Question

  

Tethering Docking Fusion TICI 100 nm MOS SNARE . CONTACTOS -WOIC 8 TBC1D DOC Esri F FB SNAPS Muncit 2 Approach to plasma membrane T-SNARE Microtubule

1. Propose an experiment to identify actin filaments and microtubules in living cells.

2. Propose an experiment that could examine whether vesicles containing glut4 move along

   microtubules near the plasma membrane.

c. Draw out a dataset that would indicate that glut4 vesicles move on microtubules that are near the plasma membrane.

4. Draw out a dataset that would indicate that glut4 vesicles move by diffusion to reach the plasma membrane.

5. Draw out a dataset that would indicate that glut4 vesicles are tethered or are docked (as opposed to moving on cytoskeletons) at the plasma membrane.

6. Propose an experiment that would test whether microtubules are required for the tethering or docking the glut4 vesicle to the plasma membrane

7. Propose an experiment that would identify whether v-SNARE and t-SNARES have formed a complex (as when a vesicle docks or had just fused).

8. Draw a dataset that would indicate that a vesicle had docked or fused.

9. Draw a dataset that would indicate that a vesicle was tethered.

10. Propose an experiment in which one could distinguish between a docked vesicle and a vesicle that

    had fused.

Answer 1

A) Experiments to identify actin filaments:

i) Visualization of nuclear actin filaments with NLS fused detecting probes:

Actin probes fused to a NLS avoid signal interference from abundant cytoplasmic actin.

Require control of expression levels to not interfere with nuclei cytoplasmic shuttling.

Addition of NES motifs allows for dynamic shuttling and avoids artefacts induced by nuclear accumulation.

Physiological assembly of nuclear F actin transiently observed upon distinct stimuli.

ii) Live cell imaging using GFP-actin derivatives:

Tagging actin with GFP derivatives extensively used for live cell analysis.

GFP actin is suitable for FRAP(Flurescence Recovery After Photobleaching) approaches to study actin dynamics and turnover within a given cellular F-actin structure including the actin cortex or protrusions such as lamellipodia.

GFP actin has been proven to be highly useful to image actin functions in living cells or organism which is mammalian cells, yeast, drosophila and Dictyostelium discodeum.

iii) Imaging F actin structures and dynamics using actin binding domains:

There are several probes available to visualize endogenous actin dynamics in living cells. With the exception of SiR actin, these are based on genetically encoded actin binding domains (ABDs) that have been derived from yeast or human proteins, which are fused to GFP derivatives.

Most popular widely used tool is LifeAct suggested to be a universal marker of actin.

Except these there are Visualizing actin dynamics using actin directed nanobodies and Nuclear probes for monitoring F actin assembly.

Experiments to identify Microtubules:

i) Microtubule dynamic instability:

Microtubles are tubular filaments assembled from αβ tubulin heterodimers arranged head to tail in 13 helically arranged protofilaments.

Microtubles grow by the addition of GTP tubulin (green) to the ends.

GTP hydrolysis occurs when β tubulin is buried in the lattice.

Catastrophe, i.e. transition from shrinkage to growth is called rescue. Note that α non exchangeable GTP trapped at the intradimer interface.

ii) Microtuble dynamics measuremant invitro:

  Typical TIRF based assay to reconstituted and measure microtuble dynamics invitro in which a GMPCPP stabilized microtuble surface that nucleates a microtubles.

The seed and free tubulin that is labelled with different flurophores. Microtubule binding proteins or small molecules can be added and the binding as well as effect on Microtubule dynamics measures in this assay.