The Overall Characteristic of the Microtubules - Essay Example

December 4 Microtubules are made up of monomer chains that have a helical shape. They arecomposed of subunits. They have a propensity for dynamic instability. Hence, they have a characteristic random growth nature. Various aspects spoil microtubules such as the rate of dehydrolisation. They play a key role in mitotic cell division and in the cell itself. By using indirect immunofluorescent labeling transient transfection of EGFP-Tubline/EGFP-CLIP-170 plasmid to epithelial cells lines and time-lapse microscop,to show that EB1 and CLIP-170 doesn’t bind only to plus end microtubules but also important in the MT organization and structural instability. Introduction The structure of Microtubule Microtubules are polymers that have elongated tube-like monomer chains. These chains are helical like. α-βtubulin subunits make up the helical tube that has 13 protofilaments that are aligned in a parallel way. The subunits enable the tube-like monomers to link with each other using vertical and lateral bonds. The α-tubulin domain bind to the β-tubulin domain forming these bonds. Hence, the mechanical strength of the microtubules is enhanced. The Microtubule Organizing Centre mediates the nucleation of the microtubules. It comprises a ring complex of γ-TURC and γ-tubulin (Atkinson, 2014, 5870). The specific ring complex serve as the template of the 13 subunits of the microtubules. Microtubules grow from the minus to the positive side. The minus side is located at the centrosome. The growth occurs towards the outer region to the cortex. Dynamics of the Microtubule The overall characteristic of the microtubules is the propensity for dynamic instability. Thus, the microtubules can shrink and grow randomly. The characteristic is associated with loss of the “GTC-cap. The loss is produced as new subunits are added. There is an ATP unit that is located in the β-tubulin part of the microtubules. The ATP unit goes through hydrolisation as the filament grows (Alieva, 2014, 670). There is a disadvantage to this growth. If the hydrolisation is more rapid than the rate of the addition of monomers, it compromises the microtubule. There is a GDP bound type of the β-tubulin that has a higher dissociation energy in comparison to the one that is bound by the ATP. It makes it more energetic for the microtubule de-polymerization. Hydrolisation enables the formation of ADP. It raises the likelihood of the microtubules being spoilt. They can be spoilt through reduction in the length and de-polymerization. The speed of spoilage of the microtubules is influenced by factors like proteins that bind along their length, and at their tips. Tao-1 is a protein that influences the mediation of the sped of spoilage of the microtubules. It is an important protein that controls plus end growth, spoilage, and stability of the microtubules. When activated, the protein leads to the de-stabilization of the microtubule plus-ends located at the cell cortex. These plus-ends are actin-rich components (Vasiliev & Samoylov, 2013, 39). Mediation is caused by this de-stabilization. The cell shape is deformed by the development of the protrusions due to the removal of the Tao-1 protein. It is phenotypically similar with the features of the SCAR knock-out cells. Hence, Tao-1 protein is an important constituent in crosstalk mediation among microtubules and cortical actin. The rate of spoilage of the microtubules is affected by the plus ends angle of the microtubules. The reason being that it results in the slow-down of the addition of new monomers onto the microtubule ends. A research comprising lengths of cells that were intrinsic shows that there was a faster rate of spoilage in microtubules that nucleated at angles that were perpendicular in relation to the cell boundaries. This is in contrast to the parallel aligned microtubules (Sambade et al, 2014, 1640). Microtubule Functions Microtubules are accountable for cell organelles positioning. They are also responsible for the intra-cellular transport. They determine the correct nucleus position during cell division. The determination is done with the aid of forces generated by their impact with the cell wall (Fishel & Dixit, 2013, 275). They are of importance to the cytoskeleton during mitosis. They form the spindle structures that align sister chromatids next to the metaphase plates (O’Rourke et al, 2014, 10). They determine the separation of sister chromatids during anaphase. Asymmetric forces generated by microtubules determine asymmetric cell division. The spindle is repositioned by the atral microtubules during symmetric division (Masoud et al, 2013, 250). Through their ability to produce force, they intrinsically control the length of the cell. Methods Cell culture The arpe-19 cells was trypsinised (5ml) and incubate at 37 c for about 5 minutes. An 8ml aliquot of media was added to the cell and centrifuged at 800 rpm for 5 minutes. Meanwhile, the cells were being counted with homocentemeter. Alcohol-sterilized forceps were used to transfer three coverslips into the base of a 6 well plate. About 150,000 cells were seeded into the 6 well plates and incubated at 37 0c overnight. Also, using the same procedure 75,000 cells were seeded in a 6 well plate and incubate at 37 A 8ml aliquot of media was added to the cell and centrifuged at 800 rpm for 5 meanwhile the cells was being counted with homocentemeter, alcohol-sterilized forceps were used to transfer three coverslips into the base of a 6 well plate. About 150,000 cells were seeded into the 6 well plates and incubate at 37 c overnight. Also, using the same procedure 75,000 cells was seeded in a 6 well plate and incubateD at 370c for 48 hours. The antibodies were mixed and incubated at the same time they are produced from different animals. For sequential labelling, the antibodies were incubated in first primary, followed by its secondary, then in 2nd primary, followed by its secondary for antibodies obtained from the same or closely related animals. The antibodies were fixed in cold methanol contained in a petridish. A plastic pipette was used to remove most of them followed by adding cold (-200C) 100% methanol and fixing the cells for 5 minutes. The cells were washed by first removing methanol into a waste container and then rehydrating them in PBS buffer with 1% serum for a minute. PBS containing 5% serum was used to block non-specific sites. A small drop of water was added to the bottom of box, followed by, placing nesco film on top. X 30µl drops of antibodies were added to the nesco film in addition to x 30µl of PBS with 1% serum. Each coverslip was drained by using forceps to place their edge on the filter paper. The filter paper was arranged around the nesco film. The secondary antibody was incubated in a moist chamber in the dark for 30m minutes. The solution was then washed to remove excess secondary antibodies in fresh PBS. 2 ml of DAPI solution per petridish was added and left for 5 minutes. The DAPI solution was removed followed by adding 2 ml of PBS. The procedure was repeated 3 times and left in PBS after the final wash. The content was mount in Hydromount + Dapco to allow viewing by a fluorescence/confocal microscope. Plasmid preparation and transfection The egfp-tublin/egfp-clip-170 was extracted using midi-prep according to the manufacture’s instruction. The bacteria cells were lysed and the isolated DNA was purified. THE ARPE-19 cells were grown to about 75% confluence and was then transfected with (….) ul EGFP-tubulin (2 ug) DNA and (…..)Ul clip=170 (2 ug) DNA, using a jetprime reagent according to the manufacture’s instruction, and incubated 37 c fir 4 hours before media was replaced. Result Function of EB1 and CLIP Figure showing function of EB1 As shown by the video, the accumulation of the CLIP was high at MT plus-ends in the control cell as compared to cells with depleted EB1. It implies that Clip accumulation depends on EB1 presence, especially with the acidic tail in question. The binding action of EB1 can be attributed to its structure, which consists of four-helix bundle and ta-dimeric parallel coiled coil (Nakamura et al, 2014, 1060). The type of structure associates with hydrophobic cavity, at its highly conserved surface patch, and a polar rim that adequately contributes to the binding of APC. The interaction, revealed by the structure, is branded the EB1-like motif. There is a possibility that the binding interaction of CLIP family depends on this. Intuitively, considering that CLIP experiences binding at the acidic tail of tubulin, it is also possible that the acidic tail of EB1 also provides the same condition allowing for binding. CLIP, acting as a plus-end tracking proteins functions in controlling dynamics of microtubule and presides over its interactions with the outside cellular components (Atkinson, 2014, 5870). However, as shown in the recorded images, in the absence of EB1 CLIP failed to track any of the ends. Instead, CLIP shows lattice diffusion that does not allow it to track. The mediating action of EB, in CLIP plus-end tracking, arises because it is able to diagnose the distinct lattice structure, thereby, probing for the tracking process. Growth traction for CLIP protein and speed Figure obtained for CLIP-170 time-lapse The tracking of CLIP protein and speed was used to explain the difference in the number of nucleation exhibited between the two ends. This is relevant, especially after considering that there was only an effect on the action formation and not on the influence the microtubules. Track Mate results: Mean comet speed = 0.364 μm/s (21.8 μm/min) Max comet speed = 0.577 μm/s (34.6 μm/min) Min comet speed = 0.210 μm/s (12.6 μm/min) Conclusion The work showed that the end tacking of microtubules plus-end with EB-1 and CLIP-17, could reveal the organization and structural dynamic of microtubules. However, although it is widely accepted that EB1 bind to the plus-ends of growing microtubules, its binding along the growing microtubules is still unclear, thus, further study could investigate exactly where EB1 bind along the microtubule. BACKGROUND: CLIP-170 CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. Work Cited http://www.ncbi.nlm.nih.gov/pubmed/15589150. Web. Read More