides started with relatively the same number of neurons, but had a smaller decrease in the number of neurons as the embryos developed.

Why is Hamburger's supernumerary limb bud experiment not consistent with the recruitment model?

The recruitment model suggests that as time progresses, more neuronal cells are "recruited" into the developing nervous system. However, the gradual decrease in number of neurons over time suggests that neurons die over time instead.

What does the competition model of neuronal outgrowth suggest?

There is a limiting amount of survival factor that is proportional to the number of target cells that need to be innervated.

In an experiment where there were twice as many neuronal cells as usual, what happened to the absolute and relative number of neurons that survived?

The number of survivors stayed constant but the percentage of survivors decreased.

In an experiment where the number of neurons arriving at a single target decreased, what happened to the absolute and relative number of neurons that survived?

The number of survivors stayed constant but the percentage of survivors increased.

What happened when a sarcoma 180 tumor was implanted into a chick embryo next to the dorsal root ganglia on one side?

The dorsal root ganglia ended up being bigger on the tumor side. This suggests that there is some sort of chemoattractant for nerves in the tumor.

What experiment was performed to determine whether the survival factor present in sarcoma 180 cells was diffusable or insoluble? What was the result?

The tumor was placed on the other side of the allantoic membrane so that there would be no direct contact with any of the dorsal root ganglia being observed. Since enlarged ganglia were still observed even without direct contact, the survival factor was determined to be a diffusible (soluble) factor.

How was the survival factor from the sarcoma 180 purified?

Fractionation

Through multiple rounds, the starting material was separated into its individual components via a "molecular cheesecloth." Each "fraction" was assayed, with the one producing the desired effect fractionated with a different resin. Eventually you should have a purified preparation of your molecule of interest.

What was the "halo assay" and what was it used for?

Chick dorsal root ganglia were isolated and then cultured overnight in the different components that were fractionated from the sarcoma 180. If the survival factor was present in the chosen mixture, lots of extended neurites would form to make a "halo". If the survival factor was not present in the chosen mixture, only dead ganglia would be observed.

What experiment was performed to determine whether the survival factor that was isolated from the sarcoma 180 was a protein or a nucleic acid? What were the expected results?

The semi-pure sarcoma 180 sample was assayed with phosphodiesterase. Since phosphodiesterase cleaves the phosphodiester bonds in nucleic acids, a halo should form if NGF is a protein but should not form if NGF is a nucleic acid.

What was the result of the addition of phosphodiesterase to an assay of semi-pure sarcoma 180?

A huge halo formed; this did not align with the hypothesis that a normal-sized halo should form if NGF is a protein and no halo should form if NGF is a nucleic acid.

What was the result of the addition of phosphodiesterase to a buffer (a negative control) instead of a semi-pure sarcoma 180 sample?

A huge halo still formed, which meant that the starting material from which the PDE was purified was likely very contaminated to begin with.

What happened when extra purified NGF from salivary gland fractions was injected into sympathetic ganglia in mice?

The experimentals demonstrated a 2.5-fold increase in the number of nerve cells in sympathetic ganglia. This is consistent with the idea that more NGF means more neuronal survival.

What happened when an NGF-antibody was added to a halo assay of NGF in neurons?

No halo was formed, meaning that anti-NGF is an effective function blocker!

What happened when Anti-NGF function blocking antibodies were added to newborn mice and chicks?

Complete destruction of the nervous system, partial destruction of the sensory nervous system, and no effect on motor neurons. This suggests that there is a level of specificity in NGF.

There are very high levels of NGF in the superior cervical ganglia and stellate ganglia, yet no NGF mRNA in them. How is this possible?

Hint - both of these ganglia innervate sympathetic/sensory targets.

The target cells are making NGF, which is getting picked up by the axons and getting transported back into the cell body where it is accumulating.

Significant amounts of NGF mRNA were found in a couple regions of the brain, including the hippocampus and neocortex. Why were there no effect on these regions during the immunosympathectomy experiment?

Due to the blood brain barrier, antibodies against NGF couldn't enter the brain.

What question was being asked in the experiment where innervating neurons for heart cells (which produce NGF) were killed with 6-OH dopamine?

Might the arrival of the first growth cones be the signal to induce NGF synthesis in the target?

What was the result of the experiment where innervating neurons for heart cells (which produce NGF) were killed with 6-OH dopamine?

The heart cells still synthesize NGF at the normal time despite no growth cone arrival. This result suggests that NGF developing heart is not induced by the arrival of growth cones.

What was the purpose of fractionating and assaying pig brain to obtain BDNF (brain derived neurotrophic factor)?

Naturally occurring cell death occurs in the central nervous system, whose neurons are not affected by NGF. As a result, BDNF was produced to see if there were other target derived trophic factors acting in the CNS.

How was it determined whether there were more members of the neurotrophin family besides NGF and BDNF? What were the findings?

Oligonucleotide primers were synthesized corresponding to the regions of homology between BDNF and NGF. These primers were then used to amplify a brain cDNA library. The products with the sequence were amplified via PCR. In addition to NGF and BDNF, homologous NT3 and NT4 products were synthesized!

How is tyrosine phosphorylation relevant to NGF?

There is a major correlation between signal transduction from NGF and tyrosine phosphorylation, suggesting that tyrosine phosphorylation may play a role in the signaling cascade that NGF induces upon binding.

How did Pamela Maher determine that tyrosine phosphorylation correlated with NGF treatment?

She took PC12 cells and treated them with NGF for various times. For each time interval, she took western blots and immunoblotted the sample with anti-PY (phosphotyrosine) probes. The presence of bands indicated that there were phosphorylated tyrosine molecules present in the samples that were treated for NGF for a given amount of time (besides 0 minutes).

What experiment was performed to determine what proteins NGF interacts with on the plasma membrane of neuronal cells? What were the results?

Radioactive NGF was added to PC12 cells, with a covalent crosslinker to ensure that the NGF would stay attached to the proteins. The proteins were then fractionated on an SDS gel, with radioactivity detected (the radioactivity implied that the protein had bound to NGF).

There were two resulting bands that formed, one at 158 and one at 98 kDa. Since the NGF weighed 13 kDa per monomer, the data suggested that there was a 145 and 85 kDa protein that NGF binds to.

What was wrong with the idea that in addition to an 85 kDa protein binding to NGF alone, an additional 60 kDa protein complex could crosslink with it to form a 145 kDa protein complex binding to NGF?

Crosslinking is very inefficient and the probability of getting two crosslinks (one between protein complexes and one between the 85 kDa complex and NGF) is very low.

What experiment was performed to determine whether the 85+60 NGF complex binding was correct or not? What would be the potential outcomes and their implications?

Non radioactive NGF was added to PC12 cells with a reversible covalent crosslinker to ensure that the proteins and NGF would stay together. All the NGF containing protein complexes were purified in an anti-NGF column. Afterwards, all the remaining NGF containing complexes were labeled with radioactivity. The crosslinks were reversed, with the resulting proteins fractionated on an SDS gel.

If the old model with a 60 kDa protein binding to the 85 kDa protein was correct, there should be two bands at each of these respective sizes.

If the old model was incorrect, bands should be seen at other sizes.

What were the results of the 85+60 NGF complex binding experiment? What were the takeaways from these results?

No 60 kDa band exists! However, there is a band at 135 kDa. This suggests that the original model was wrong and that instead, there was a 135 kDa protein that is either an NGF receptor all by itself, or is part of a larger receptor complex.

What was responsible for the 135 kDa band discovered in the NGF receptor radioactivity experiment?

TrkA - a transmembrane protein that has an intracellular tyrosine kinase domain. It has the same distribution throughout the body as NGF.

TrkA is an orphan receptor. What does this mean?

It is an identified "receptor" with no known ligand. It is still considered a "receptor" because it looks like a receptor based upon its known structural and cell biological properties.

What were the results of the following experiment of adding NGF to PC12 cells for a certain amount of time and immunoprecipitating TrkA or PY out of the cells onto a western blot with SDS gel?

The 135 kDa protein blot was found on all time periods when immunoprecipitated with anti-TrkA and the 135 kDa protein blot was found on all time periods besides zero minutes when immunoprecipitated with anti-PY.

What is required to tyrosine phosphorylate TrkA?

NGF

When non-NGF responsive PC12 cell mutants are cultured in NGF, no neurite outgrowth is recorded and an immunoblot of cell proteins with anti-TrkA shows no bands. What conclusion can be drawn from this?

These cells lack TrkA.

When non-NGF responsive PC12 cell mutants are transfected with TrkA cDNA and then cultured in NGF, good neurite outgrowth is observed. An anti-TrkA and anti-PY immunoprecipitation of this sampleshow bands at the 135 kDa size. What conclusions can be drawn from this?

TrkA "rescues" mutant PC12 cells unable to respond to NGF, and TrkA is a functional NGF receptor.

What experiment was performed to demonstrate that NGF signaling can be acquired at the tips of growing neuronal processes to promote neuronal cell survival? What was the result?

Neuronal cells were placed in a culture dish with two chambers; one had the axon terminals and the other had the cell bodies. In the control, Anti-NGF was placed in the same chamber as the cell bodies. In the experimental, Anti-NGF was placed in the same chamber as the cell bodies and NGF was placed in the other chamber as the axon terminals. There was a significant increase in the amount of neuronal survival in the experimental, suggesting that NGF at the neuronal terminus is sufficient to confer neuronal survival.

Does the following occur in necrosis or apoptosis:

Initial swelling of the cell

Necrosis

Does the following occur in necrosis or apoptosis:

Minimal effects on chromatin

Necrosis

Does the following occur in necrosis or apoptosis:

Metabolically active suicide process

Apoptosis

Does the following occur in necrosis or apoptosis:

Cells shrink in size, with no swelling

Apoptosis

Does the following occur in necrosis or apoptosis:

Organelle breakdown

Necrosis

Does the following occur in necrosis or apoptosis:

Cell membrane rupture

Necrosis

Does the following occur in necrosis or apoptosis:

Chromatin condenses against nuclear envelope

Apoptosis

Does the following occur in necrosis or apoptosis:

Nuclear DNA fragmentation

Apoptosis

Does the following occur in necrosis or apoptosis:

Major inflammatory response - macrophages invade; phagocytosis of debris

Necrosis

Does the following occur in necrosis or apoptosis:

Cytoplasm and nucleus break up into small bodies and can be phagocytosed by adjacent cells.

Apoptosis

How does NGF prevent apoptosis?

Since cell death is an active process, NGF is repressing a death program by blocking the synthesis of certain proteins involved.

What do ced-3 and ced-4 do?

Transcription of proteins that promote cell death.

What does ced-9 do?

Transcription of proteins that prevent cell death.

What is the mammalian protein homolog to ced-3?

ICE (interleukin converting enzyme)

When taking fibroblasts in culture:

- No manipulation lead to 1% cell death

- Addition of a vector alone lead to 1.4% cell death

- Addition of mouse ICE cDNA lead to 93% cell death

- Addition of worm ced-3 cDNA lead to 93% cell death

- A point mutation of the active site of the cysteine protease lead to a 3.7% cell death

What conclusions can be taken away from this data?

- The data strongly suggests that ced-3/ICE can mediate cell death via its protease function in mammalian cells.

- Cell death machinery is in place and just needs a trigger or switch to be activated.

What is the mammalian protein homolog to ced-9?

Bcl-2

Low amounts of ___ must be a signal that growth cones are near to the their target.

FGF

Collapsin

A protein that acts as a repellant for DRG neurons.

DRG sensory neurons normally innervate the skin. What happens when collapsin is knocked out?

Neurons migrate into adjacent and inappropriate locations.

Ephrin usually acts as a border for the tectum which growing retinal ganglion cells grow into during development. What happens when ephrin A5 is knocked out?

They migrate right past the normal stop site / target.

When the optic nerve of a frog is cut and the retina is rotated 180 degrees, how will the ganglion cells regrow when they move back into the tectum?

The frog's system is 180 degrees inverted (rejecting Weiss's hypothesis). Therefore, we do not have functional molding.

Ephrins are ligands for Eph receptors, which are membrane bound proteins in a ________ system for developing nasal and temporal nerves.

Repellant

Reticular Theory

An obsolete theory that states that all neurons are fused together into one large "synctium".

Neuron Theory

A theory that states that there are many individual nerve cells with "synapses".

What experiment was performed to prove that neurexin and neurolignin were inductive and not just adhesive?

Non neuronal cells were transfected with neurolignin and co-cultured with growing neurons. The neuronal growth cones started making pre-synaptic proteins like synaptotagmin, which suggests that synaptotagmin is a pre-synaptic cell protein involved in vesicle release / exocytosis!

What is the regeneration experiment?

In adult frogs, the motor axons were cut and muscle cells were destroyed so that only the basal lamin (AKA ECM) was remaining. New myofibers were generated via cell division. ACh receptor formed directly on the regenerated muscle fibers beneath the synaptic portion of the old basal lamina on the same site as before!

Agrin

- Growth factor that promotes the development of a motor end plate in skeletal muscle

- Released by somatic nerve

- Clusters together & maintains ACh receptors in muscle development

- Creates an elaborate neuromuscular junction

- Does NOT change myoblasts in smooth muscle