2. Microtubules are critical to a number of cell processes including serving as

Question

2. Microtubules are critical to a number of cell processes including serving as a guide for the movement of vesicles and organelles to being the key part of 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?

                c. Paclitaxel, otherwise known as Taxol or Taxotere, was discovered over 50 years ago as a result of an NCI-sponsored screening program. It is one of the most commonly used and effective chemotherapeutic drugs that is embedded in a number of chemotherapy programs to treat many types of cancers that include ovarian, melanoma, esophageal, bladder, prostate, breast, lung and others. In fact, it is listed by the World Health Organization List of Essential Medicines. How does it work and how does it differ from another commonly used breast cancer drug, Herceptin?

                        d. Opinion: (extracted from https://www.spglawfirm.com/defective-drugs/taxotere-permanent-hair-loss-lawsuit/?utm_source=PPC&utm_medium=TAXOTERE&utm_campaign=CID-715&gclid=CO3B_PqcutACFZJMDQodenMNzQ ). A federal lawsuit has been filed in the state of California in March, 2016 by a woman who blames her permanent hair loss on the popular breast cancer chemotherapy drug, Taxotere. Ami Dodson claims the drug manufacturer and corporate giant, Sanofi-Aventis, engaged in a fraudulent marketing scheme to drive sales while hiding this serious side effect of the drug. With prescriptions written for nearly 75% of all breast cancer patients, Taxotere has been noted as the most frequently used breast cancer drug in its class. Since 2004, drug sales have skyrocketed to about $1.4 billion annually for the company. Permanent hair loss is considered uncommon in chemotherapy treatment, as in most cases, hair grows back 3-6 months after treatment. Studies show, however, that with Taxotere there is an increased risk of permanent hair loss that can occur anywhere on the body. Ami Dodson, plaintiff, claims in a lawsuit filed in March that the company has known since possibly the late 1990’s that 9.2 percent of people who used the drug had experienced hair loss for a duration of more than 10 years. Dodson says these study results were deliberately hid from the public and instead the company kept the warning vague, stating instead that “hair generally grows back.” Assume that you are the judge and jury for this lawsuit. How might you rule and why? What additional information might you require from the lawyers to validate your decision on this lawsuit?

Explanation / Answer

Ques-1: 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?

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.

Ques-2. What is the function of kinesin and dynein and how do their roles differ in axonal transport?

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 -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

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