Lecture 6: fluorescence microscopy - research C. elegans Flashcards
Note: based on lecture slides and not on lecture itself Note2: sorry I don't understand half of this lecture, I lost it at powerpoint slide 13/14
What is meant by the fact that cilia are the cell’s antenna?
Cilia are microtubule-based organelles that project to the outside of the cell surface and sense the chemical and/or physical extracellular environment. (Depending on whether cilia are motile, they can be responsible for locomotion)
What are amphid sensilla?
Amphids are a pair of laterally located sensilla in the head that are open to the oustide at the sides of the lips. They are the largest chemosensory organs of C. elegans. As can be seen in the picture.
The cilia of C. elegans are composed of several segments. Name these.
- Base, located towards the cell body.
- Transition zone
- Proximal segment
- Distal segment
- Tip, located at the end of the cilia
How is this chemosensory organ, the amphid sensilla built up?
The amphid neurons cell bodies are located anteriorly and posses axons that associate with the cell body (soma). The dendrites of these neurons extend anteriorly and terminate with diverse cilated structures. Study this picture closely.
What is intraflagellar transport (IFT)?
Bidirectional motility along axonemal microtubules. It is essential for the formation and maintenance (ciliogenesis) of cilia and flagella.
What proteins are important in intraflagellar transport (IFT)?
- Three motor proteins: kinesin-II, dynein, OSM-3
- IFT particles → IFT-A and -B
Explain the general mechanism of intraflagellar transport in C. elegans chemosensory cilia.
IFT particles A and B are transported and moved along microtubuli with the help of motor proteins that are attached to the microtubuli and IFT particles. There are two directions IFT particles can be moved, anterograde (towards the flagellar tip/positive end) and retrograde (towards the cell body/negative end). IFT particles are moved anterograde with the help of kinesin-II, where cargo is handed over to OSM-3. IFT particles are moved retrograde with the help of Dynein.
What is researched here in regard to intraflagellar transport (IFT) in C. elegans chemosensory cilia?
What the dynamics of the IFT machinery consist of and what cargo membrane proteins are involved in sensing.
What is seen here?
What you see are blobs of the cargo trains moving together. There are too many to discriminate them. The cargo’s are transported from base to tip, but also there’s movement in the opposite direction.
What is meant by IFT-trains and by IFT-train velocity?
- IFT-train: ciliary cargo, i.e. IFT-A and IFT-B
- IFT-train velocity: the speed of cargo movement.
What determines the IFT-train velocity?
The location on the microtubuli → the velocity increases as the distance from base increases. (Fluorescent intensity stays constant indicating intact cargo movement).
When cargo is moved anterograde, kinesin-II hands over its cargo to OSM-3. What is the difference between these two?
Kinesin-II is a slow motor protein, while OSM-3 is a fast motor protein.
Here is a picture of individual kinesin-II and OSM-3 proteins. The picture is made by photobleaching GFPs. Why is photobleaching necessary?
Because the fluorescence intensity is too high to look at individual motor proteins. So if you photobleach the GFP on these motor proteins, you will randomly destroy them. If you do this long enough you will end up with a few GFP particles, which are still fluorescent. In this way you can analyze single motor proteins
What is seen in this picture of individual motor proteins?
What you can see is that two kinesin-II molecules make a U-turn. Kinesin-II can only walk to the plus end direction. What needs to happen here is that the motor protein falls of the train and docks to another train which is moving in the opposite direction. The kinesin-II is first the driver of the train and after falling of it will turn into a passenger, so it binds to the cargo. OSM-3 acts in the same way.
What is meant by the fact that drivers turn into passengers and vice versa?
When motor proteins kinesin-II hands over its cargo to OSM-3 and reaches the tip, it will flip over to the opposite side of the microtubuli so that the cargo can be moved retrogradely. With this also the motor protein OSM-3 is flipped. Now motor protein Dynein drives the cargo, while OSM-3 is still attached to the cargo as a passenger.
There is a specific place where kinesin-II hands over its cargo to OSM-3. Where?
Kinesin-II carries cargo between the base and start of the proximal segment. Kinesin-II hands over its cargo after this, where OSM-3 will carry the cargo from the transition zone to the tip.
Study this picture. This picture tells us that the different motor proteins have different roles. Briefly describe the roles of kinesin-II, OSM-3 and dynein.
- Kinesin-II: import and navigate in the transition zone which is the first µm of the cilium. Here, there are many proteins that are connecting the microtubules to the membranes. This is a filter for proteins which are allowed to go into the cilium.
- OSM-3: long distance transporter
- Dynein: multipurpose and can do everything on its own.
OCR-2 is a transmembrane cargo that is transported through intraflagellar transport (IFT). What is OCR-2?
OCR-2 is a TRPV (transient receptor potential) cation channel, an ion channel that is highly calcium selective and involved in signaling. It is important in sensation of pain, non-neuronal pressure sensation and osmosensation. Binding of ligands to the TRPV channel will cause Na+/Ca+ depolarization, i.e. neuronal stimulation.
How is OCR-2 normally distributed over the cilium?
It’s present all over the cilium, but around the transition zone and distal segment it’s present in higher concentrations.
What is seen when BBS-8 is knocked down in C. elegans in regard to OCR-2 distribution?
OCR normally is distributed around the transition zone and distal segment. When BBS-8 is knocked down, the highest concentration of OCR-2 can be found around the proximal segment.
