Lecture 12- Cell Biology of Neurons 2 Flashcards
What is the central dogma of biology?
- DNA is transcribed to mRNA (messenger RNA)
- mRNA three-part code is translated to an amino acid by ribosomes
- Amino acids are assembled into chains to form proteins
(basically DNA to RNA, RNA to protein)
What goes a Nissl stain show?
- Nissl was found to be selective for RNA and so Nissl granules (dark patches) represents large stacks of rough endoplasmic reticulum.
- These stacks are close to the soma of the cell therefore Nissl cells are good for visualizing cell bodies
What is meant by the term phospholipid bilayer?
- This is what the cell membrane is
- Hyrophobic ends in middle, Hyrophilic ends inside/outside
- Not permeable to ions therefore need proteins to allow function (transport/ channels allow ions to cross)
What is the structure of the proteins embedded in the cell membrane?
- May span membrane, or be inserted partway
- Complex folded 3D structure
- Composed of subunits (same/different)
Are Synaptic vesicles (SSV and LDCV) simple phospholipid spheres?
No have multiple membrane associated proteins
What is an essential feature of a neuron’s membrane in allowing it to be part of a communication network?
- Needs to be polarized (different charges indifferent parts)
- Proteins associated with the neuronal membrane are essential for this as allow specificity
What is the result of a neuron having a large membrane area?
- 200x greater membrane area than the diameter of a neurons soma
- Therefore need to make lots of proteins to maintain this area and so need lots of RER and specialized vesicles
What is the cytoskeleton made of?
Proteins
What can changes to membrane proteins cause?
- Proteins are essential in neuronal function and need to be in the ‘right’ place
- Changes to the location/ shape of these proteins causes dysfunction
How do kinases and phosphates work to create cellular energy?
- Protein Kinases add a phosphate group from ATP activating the target and forming ADP (byproduct)
- Protein Phosphatases remove a phosphate group deactivating target
What is the general method for identifying and locating proteins?
- Called immunohistochemistry
- Have protein of interest (antigen: receptor, vesicle etc.) which we manufacture a primary antibody for in a species like a goat or rabbit. The secondary antibody is what then binds to the primary antibody (often called an anti-goat or anti-rabbit depending on the original species the antibody was made in).
- It’s a marker that binds to the secondary antibody that can be visualized and allows us to locate the protein of interest in electron microscopy. The marker can either be a fluorescent molecule (shine light in order to see) or enzyme (creates a coloured by product via a substrate)
What can the location of proteins (obtained via immunohistochemistry tell us)?
- About their function/ precision of function and structure
- Also can help us tell the difference between axons and dendrites
What is ankG and where is it found?
- scaffolding protein
- proximal axon
- nodes
What is Caspr and where is it found?
- transmembrane protein
- member of neurexin family
- cell adhesion
What is MAP2 and where is it found?
- microtubule associated protein
- absent in most astrocytes
What is PO and where is it found according to immunohistochemistry?
Myelin protein
What does immunohistochemistry show in regards to dendrites?
- Dendrites come up as green and F-actin as red
- When merge/ both present get yellow
- Picture shows yellow only in the spines
- This matches what we know about the function of actin as it helps to grow/ stabilize the dendritic spines
What is important when using immunohistochemistry?
Need to have some idea of a proteins function/ location before you then go and stain for it as you can’t stain everything only specific things. Not an exploratory thing. Need to use it to confirm hypotheses.
Is a synaptic clef just a space?
- No, its a very dense fibrous matrix of associated proteins (Appears dark in TM)
- Bunch of proteins stick through both membranes (the pre and post synaptic membranes) to hold them together preventing widening of the synapse/ gap
What is the difference in specialization of the pre and post-synaptic membranes?
-Pre= specialized for neurotransmitter release (contains active zones).
Contains: Calcium channels, Docking proteins for vesicles (SNAREs etc), Neurotransmitter autoreceptors, Reuptake channels, Vesicle accessory proteins
-Post= specialized for receiving neurotransmitters (contains receptors).
Contains: Voltage gated ion channels. G-Protein coupled receptors.
Why is does the soma use so much energy/ ATP?
Because of the major requirement to make many diverse synaptic proteins
What is PSD-95? What does it illustrate?
-Scaffold protein, under the plasma membrane for the clustering of
receptors, ion channels etc.
-Shows the importance of synapse proteins remain in the correct location
Complex neuron shapes need to…
How is this accomplished?
- Maintain their shape
- Stay connected to next neuron (at synaptic cleft)
Accomplished via:
-Clef proteins (trans-synaptic) which are secreted from the presynaptic membrane and adhere/ interact with molecules on the surface of adjacent cells.
How do neurons utilize myosin?
Associate with actin, similar to function in muscle. Help movement/ trafficking.
What is the purpose of synaptic tagging?
-To let the actin/ trafficking mechanism know which synapse to drop cargo at (hypothesized mechanism)
What is local protein synthesis?
- mRNA transported to synapse and only transcribed into protein on synaptic activation
- Location of polyribosomes at the post synaptic membrane supports this idea
What is FISH?
- Fluorescence in situ hybridization
- Have target RNA. A fluorescent tag tells us where the RNA is as turns on when bound to target.
- This can be useful as it can indicate what proteins are being formed.
What does puromycin labelling tell us?
- Allows the identification of newly synthesized proteins (contain puromycin)
- Antibodies against puromycin + target (means we identify the proteins we are interested in not just all proteins).
- Cause a PLA signal
Why does there need to be local protein synthesis?
Because there is so many dendritic branches and neurons are big. If all proteins were made in the soma and were trafficked would take too long + too much energy.
What is a case where protein synthesis goes wrong?
- Alzheimer’s disease (Tau, amyloid beta)
- Treatments could target this
