Question

4. Microtubules are critical to a number of cell processes including serving as a guide for the movement of vesicles and organelles to playing a critical role in mitosis that is responsible for the separation of the two daughter cells.

                  a. Challenge: You are studying the movement of a particular type of vesicle in a cell and want to determine if its movement is being mediated by microtubules and its associated molecular motors. How could you design an experiment to determine if microtubules are involved in this process?

                  b. What is the function of kinesin and dynein and how do their roles differ in axonal transport?

Answer 1

Answer"

Experimental design to study whether microtubules are involved in the movement of vesicles in a cell:

• In vitro assays are designed to study the involvement of motor protein and microtubules in the movement of cytoplasmic vesicle movements using video-enhanced microscopy.
• Initially Squid axons with a cell-free system are chosen for in vitro assays followed by use of a video camera to obtain images via detecting the small objects & movement to vesicles in living cells of squids.
• The plasma membrane should be removed from the cell using detergents followed by isolation of cytoplasmic extract.
• This has to be spread on the glass slide to detect the cellular kinesin, microtubule proteins binding to tubulin at plus ends & minus ends by gel filtration or SDS-PAGE, to promote vesicle movement finally results have to be compared toc control samples after gel filtration.
• Immunohistochemistry should be performed to examine the specific protein expression in the cells followed by imaging
• Total amount of tubulin proteins are assembled together in both control samples as well as kinesin heavy chain sample (KHC), it is profoundly due to existence of pallet fractions of tubulin (SDS-PAGE, and the gel was stained to ​reveal the position of tubulin).
• if there is no assembly of microtubule then it has observed in the supernatant solution during gel filtration thereby it is found that in presence of adenosine triphosphate the Kin I motor enabled depolymerization of microtubules.
• Here in this process it is observed considerably usage of adenosine triphosphate during experiment for the disassembly of microtubules.    
• Apply taxol to the suid axon durin the experiment, If there is no enhancement of GTPase using tubulin dimmers or even considerably using kin I because microtubules stabilization was completely associated with nonhydrolysable guanosine triphosphate (GTP) relatively than taxol.
• Thereby it is illustrated that the microtubule destabilization by Kin I is not associated with GTPase.

b):

• Microtubules are made of single tubulin protein and it has a dimer i.e. alpha-tubulin & beta-tubulin.
• These microtubules are going to grow unequally in which plus end is going to grow faster whereas minus end is going o grow slower.
• The alpha- tubulin and β-tubulin are going to bind with GTP to promote polymerization
• Microtubules are the main cytoskeletal filaments, which are going to promote the growth of spindle fiber during formation of metaplate using kinesin & dyenin motor proteins.
• Therefore, kinetochore is going to microtubules through plus ends & minus ends.
• Minus ends are going to shrink due to a dyenin whereas microtubules toward plus ends with growing due to kinesin protein.
• However, if at plus end catastrophin binding proteins are going to bind to initiate dynamic instability of the microtubules result in GTP hydrolysis at the plus ends.

Please Rate My Answer...........Thank..........u...