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23. What are filipodia and how do they relate to cytonemes? Another why to phrase this would be, compare and contrast filipodia and cytonemes, both structurally and functionally.
- Filipodia: Bundle of parallel actin filaments in actively elongating cells, extending and contracting. Role in cell movement by DIRECTING NERVE CONE outgrowth.
- Cytoneme: A type of filipodia that forms on STATIONARY cells. Role in sensory function, in their ability to SENSE morphogens via their RECEPTORs.
24. What are imaginal discs? When do they form? What do they form? What is the significance of the morphogenetic furrow in the eye disc and the AP boundry in the wing disc?
- Imaginal discs: Two cell layers, a columnar layer and a squamous perpipodial layer. Forms in the larvae and evert between the the larval EPIDERMIS and larval CUTICLE.
- Discs make filipodial extensions that attach to the monolayer, termed cytonemes, that orient towards morphogen producing cells.
- The Morphogenetic furrow in the eye disc: A wave of differentiation with the differentiated photoreceptors located posteriorly, and the undifferentiated cells ANTERIOR…only these have cytonemes associated with them.
- AP boundary in wing disc: Here cytonemes of these future blade cells orient towards the AP boundary in the wing disc, where morphogens are located.
25. What was the primary question being addressed in the Roy et. al. (2011) paper, what methods did they use and what were their primary conclusions?
- They wanted to see if any patterns emerge regarding the distribution and orientation of cytonemes in various cells: Found that cytonemes do orient towards the source of known morphogens, where they interact with them via their receptors.
- A second surprise finding: Each cytoneme has its own receptors, they do NOT co-express receptors
- Looked at eye and wing imaginal discs, which were isolated from larvae, then placed on depression in a slide to observe.
- Clones made that were then labeled with CD8:GFP (CD8 used to label the entire membrane, to identify ALL cytonemes present).
- Constructs made to express various morphogens, such as Dpp, Bnl,
- Constructs also made to tag various Morphogen receptors, such as thick vein (Tkv) harbored by Dpp, and breathless (Btl) the receptor for Bnl
- Using recombination, made EGFreceptor dominant negatives which KO all receptor function, and HeatShock and Spi: all led to loss of directionality and orientation with cytoneme
- 1. Tissue specific responses were seen with different morphogen expressions. Ie Dpp expression affects cytonemes in wing BLADE cells, but not in eye disc or ASP
- 2. Cytonemes with multiple receptors are found on the same cell, but each cytoneme only contains ONE type of receptor. Seen when Tkv (GFP) was labeled with Btl (mCherry), no co-localization observed on the cytonemes.
26. Compare & contrast primary cilia and cytonemes. Be sure to include information about structure, number per cell, and function.
- Primary cilia and cytonemes
- Both protrusions out from the cells, cytonemes being a type of filopodia, and primary cilia being composed of axonemes.
- Both long shaped, signals transmit to and from the tip of primary cilia via motor proteins along its MICROTUBULEs.
Signals with the cytonemes are recognized via various receptors found on cytonemes, which are composed of bundled parallel ACTIN filaments
- Number per cell?
- Both involved with cell movement and directionality.
- Both are involved in transmitting signals and sensory functions.
27. What are scaffold proteins, where are they found, and in what species were they first described?
Scaffold proteins: Structures that provide multiple binding sites for various responding proteins (kinases). They are found near the membrane of the cell. They were first found in yeast S. cerevisiae, where the scaffold Ste5 was studied.
28. What is/are the proposed function(s) of scaffold proteins in intracellular signaling processes?
- Scaffold proteins hold each of the kinases in close proximity so the signaling cascade can occur via phosphorylation of various kinases. Scaffold protein aids in:
- 1. Preventing inappropriate signaling responses don’t occur: Ie, if there are other proteins that Ras can activate, for example, the scaffold may limit the response because only specific mitogens can bind to the receptors. Makes SURE the appropriate proteins are activated to give appropriate response.
- 2. Prevents Phosphatase from acting: By having the proteins all congregate on the scaffold, they are no longer floating freely in the space around, therefore increasing their ability for signal to be transmitted BEFORE phosphatase inactivates the proteins.